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Effects Of Acupuncture And Electroacupuncture On Immune Responses And Pulmonary Functions In Horses

Permanent Link: http://ufdc.ufl.edu/UFE0024888/00001

Material Information

Title: Effects Of Acupuncture And Electroacupuncture On Immune Responses And Pulmonary Functions In Horses
Physical Description: 1 online resource (220 p.)
Language: english
Creator: Tangjitjaroen, W
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: acupoint, acupuncture, airways, antiinflammation, antiinflammatory, artificial, bal, balf, bronchoalveolar, bronchoprovocation, disease, electroacupuncture, endotracheal, equine, expiration, fev0, fev1, forced, fvc, histamine, horse, horses, iad, immune, inflammation, inspiration, lung, medicine, mef25, mef50, mef75, neutrophil, novel, pulmonary, rao, respiratory, ros, spaopd, tcvm, tlc, tnf, veterinary, volume
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Using acupuncture (AC) and electroacupuncture (EA) as alternative therapies to conventional equine medical practice is increasing worldwide. The benefit of these therapies for treating chronic musculoskeletal disorders such as pain in the thoracolumbar area is well documented and seems to be superior to that of conventional treatment alone. Acupuncture and EA also are used for treating other medical problems such as gastrointestinal, ophthalmic, and respiratory disorders. However, modern scientific evidence supporting their use for treating these diseases in horses is limited. This study investigates the effects of AC and EA on immune responses and pulmonary functions of Thoroughbred horses. These two topics were chosen because a healthy pulmonary system is vital to improving athletic performance, and inflammation of the lower airways occurs commonly in horses. Several forms of inflammation of the lower airways have been described, including inflammatory airway disease (IAD), recurrent airway obstruction (RAO), and summer pasture associated obstructive pulmonary disease (SPAOPD). These diseases are thought to be caused by dysregulation of immune responses, and investigation of the effects of AC an EA on the immune system might help explain how AC and EA contributes to treatment of these diseases. The initial investigation compared effects of AC and EA at acupoints LI-4, LI-11, and GV-14 on immune functions. Results indicated that only EA significantly induced anti-inflammation as demonstrated by in vitro suppression of TNF-? production in antigen-stimulated whole blood. Electroacupuncture at acupoints commonly used for treating equine chronic respiratory diseases (GV-14, CV-22, BL-13, Ding-chuan, Fei-men, Fei-pan, and Fei-shu) produced similar results. This in vitro anti-inflammation was likely governed by modulation of a cellular component of the innate immune system by altering the production of inflammatory cytokine upstream of the inflammatory cascade. Effects of EA on pulmonary functions were investigated using the rapid partial forced expiration maneuver. Results showed that EA produced no significant change in pulmonary functions in clinically normal horses. The rapid partial forced expiration maneuver is an emerging technique for measuring biomechanical properties of the lung. Additional studies using this technique in horses of different breeds, ages, body weights, and horses with known lung disease are needed.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by W Tangjitjaroen.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Colahan, Patrick T.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024888:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024888/00001

Material Information

Title: Effects Of Acupuncture And Electroacupuncture On Immune Responses And Pulmonary Functions In Horses
Physical Description: 1 online resource (220 p.)
Language: english
Creator: Tangjitjaroen, W
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: acupoint, acupuncture, airways, antiinflammation, antiinflammatory, artificial, bal, balf, bronchoalveolar, bronchoprovocation, disease, electroacupuncture, endotracheal, equine, expiration, fev0, fev1, forced, fvc, histamine, horse, horses, iad, immune, inflammation, inspiration, lung, medicine, mef25, mef50, mef75, neutrophil, novel, pulmonary, rao, respiratory, ros, spaopd, tcvm, tlc, tnf, veterinary, volume
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Using acupuncture (AC) and electroacupuncture (EA) as alternative therapies to conventional equine medical practice is increasing worldwide. The benefit of these therapies for treating chronic musculoskeletal disorders such as pain in the thoracolumbar area is well documented and seems to be superior to that of conventional treatment alone. Acupuncture and EA also are used for treating other medical problems such as gastrointestinal, ophthalmic, and respiratory disorders. However, modern scientific evidence supporting their use for treating these diseases in horses is limited. This study investigates the effects of AC and EA on immune responses and pulmonary functions of Thoroughbred horses. These two topics were chosen because a healthy pulmonary system is vital to improving athletic performance, and inflammation of the lower airways occurs commonly in horses. Several forms of inflammation of the lower airways have been described, including inflammatory airway disease (IAD), recurrent airway obstruction (RAO), and summer pasture associated obstructive pulmonary disease (SPAOPD). These diseases are thought to be caused by dysregulation of immune responses, and investigation of the effects of AC an EA on the immune system might help explain how AC and EA contributes to treatment of these diseases. The initial investigation compared effects of AC and EA at acupoints LI-4, LI-11, and GV-14 on immune functions. Results indicated that only EA significantly induced anti-inflammation as demonstrated by in vitro suppression of TNF-? production in antigen-stimulated whole blood. Electroacupuncture at acupoints commonly used for treating equine chronic respiratory diseases (GV-14, CV-22, BL-13, Ding-chuan, Fei-men, Fei-pan, and Fei-shu) produced similar results. This in vitro anti-inflammation was likely governed by modulation of a cellular component of the innate immune system by altering the production of inflammatory cytokine upstream of the inflammatory cascade. Effects of EA on pulmonary functions were investigated using the rapid partial forced expiration maneuver. Results showed that EA produced no significant change in pulmonary functions in clinically normal horses. The rapid partial forced expiration maneuver is an emerging technique for measuring biomechanical properties of the lung. Additional studies using this technique in horses of different breeds, ages, body weights, and horses with known lung disease are needed.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by W Tangjitjaroen.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Colahan, Patrick T.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024888:00001


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EFFECTS OF ACUPUNCTURE AND ELECTROACUPUNCTURE ON IMMUNE
RESPONSES AND PULMONARY FUNCTIONS IN HORSES




















By

TANGJITJAROEN WEERAPONGSE


A DIS SERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA INT PARTIAL FULFILLMENT
OF THE REQUIREMENT S FOR THE DEGREE OF
DOCTOR OF PHILO SOPHY

UNIVERSITY OF FLORIDA

2009


































O 2009 Tangjitj aroen Weerapongse

































To my Mum and my Dad
To all who nurtured my academic interest and have made this mile stone possible









ACKNOWLEDGMENT S

Completing the experiments and writing this dissertation has been both academically

challenging and rewarding. Without the inspiration, support, encouragement, and guidance of the

following people, this dissertation would have never have been completed. I wish to express my

deepest gratitude to the following.

At the very first, I gratefully acknowledge the Royal Thai Government for financial

support of my graduate study. Thank you to Dr. Chamnan Trinarong, Chair of the Equine Clinic

at the Faculty of Veterinary Medicine, Chiang Mai University, for giving me an extraordinary

opportunity to pursue overseas graduate study.

I wish to deeply thank Professor Dr. Patrick T. Colahan for accepting me as his graduate

student. His academic and professional commitment inspired and motivated me. Without his

patient academic guidance and support, this dissertation could not have been completed.

I sincerely thank Dr. Huisheng Xie and his family for inspiration, support, and steady

encouragement. His excellence in the traditional Chinese veterinary medical profession and

teaching skills are extraordinary. Through his kindheartedness, I shared a small part of his

wisdom, and I am grateful.

I also sincerely thank Dr. David J. Hurley for providing a foundation and guidance in

immunological assay. The importance of his mentoring in the immunological aspect of my study

made his importance to the completion of my dissertation second to no one.

I thank Dr. Richard D. Johnson for motivating me to achieve high academic standards,

and to have given me the opportunity to learn equine anatomy in his class. A thorough

knowledge of anatomy has served as a foundation for every aspect of my studies.

I thank Dr. Daiqing Liao for accepting the invitation to join my committee in the last

period of my program. Even though I did not have many opportunities to interact with him, I










greatly appreciate his taking on a responsibility that added to his already full-time academic

work.

I thank Professor Dr. James H. Jones, Chair of the Department of Surgical and

Radiological Science at the School of Veterinary Medicine, UC Davis, for his guidance and

excellent technical support in helping me build the forced expiration device. It is a marvel for

me, who has no engineering background, to have built this machine. His guidance and input in

the engineering aspect of the forced expiration device made his contribution to the completion of

my dissertation second to no one.

I thank Dr. Steve Giguere for his guidance in the field of equine lower airway

inflammatory disease, and allowing me to used the inductance plethysmography and

pneumotachograpgy machine and other facilities of the Internal Medicine Clinic in my research.

I also sincerely thank Brett Rice, Stacie Atria, Jennifer A. Claflin, Ted Broome, and other

members of the staff of the Equine Performance Laboratory at the University of Florida. The

assistance I have received from them was invaluable. They were essential to every research

proj ect I accompli shed.

I gratefully thank my family; Dad and Mum for nourishing my life and for encouraging

me to achieve this academic work, and my sisters and brother who cared for my Dad and Mum

while I am studied overseas.

Last but not least, I sincerely thank everyone who contributed to my research but whom I

have forgotten to mention in thi s acknowledgement.











TABLE OF CONTENTS
IM Le

ACKNOWLEDGMENT S ........._._ ...... .... ............... 4...

LIST OF TABLES................................. 10

LIST OF FIGURE S ............... .................... 14

LIST OF ABBREVIATIONS ........._._ ...... .... ............... 16..

CHAPTER

1 HISTORY OF TRADITIONAL CHINESE VETERINARY MEDICINE AND A
REVIEW OF MODERN SCIENTIFIC RESEARCH ON EQUINE ACUPUNCTURE....... 23

Introduction ............... ..... ...... .. .. ... ............... 23
History of Traditional Chinese Medicine and Traditional Chinese Veterinary
M medicine ............... .. ..... .. ... ..... . ............. 23
Origin of Traditional Chinese Veterinary Medicine and Equine Acupuncture
Research................ ... ............. 26
Equine Acupuncture Research ........._..._... .. ...... ...._... ............. ... ...........2
Research in the Field of Analgesia and Musculoskeletal Pain ................. ................ ...29
Acupuncture for Managing Ocular Problems ................. ...............34........... ...
Acupuncture Research in Gastrointestinal Disorders ................. .......... ................3 6
Acupuncture Research in Respiratory Disorders ................. ...............39...............
Acupuncture Research in Other Medical Problems ............... ...............41....
AC Research in Reproductive Sy stem ............... ... .... ......... ...............42. ...
Research in Diagnostic Potential of Acupoints and Meridians ............... ............._..46
Sum m ary ................. ................. 48........ ....

2 MODULATION OF IMMUNOLOGICAL RESPONSE BY ACUPUNCTURE AND
ELECTROACUPUNCTURE AT LI-4, LI-11, AND GV-14 IN CLINICALLY
NORMAL HORSE S. .............. .................... 5 1

Introduction ............... .................... 5 1
M ethod s ............... .................... 5 2
Anim al ......................... .... ... ......... 52
Acupuncture and Electroacupuncture................ .......... 53
Source of Fungal Anti gens ........._._.._......_.. ..............._ 53..
Leukocyte Separation ............... ..... ......................... 54
Reactive Oxygen Species Generation of Neutrophil ................. ...........................5 5
Heparinized-bl ood Stimulation.................. ................. 5
Equine Tumor Necrosi s Factor Alpha (TNF-oc) Assay ........................... ...............5 7
Immunoglobulin Assay ............... .................... 58
Statistical Analysis................ ............... 59











Results ................. ................. 59..............
A nim al s ....................... ...... ... .. ........... 59
Acupuncture and Electroacupuncture Procedures .................. ............ ........59
Homogeneity of Samples Before Electroacupuncture and Acupuncture ................... ......60
Plasma Immunoglobulins ................. ............... ... ................60.....
Reactive Oxygen Species Generation of Neutrophil .................. .. .... ........ ...................6 1
Heparinized-blood Stimulation and Equine Tumor Necrosis Factor Alpha (TNF-oc)
A ssay ............ ..... ._ ...............62....
Discussion............... ...............63

3 COMPARISON OF INDUCTANCE PLETHYSMOGRAPHY AND
PNEUMOTACHOGRAPHY AND THE RAPID PARTIAL FORCED EXPIRATION
MANEUVER FOR DIAGNOSIS OF EQUINE LOWER AIRWAY INFLAMMATORY
DISEASE. ................................... 77


Introduction ............... .................... 77
M ethod s ............... .................... 8 0
Subj ect ........._...... ................ 8..... 0....
Study D esign ................ .... .... ...... ......... ........... ..............8
Histamine Bronchoprovocation and Respiratory Inductance Plethysmography and
Pneomotachography (Open PlethThf) ...._.._.._ ..... .._._. ....._.._............8
Bronchoalveolar Lavage. .................... ............... 8 2
Rapid Partial Forced Expiration Maneuver................. ............... 84
Negative pressure generator and vacuum reservoir ............... ....................85
System for artificial inspiration ........._._._ ...._. ............... 86..
Airflow measurement apparatus................ ............... 87
Airflow direction control system................ ................ 89
Data Acquisition sy stem ............... .... .... ............. 90
Animal Preparation for rapid partial forced expiration. maneuver. .........._.... ..............90
Induction of Rapid Partial Forced Expiration ................. ...............91...............
Calculation of the Pulmonary Function Test Parameters ................. .... ... ................... 92
Reset the calibrated pressure differential of LFE with pressure/temperature
corrected flow rate ................. ... ........... ............ ............9
Calculation of expiratory volume from airflow data ............... ....................93
Determination of pulmonary function test parameters ................ ... .. ................93
Determination of suitable negative pressure for the rapid partial forced
expiration maneuver ................. ................. 93..............
Statistical Analy si s................ ............... 93
Results ................. ................ 94..._._. ....
Subj ect ........._.... ... .... .._. .. ......_. ... .. ... ... ...............9
Histamine Bronchoprovocation and Respiratory Inductance Plethysmography and
Pneumotachography (Open PlethTM> f)............... ................. 95...............
Broncho-alveolar Lavage ............ ................... .... ............... 96....
Rapid Partial Forced Expiration (RP-FE) Maneuver ................. ................. ........ 97
Discussion............... ............... 99












4 EFFECTS OF ELECTRO ACUPUNCTURE ON PULMONARY FUNCTION AND
IMMUNE RESPONSE IN HORSES ............... ....................121


Introduction and Back ground ............... .................... 121
Material s and Method s............... ................ 127
A nim als ............... .................... 127
Electroacupuncture ............... .................... 128
Data and Sample Collection............... ............... 129
Rapid Partial Forced Expiration Maneuver................ ............... 130
Arterial Blood Gas Analysis During RP-FE ........................... ........133
Calculation of the Pulmonary Function Test Parameters ................. ......___............13 3
BAL f Collection and Preparation.............. ........ .................. .. ................. .....13
Tumor Necrosis Factor Alpha (TNF-u) Production of Whole Blood and TNF-a
A s say ............ _... .... ._ .............. 13 4...
Immunoglobulin Assay ............... .................... 135
Cortisol As say ................. ................. 13......_. 5....
Stati sti cal Analy si s................ ............... 13 6
Results ................. ................. 137........ .....

Subj ect ................ .......... ................ 137....
EA and Sham Treatments................ ... .... ................. 137
Complete Blood Count and Other Hematological Parameters ................... ............... 138
Broncho-alveolar Lavage and BALf Cytology ................. ...............139........... ...
Immunoglobulins ........._....... .... .. .._.._ ..... ............... 140_
TNF-a Production of Whole Blood Stimulation ......____ ..... ... .__ ..........__..... 141

Rapid Partial Forced Expiration Maneuver................ ............... 142
Arterial Blood Gas Analysi s During RP-FE ......___. ....... __. .....__..........4
Serum Corti sol ............... .................... 144
Di scu ssi on ................ ................. 145........ ...


5 SUMMARY AND CONCLUSIONS ............... ....................182


Introduction ............... .......... ... .. .. .. ........ .. ......... 182
Histamine Bronchoprovocation as a Test for Equine Airways Hyper-sensitivity ........._......183
Rapi d Partial Forced Expiration for T testing Equine Pulmonary Functi on. ........._.._...............18 5
Acupuncture and Electroacupuncture Efects on Equine Immune Response and
Pulmonary Function ............... .......... ... .. .... .. ........190
Improved Mucociliary Action of the Airway Epithelium ...........__....... ..._.._. ......... 1 91
Reduced Airway and Pulmonary Tissue Inflammation ............... .....................192
Activation of Cholinergic Anti-inflammation ............... .................... 193
Alteration of Immune Responses................ ............... 193
Modulati on of Autonomi c Nervous Sy stems ................. .. ........... .... ............. ....19
Alteration in the Peripheral Sensory Input From Inflamed Pulmonary Tissues............ 196
Other M echani sm s........._.. ..... ._ ............... 197...
Summary ........._.. ..... ._ ............... 198....













LIST OF REFERENCES ........._...... ...............200._.._. ......


BIOGRAPHICAL SKETCH ........._...... ................ 220.._... .....










LIST OF TABLES


Table page

1-1 Treatment efficacy of AC and its related technique in equine thoracolumbar pain. .........50

1-2 Treatment efficacy of AC and its related techniques in equine lameness originating
from j oint problems. ........._._.._ ...._... ...............50...

2-1 Anatomical location of acupoints and treatment indications. ........._._ ..... ..._..__.........70

2-2 Demographic information on horses in the EA and AC groups. ........._..... ........._.......70

2-3 MeantSE concentration of immunoglobulin isotypes (x105 ng/ml) from pre-
treatment samples of EA and AC groups. .............. .....................70

2-4 MeantSE neutrophil-ROS response ratio from pre-treatment samples of EA and AC
groups s ................. ................. 7......... 1.....

2-5 MeantSE TNF-a concentrations (pg/ml) from pre-treatment samples of EA and AC
groups. ........._... ...... ___ ...............72.....

2-6 Mann-Whitney Rank Sum test statistics between-group comparisons of pre-treatment
samples in EA and AC groups. .............. .....................73

2-7 Meanf SE immunoglobulin isotype concentrations (x105 ng/ml) of pre- and post- EA
and AC treatments, and test statistics of within-group comparisons. .............. .... ........._... 73

2-8 Meanf SD neutrophil-RO S response ratios of pre- and post- EA and AC treatments,
and test stati stics of within-group compari sons. ................. ...............74..............

2-9 MeanfSD TNF-a concentration (in pg/ml) of pre- and post- EA and AC treatments,
and test statistics of within-group comparisons. .............. .....................75

2-10 Mann-Whitney Rank Sum test statistics for between-group comparisons of post-
treatment data in EA and AC groups. .............. .................... 76

2-1 1 Cutaneous and muscle innervations of acupoints being stimulated. .............. ...............76

3-1 Interpretation of histamine concentrations that cause 3 5% increase in delta flow
(modified from operation manual of Open PlethTIL System). .............. .....................114

3-2 Histamine concentration causing PC 3 5 delta flow and their interpretations from the 1 st
and the 2nd HB tests. ........................... ........114

3-3 Descriptive statistics of the degree of airway hyper-sensitivity based on results from
the 1st and 2nd tests of hi stamine bronchoprovocation. ..........___......___ ..............1 15










3-4 P-values from comparisons of the HB results between the 1st and 2nd tests and their
correlations. .............. .................... 115

3-5 MeantSD of BALf cytology results of the 1st and 2nd HB test and p-value of
Wilcoxon Signed Rank test and Correlations between the tests. .............. ....................115

3-6 MeantSD and Mann-Whitney U statistics of BALf cytology from horses with hyper-
sensitive and normal airways................ ................116

3-7 MeantSD of PFTPs derived from RP-FE at negative pressures of 25, 50, 75, 1 00,
125, 150, 200, and 250 Torr. ........................... ........117

3-8 Mean +SD of PFTPs derived from the 1st and the 2nd RP-FE, when 150, 200, and 250
Torr were used to induced RP-FE. ........................... ........118

3-9 Wilcoxon Signed Rank test statistics (p-value) of pulmonary function test parameters
(PFTPs) from the 1st and the 2nd RP-FE maneuver at 150, 200, and 250 Torr. ..............119

3-10 Meant SD of percentage of BALf neutrophils of horses with low % Neu and horses
with high % Neu, and Mann-Whitney U test statistics. .................. ................119

3-1 1 Meant SD of PFTPs of horses with low percentage of BALf neutrophils and horses
with high percentage of BALf neutrophil and Mann-Whitney U test stati stics..............__120

4-1 Anatomical location of acupoints, their Western medical indication, needle size and
method of insertion. .............. .................... 164

4-2 Numbers of horses categorized by degree of reaction to EA and to sham treatments
in the 1 st and 2nd trials prior to eliminating the horse that did not accept EA
treatment and horses receiving NSAID s. ....._ .....___ ........__ ..........16

4-3 P-values from the Type III sum of squares test on main effects and their interactions
for white blood cell indices. .............. .................... 165

4-4 MeantSE white blood cell indices, by treatment and sampling time. Normal
reference values in parentheses. .............. .................... 166

4-5 P-values from pairwi se compari sons of white blood cell indices, by treatment and
sampling tim e. .............. .................... 167

4-6 P-values from the Type III sum of squares test on main effects and their interactions
for red blood cell indices. .............. .................... 167

4-7 MeantSE red blood cell indices, by treatment and sampling time. Reference values
in parentheses. .............. .................... 168

4-8 P-values from pairwise comparisons of the red blood cell indices, by treatment and
sampling tim e. .............. .................... 169










4-9 Meant SD and range (in parenthesi s) of percentages of recovered B ALf and
percentages of ELF in BALf samples determined by urea dilution technique. ..............169

4-10 P-values from the Type III sum of squares test on main effects and their interactions
for broncho-alveolar lavage fluid cytological parameters. ................ ...................170

4-1 1 MeantSE ELF-corrected TNC and differential counts of BALf cells, by treatment
and sampling time. .............. .................... 170

4-12 P-values from paired samples t-test of BALf cytological parameters by treatment........ 171

4-13 P-values from the Type III sum of squares test on the main effects and their
interactions for plasma concentration of immunoglobulin isotypes. .............. .................171

4-14 MeantSE concentrations of plasma immunoglobulin isotypes (x105 ng/ml), by,
treatment and sampling time. .............. .................... 171

4-15 P-values from pairwise comparisons of concentration of plasma immunoglobulin
isotypes, by treatment and sampling time. .......................... ......... ........... 7

4-16 P-values from the Type III sum of squares test on main effects and their interactions
for concentration of immunoglobulin isotypes in ELF-corrected BALf ..........................172

4-17 Meanf SE concentrations of immunoglobulin i sotypes in ELF-corrected BALf (x1 05
ng/ml), by treatment and sampling time. ........................... ........172

4-18 P-values from pairwise comparisons for concentrations of immunoglobulin isotypes
in ELF -corrected BALf ................. ................. 173........ ....

4-19 P-values from the Type III sum of squares test on main effects and their interactions
for TNF-a production from stimulated whole blood by stimulants. .............. .................173

4-20 MeantSE TNF-a concentrations in whole blood after stimulation, by treatment and
sampling time.. ............. ..................... 174

4-21 P-values from pairwise comparisons of TNF-a concentrations in whole blood after
stimulation, by treatment. .............. .................... 175

4-22 P-values from the Type III sum of squares test on the main effects and their
interactions for FEVx, F VC, and PEF. .............. .................... 176

4-23 P-values from the Type III sum of squares test on the main effects and their
interaction for MEFx% and FEVx/FVC ratio. ........................... ........176

4-24 MeantSE FEVx, FVC, and PEF obtained by the rapid partial forced expiration
maneuver, by treatment and sampling time. ........................... ........177










4-25 Mean+SE MEFx% and FEVx/FVC ratio obtained by the rapid partial forced
expiration maneuver, by treatment and sampling time. .................. ................178

4-26 P-values from pairwise comparisons of the FEVx, FVC, and PEF obtained by the
rapid partial forced expiration maneuver. .....__.....___ ..........._ ............ 7

4-27 P-values from pairwise comparisons of the MEFx% and FEVx/FVC ratio obtained
by the rapid partial forced expiration maneuver. .............. .....................179

4-28 P-values from the Type III sum of squares test on main effects and their interactions
for serum cortisol concentration ................. ................. 180........ ...

4-29 Mean+ SE concentrations of serum cortisol, by treatment and sampling time. ................ 180

4-3 0 Details of cutaneous and muscle innervations at acupoints used in thi s study. ............... 18 1

5-1 Acupoints commonly used to treat chronic respiratory diseases of horses. ................... .. 199










LIST OF FIGURES


FiMr page

3-1 Variable transformer. .............. .................... 107

3-2 Diagram of device setup to perform rapid partial forced expiration maneuver in
horses and component symbols. ................................... 108

3-3 Mouth gag made from an 8" x 1 1/2" NPT PVC pipe. Hand guard made from black
rubber curry comb. .............. .................... 109

3-4 Flow-volume loops derived from average flow data and volume data of horses with
normal airways and horses with hyper-sensitive airway. ................. ...................110

3-5 Relationship of airflow rates measured by NIST-calibrated mass flow element
(MFE) and differential pressure generated before and after rapid partial forced
expiration (RP-FE). .............. .................... 110

3-6 Example of data acqui sition window of Windaq Pro+ software. ........._._. .........._._.....11 1

3-7 Example of forced expiratory flow rates generated by different negative pressures....... 112

3-8 Example of flow-volume loops generated by different negative pressures. ...................1 12

3-9 Example of flow-volume loops generated by negative pressures at 150, 200, and 250
Torr. ........... ..... .._ .............. 113...

3-10 Flow-volume loops derived from average flow data and volume data of horses with
low percentages of BALf neutrophils and horses with high percentages of BALf
neutrophil s. .............. .................... 113

4-1 Electroacupuncture ....._. ................. ................. 152....

4-2 Sham electroacupuncture. ........................... ........153

4-3 A pressure regulator and a set point regulator installed on the manifold of the
artificial inspiration system. .............. .....................154

4-4 Solenoid valve used for controlling the air from the air blower in the artificial
inspiration system and for isolating the LFE from the artificial respiration manifold
and the negative pressure reservoir. ........................... ........155

4-5 Diagram of the rapid partial forced expiration apparatus. ................ ..................156

4-6 Component symbol s. ........._.__........__. ............... 157..

4-7 Apparatus setup for laminar flow element (LFE) calibration using NIST-traceable
mass flow element (MFE). .............. .................... 158










4-8 The 12.829-liter syringe used for testing an accuracy of integrated airflow volume
compare with a 60 ml disposable syringe. ........................... ........159

4-9 Linear relationship of airflow rate to AP before RP-FE and after RP-FE. .............._........160

4-10 MeantSD arterial blood pH during RP-FE maneuver (data were obtained from 8
horses). .............. .................... 160

4-11 MeantSD arterial blood pCO2 during RP-FE maneuver (data were obtained from 8
hor se s). .............. .................... 16 1

4-12 MeantSD arterial blood pO2 during RP-FE maneuver (data were obtained from 8
hor se s). .............. .................... 16 1

4-13 MeantSD arterial blood HCO3 during RP-FE maneuver (data were obtained from 8
horses). .............. .................... 162

4-14 Mean+SD TNF-oc production in electroacupuncture (EA) and sham-EA (sham)
groups when whole blood was stimulated with Zymosan. ................ ...................162

4-15 Airflow rates from five artificial forced expirations (FE) during rapid partial forced
expiration maneuvers on one horse in 20 July 2008 ................. .............................163

4-16 Flow-volume loops from five artificial forced expirations (FE) during rapid partial
forced expiration maneuvers on one horse in 20 July 2008.. .............. .....................163









LIST OF ABBREVIATIONS

OC Degree Celsius

r Fluid viscosity

TI Mathematical constant (approximately 3.141592654)

AP Pressure differential

ABTS 2,2'-Azi no-bi s(3 -Ethylb enzthiazoli ne-6 -Sulfonic Aci d)

AC Acupuncture

ACTH Adrenocorticotropic hormone

AFU Arbitrary fluorescent units

AID Airway inflammatory disease

AquA Aquaacupuncture

AurA Auricular acupuncture

AVMA American Veterinary Medical Association

AVMA The American Veterinary Medical Association

BAL Broncho-alveolar lavage

BALf Broncho-alveolar lavage fluid

B.C.E. Before current era

BL Bladder meridian

BP Barometric pressure

BSA Bovine serum albumin

BT Body temperature

CA Aspergillus fumiga~tus cellular antigen system

C.E. Current era

CE Aspergillus fumiga~tus culture filtrate antigen

CFA Complete Freund' s adjuvant









cm Centimeter

cm H20 Centimeter of water

CNS Central nervous system

COPD Chronic obstructive pulmonary disease

CRH Corticoid releasing hormone

CSF Cerebrospinal fluid

CPT Cutaneous pain threshold

CV Conception vessel meridian

DNIC Diffuse noxious inhibitory control

EA Electroacupuncture

EHVl Equine herpes virus type 1

ELISA Enzyme-linked immunosorbent assay

ELF Epithelial lining fluid

ET Endotracheal

FE Forced expiration

FEV Forced expiratory volume

FEVx Forced expiratory volume at x seconds (such as FEV0.5, FEV1.0)

FSH Follicle stimulating hormone

FVC Forced vital capacity

FV loop Flow volume loop

GB Gall bladder meridian

GDNF Glial cell line-derived neurotrophil factor

GnRH Gonadotrophin releasing hormone

GV Governing vessel meridian

HA Hemoacupuncture









HB Histamine bronchoprovocation

HD Histamine diphosphate

HPOA Hypothalami c-pituitary ovarian axi s

HWRI Hoof withdrawal reflex latency

Hz Hertz

ID Internal diameter

IVAS International Veterinary acupuncture Society

IL-10 Interleukin 10

EL-18 Interleukin 18

EL-1B Interleukin 1 beta

IL-6 Interleukin 6

INOS Nitric oxide synthase

IVAS International Veterinary Acupuncture Society

kg Kilogram

KID Kidney meridian

L Length of the pipe

LAC Laser acupuncture

LAurA Laser auricular acupuncture

LFE Laminar flow element

LH Lutnizing hormone

LI Large intestine meridian

LIV Liver meridian

LPS Lipopolysaccharide

LU Lung meridian

mA Milliampere









MEFx%

MFE

mg

ml

mm

mRNA

NAA

NF-cB

NIST

nm

NPT

NSAIDs

NSC

PBS

PC

PC3 5 delta flow


pCO2

PEF

PFTPs

PMA

pO2

PVC

Q

r

RAO


Expiratory flow rate when x% of forced vital capacity has been expired

Mass flow element

Milligram

Milliliter

Millimeter

Messenger ribonucleic acid

National Acupuncture association

Nuclear factor kappa B

National institute of standard technique

Nanometer

National pipe thread

Non-steroidal anti-inflammatory drugs

Neuronal stem cell

Phosphate buffered saline

Pericardium meridian

Concentration of histamine that results in a 3 5% reduction in dynamic
compliance of the airway

Partial pressure of carbon dioxide

Peak expiratory flow

Pulmonary function test parameters

Phorbal 12-myristate 13 acetate

Partial pressure of oxygen

Polyvinylchloride

Volumetric flow rate

Radiu s

Recurrent airway obstruction









RFC Relative centrifugal force

ROS Reactive oxygen species

RP-FE Rapid partial forced expiration

RPM Round per minute

SI Small intestine meridian

SLPM Standard liter per minute

SP Spleen meridian

SPAOPD Summer pasture associated obstructive pulmonary disease

ST Stomach meridian

TCM Traditional Chinese medicine

TCVM Traditional veterinary medicine

TH Triple heater meridian

TLC Total lung capacity

TMB 3,3',5,5 '-tetramethylbenzidine

TNF-oc Tumor necrosis factor alpha

WHO World Health Organization

Zym Zymosan









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

EFFECTS OF ACUPUNCTURE AND ELECTROACUPUNCTURE ON IMMUNE
RESPONSES AND PULMONARY FUNCTIONS IN HORSES

By

Tangj itj aroen Weerapongse

August 2009

Chair: Patrick T. Colahan
Major: Veterinary Medical Sciences

Using acupuncture (AC) and electroacupuncture (EA) as alternative therapies to

conventional equine medical practice is increasing worldwide. The benefit of these therapies for

treating chronic musculoskeletal disorders such as pain in the thoracolumbar area is well

documented and seems to be superior to that of conventional treatment alone. Acupuncture and

EA also are used for treating other medical problems such as gastrointestinal, ophthalmic, and

respiratory disorders. However, modern scientific evidence supporting their use for treating these

diseases in horses is limited.

This study investigates the effects of AC and EA on immune responses and pulmonary

functions of Thoroughbred horses. These two topics were chosen because a healthy pulmonary

system is vital to improving athletic performance, and inflammation of the lower airways occurs

commonly in horses. Several forms of inflammation of the lower airways have been described,

including inflammatory airway disease (IAD), recurrent airway obstruction (RAO), and summer

pasture associated obstructive pulmonary disease (SPAOPD). These diseases are thought to be

caused by dysregulation of immune responses, and investigation of the effects of AC an EA on

the immune system might help explain how AC and EA contributes to treatment of these

diseases.









The initial investigation compared effects of AC and EA at acupoints LI-4, LI-11i, and

GV-14 on immune functions. Results indicated that only EA significantly induced anti-

inflammation as demonstrated by in vitro suppression of TNF-oc production in antigen-stimulated

whole blood. Electroacupuncture at acupoints commonly used for treating equine chronic

respiratory diseases (GV-14, CV-22, BL-13, Ding-chuan, Fei-men, Fei-pan, and Fei-shu)

produced similar results. This in vitro anti-inflammation was likely governed by modulation of a

cellular component of the innate immune system by altering the production of inflammatory

cytokine upstream of the inflammatory cascade.

Effects of EA on pulmonary functions were investigated using the rapid partial forced

expiration maneuver. Results showed that EA produced no significant change in pulmonary

functions in clinically normal horses.

The rapid partial forced expiration maneuver is an emerging technique for measuring

biomechanical properties of the lung. Additional studies using this technique in horses of

different breeds, ages, body weights, and horses with known lung disease are needed.









CHAPTER 1
HISTORY OF TRADITIONAL CHINESE VETERINARY MEDICINE AND A REVIEW OF
MODERN SCIENTIFIC RESEARCH ON EQUINE ACUPUNCTURE

Introduction

History of Traditional Chinese Medicine and Traditional Chinese Veterinary Medicine

Acupuncture (AC), a branch of traditional Chinese medicine (TCM), has been practiced

worldwide for thousands of year. Its clinical benefits have been demonstrated in both human and

veterinary medicines. It is clear that AC originated in ancient China, as the earliest document of

AC has been found as part of archeological explorations of very early Imperial sites within

modern China. These materials include the most important TCM manuscript "Huangdi Neifing."

which also i s known as The Inner Canon of Huangdi or Emperor Huang' s Inner Canon. Huangdi

Neifing contains two texts presented in a question and answer format between Huangdi (Emperor

Huang) and six of his medical ministers.l The first text, Su~wen (basic question), contains the

theoretical foundation of TCM and a method of diagnosing diseases. The second text, Lingshu

(spiritual pivot), explains AC treatment methods.

Joseph Needham (1900-1995) and Lu Gwei-Djen (1904-1991), highly respected scholars

in TCM, believed that Suwen was composed during the 2nd to 4th centuries B.C.E.2 The version

ofHuangdi Neifing Su~wen used today is called Chong Guang Bu Zhu Huangdi Neifing Suwen

(Huangdi Neifing Suwen; Again Broadly Corrected [and] Annotated), and is derived from the

Imperial Editorial Office of the Song Dynasty (960-1279 C.E), and is based on a revision of

Wang Bing' s Suwen manuscript (762 C.E ).3 The Inner Canon ofHuangdi has played an

important role in the modernization of TCM, including traditional Chinese veterinary medicine

(TCVM). It has served as a primary reference for the foundations and doctrines of both TCM and

TCVM.









Over time, AC has spread to other Asian countries giving rise to new forms of AC

techniques such as Hari (meridian therapy) and Sujok (hand and foot acupuncture) of Japan and

Korea respectively. Even so, the practice of AC was almost abandoned in China during the early

20th century. This was due in large part to the Boxer Protocols, a treaty signed on 7 September

1901 between the Qing Empire of China and the Eight-Nation Alliance (Russia, United States,

and several European countries), and imposed upon China subsequent to the failed of Boxer

Rebellion in 1900.4 This formal agreement mandated the Chinese Imperial government of the

Qing Dynasty to transform and adopt Western ideas. This also cause to a declining in the

imperial power of the Qing Dynasty, which later overthrown by the revolution in 1911.

Following the revolution, traditional medicine and AC were blamed for their antiquity,

and their practice almost was restricted in 1914. Despite traditional medicine was not totally

banned, by the mid 1949, the number of TCM doctors trained in China declined to 270,000,

while the number of Western-trained doctors increased to more than 1.7 million.' Then, Mao

Zedong, the leader of the People' s Republic of China from 1949 until his death in 1976, lead a

campaigned to revitalize TCM and to promote an integration of TCM into Western medicine.6 I

is not obvious why Mao chose to promote an integration of TCM and Western medicine rather

than continuing the development of Western medicine alone in China. However, many historians

believe that conventional medical care was not widely available during the time of his

nationwide campaign due to socioeconomic conditions. Further, TCM seemed to be the only

affordable medical care that could accommodate the large Chinese population. After the civil

war, Mao played an important role in revitalizing TCM and AC by encouraging the Chinese

medical community to study and incorporate TCM into mainstream medical care. Promoting









TCM in China was not problematic since the basic concept of TCM was deeply rooted in

Chinese culture.

Although European physicians have known about AC since at least thel7th century, it

was not until the late 20th century that American physicians were motivated to consider its use.

During U.S. President Richard Nixon' s diplomatic trip to China in 1971, James Reston, a

reporter for the New York Times traveling with Nixon, received AC treatment after undergoing

an emergency appendectomy. After returning to the United States, Reston wrote about his

experience with post-operative pain relief following AC treatment.' B because of hi s story, the

American medical community became interested in AC and initiated serious scientific

investigations of its clinical benefits.

In 1974, in cooperation with the National Acupuncture Association (NAA), the

International Veterinary Acupuncture Society (IVAS) was founded in the United States. The

organization promotes the practice of veterinary AC and encourages addition of AC into modern

veterinary medical practices.8 In response to a worldwide increase in AC practice, the World

Health Organization (WHO) hosted the National Symposia of Acupuncture and Moxibustion and

Acupuncture Anesthesia in 1979 in Peking, China. Acupuncturists from various countries were

invited to identify conditions that they believed might benefit from AC treatment. The

symposium concluded that 43 diseases could be treated by AC.9 As most of the published data

were derived from cases reports in this symposium. Therefore, the creditability of this body of

data has been questioned. However, subsequent new data from controlled clinical trials, that

were reviewed by WHO in 2003 confirmed the clinical benefits of AC.9 This review classified

the benefits of AC for treating diseases into four groups.

*Diseases, symptoms or conditions for which the effectiveness of AC treatment has been
proven through controlled trials.










* Diseases, symptoms or conditions for which the therapeutic effect of AC has been shown,
but for which further proof is needed.

* Diseases, symptoms or conditions for which there is only one controlled trial reporting
some therapeutic effect, but for which AC is an alternative because conventional medicine
provides no or little benefit.

* Diseases, symptoms or conditions for which AC may be tried when the practitioner has
special modern medical knowledge and adequate monitoring equipment.

Origin of Traditional Chinese Veterinary Medicine and Equine Acupuncture Research

How TCVM developed is unclear. However, it can be postulated that it may have

originated from experimental AC treatments in horses and other farm animals. These animals

played important role as family asset in ancient Chinese cultures as food sources than did

companion animals like dogs and cats, and for transportation. Horses were particularly important

and widely used in ancient Chinese warfare. Their importance is demonstrated by the inclusion

terra-cotta army in the tomb of emperor Qin-shi, of a four-horse war chariot, a soldier leading a

horse, and a stable boy.

Equine acupuncture has played an important role in Chinese civilization since ancient

times. Bole' s Cannon of Veterinary Medicine (659-621 B.C.), written by Sun-yang during Spring

and Autumn periods (770-476 B.C.) of the Eastern Zhou Dynasty (770-221 B.C.) demonstrated

that TCVM extends well back in ancient China.l0 In his text, Sun-yang describes diseases of

livestock and horses, and provides some of the therapeutic foundations used in modern practice,

including methods for AC treatment for illnesses in horses. Unfortunately, the text contains no

explanation as to how the AC system was discovered or how TCVM developed, and no date has

been established for the foundation of ancient equine medical practices. However, TCVM shares

many similarities with TCM, and it is likely that TCVM was derived from TCM. The application

of traditional Chinese medical doctrines (Qi, Yin Yang, Wu Xing or Five Elements, Eight









Principles, Zang-Fu or organ physiology, and meridians), in historical philosophical frameworks

in TCVM is similar to those of in TCM.

The discovery of equine acupoints is thought to have been accidental. For example, a

chronic illness of a horse may have disappeared after an injury at a certain spot on the body was

treated with a sharp obj ect (e.g., an arrow, spear, or sword). If this same "treatment" seemed to

have a beneficial effect in several horses, over time it likely inspired ancient Chinese

veterinarians and horsemen to conclude that a specific point, even remote to the site of injury,

could be used to treat disease.

With the spread of evidence-based knowledge from the scientific revolution, philosophy-

based and experiential knowledge has been replaced by newer concepts. That modern evidence-

based information derived from controlled experiments have replaced the understanding based

on philosophy and simple historical observation as a basis for practice. Medicine is one of the

fields that has been most affected by this transformation. Despite this recent trend, the practice of

TCVM is still based mostly on traditional doctrines and principles. To become fully integrated

into modern medical practice, TCVM needs scientific evaluation to determine if it possesses real

clinical benefits. Further study will also be required to fully explain the physiological

mechanisms.involved in mediating those clinical benefits. Most current research is investigating

the effects of AC practices on the nervous system and humoral substances via an assessment of

the level of endogenous opioids and substance-P relative to treatment. Scientifically elucidating

how AC works is necessary before TCVM can be widely integrated into modern veterinary

practice. A better understanding of the basis of AC will facilitate development of more specif ic

and more effective therapeutic strategies.









Even though AC and herbal medicine have been used for treating animals in China for

more than 1,000 years, their application to treat animal diseases in modern veterinary practice is

relatively new in the United States and Europe. Early research and reports of clinical benefits

were published in Chinese and were associated with cases being treated in Chinese academic

institutions. These publications were generally available only in China and usually were often

overlooked by Western veterinary medicine.

Early scientific investigations in dogs and horses by Western-trained veterinarians

provided promising results for AC, especially in chronic musculoskeletal pain such as equine

back pain."l These scientific investigation brought AC to the attention of conventional

practitioners. They noted that various AC techniques, often in combination with herbal medicine,

were useful for treating chronic diseases that respond poorly to conventional medical

management. The clinical benefits of AC have impressed many conventional veterinary

practitioners who struggled when treating chronic diseases that respond poorly to conventional

medical treatment. This experience has produced a strong incentive for veterinarians to pursue

AC training and incorporate TCVM into their practice. In the United States, it has been estimated

that approximately 2,000 veterinarians are certified as equine or small animal acupuncturist.

Currently, veterinary AC training in the United States is offered by three major institutions, Chi

Institute of Traditional Chinese Veterinary Medicine, International Veterinary Acupuncture

Society (IVAS), and the University of Colorado. It is predicted that the demand for veterinary

AC and other forms of complementary medicine will steadily increase due to the increasing

popularity among pet owners of holistic medicine and natural products.

The American Veterinary Medical Association (AVMA) has provided a guideline for

complementary and alternative veterinary medicine and has stated that:









Veterinary acupuncture and acutherapy are considered an integral part of veterinary
medicine. These techniques should be regarded as surgical and /or medical procedures
under state veterinary practice acts. It is recommended that educational programs be
undertaken by veterinarians before they are considered competent to practice veterinary
acupuncture.1

Acupuncture was first introduced to modern veterinary medicine as an alternative therapeutic

option for chronic pain management. Its therapeutic benefit for treating chronic musculoskeletal

pain has been shown to be superior to that of the conventional therapeutic regime.

Neurohormonal explanations of the therapeutic mechanisms of AC provide an understanding

into how AC works and promote the integration of AC into general practice. A rapid increase in

the use of TCVM to treat animals has resulted in several veterinary colleges around the world

integrating AC and other types of complementary medicine into their curricula.

The increase in popularity of AC and related modalities is facilitated by increased

numbers of veterinary acupuncturists, now estimated at about 2,000 in the United States, The

popularity of AC is also buoyed by a positive public perception of complementary medical

practices seen as "natural or holistic"alternative to Western medicine. However, the academic

and scientific veterinary community is split over the perceived benefits of AC. There exists a

disparity, although narrowing, between the experience-based medicine touted by acupuncturists

and the evidence-based medicine required for widespread acceptance by the veterinary

community. In this review, a critical analysis of available literature has been attempted to

highlight areas of experimental and clinical research in the horse. Due to limited research

available on horses, relevant studies on humans and other species are included.

Equine Acupuncture Research

Research in the Field of Analgesia and Musculoskeletal Pain

Standard medical management for chronic musculoskeletal pain, including lameness,

back pain, laminitis, and navicular disease, includes long-term prescription of non-steroidal anti-









inflammatory drugs(NS AIDs), symptomatic treatment, and supportive therapy. In diagnoses

carrying a poor prognosis, such as chronic laminitis, most of the affected horses will be subj ected

to euthanasia. This may be due to a lack of improvement in clinical signs after a long period of

treatment or a decision of the owner to terminate the treatment due to economic constraints. Even

with a better prognosis, intensive nursing care until the animal is able to survive independently

often is essential. Hospitalization care usually is followed by a long period of rest, which

sometimes may last a year or more. Consequently, any forms of adjunctive therapy that reduces

the hospitalization period or improves clinical signs are beneficial. Acupuncture and EA may be

a suitable modality for managing pain, reducing inflammation and, ultimately, improving the

outcome in horses with these diseases.13-15

Chronic back pain is one of the most common musculoskeletal problems in horses and is

a leading cause of poor performance. Diagnosis is based on the performance history and a

finding of hyperalgesia or hypersensitivity in the thoracolumbar area during digital palpation.

Due to a lack of other specific clinical signs, the condition can be overlooked or incorrectly

diagnosed. Without adequate treatment, the horse may suffer from chronic pain.14

AC and EA are highly recommended for treating equine chronic back pain, as confirmed

by recent investigations and reports.14,16-19 Application of AC and EA for back pain was also

strongly supported by clinical studies in people.2021 Several methods of treatment have been

employed, with no statistical differences in results among needle stimulation, electrical

stimulation, acupoint inj section (saline or inj ectable vitamin Bl2), and low-power infrared

stimulation.22'23 Acupoints for treatment of back pain include Bai-hui, back-associated acupoints

along the Bladder meridian, Hua-tuo-jia -ji, .1/tenl-l/ni, .1/lr'n-pen~g, and \1/ten-panI .24,25 With a

success rate of the AC treatment and related techniques was 98% (Table 1-1).









Local analgesia has been produced after EA with 40-80 Hz stimulation at the Yan-chi~YYY~~~~YYY~~~YYY

acupoint. After treatment, the local pain threshold was increased 1.7 and 1.4 times for horses and

mules, respectively.26 These results suggest that the analgesic effect is derived from diffuse

noxious inhibitory control (DNIC), in which neuronal signals originating from noxious

stimulation in their receptive fields are inhibited by other pain sensations.27 The local increase in

pain threshold demonstrated an immediate segmental analgesic benefit from the treatment.

Bilateral EA and AC at BL-18, 23, 25, and 28 on horses significantly increased skin

temperature and the cutaneous pain threshold (CPT) over the lumbar area when measured by the

radiant heat-evoked skin twitch reflex.28 Electroacupuncture produced a greater increase in CPT

at all times measured (3 0, 60, 90, and 120 minutes after treatment) than did AC, and the increase

in CPT of the AC group was greater than that in the control group. The researchers also

demonstrated that the skin temperature and CPT of the EA group reached their maximum level at

30 minutes post treatment. Moreover, the P-endorphin concentration of the spinal cord

cerebrospinal fluid (CSF) collected from the spinal cord was increased at 30, 60, 90, and 120

minutes post treatment. These increases in CSF P-endorphin concentrations in the EA group

were significantly greater than the concentrations prior to the treatment for all post treatment

measurements. Acupuncture treatment also increased the CSF P-endorphin at all the sampling

points, but reached its highest concentration at 60 minutes post treatment. However, the AC

induced increases in P-endorphin were not statistically significant. In the EA group, the

concentration of P-endorphin was highest at 120 minutes after treatment. The plasma P-

endorphin concentration in horses that received EA and AC tended to increase, but with no

significant difference from the concentration prior to the treatment observed28 In this study, the

skin temperature and CPT reached their maximum value sooner after treatment than the










maximum concentration CSF P-endorphin were reached. These results suggest that in addition to

the opioid-mediated analgesic pathway, AC and EA also induces an immediate effect that alters

a local neuronal circuit, leading to an immediate increase in skin temperature and CPT. The best

known mechani sms proposed to explain the rebound inhibition of neuronal transmi ssion in AC

and EA are the Gate Control Theory proposed by Melzack and Wall and the DNIC described by

Le Bars.27,29,30

Electroacupuncture alters hoof withdrawal reflex latency (HWRL).ls HWRL is defined as

the duration between the initiation of radiant heat lamp illumination and retraction of the hoof. In

this study, either two or four acupoints, in combination, and selected from among Bai-hui, SI-9,

San-yang-lou, Qian-chan-wan, and Qian-Jiu, increased HWRL regardless of the frequency of

the electrical current being used when, compared to a negative control treatment (2 ml of a single

saline inj section subcutaneously). However, HWRL increases were not greater than a positive

control treatment (2 ml of 0.5% bupivacaine hydrochloride inj section subcutaneously).

Electroacupuncture applied at SI-9, San-yang-lou, Qian-chan-wanh~~~hh~~~hh~~ and Qian-jiu at 80-120 Hz

caused a greater increase in HWRL than EA at 20 Hz alone. These results suggested that EA at

80-120 Hz is more effective at reliving pain than EA at 20 Hz, and that dynorphins, rather that

mu-acting P-endorphins, are responsible for the observed local analgesia.l

A clinical response after treatment of equine facial nerve paralysis with AC and EA also

has been reported. This finding is consistent with the purported success of AC and EA in neural

preservation and regeneration. The overall success rate was greater than 95%.31-35 Other nerve-

related disorders for which AC and related treatment techniques may be used as an alternative

therapy include paralysis of the supra-scapular nerve and recurrent laryngeal nerve paralysis.

Studies on rats demonstrated that local EA stimulation of the lumbar muscles increased blood









supply of the sciatic nerve more than 50%, while EA at the lumbar nerve root and at the

pudendal nerve increased local blood flow by 100%.36

Similar clinical effectiveness of AC and EA for treating dogs with hind limb weakness or

paralysis has been shown.37-40 The causes of canine hind limb weakness and paralysis are usually

related to a focal disruption at some levels of the spinal cord. Electroacupuncture at GV-1, GV-2,

GV-6, and GV-9 has been shown to improve survival rate of neuronal stem cells (NSC) being

transplanted into a fully transected spinal cord in rats, and increased their migration distance

toward the caudal part of the transected spinal cord when compared to the non-treatment group.41

Electrical stimulation of the peripheral nerves has been shown to increase proliferation and

differentiation of neural progenitor stem cells.42 It also promoted re-myelination and neuronal

repair. The neuronal plasticity following EA may be partly due to a modulation of factors

involved in gene transcription, including c-Fos and c-Jun.43 Studies of cats with dorsal rhizotomy

at the lumbosacral region of the spinal cord demonstrated that EA significantly increased the

neurotrophin-3 and the expression of endogenous nerve growth factor at both protein and mRNA

levels.44 These data strongly suggest that AC and EA may also augment the treatment of a

variety of nerve-related disorders.

Arthritis is a maj or cause of lameness in performance horses. In degenerative

osteoarthritis, AC may not reverse pathological changes within synovial structures, but it

provides prolonged symptomatic relief.45 Research in China indicated that AC, EA, and their

related treatment techniques (hemoacupuncture and aquapuncture) can effectively treat j oint-

associated lameness originating from the forelimb and general arthritis in horses.46-49 Details of

this research are shown in Table 1-2. A review of 10 randomized, controlled trials in humans,

which included 1,456 participants, demonstrated that AC is an effective treatment for pain and









physical dysfunction associated with osteoarthritis of the knee.5o It also significantly improved

the quality of life compared to that of patients who received only conventional treatment." Other

smaller clinical trials for this clinical problem also demonstrated the same positive result.52-56

Pain relief has been thought to depend on either opioids or non-opioids analgesic mechanisms."

Electroacupuncture has been shown to suppress the expression of the glial cell line-

derived neurotrophic factor (GDNF) immunoreactivity of cells from the dermal and

subcutaneous ti issues of the Complete Freund' s adjuvant (CFA)-induced arthriti s rat.'" Studies

demonstrated that EA not only significantly suppressed nocicepive behavior, but also inhibited

spinal microglia activation induced by intra-articular inj section of CFA.59 Suppression of spinal

microglia activation by EA also resulted in a down regulation of the inflammatory cytokines in

the spinal cord. This finding suggested that EA might have an important anti-neuroinflammatory

effect. In addition to the therapeutic mechanisms discussed above, activation of the muscarinic

cholinergic receptors and serotonergic receptors also has been demonstrated.60

Acupuncture for Managing Ocular Problems

In human medicine, AC and EA have been used to treat ocular pain, dry eye syndrome,

ocular hypertension, and nerve-associated ocular disorders.61-66 Their use for ophthalmic

treatment in conventional equine medicine is an emerging field. In combination with NS AIDs,

they effectively alleviate ocular inflammation and ocular pain. Results from clinical practice and

laboratory animal experiments are promising. However, scientific data for horses are limited.

Most of the data were obtained from case reports, clinical trials in humans, and research

conducted on laboratory animals such as dogs and rabbits. Thirty minutes of AC at Tai-yang,

BL-2, and TH-23 significantly increased tear production in rabbits as measured by the

Schirmer' s Test.67 Subsequent histological examination of the lacrimal gland indicated that an

immediate increase in tear production after AC was due to an increase in the secretary activity of









the gland. Moreover, after ten consecutive days of AC, tear production remained greater than the

value obtained prior to the beginning of AC treatment, and the histological structure of the gland

indicated an increase in glandular synthesis and secretary activities.67

Increase in the intraocular pressure is a sign of glaucoma, a leading cause of ocular pain.

Previous research strongly indicated that AC and EA at locations distant from the eyes and

selected according to TCVM principles, reduced intraocular pressure in several animal species.

A single session of AC at LIV-4, LIV-3, and GB-37 significantly reduced intraocular pressure in

healthy dogs.68 A similar study in rabbits has been conducted using a different acupoint; an hour

ofEA at GB-30 reduced intraocular pressure, systemic blood pressure, and aqueous humor flow

rate.69 These reductions were concurrent with a significant increase in endorphin in the aqueous

humor, while the concentrations of the sympathetic neurotransmitter, norepinephrine, and

dopamine were decreased. Moreover, the effect of intraocular hypotension from a single EA

treatment lasted longer than 9 hours. This intraocular hypotensive effect could be prevented by a

pre-treatment with an opioid antagonist.

An involvement of sympathetic innervation in the EA-induced intra-ocular hypotension

has been demonstrated in rabits.70 In this model the cervical sympathetic trunk and superior

cervical ganglion were surgically excised. After sympathetic denervation, the EA-induced

intraocular hypotension was significantly reduced. Further study indicated that this EA-induced

intraocular hypotension was associated with activation of ic-opioid receptors in the intraocular

tissues, which require intact sympathetic innervation.70

It is clear that the mechanisms of AC and EA in treating ocular disorders depend on the

autonomic nervous system and the endogenous opioids. However, an involvement of the rebound

analgesia pathway such as the Gate Control Theory or DNIC is highly feasible. Both tactile and









pain sensations arising from ocular and surrounding tissues are relayed at the trigeminal ganglion

and transmitted to the ventral posterior medial nucleus of the thalamus via the trigeminothalmic

tract before being relayed to the primary somatosensory cortex. Sensations generated by AC and

EA are transmitted via the AP nerve fibers to the spinal cord and the trigeminal ganglion at a

faster rate than those of pain sensation, transmitted via the C nerve fibers. Activation of the

inhibitory interneurons leads to an inhibition of pain sensation transmitted by the C nerve

fibers.n Even though it is unknown how AC and EA suppress ocular inflammation, it is possible

that they reduce intraocular pressure and pain, thereby constraining inflammation and providing

for ocular tissue healing. However, the increase in P-endorphin in the aqueous humor after AC

suggests that this endogenous opioid substance might play an important role in how AC reduces

inflammation ofthe eyes.

Based on available research data, it seems that AC and EA at acupoints located around

the eyes such as BL-2, Tai-yang and TH-23 are suitable for treating ocular pain and stimulating

tear production, while AC and EA at acupoints remote from the eyes such as GB-3 0, GB-36,

LIV-3, and LIV-4 are suitable for controlling the fluid dynamics of aqueous humor.

Acupuncture Research in Gastrointestinal Disorders

Disorders of the gastrointestinal tract that can be treated by AC and EA include

indigestion, diarrhea, and colic. It is generally accepted that AC and EA are not recommended in

cases that require surgical intervention such as intussusception and torsion of the large intestine.

Previous research suggested that therapeutic effects of AC and EA for treating colic may be

complex and may not be explained by a single mechanism. Bilateral application of EA at BL-21,

BL-25, BL-27, ST-36, and Bai-hui has been shown to increase the rectal pain threshold in a

controlled rectal distension model.72 Although the increase in pain threshold was not as great as









with butorphanol, the result suggested that EA at those acupoints could be used to suppress pain

originating from the large intestine.

Chronic diarrhea and chronic inflammatory bowel diseases are other disorders for which

AC and EA therapeutic efficacy have been frequently investigated. GV-1 is the most important

single acupoint for treating diarrhea in humans and animals. The mechanism of GV-1 AC for

stopping diarrhea is unclear. GV-1 AC has been shown to stop diarrhea as well as significantly

reduce colonic motility and inflammation in rats with experimentally induced colitis.7 Pre-

treatment of these rats with naloxone, an opioid antagonist, prior to the GV-1 AC application

abolished the AC effect. This result suggested that the anti-diarrhea and anti-colitis action of

GV-1 may be due partly to the endogenous opioid anti-inflammation pathway.73 In humans

suffering from hemorrhoids, EA at GV-1 reduced pain sensations during defecation.74 The

analgesic effect was as potent as the standard conventional treatment evaluated with the pain

visual analog scale. Local analgesia following EA at GV-1 may be explained by the Gate control

theory and DNIC mechanism.

In TCVM practice, LI-4 and ST-36 also are commonly used in conjunction with GV-1 to

treat diseases associated with the gastrointestinal system. Research has demonstrated that AC

and EA possess therapeutic effects via a down regulation of the production of inflammatory

cytokines such as IL1-P, IL-6 and TNF-u.7 Electroacupuncture at ST-36 has been shown to

decrease plasma TNF-a concentration and down regulate its mRNA expression in the colonic

tissue of rats with experimentally induced colitis.76 Moreover, EA at LI-4 and ST-3 6

significantly reduced the macroscopic lesions caused by colitis and significantly reduced

myeoperoxidase activity of the inflamed colonic tissues. In this study, the researchers

demonstrated that the anti-inflammatory activity of EA on LI-4 and ST-36 was suppressed by the









administration of a P-adrenoceptor antagonist.76 Therefore, EA at these acupoints may activate

the sympathetic anti-inflammatory pathway, mediated through P-adrenoceptors.7

M~i-jiao-gan, an acupoint, can be translated as vagosympathetic trunk. This acupoint is

recommended for treating poor appetite, diarrhea, and indigestion.'" The point is located at the

junction of the cranial and middle one-third of the neck and dorsal to the jugular vein. The vagus

nerve is located in this area, and is a maj or parasympathetic nerve that contains both afferent and

efferent nerve fibers for both somatic and visceral tissues.79,80 Acetylcholine is a maj or

neurotransmitter in both preganglionic and postganglionic parasympathetic nerve synapses. This

acupoint is a good example of an AC application that is supported by a related anatomical

structure. Direct electrical stimulation of the vagus nerve attenuates the release of TNF-oc and

prevents LPS-induced endotoxic shock.81 Moreover, acetylcholine attenuates the in vitro release

oflIL-1P, IL-6, and IL-18, but not the anti-inflammatory cytokine, IL-10, in LPS-stimulated

human macrophage culture. Thi s cholinergi c-dependent anti -inflammatory pathway suppressed

the non-specific innate immune response and may explain how AC and EA function in treating

diseases of the gastrointestinal system and other visceral organs.

A study of the visceral analgesic effects of EA at Guan-yuan-shu (BL-21i) on horses with

colic, experimentally induced by duodenal balloon distension, did not demonstrate an

improvement in the clinical signs of colic.82 I, COntrast, early clinical practice in China indicated

that AC at Jiang-ya can be used effectively to control clinical signs of colic.83,84 Moreover, data

from a case report indicated that AC and EA may possess positive clinical benefits in treating

chronic recurrent colic.85 To confirm a therapeutic benefit of AC and EA, additional studies with

modern research design are necessary. Until then, AC and EA may not be a treatment of choice









when colic horses are admitted to veterinarians, since the vast maj ority of the cases are acute,

often emergencies, and a delay in diagnosis and treatment may negatively affect their prognosis.

Acupuncture Research in Respiratory Disorders

Chronic respiratory diseases such as recurrent airway obstruction (RAO) and summer

pasture associated chronic obstructive pulmonary disease (SPAOPD) are chronic debilitating

respiratory disorders. Conventional treatment includes improving the stable environment to

eliminate potential inciting causes and symptomatic therapy.86 Long-term medical management

is required in most cases. When the inciting cause is re-introduced, clinical signs of a respiratory

problem reappear. Besides the conventional medical managements, AC, EA, and Chinese herbs

have been used as adjunctive therapies to control clinical signs. They are intended to decrease the

dosage requirements of bronchodilators and anti-inflammatory agents, and improve the quality of

life of animals suffering from chronic respiratory diseases.

Several studies indicated that AC and EA induce immediate mild to moderate

bronchodilator effects. A clinical study in human asthma found that AC at LU-7, LI-4, PC-6, ST-

40, LI-11, and PC-3 for 15 minutes improved the forced expiratory volume in the first second

(FEV1).87 Improvement in pulmonary function parameters (transpleural pressure, tidal volume,

minute ventilation, peak inspiratory flow, and peak expiratory flow, in RAO-affected horses also

has been demonstrated after a single AC treatment.88 However the increases were not statistically

significant, and the researchers concluded that the improvements in those pulmonary function

parameters were due partly to animal handling.

Analgesic and anti-inflammatory effects of AC and EA are well recognized in both

somatic and visceral tissues.60,89 Maj or acupoints that are indicated for these purposes include LI-

4, IL10, and ST-36. 60,90,91 These acupoints are commonly included in the treatment regime of

equine respiratory disorders. Their analgesic and anti-inflammation mechanisms have been









linked to a release of endogenous opioid substances in the central nervous system (CNS).28

These endogenous substances activate opioid receptors in CNS tissues, such as the substantial

gelatinosa of the spinal cord and the periaqueductal grey of the midbrain, and produce

analgesia.92,93 Immunocytes such as macrophages, monocytes, and polymorphonuclear cells also

possess opioid receptors predominately C1-opioid receptors on their cell surfaces.94 Once

activated, the receptor induces an anti-inflammatory response via the down regulation of the

transcription factor, nuclear factor kappa-B (NF-rB).95 NF-KB down regulation has been

demonstrated to reduce mRNA expression of other inflammatory cytokines, including TNF-ot,

I-1P, IL-6, nitric oxide synthase (iNOS), and metalloproteinase.96 Carneiro et al.97

demonstrated that, in rats with ovalbumin-induced bronchial asthma, EA reduces the

inflammatory cell infiltration in the peribronchial tissue and in the pulmonary perivascular

spaces.97 Moreover, the number of total nucleated cells and the percentages of neutrophil and

eosinophil leukocytes in bronchoalveolar fluid (BALf) are significantly decreased compared to

control and sham EA groups. In this study, EA was performed on acupoints mimicking the

treatment regime for human asthma, including GV-14, BL-13, LU-1, CV-17, ST-36, SP-6, and

Ding-chuan.

An increase in mucus production is a common consequence of airway inflammation.

Until recently no scientific evidence has directly demonstrated the effect of AC or EA on

mammalian mucociliary clearance. Disruption of normal mucocilliary clearance is a

consequence of chronic airway inflammation and was hypothesized to be caused by neutrophil-

derived elastase.98 The increased production and accumulation of mucus decreases airway caliber

and increases total airway resistance. Tai et al.99 demonstrated that EA at acupoints LU-1 and

CV-22 significantly increased the rate of tracheal mucociliary transport in treated quails









compared to a control group.99 Moreover, EA at these acupoints significantly reversed the

decrease in mucociliary transport caused by the administration of human neutrophil-derived

elastase.

Acupuncture Research in Other Medical Problems

In combination with appropriate Chinese herbal formulas, AC and EA are safe for long-

term medical management for geriatric animals, behavioral problems, Cushing' s disease, and

anhydrosis. Unfortunately, the scientific data on the therapeutic effects of AC and related

techniques on these diseases are limited.

A positive clinical response of horses (N=4) suffering from anhydrosis to AC has been

demonstrated.100 A retrospective study of 24 horses affected by anhydrosis and treated with

TCVM indicated that using a combination of AC, EA, and a Chinese herbal formula (New Xiang

Ru San) was an effective therapeutic approach. 101 In this study, owners of 25 horses were

interviewed by telephone and asked to grade improvement in clinical signs after their horses

were treated with TCVM. The improvement grading system was developed by the authors and

was based on a visual analog scale. Thirty-six percent of the clients reported complete recovery,

32% reported significant improvement, 28% reported slight improvement, and 4% reported no

improvement. The positive clinical benefit may be due partly to AC/EA or supplementation with

New Xiang Ru San, or a combination of the treatments. Although it is uncertain as to which

therapeutic modality is more beneficial for the treatment of anhydrosis, the finding is significant

since conventional medicine provides no therapeutic benefit for affected horses.

Electroacupuncture significantly increased blood cortisol concentrations of horses when

compared to sham treatment.10 This increase was hypothesized to be mediated through

activation of the hypothalamic pituitary adrenal axis causing a release of P-endorphin and









ACTH.102 COrtisol or endogenous glucocorticoid, is produced by the adrenal cortex. It is an

important steroid hormone, which regulates the immune response, glucose metabolism, and

amino acid mobilization.

Electroacupuncture at Er-jian or Qun-hui acupoints has been used to produce analgesia

for surgery in horses (n=62).103 The degree of analgesia was adequate to allow a veterinary

surgeon to perform a standing operation.10 Another study in dogs demonstrated that EA at ST-

36 and GB-34 produced greater analgesic effect than EA performed at ST-36 and SP-6. In dogs

in which analgesia has been induced, it was feasible to perform a simple laparotomy. 104 Using

AC and EA analgesic to control pain during operations on horses seems far from realistic. Since

horses respond unpredictably and violently to nociceptive stimuli, and without appropriate and

dependable control of anesthetic depth, a life threatening injury to the surgeon, damage to the

surgical facility and equipment, or injury to the patient may occur.

AC Research in Reproductive System

Application of AC and EA for treating disorders of the reproductive system is another

field in which the positive clinical response rate is significant. Among all reproductive system

problems, infertility is the most common disorder treated by AC. Research in humans has

demonstrated that AC is an effective therapy for women with infertility. Following in vitro

fertilization and embryo transfer, AC has been used to improve the pregnancy rate.105-107 Causes

of equine infertility may be simply categorized as due to infection or non-infection. Common

infectious agents that can cause infertility include viruses, bacteria, and fungi. Non-infectious

causes range from anatomical defects and lameness to stress-induced infertility. In some cases

the problem may be multi-factorial. Examples of common non-infectious causes of equine

infertility include hormonal imbalance, excess uterine fluid, endometrial cysts, and lameness.









The cause of infertility requires careful diagnosis before an appropriate treatment, whether

conventional or integrative, is implemented.

In TCVM, mare reproductive function is related to the Chinese parenchymatous visceral

organs Kidney and Liver. It also depends on the Conception vessel and the Penetrating channel.

Therefore, acupoints commonly stimulated for treating mare infertility include Bai-hui, Yan-chi,

BL-23, .1hen~l-l/ni, .1hen'l-peng, KID-3, KID-7, GV-3, GV-4, CV-4, CV-6, and LIV-3.108,109 A

TCVM pattern diagnosis provides the core of any acupuncture treatment.

In Thoroughbred mares with a history of excess uterine fluid and/or uterine pooling that

did not respond to the conventional treatment, adjunctive AC treatment significantly reduced

uterine fluid accumulation as determined by transrectal ultrasonography.110 A high percentage

(8 1%) of thi s group of mares was later able to get pregnant."1 Another report using EA for the

same purpose has reported a significant increase in uterine tone within 24 hours after the

treatment." The decrease in the uterine fluid accumulation may be due partly to an

improvement in local circulation and the uterine blood flow mediated by central inhibition of

sympathetic nerve activity.112

Additional studies in other animals also support the benefit of AC and EA for treating

infertility. A small-sample study of anestrous sows indicated that AC at Bai-hui and Wei-ken

could be used to induce estrous within 14 days after the treatment, but not in a sham AC group

when needles were used to stimulate Chian-feng and Chou-shu acupoints. The induction of

estrous in sows using AC was superior to treatment with gonadotrophin releasing hormone

(GnRH) alone.113 A similar study in repeat-breeder cows that failed to respond with GnRH

treatment and failed to become pregnant after more than three inseminations showed the same

positive result.114 In this study aquapuncture with 10 ml and 5 ml of 50% glucose solution were









inj ected into Bai-hui and .1hen~l-peng acupoints, respectively. The authors reported that most of

the cows exhibited estrous signs within 14 days and were artificially inseminated. Later

pregnancy diagnosis by rectal palpation revealed that 66% of the treated cows were pregnant.

However, the percentage of cows that carried their pregnancy to a full term and successfully

calved was 44%.114 A lower rate of successful calving may be due partly to the fact that the

repeat-breeder cows are likely to be predisposed to other stress factors and disorders such as

metabolic acidosis and hot climate, which may contribute to a loss of their pregnancy. A positive

clinical benefit of EA also has been shown in cows with ovarian cysts. 1

Aquapuncture at Bai-hui with prostaglandin induces luteolysis of the corpus luteum in

mares.116 The luteolytic effect and the ability to induce estrous cycle were as effectively as

intramuscullar admini station of prostaglandin at the recommended dosage. Bai-hui

aquapuncture with micro-dose of prostaglandin was still effective, and caused less systemic

adverse effects than prostaglandin alone at the recommended dosage.116 However, a later study

refuted these claims. "7 Further scientific scrutiny on this topic is required.

Electroacupuncture at appropriate acupoints reversed abnormal functions of the

hypothalamic-pituitary-ovarian axis (HPOA).""s A series of AC treatments at BL-18, BL-23,

CV-3, CV-4, and SP-6 in women with ovulatory dysfunction has been shown to improve the

ovulation rate over 80%. 119 Hormonal profiles also showed that AC and EA were capable of

adjusting the level of the follicle stimulating hormone (FSH), lutinizing hormone (LH), estrogen,

and progesterone to their normal phy siol ogi cal concentrate ons.119120

A study on rats demonstrated that EA at CV-4 increased the immunohistochemical

reactivity for GnRH in the medial pre-optic area, the arcuate nucleus, and the nucleus

periventricularis of the hypothalamus.121 Ovariectomized rats possess abnormal reproductive









profiles and have been used as an experimental model to study abnormalities of reproductive

hormones. Based on this experimental model, EA has been shown to restore the function of the

HPOA.122 Electroacupuncture significantly increased the number of GnRH positive neurons in

the hypothalamus of ovariectomized compared to both ovariectomized and normal rats.123 GnRH

positive neurons in the nucleus paraventriculari s also have been found to co-localize with the

corticoid releasing hormone (CRH)-positive neurons identified by immunofluorescent double-

labelling histochemistry and laser confocal scanning microscopy.123 This data suggested that AC

and EA may modulate the hypothalamic pituitary gonadal axis by altering the synthesis and

secretion of regulatory hormones GnRH and CRH from the hypothalamus, and modulate the

reproductive hormones and homeostasis. Release of GnRH after EA also has been show in

rabbits.124

Even though the benefits of AC and EA have been confirmed in clinical practice, their

mechanisms remain unclear. Sensory signals generated by AC and EA may modulate either

neuroendocrine control of the hypothalami c-pituitary-gonadal axi s or modulate the peripheral

autonomic neural control of the reproductive tract, or both. A more thorough understanding of

their mechanisms will allow the practitioner to specify the action of each acupoint and improve

the effectiveness of the treatment.

A positive clinical benefit of AC and EA in the treatment of male infertility has been

demonstrated in humans. Acupuncture decreased the number of defective spermatozoa and

improved their motility.125-127 Improvement in acrosome ultrastructure, nuclear shape, axonemal

pattern and shape, and accessory fibers of sperm organelles also has been shown.125 Acupuncture

also increased sperm count per ejaculate, especially in subjects with a history of genital tract









inflammation.128 Even though the results in humans seem promising, a placebo effect was not

ruled out.

Research in Diagnostic Potential of Acupoints and Meridians

In TCVM, acupuncturists can use Ah-shi point to diagnose animal disorders. Ah-shi point

is also known as trigger point and as myofascial pain point. Trigger points are defined as

locations of pain during pressure and palpation. These locations may be obj ectively detected by

pressure algometry, pressure threshold measurement, magnetic resonance, thermography, and

history of illness. The associated acupuncture principle states that "where there is a pain, there is

an acupoint". Therefore, occurrence of a trigger point may be at the same location as an ordinary

acupoint or at a non-specific location on the body surface. Hypersensitivity upon palpation at

trigger point may indicate underlying local tissue inflammation or a specific pathology ofa

certain tissue.

The presence of a trigger point in horses has been demonstrated by using sensitivities

within the cleidobrachialis muscle. Horses with chronic musculoskeletal pain possessed an

obj ective sign of spontaneous electrical activity, spike activity and local twitch response at the

myofascial trigger point location.129 McCormick demonstrated a relationship between five

diagnostic trigger points and the cause of a fore limb lameness.13 In his study, acupoints LI-1 8,

SI-16, TH-15, BL-42, and BL-14 were used to represent channel diagnosis for the Large

intestine, Small intestine, Triple heater, Lung, and Pericardium channels, respectively.

Hypersensitivity of these acupoints has been proposed to be a result of imbalance in the channel

they represent. The pathways of these channels are located in the fore limb, and their imbalances

are thought to be caused by pathology in the fore limb. The study found that the evidence of

hypersensitivity among these trigger points was significantly greater in heel lameness and

laminitis, but not lameness originating from sub-solar regions. Pericardium channel imbalance









caused by heel lameness was significantly greater than channel imbalance caused by laminitis.130

The result also showed that pain originating in the toe region is highly correlated with the

imbalance of the Lung and the Large intestine channels. Subsequent intra-articular anesthesia of

the distal interphalangeal joint abolished the Lung and the Large intestine channel imbalance in

all of these horses. Moreover, lameness and response to intra-articular therapy of the proximal

interphalangeal j oint (fetlock j oint) may be evaluated by a sensitivity of SI-16, TH-15, BL-14,

LI-18 and BL-42.131 The sensitivity of these acupoints was demonstrated in 54% of horses (176

of 327 horses) in which the clinical signs and medical history suggest that the cause of lameness

originates from the fetlock j oint. Sensitivity of LI-18 and BL-42 was detected in all 176 horses.

Sensitivity of SI-16, TH-15, and BL-14 was also increased, but not consistently among these

horses. Subsequent intra-articular inj section therapy reduced the sensitivity of those acupoints in

54% of these horses, and 63% of them became sound. Horses with no change in their sensitivity

to acupoints or still unsound after intra-articular therapy, were later demonstrated to have

concomitant sources of pain, such as interphalangeal j oint, carpal joint, and pain originating from

hind limbs.131 These results demonstrate the presence of trigger points in fetlock pain and the

diagnostic value of those trigger points. Therefore, it is suggested that the hypersensitive reaction

at LI-18 and BL-42 may be caused by pathology within the distal interphalangeal joint.

The benefit and specificity of BL-18, BL-19, BL-3 6 Xie-qi, and .1/teit-\lri acupoints for

diagnosing hind limb lameness caused by tarsal joint and metatarsophalangeal joint also have

been demonstrated.132 Using sensitivity of acupoints for diagnosing equine herpes virus type 1

(EHVl) and equine protozoal myelitis also has been proposed. However, results from standard

diagnosis did not confirm the researchers' supposition.133,134









Tenderness and neurologic inflammation at GV-1 also has been demonstrated in rats with

induced-colonic inflammation.73 GV-1 is an important acupoint for treating diarrhea, bloody

defecation, constipation, and perineal problems.135

Summary

Current research has confirmed certain therapeutic benefits of AC and EA. In

musculoskeletal disorders, the success rate of the treatment was nearly 100% for thoracolumbar

pain. For this type of disorder, AC, EA, aquapuncture, or laser AC can be used with no

significant difference in their therapeutic efficacy. For treating nerve-associated disorders, EA

seems to be the best method, and electrical stimulation has been shown to be or beneficial in the

promotion of nerve repair, re-myelination, and neuronal plasticity. Little modern research on

equine arthritis has been conducted, and clinical application of AC and EA to treat equine

arthritis is still based on empirical and personal experience. However, results from human

research seem promising, and research on their therapeutic effects for treating equine arthritis

may be worthwhile. For ophthalmic treatment, local acupoints around the eyes are appropriate

for ocular pain and inflammation. Possible therapeutic mechanisms include opioid analgesia and

anti-inflammation. Using AC and EA for treating an increased intra ocular pressure also is

promising. The therapeutic mechanism is thought to be opioid and sympathetic innervation

dependent.

In the field of reproductive di orders, AC and EA therapeutic effects are thought to be

mediated by the ability of AC and EA to restore balance of the hypothalamic pituitary gonadal

axi s and to promote uterine tone by the central inhibition of the sympathetic nerve activity.

Currently available research data were obtained from studies on humans. Interpretation of results

should be done with care, since the positive results of treatments on humans may by due to the

placebo effect. Additional studies in animals may eliminate this confounding positive










psychological effect, and demonstrate the true effects of AC and EA. To date, scientific scrutiny

of the mechani sms of AC and EA for treating reproductive problems in horses has been limited

to studies on mares, and is absent for stallions.

Acupuncture and EA provide mild to moderate analgesia and are not the anesthetic

methods of choice when surgical intervention is required. Because the vast maj ority of colic

cases are acute and rapidly progress, delay in diagnosis and treatment may affect their prognosis.

Therefore, all cases of colic presented to veterinarians are likely to be treated first with

conventional medicine, and the clinical benefits of AC and EA are rarely investigated. However,

for chronic gastrointestinal problems, treatment efficacy for both AC and EA has been

demonstrated. Treatment benefit was thought to be due party to an activation of the cholinergic

anti-inflammatory pathway, leading to a down regulation of inflammatory cytokines such as 1L1-

p, IL-6 and TNF-oc.

In respiratory disorders, EA might be beneficial for treating RAO. Results from

laboratory animals suggested that the possible treatment mechanisms include anti-inflammation,

bronchodilation, and increased mucociliary clearance. Anhidrosis is another medical problem for

which AC and EA is useful. This is important because TCVM seems to be a treatment of choice.

The only other effective treatment is relocation of the affected horses to a more temperate

environment. Finally, diagnostic and therapeutic values of trigger points have been confirmed

and, for some, an increase in sensitivity is highly positively correlated with a suspected disorder.

Even though previous research supports using AC and EA for veterinary medicine, more

carefully controlled research in every aspect of TCVM for the horse is necessary.










Table 1-1. Treatment efficacy of AC and its related technique in equine thoracolumbar pain.
Investigator. AC Technique (s) Total Number of cases Number of
cases with positive cases not
response to response to
treatment treatment
Klide 19841 AC 15 13 2
Petermann 200216 AC+AurA+ 512 502 10
LAurA+LAC
Xie 20051 AquA+ Herbal *4 4 0
medicine
Rungsri(Kulchaiwat) 200919 EA **16 16 0
Total 547 535 12
AC = acupuncture, AurA = auricular acupuncture, LAurA = laser auricular acupuncture, LAC =
laser acupuncture, EA = electroacupuncture, = Effectiveness of EA treatment on these horses
was compared to the treatment efficacy of oral administration with phenylbutazone and normal
saline in others 1 1 horses, ** = Effectiveness of EA treatment on these horses was compared to
the treatment efficacy of sham EA in others 7 horses.

Table 1-2. Treatment efficacy of AC and its related techniques in equine lameness originating
from j oint problems.
Locati on.Reference AC technique Total cases Sound Improve Failure
All four limbs.46 EA 198 174 21 3
Forelimb.47 AquA+HA 85 83 2 0
Forelimb or AquA+Herbal 161 136 15 10
hindlimb.48 medicine
Rhumati sm.49 EA 153 143 0 10
EA = electroacupuncture, AquA = aquapuncture, HA = hemoacupuncture.









CHAPTER 2
MODULATION OF IMMUNOLOGICAL RESPONSE BY ACUPUNCTURE AND
ELECTROACUPUNCTURE AT LI-4, LI-11, AND GV-14 IN CLINICALLY NORMAL
HORSES.

Introduction

Application of alternative medicine, such as traditional Chinese veterinary medicine

(TCVM), in treating diseases caused by disorders of the immune system is both an ancient and a

novel, at least in Western cultures, approach to veterinary medicine. Acupuncture (AC), a part of

TCVM, was developed in ancient Chinese culture and has been practiced in China for more than

a thousand years. With a long history of clinical benefits, AC is now being integrated into

Western veterinary medicine, and is being adopted as an alternative therapy for a number of

diseases. Interestingly, AC has changed only slightly since the ancient Chinese texts described

the methods for treating illness. Electroacupuncture (EA) is a form of treatment that was derived

from and remains closely related to AC. It was developed by an integration of knowledge about

electrophysiology of body tissues that was merged with the classical practices of AC. In human

medicine, both AC and EA have been used as adjunctive therapies for several diseases, including

asthma, human immune deficiency virus (HIV), allergic disease, and disorders of the immune

system.87,136,137 A common pathophysiologic aspect of each of these diseases is a dysregulation

of immunological function. Under TCVM treatment principles, several acupoints, including LI-

1 1, ST-3 6, LI-4, and GV-14, have been used for improving health and modulating the immune

sytm135,137

Multiple AC treatments at LI-11, GV-14, SP-10, and ST-36 have been shown to

significantly decrease the number of peripheral circulating leukocytes (including lymphocytes) in

healthy humans.138 It is possible that the alterations in these hematological parameters were due

to movements of immune cells between cell storage compartments.138 Modulation of the cellular









immune response was demonstrated in AC performed at LI-1 1 twice a week for a total of eight

treatments.13 After the fourth treatment, the increase in reactive oxygen species (ROS)

production by neutrophils was greater in the AC group than in the placebo group.

In human medicine, ulcerative colitis is another chronic inflammation for which AC

treatment possessed a positive clinical benefit. According to the randomized single-blind study,

AC performed at the acupoints selected according to the traditional Chinese medical diagnosis

significantly reduced the colonic activity index and improved the quality of life of the patients.140

Further study in the rat indicated that the mechanism of the treatment may be partly associated

with a down regulation of colonic tumor necrosis factor-alpha (TNF-u) mRNA expression.76

In veterinary medicine the LI-4, LI-11, and GV-14 acupoints are commonly included in

treatment protocols of several aliments, including fever, immunodeficiency, and dental pain.135

This study compares the effects of AC and EA on circulating immunoglobulins, ROS production

by circulating neutrophils after in vitro stimulation, and TNF-a production in stimulated whole

blood cultures in vitro, as evidence of in vivo immune modulation.

Methods

Animal

Twenty-four mature Thoroughbred horses with no history of illness or hospitalization

during the past month were randomly assigned for AC (n=12) and EA (n=12) treatments. All

horses were kept in paddocks in groups of 2 or 4. Shelter was available in each paddock. The

horses were fed twice a day with balanced commercial feed. Clean water and good quality hay

were available ad libidum. Numbers from 1-2 and 1-4 were randomly assigned to the horses in

the groups of 2 and 4 horses, respectively. Horses with odd numbers were assigned to EA

treatment. Horses with even numbers were assigned to AC treatment. Protocol for animal use









was approved by the University of Florida Institutional Animal Care and Use Committee (Permit

A-130).

Acupuncture and Electroacupuncture

Acupoints used in our study were LI-4, LI-11, and GV-14. These acupoints were selected

because, according to the TCVM, they are recommended for tonifying Qi (defined as the life

energy that nourishes and propels physiological activities of living organisms). LI-4 is

commonly used in treating inflammation of the front leg and is known to possess analgesic

properties. LI-11 is an immune-stimulation acupoint. According to acupuncture treatment

protocol, GV-14 is recommended for treating fever and excessive sweat and other disorders that

are diagnosed as excess Heat pattern in TCVM.135 Detailed descriptions of each acupoint and

TCVM indication are shown in Table 2-1.

A 0.35 mm diameter x 50 mm disposable sterile stainless steel acupuncture needle

(Kingli, China) was used at LI-1 1 and GV-14 acupoints. A 0.35 mm x 25 mm acupuncture

needle was used at LI-4. For EA, acupoints were stimulated with an electrical stimulator

(Pantheon Research). Intensity and frequency were set at 3 -5 mA of 15 Hz for 10 minutes and

immediately followed by 3 -5 mA 200 Hz for 10 minutes. The needles were secured in place with

superglue. Horses assigned to the AC were treated the same way except needles were not

connected to the electrical stimulator, and needles were left in each acupoint for 20 minutes.

After AC and EA, needles were gently removed. Electroacupuncture and AC were performed

once a day for 3 consecutive days.

Source of Fungal Antigens

Aspergillus femiga~tus antigens used in this study were obtained in the form of sterile

commercial products available for immunological testing in humans and animals (Greer source

materials). The manufacturer prepared the cellular antigen (CA) from a defatted dry powder of









A. fumigatus, ATCC strain 1022, obtained from a 20-day static culture (#XPM3X1A5). Fungal

cellular antigen was extracted using 0.01M ammonium bicarbonate at 1:20 weight/volume. The

extract was then concentrated, and dialyzed with an AmiconTM f11tration/dialysis system. Finally

the fluid was passed through a 0.2 Clm sterile filter and was lyophilized. Culture filtrate antigen

(CE), (#XPM3-Fl6-50.0) was obtained from a 20-day old static culture medium. The culture

fluid was concentrated and dialyzed with an AmiconTM f11tration/filtration system. The fluid was

then passed through a 0.2 Clm sterile filter and was lyophilized. Protein content of each antigen

was measured using the Bradford protein assay.

Leukocyte Separation

Sixty ml of peripheral blood was collected from the left external jugular vein into a pre-

heparinized sterile syringe. Sodium heparin (Abraxis Pharmaceutical) was used as an

anticoagulant and its final concentration was 10 unit/ml. Polymorphonuclear leukocytes were

isolated from leukocyte rich plasma prepared by the method that has been previously described

by Sun et al.141 To obtain leukocyte rich plasma, the heparinized blood was allowed to settle for

15-20 minutes. Neutrophils were separated from mononuclear cells by the single step gradient

method using lymphocyte separation medium, density 1.077 g/cm3 (Mediatech #25-072-CV).142

Leukocyte rich plasma from the blood sample was then carefully layered over 10 ml of leukocyte

separation medium in a 60 ml sterile conical centrifuge tube (Fisher #06-443-18). The

mononuclear cells were separated from the polymorphonuclear leukocytes and contaminated red

blood cells by centrifugation at 800 RCF for 30 minutes and 21 Co. After centrifugation, the

polymorphonuclear leukocytes were recovered from the cell pellet. Contaminated red blood cells

were removed by hypotonic lysis with 20 ml of sterile distilled deionized water (Mediatech #25-

05 5-CV) and vortexed for 30 seconds. The osmolarity of the cell suspension was immediately









restored to isotonic condition by adding 20 ml of sterile double strength phosphate buffer saline

(2 x PBS). Cell suspensions were then adjusted to 45 ml with 1 x sterile PBS for washing

(Mediatech #21-040-CV). The tubes were centrifuged at 800 RCF at 21Co for 10 minutes. In

total, the neutrophils were washed three times with 40 ml of sterile 1 x PBS and centrifuged at

800 RCF for 5 minutes at 21Co. Before the final wash, the cell concentration was determined by

counting with a hemocytometer, and cell viability was determined by Trypan blue exclusion.

After the final wash, concentration of polymorphonuclear leukocytes was adjusted to 3 x1 06

cell/ml with media containing 10% fetal bovine serum (FBS, Hyclone #SH30070), 1% L-

glutamine (Mediatech #25-005-CL), 2% sodium pyruvate (Mediatech #25-000-CL), and 0. 1%

gentamicin sulfate (Mediatech #30-005-CR) in RPMI 1640 without phenol red and L-gultamine

(Mediatech #17-105-CV).

Reactive Oxygen Species Generation of Neutrophil

The assessment of equine neutrophil ROS generation was modified from the method

described by Donovan et al.143 Briefly, 100 Cll of neutrophil suspension in media or of media

alone were added to each well of a 96-well black opaque flat bottom plate (Costar #3915).

Solutions of stimulants were prepared in the media, including purified E coli 01 11 :B4

lipopolysaccharide (LPS) (InvivoGen # tlrl-eblps), zymosan (Zym) (Molecular Probes # Z2849),

Aspergillus f emigatus CA, A. fumigatus CE, and phorbal 12-myristate 13 acetate (PMA) (Biomol

#PE-160). To each designated well, in triplicate, were added 10 Cll of media alone, 10-6 M PMA,

LPS (10, 1, 0.1 Clg/ml), Zym (10 Clg/ml), CA (10 Clg/ml), or CE (10 Clg/ml), and 10 Cll of 100 CIM

of DHR-123 (Invitrogen #D632). Lipopolysaccharide, CA, and CE were used because they have

been suspected of being involved in the pathogenesis of equine acute abdomen, laminitis, and









recurrent airway obstruction.144-14 Zym is a standard fungal antigen, which is routinely used in

general immunological testing.

After adding DHR-123, the plate was shaken with a plate shaker (MS 1 Minishaker) for

30 seconds and incubated at 370C in 5% CO2 for 3 hours in the dark. Each plate was evaluated

by using a fluorescent plate reader (Bioteck) with 485 nm excitation and 538 nm emission to

monitor conversion of the dye from the non-fluorescent to the fluorescent form in arbitrary

fluorescent units (AFU). Phorbal 12-myristate 13 acetate was used to generate the maximum

stimulation of ROS production and was used as a positive control for each sample. The ROS

production of neutrophils in response to each stimulant was determined from a calculation of the

response ratio using the following formula:

ROS response ratio = AFU of stimulated cells/AFU of non-stimulated cell s (2-1)

Heparinized-blood Stimulation

A 1440 Cll of heparinized-blood was transferred into 10 sterile pyrogen-free microfuge

tubes (Fisher #05 -408-129). Sterile solutions of each stimulant, including LPS, Zym, CA, and

CE, at concentrations of 1 Clg/ml (10X), 2 Clg/ml (20X), and 3 Clg/ml (30X), were prepared with 1

x PBS. These concentrations were used for single, double, and triple stimulations, respectively.

For a single stimulation, 160 Cll of 1 Clg/ml of each stimulant was added to the microfuge tube.

For double stimulations, 80 Cll of 2 Clg/ml of 2 combinations among LPS, CA, and CE were

added. For triple stimulations, 53.3 Cll of 3 Clg/ml of LPS, CA, and CE were added to the tube.

Microfuge tubes were incubated in a 150 rounds/minute shaker for 6 hours at 370C. At the end of

incubation, the microfuge tubes were placed in ice for 5 minutes prior to centrifugation at 14000

RCF for 1.5 minutes. Two 250 Cll aliquots of plasma were collected in microfuge tubes and

stored in -200C for TNF-oc analysis.










Equine Tumor Necrosis Factor Alpha (TNF-cl) Assay

Tumor necrosis factor-alpha in plasma collected from stimulated-heparinized blood was

analyzed with ELISA (protocol was kindly provided by Dr. David J. Hurley, College of

Veterinary Medicine, University of Georgia). Briefly, 100 Cll of 3 Clg/ml of anti-equine TNF-a

polyclonal antibody (Endogen #PETNFAI) in a carbonate bicarbonate coating buffer at pH 9.7

(Sigma Aldrich #C-3 041) was added to the 96-well plate (Nunc-ImmunoTM Modules #469949).

The plate was incubated overnight at 40C. After coating, the coating buffer was removed, and

200 Cll of blocking buffer containing 1% BSA (Sigma Aldrich #B4287) in PBS was added. The

plate was then incubated at room temperature for one hour. After incubation, each well was

washed three times with 250 Cll of washing buffer containing 0.05% Tween 20 (FisherBiotech

#BP3 37-5 00) in PB S. A series of two-fold dilutions of equine recombinant TNF-a (ErTNF-u)

standard (from 10000 pg/ml to 39.05 pg/ml) was prepared with diluent containing 1% BSA and

0.05% Tween 20 in PBS. Plasma was diluted 1:5 with the diluent. Then 100 Cll of each

concentration of standard and diluted plasma was added to a designated well, in duplicate, and

the plate was incubated at 370C for two hours. After incubation, each well was washed three

times with 250 Cll of washing buffer. After washing, 100 Cll of equine TNF-a biotin-labeled

polyclonal antibody (Endogen #PETNFABI) at a concentration of 0.9 Clg/ml was added to each

well and the plate was incubated for 90 minutes at 37oC. After incubation, the washing step was

repeated three times and 100 Cll of 1 :5000 of Avidin-HPR (BD Bioscience Pharmingen #554058)

was added to each well. The plate was incubated at 37oC in the dark for one hour. After

incubation, the washing step was repeated five times, and 100 Cll ofABTS peroxidase substrate

system for HRP (KPL #50-62-00) was added to each well. The plate was then incubated for 30

minutes at room temperature. The plate was evaluated with a universal microplate reader










(Elx800, Bio-Tek) at a wavelength of 405 nm. TNF-oc concentration in the plasma was

calculated based on a four-parameter logistic curve-fit generated by KC4 software.

Immunoglobulin Assay

Plasma immunoglobulins (IgA, IgM, IgGa, IgGb, and IgG(T)) were determined with

horse immunoglobulin ELISA Quantitation Kits (Bethyl laboratories, inc. Texas #E70-116,

#E70-114, #E70-124, #E70-127, and #E70-105). Assays were performed according to the

protocols provided by the manufacturer. Briefly, Nunc MaxiSorp C bottom well modules in a 96-

well frame were coated with 100 Cll of goat anti-horse immunoglubumin diluted in carbonate

bicarbonate coating buffer (Sigma #C3 041). After 1 hr of incubation, the buffer was removed

and each well was washed three times with 200 Cll of washing buffer (Sigma #T903 9). After

washing, 200 Cll of postcoat solution (T6789) was added to each well and the plate was incubated

for 30 minutes. After incubation, the washing step was repeated three times. Dilutions of

immunoglobulin standard were prepared from the standard serum according to the protocol.

Plasma samples were diluted with sample/conjugate diluent. A suitable dilution of sample was

predetermined with equine plasma with the same assay. 100 Cll of each dilution of standard and

diluted plasma was added to a designated well in duplicate. The plate was incubated for one hour

at room temperature. After incubation, the washing step was repeated three times, and 100 Cll of

diluted goat anti-horse Ig-HRP conjugate was added to each well. The plate was then incubated

for 1 hour. After incubation, the excess HRP conjugate was removed, and the washing step was

repeated five times. After washing, 100 Cll of TMB peroxidase substrate was added to the each

well, and the plate was incubated for 15 minutes. The peroxidase reaction was stopped by adding

100 Cll of 2M of H2SO4 (Fisher #SA8 18-500) to each well. The plate was evaluated with a

universal microplate reader at a wavelength of 450 nm. The concentration of immunoglobulin in










the sample was calculated based on a four-parameter logistic curve-fit generated by KC4

software.

Statistical Analysis

Immunoglobulin concentrations, ROS generation ratios, and TNF-oc concentrations were

first evaluated by box plot. Outlier and extreme outlier data in each data set were excluded from

further statistical analysis. Differences between pre-EA and pre-AC treatment groups were

compared using Mann-Whitney Rank Sum test. Differences between pre- and post-within-group

treatments were compared using Wilcoxon Signed Rank test. Differences between post-EA and

post-AC treatment groups were compared using Mann-Whitney Rank Sum test. Because of

presumed genetic differences and variation in housing environments, a p valtfd.1 was used

for determining significance. Statistical analysis was performed with SPS S. 17 for Windows.

Results

Animals

Twenty-one geldings and 3 mares were included in this study. The EA group contained

12 geldings, while the AC group contained 9 geldings and 3 mares. MeantSD age of subjects in

EA and AC groups were 8.211.1 years (ranged from 6-10) and 7.412. 1 years (ranged from 3-10),

respectively. MeantSD body weight of subj ects in EA and AC groups were 54613 5 kg (ranged

from 490-590) and 539146 kg (ranged from 473-615), respectively. Demographic information

on horses in the EA and AC groups is presented in Table 2-2.

Acupuncture and Electroacupuncture Procedures

With physical restraint by a halter and leading rope held by a handler, most of the horses

accepted the AC and EA procedures. One horse in the AC group showed signs of mild

discomfort, including restlessness, and continuously contracted its cutaneous muscles at the first

AC treatment. The signs of discomfort decreased during the second and the third AC treatments.









Two horses in the EA group showed signs of severe discomfort when acupuncture needles were

stimulated with electricity. The signs of discomfort included restlessness and twitching of the

cutaneous muscles. However, EA was completed on all horses without additional physical

restraint. Rhythmic muscle contraction during the low frequency stimulation was not observed in

our study.

Homogeneity of Samples Before Electroacupuncture and Acupuncture

Mean+SE concentrations of immunoglobulins, ROS ratios generated by circulating

neutrophils, and TNF-a production of whole blood in pre-treatment samples of EA and AC

groups are given in Tables 2-3 to 2-5. Concentrations of IgA, IgM, and IgGb between EA and

AC groups before treatment were quite uniform. Reactive oxygen species ratios generated by

circulating neutrophils following stimulation with PMA, LPS, Zym, CA, and CE between EA

and AC groups before treatment were also essentially the same. Tumor necrosis factor alpha

production of whole blood following LPS, Zym, CA, CE, and CA+CE stimulations between EA

and AC groups before treatment were not significantly different. However, concentrations of

IgGa, IgG(T), and the TNF-a production of whole blood stimulated with CA+LPS, CE+LPS,

and CA+CE+LPS were significantly different in pre-treatment samples of the two groups of

horses (Table 2-6).

Plasma Immunoglobulins

After three treatments of AC and EA, plasma concentrations of the five immunoglobulin

isotypes measured in this experiment were not significantly different from the levels prior to

treatment (Table 2-7). Between groups comparisons of post-treatment samples of EA and sham

groups were not significantly different (Table 2-10).









Reactive Oxygen Species Generation of Neutrophil

The viability of isolated neutrophils was greater than 96% in all samples prior to the final

wash. The assessment of RO S was done using a fixed number of neutrophils recovered from

blood samples (3 00,000 cells per sample); thus, it represents a measure of an average "per cell

response" rather than a direct measure of the change in the total circulating ROS production

capacity of the horse. The change in the number and fraction of circulating neutrophils would be

required to generate an assessment of the total impact in the horse.

Mean+SD ROS ratios generated by each stimulant and test statistics from within-group

comparisons are presented in Table 2-8. Phorbal 12-myristate 13 acetate generated the greatest

ROS production in all samples. Electroacupuncture treatment induced a significantly increased

neutrophil-ROS response when the cells were stimulated with PMA. Phorbal 12-myristate 13

acetate induces ROS production by maximal intracellular simulation of enzymatic action,

primarily through complete activation of protein kinase C at the concentration tested.

However, EA treatment significantly reduced ROS production by neutrophils when the

cells were stimulated with 1Clg/ml of LPS, but not with lower concentrations (0. 1 and

0.0 1Cg/ml). Lipopoly saccharide induces RO S through TLR4-mediated signaling and its

associated inflammatory activation. Neutrophil-ROS response ratios generated by neutrophils

when the cells were stimulated with Zym, CA, and CE were not affected by EA. Acupuncture

treatment did not affect the production of ROS by any of the stimulants utilized in these trials

(Table 2-8). Between-group comparisons of post-treatment samples of EA and sham groups were

not significantly different (Table 2-10).










Heparinized-blood Stimulation and Equine Tumor Necrosis Factor Alpha (TNF-cl) Assay

Tumor necrosis factor alpha production in whole blood cultures is a measure of the

capacity of all the cells in a fixed volume sample of blood to mount a specific inflammatory

response under conditions directly simulating blood in the circulation. The TNF-a production

response is impacted by both the circulating numbers and fraction of monocytes (the cells

primarily responsible for TNF-a production) and their fraction of circulating cells.

After in vitro incubation of whole blood with only PB S added, TNF-a production in the

blood samples obtained from two horses receiving AC treatment were greater than 500 pg/ml.

The high concentrations of TNF-a in the absence of an inducing stimulant may indicate

contamination of the blood samples. Therefore, data derived from these horses were excluded

from the statistical analyses.

For all horses, production of TNF-a was significantly greater in whole blood cultures

stimulated with LPS, Zym, or CA than for cultures containing only PBS. However, CE proved

to be an ineffective stimulant for TNF-a production in this whole blood culture system. Thus, the

data from CE stimulated whole blood cultures were excluded from within-group analysis.

Electroacupuncture treatment significantly reduced TNF-a production in response to

Zym, CA, CA+CE, CA+LPS, CE+LPS, and CA+CE+LPS (Table 2-9). However, AC treatment

did not significantly reduce TNF-a production with any stimulants used (Table 2-9). TNF-a

production by whole blood culture containing Zym, CA+LPS, CE+LPS, and CA+CE+LPS of

horses receiving EA treatment was significantly lower than that receiving AC treatment; p-values

were 0.03, 0.06, <0.01, and 0.06, respectively. Moreover, there were no significantly differences

in TNF-a production by whole blood culture containing LPS, CA, CE, and CA+CE after EA and

AC treatment (Table 2-10).









Discussion

Acupoints that possess immune stimulatory properties include LI-4, LI-11, ST-36, GB-

39, SP-6, GV-14, BL-11, BL-20, BL-23, BL-24, BL-25, BL-27, BL-28, and CV-12. 137 Our

study investigated the immune modulatory effects of AC and EA when stimulations were

performed at LI-4, LI-11, and GV-14. These acupoints have been demonstrated to possess anti-

inflammatory and analgesic properties in rats.147 They are located on the cranial part of the body

including the front limbs. Their locations offer a safe and easy approach for the veterinary

acupuncturi st to insert an acupuncture needle.

LI-4, also known as He-gu, is one of the most studied acupoints in humans and laboratory

animals. It has been used for reducing pain and alleviating inflammation originating in the face

and j aw regions.148,149 An increase in the neural activity of the spinal cord at the 6th COTVICal

vertebra to the 1st thoracic vertebra after AC at LI-4 indicated that sensory input originating from

AC is partly involved in the analgesic and therapeutic mechanisms of this acupoint.lso During

acupoint stimulation, cutaneous and somatic sensory inputs were transmitted to the spinal cord.

Sensory inputs generated from LI-4 and LI-11 were transmitted through the brachial plexus,

while the sensory input originating from GV-14 was transmitted to the spinal cord via the dorsal

cutaneous branches of the dorsal branches of the cervical and thoracic nerves. Details of the

cutaneous and somatic sensory innervations are shown in Table 2-11.

Electroacupuncture at LI-4 has been shown to increase the functional magnetic resonance

imaging (fMLRI) signal in several parts of the brain including, the pre-central gyrus, postcentral

gyrus, and putamen, and insula, while AC decreased the fMRI signal in the posterior cingulate,

superior temporal gyrus, putamen, and insula.l5 These results suggested that EA and AC

recruited different neural networks for their treatment mechanisms.l5









GV-14, also know as Da-zhui, is recommended in TCVM for treating disease patterns

caused by Excess Heat, including fever, skin rash, seizure, sweat, and other abnormalities such

as cough and cervical pain.135 Recent research showed that a series of AC at GV-14, LI-11, SP-

10, and ST-36 decreased the total number of peripherally circulating leukocytes and the number

oflymphocytes.138 Acupuncture has been shown to decrease the level of serum IgM in humans

suffering from Behcet' s disease.152 The disease is characterized by a chronic inflammation of

blood vessels throughout the body. This inflammation is likely caused by an anti-endothelial

antibody (i.e., IgM). Patients receiving AC treatment also benefited from a lower recurrence rate

in clinical signs than did patients receiving conventional treatment. However, how AC reduced

serum IgM was unknown.152 Decreases in serum IgM and increases in serum IgG after AC also

have been demonstrated in human asthma. 153 AC at CV-3, GV-4, GV-14, GV-20, SP-6, SP-9,

LI-4, LI-11, ST-30, ST-36, KI-3, KI-5, TH-5, LIV-2, BL-28, BL-31, BL-43, and PC-6 for 12

treatments over a period of one month has been shown to be beneficial in the treatment of

chronic pelvic inflammatory disease in women.154 After the last treatment, serum IgM was

decreased, while total serum y-globulin was increased.

The benefit of AC to immune modulation was demonstrated in stress-related immune

suppression in competing athletes. Daily AC at LI-4, ST-36, ST-6, and LU-6 for 15 minutes has

been shown to inhibit exercise-induced decrease of salivary IgA, and exercise-induced increase

of salivary cortisol. "5 After the treatments, subj ects reported high scores for their mental

wellness. Acupuncture inhibition of exercise-induced alteration of immunological and

endocrinological parameters in this study was positively correlated with psychological

parameters. Therefore, the results were potentially due to placebo effects. Electroacupuncture

and AC treatment regimes in our study did not alter the levels of circulating immunoglobulin.









This may be due to the fact that immunoglobulin is a product of the secondary immune response,

and a longer period of time for the immunoglobulin-producing cells to adapt to the

immunological stimulation might be required.

Phorbal 12-myristate 13 acetate caused maximum increase in ROS production by

mimicking the action of diacylglycerol which activates protein kinase C (PKC), the maj or

cellular phorbal ester receptor.156,157 Lipopolysalccharide induces ROS production through the

binding of the Toll-like receptor 4 (TLR4) receptor and its associated signaling cascade.

Electroacupuncture significantly increased the neutrophil-ROS response when cells were

stimulated by PMA, but significantly decreased the response when cells were stimulated with

LPS at a concentration of 1 Clg/ml. Phorbal 12-myristate 13 acetate induces maximal ROS

responses in horse neutrophils, essentially representing the total enzymatic capacity of cells to

produce ROS. In contrast, LPS induces ROS production based on extracellular signaling and is

limited by the level of TLR4 receptor expression, and the capacity of neutrophils to amplify

TLR4 mediated signaling. Thus, the enhancement of PMA induced ROS and reduction in

maximal LPS induced ROS may represent a down-modulation in the level of pro-inflammatory

receptors (such as TLR4), but an increase in the general enzyme levels needed to produce ROS.

This suggests that a complex set of regulatory processes in the function of neutrophils are

induced by EA, but the same regulatory level is not reached by simple AC stimulation. However,

there is no research evidence demonstrating up regulation of PKC by either EA or AC. Previous

research in rats showed that EA stimulation at ST-3 6, GV-20, Yin-tang, and ST-40 could reverse

an over-expression of PKA and PKC in the hippocampus.l5

Neutrophil-ROS responses to other stimulants did not differ relative to EA treatment.

Each was due to signaling through different families of cell surface receptors. The response to









zymosan is regulated by several receptors and the lack of significant change in the overall

response may have been due to a shift in the expression of individual receptors that gave an

equivalent balance in the response. The responses to both the fungal antigens utilized were rather

weak, so a larger enhancement or depression of the response would have been needed to measure

a significant level of change.

The EA-induced increase in ROS production of neutrophils when stimulated with PMA

corresponded to the results of earlier research, which demonstrated an increase in reactive-

oxygen burst (RB) activity of neutrophils following a series of AC treatment.139 In the previous

research, RB of neutrophils was induced by either priming with initial incubation with

recombinant TNF-oc followed by receptor stimulation with N-formyl-methionyl -leucyl

phenylalanine (FMLP), FMLP activation alone, or by phagocytosis of E. coli. N-formyl-

methionyl-leucyl phenylalanine is derived from bacterial protein degradation.159 Receptors for

FMLP can be found on the surface of granulocytes.160 Binding of FMLP receptors activates

phospholipase C and causes increase production of diacylglecerol and inositol triphosphate.

These secondary messengers later activate the PKC, which is also activated by PMA.

Lipopolysaccharide activates the TLR4 present on the cell surface, which activates MAP

kinase (Erk) and nuclear factor KB (NF-KB), which in turn, control transcription of target

genes.161 Decrease in ROS production by neutrophils after the cells were stimulated with LPS

suggested that EA might possess an anti-inflammatory effect. However, its mechanism is still

inconclusive. Previous research on the effects of EA at ST-3 6 in experimentally induced

cutaneous anaphylaxis in mice demonstrated that EA inhibited cutaneous anaphylaxis and

inhibited the release of 1-6.162 The anti-anaphylactic activity of EA was associated with a









reduction in DNA binding activity of NF-icB, as determined by Western blot analysis and

transcription factor enzyme-linked immunoassay.

In our assessment of TNF-a production in whole blood cultures, we found that LPS was

the most potent stimulant. Lipopolysaccharide was more potent than CA, CE, or Zym. Culture

filtrate antigen of A. fumigatus was the weakest stimulant of TNF-a production in whole blood

cultures. TNF-a production induced by Zym was inconsistent, and only two horses from each

group responded to Zym. The cause of inconsistent responses to Zym is unknown, but may be

due partly to individual subj ect differences in expression of the family of receptors regulating

Zym response. There was evidence of a synergistic effect when CA or CE was individually, or if

both were added to LPS. Electroacupuncture generally suppressed TNF-a production in whole

blood cultures, except when LPS was added alone. Results of cultures with multiple stimulants

combined with LPS (i.e. CA+LPS, CE+LPS, and CA+CE+LPS) indicated that EA generally

suppressed TNF-a production.

In the AC group, TNF-a production in whole blood cultures was not significantly

different from the baseline samples prior to treatment. Tumor necrosis factor alpha is an

inflammatory cytokine. It is an important component of the early response of the innate immune

system. Therefore, suppression of TNF-a release from immune cells, as demonstrated in this

study, may partly play a role in how EA exert its anti-inflammatory action.

Until recently, it has not been clear how AC and EA modulate immune functions.

Previous research in electrical stimulation of the vagus nerve has been shown to attenuate

endotoxin-induced shock in rats. This modulation was characterized by an inhibition of TNF-a

synthesis in the liver and a reduction of the TNF-a concentration in plasma.81 The inhibition of

endotoxic shock was suggested to be partly due to the ability of EA to restore the hepatic









metabolism of rats subj ected to LPS-induced shock. The restoration of hepatic metabolism was

characterized by the ability of the hepatocytes to maintain their glycogen storage level and the

activity of the enzyme sorbital dehydrogenase (SDH), glucose-6-phosphate, and ATPase.163

The vagus nerve is a maj or part of the cholinergic nervous system and contains both

afferent and efferent nerve fibers for both somatic and visceral tissues.79'80 The cholinergic

efferent signals have been demonstrated to cause vasodilatation in visceral tissues and alter the

contractility of vi sceral smooth muscle, including intestines and uterus.164,165 Electrical

stimulation of the efferent vagus nerve attenuated macrophage-dependent intestinal inflammation

in the post-operative ileus in mice.166 This attenuation has been suggested to be due in part to the

activation of the alpha-7 nicotinic acetylcholine receptors on the macrophage. Activation of

endothelium nicotinic acetylcholine receptors also has been shown to reduce the expression of

the adhesion molecule and chemokine release.167 Therefore, cholinergic dependent anti-

inflammation may be due partly to an inhibition of inflammatory cell migration to the site of

tissue damage, and down regulation of the production of pro-inflammatory cytokines that cause

inflammation. This finding may be used to explain how EA reduces inflammation in diseases of

the visceral organs.76 Whether AC possesses the same therapeutic mechanism or not is still

inconclusive and needs further investigation.

Electroacupuncture and acupuncture in our study did not produce immunological

modulation that was fully comparable with those of previous studies. In these studies, EA

treatment, but not AC treatment, significantly modulated neutrophil production ofROS induced

with either PMA or LPS, and modulated TNF-oc production by blood cells stimulated with LPS,

both alone and in combination with other stimuli tested. This could be because the horses used

in our study were healthy. Thus, they might not gain as much benefit from either EA or AC










treatment as sick people or animals. According to one of the TCVM treatment principles, the AC

and EA treatment obj ective is to cure an imbalance of the health status. Healthy horses can be

assumed to able to maintain their health. Thus, the treatment should do neither harm, nor provide

significant benefit to them. Moreover, in a real clinical TCVM treatment, multiple EA or AC

treatments are performed over a long period of time at a pre-defined interval, such as weekly

treatment for five to ten consecutive weeks. The clinical AC/EA treatment also differs from our

AC/EA treatment in terms of the numbers of acupoints being stimulated, and how each acupoint

was selected. However, our study suggested that short-term aggressive stimulation at LI-4. LI-

11i, and GV-14 has a potential impact on immunological function. Despite the lack of a clear

impact on neutrophil-ROS response to EA and AC treatments, both EA and AC reduced the level

of TNF-oc production in whole blood culture after exposure to individual and combinations of

microbial stimulants. Our results also suggest that the immunomodulatory effect of EA and AC

may be partly due to a modulation of the innate immune response mediated primarily by

monocytes, and that EA is more effective than AC.




































E


AC (x10' ng/ml)
11.410
11.312
7.326
11.120
28.701
+2.088
57.810
+4.057
39.125
12.938


IgA

IgM

IgGa

IgGb

IgG(T)

EA = electroacupuncture, AC =


Table 2-1. Anatomical location of acupoints and treatment indications.
Acupoints Anatomical location Treatment indications
LI-4 Distal and medial to base of second Facial paralysis, dental pain, sore throat,
metacarpus. anhydrosis, fever, and
immunodefi ci ency.
LI-11 In depression cranial to elbow in the Fever, dental pain, uveitis, sore throat,
transverse cubital crease. seizure, abdominal pain, diarrhea, and
paralysis of front limb.
GV-14 Dorsal midline at depression in Fever, cough, heaves, cervical stiffness,
cervicothoracic vertebral space skin rash, and seizure.
(C7-T 1)
(Source: Xie H, Trevisanello L. Equine Transpositional Acupoints In: Xie H,Preast V, eds. Xie's
veterinary acupuncture. 2007; page 27-87.135)

Table 2-2. Demographic information on horses in the EA and AC groups.
Group Gelding Mare Total Age in year (meanfSD) Age range (years)
EA 12 0 12 8.2 f 1.1 6 10
AC 9 3 12 7.4 f 2. 1 3 10
EA = electroacupuncture, AC = acupuncture.

Table 2-3. MeantSE concentration of immunoglobulin isotypes (x105 ng/ml) from pre-treatment
samples of EA and AC groups.


Group


Ig isotype


A (x10'ng/ml)
10.761
~0.877
6.773
~0.701
33.603
~2.183
55.046
~3.489
46.295
~2.228
acupuncture.










Table 2-4. MeantSE neutrophil-ROS response ratio from pre-treatment samples of EA and AC
groups.
Group
Stimulant EA AC
PMA 20.60 20.89
~0.80 11.03
LPS (1.0 Gig/ml) 1.52 1.69
~0.13 10.15
LPS (0.1 Clg/ml) 1.33 1.54
~0.08 10.13
LPS (0.01 Clg/ml) 1.26 1.40
~0.06 10.08
Zym 1.79 1.95
~0.13 10.09
CA 1.19 1.34
~0.06 10.11
CE 1.10 1.16
~0.03 10.02
ROS = reactive oxygen species, EA = electroacupuncture, AC = acupuncture, PMA = phorbol
12-myristate 13-acetate, LPS = lipopolysaccharide, Zym = zymosan, CA = cellular antigen ofA.
jitmigatus, CE = culture extract antigen from static growth ofA. fumigatus.










Table 2-5. MeantSE TNF-oc concentrations (pg/ml) from pre-treatment samples of EA and AC
groups.
Group
Stimulant EA AC
None 0 0
0O 10
PBS 0 0
0O +0
LPS 3893 5647
~576 1915
Zym 530 1029
~214 1264
CA 4487 4492
~891 +464
CE 0 0
+0 +0
CA+CE 4309 5715
~737 1938
CA+LPS 4801 7004
~668 1963
CE+LPS 3759 6595
~344 1896
CA+CE+LPS 5282 8008
~605 11064
EA = electroacupuncture, AC = acupuncture, PMA = phorbol 12-myristate 13-acetate, LPS =
lipopolysaccharide, Zym = zymosan, CA = cellular antigen ofA. jitmigatus, CE = culture extract
antigen from static growth ofA. fumigatus.





















































Ig = immunoglobulin isotype, EA = electroacupuncture, AC = acupuncture.


Treatment
EA 12 2n 2 10 AC (12 2n 2 10)
Pre Post Pre Post
Ig isotype (x105 ng/ml) (x105 ng/ml) P-value (x105 ng/ml) (x105 ng/ml) P-value
IgA 10.761 10.820 0.57 11.410 11.020 0.38


Table 2-6. Mann-Whitney Rank Sum test statistics between-group comparisons of pre-treatment
samples in EA and AC groups.


TNF-oc production of
whole blood stimulation
by stimulant
None
PBS
LPS
Zym


ROS of neutrophils
by stimulant
PMA
LPS 1.0
LPS 0.1
LPS 0.01


Ig isotype
IgA
IgM
IgGa
IgGb


P-value
0.843
0.977
*0.089
0.843


P-value
0.608
0.468
0.401
0.217


P-value
1.000
1.000
0.148
0.180


IgG(T) *0.089 Zym 0.316 CA 0.624
CA 0.562 CE 1.000
CE 0.169 CA+CE 0.219
CA+LPS *0.093
CE+LPS *0.006
CA+CE+LPS *0.059
ROS = reactive oxygen species, PMA = phorbol 12-myristate 13-acetate, LPS =
lipopolysaccharide, Zym zymosan, CA cellular antigen of A. fumigatus, CE culture extract
antigen from static growth ofA. fumigatus, # = outlier and extreme outlier data determined by
box-plot were excluded from the analysis, = significant difference was accepted at p < 0.1.

Table 2-7. Mean+SE immunoglobulin isotype concentrations (x105 ng/ml) of pre- and post- EA
and AC treatments, and test statistics of within-group comparisons.


11.312
7.326
11.120
28.701
+2.088
57.810
+4.057
39.125
12.938


11.185
7.2 058
11.141
28.345
12.105
58.868
14.945
40.597
13.172


~0.877
6.773
~0.701
33.603
~2.183
55.046
~3.489
46.295
~2.228


10.966
6.654
10.699
32.845
+2.041
54.596
13.482
46.657
12.129


IgM

IgGa

IgGb

IgG(T)


0.52

0.20

0.47

0.69


0.42

0.47

0.83

0.69










Table 2-8. Mean+SD neutrophil-ROS response ratios of pre- and post- EA and AC treatments,
and test statistics of within-group comparisons.
Treatment
EA 12 2n 2 10) AC (12 2n 210)
Stimulant Pre Post P-value Pre Post P-value
PMA 20.60 23.19 <0.01 20.89 22.10 0.55
~0.80 11.13 11.03 11.34
LPS1.0 1.52 1.23 0.05 1.69 1.47 0.19
(Cig/ml) 10.13 10.03 10.15 10.12
LPSO.1 1.33 1.25 0.43 1.54 1.38 0.19
(Cig/ml) 10.08 10.06 10.13 10.09
LPSO.01 1.26 1.33 0.58 1.40 1.27 0.27
(Cig/ml) 10.06 10.13 10.08 10.06
Zym 1.79 2.11 0.15 1.95 2.06 0.27
~0.13 10.06 10.09 10.08
CA 1.19 1.16 0.72 1.34 1.17 0.19
~0.06 10.02 10.11 10.04
CE 1.10 1.15 0.23 1.16 1.15 0.57
~0.03 10.02 10.02 10.03
EA = electroacupuncture, AC = acupuncture, PMA = phorbol 12-myristate 13-acetate, LPS =
lipopolysaccharide, Zym = zymosan, CA = cellular antigen of A. jitmigatus, CE = culture extract
antigen from static growth ofA. fumigatus.










Table 2-9. Mean+SD TNF-oc concentration (in pg/ml) of pre- and post- EA and AC treatments,
and test statistics of within-group comparisons.
Treatment
EA (12 > n 2 10) AC ( 10> n 2 8)
Stimulant Pre Post P-value Pre Post P-value
None 0 0 1.00 0 0 1.00
0O 10 10 10
PBS 0 0 1.00 0 0 1.00
0O +0 +0 +0
LPS 3893 3834 0.97 5647 5014 0.19
~576 1611 1915 1643
Zym 530 14 0.06 1029 706 0.54
~214 114 1264 1261
CA 4487 2675 0.06 4492 4292 0.64
~891 1713 +464 1783
CE 0 41 1.00 0 0 1.00
0O 133 10 10
CA+CE 4309 2889 0.07 5715 4594 0.30
~737 162 1938 1908
CA+LP S 4801 3646 0.06 7004 5821 0.49
~668 1468 1963 1766
CE+LP S 3759 2851 0.08 6595 5578 0.49
~344 1319 1896 1735
CA+CE+LP S 5282 4125 0.03 8008 5873 0.20
~605 1693 11064 1661
EA = electroacupuncture, AC = acupuncture, PMA = phorbol 12-myristate 13-acetate, LPS =
lipopolysaccharide, Zym = zymosan, CA = cellular antigen of A. fumigatus, CE = culture extract
antigen from static growth ofA. fumigatus.









Table 2-10. Mann-Whitney Rank Sum test statistics for between-group comparisons of post-
treatment data in EA and AC groups.
TNF-oc production of
ROS of neutrophils whole blood stimulation
Ig isotype P-value by stimulant P-value by stimulant P-value
IgA 0.79 PMA 0.56 None 1.00
IgM 1.00 LPS 1.0 0.19 PBS 1.00
IgGa 0.114 LPS 0.1 0.30 LPS 0.16
IgGb 0.608 LPS 0.01 0.65 Zym *0.03
IgG(T) 0.123 Zym 0.59 CA 0.12
CA 0.84 CE 0.57
CE 0.97 CA+CE 0.19
CA+LPS *0.06
CE+LP S *<0.0 1
CA+CE+LPS *0.06
ROS = reactive oxygen species, PMA = phorbol 12-myristate 13-acetate, LPS =
lipopolysaccharide, Zym zymosan, CA cellular antigen of A. fumigatus, CE culture extract
antigen from static growth ofA. fumigatus, # = outlier data determined by box-plot were
excluded from the analysis, = significant difference was accepted at p < 0.1i.

Table 2-11. Cutaneous and muscle innervations of acupoints being stimulated.
Acupoint Cutaneous nerve Muscle / innervation
LI-4 Medial cutaneous antebracheal of None / medial palmarmetacarpal
the musculocutaneous nerve
LI-11 Cranial cutaneous antebrachial, Triceps (lateral head) / radial Brachialis /
lateral cutaneous antebrachial musculocutaneous and radial
nerve of the superficial branch
of the radial nerve, and lateral
cutaneous branch of 2nd thoracic
nerve (component of the
intercostobrachial nerve)
GV-14 Dorsal branch of local cervical and
local thoracic spinal.
(Source: Budras K-D, Sack WO, Roick S, et al. Anatomy of the horse : an illustrated text. 4th ed.
Hannover: Schltitersche, 2003. Blythe LL, Kitchell RL. Electrophysiologic studies of the
thoracic limb of the horse. Am J Vet Res 1 982;43:.15 1 1-1 524 168,169$









CHAPTER 3
COMPARISON OF INDUCTANCE PLETHYSMOGRAPHY AND
PNEUMOTACHOGRAPHY AND THE RAPID PARTIAL FORCED EXPIRATION
MANEUVER FOR DIAGNOSIS OF EQUINE LOWER AIRWAY INFLAMMATORY
DISEASE.

Introduction

Chronic lower airway inflammatory disease is one of the most common causes of poor

performance in horses. These include inflammatory airway disease (IAD), recurrent airway

obstruction (RAO) or heaves, and summer pasture associated obstructive pulmonary disease

(SPAOPD). Inflammatory airway disease is diagnosed in young training racehorses, while the

other two diseases are commonly diagnosed in older horses and usually associated with poor

stable management."o It is still unclear whether horses that have been diagnosed for IAD at on

early age may progress to RAO or SPAOPD. Horses that are being kept in a poorly ventilated or

dusty environment are at a higher risk of developing the diseases. Affected horses show signs of

chronic coughing, dyspnea, exercise intolerance, and poor performance. There are also increases

in respiratory rate, nasal discharge, and neutrophils in broncho-alveolar lavage fluid (BALf).86

Inflammatory airway disease, RAO, and SPAOPD are thought to share many similarities in their

disease pathogenesis. In chronic long-standing cases, alteration of the pulmonary and airway

histological structures in response to the persistent exposure of a causative antigen results in

increased airway resistance and a decreased gas exchange capacity of the lung."'

Until recently, there has been no single diagnostic procedure that can be used to

accurately distinguish these diseases. Current diagnosis is based on case history, clinical signs,

broncho-alveolar fluid (BALf) cytology, and response to the treatments. The ability to recognize

these diseases at an early stage is important. However, this is difficult due to the lack of specific

clinical manifestations and, more importantly, affected horses may not show clinical signs of

respiratory problems until the amount of pulmonary tissue affected is greater than the functional









reserve. For these reasons, many affected horses are undiagnosed due to the sub-clinical nature

of these diseases. Several progressive diagnostic methods have been developed in attempting to

identify early stages of the disease, including intra-pleural pressure, histamine

bronchoprovocation, and forced expiration. 172-175

Airway hyper-responsiveness is thought to be a sequel to chronic airway inflammation.

Inflammation of airways increases mucus secretion and accumulation in the airways.176 It also

increases the respiratory resistance caused by contraction of the airway smooth muscles; this is

triggered by inflammatory cytokines being released locally by infiltrated leukocytes. Airway

hyper-responsiveness can be provoked by exposing the affected airways to a known irritant or by

an administration of exogenous histamine.144, 171,177 In human medicine, testing a physiological

response of the airway with histamine is called histamine bronchoprovocation (HB) or histamine

challenge. Together with the pulmonary function test, HB has been used in the diagnosis and

determination of the severity of chronic respiratory problems like bronchial asthma and chronic

obstructive pulmonary disease (COPD).17 Comparison of a series of HB tests over time has been

suggested to be useful for obj ectively determining improvement in clinical signs and response to

therapy.

The effects of HB were determined by pulmonary function testing, conducted

immediately after HB. The most commonly performed test in human respiratory clinics is forced

expiration (FE).179 COmputed parameters derived from the test are collectively called pulmonary

function test parameters (PFTPs). Commonly computed PFTPs of FE include forced expiratory

volume in x second (FEVx), forced expiratory volume (FEV) or forced vital capacity (FVC),

peak expiratory flow (PEF), and forced expiratory volume in 1 second/FEV ratio (FEV1/FEV).

The most commonly reported FEVx is the FEV1. Expiratory flow rates at 25, 50, and 75% of









FVC has been expired (MEF25%, MEF50%, and MEF75%) also can be computed.17 These

PFTPs reflect biomechanical characteristics of the airways and pulmonary tissues, including lung

volume, airway resistance, and lung compliance. Deviation of these parameters from reference

values can be used as indicator of pathology in pulmonary tissues.

Pulmonary disease produces alteration in PFTPs at the end of expiration. When the

airflow rate and expiratory volume are plotted, flow volume (FV) loops are generated.

Pulmonary pathology affects both PFTPs and the FV loop. The FV loop of a patient suffering

from obstructive pulmonary disease demonstrates a scoop out characteristic after peak flow. This

scooped out characteristic indicates decreased airflow rate caused by narrowing of the small

airways, is a common pathological change found in lower airway inflammatory diseases."so,1s

The forced expiration test procedure in humans is self-induced and voluntary. It is

accomplished with coaching by respiratory therapist or diagnostician. The test procedure is

explained to the patient before the recording is made. In some instances, a patient may be asked

to practice the test before recording of the data is attempted. The quality of the test depends on

good coaching and the cooperation of the patient. Explaining the details of the test to the patient,

and emphasizing that the reliability of the test depends exhaling as rapidly and as fully as

possible is critical.182 Forced expiration is considered a superior method of testing pulmonary

function. It is reliable and consistent.18

In equine medicine, HB also has been described.174,184,185 By comparing respiratory

function following admini station of various concentrations of hi stamine to the airways, a

histamine response curve has been generated. Results suggested that the technique provides a

non-invasive alternative diagnostic technique for clinical practices.17









In equine practices, the measurement of FE and computation of the associated PFTPs

have been described."' The maneuver has been accomplished by exposing an airtight-sealed

airway and maximally inflating the lung to its total lung capacity (TLC) to a negative pressure. A

90 cm long by 22 mm internal diameter nasotracheal tube with inflatable cuff was used to

connect the lower airways with the negative pressure reservoir. Airflow and respiratory volume

were indirectly computed by measuring the instantaneous change in pressure in a vacuum

reservoir. The flow volume (FV) loop thus derived was diagnostic for obstructive pathologies of

airways."' Reproducibility of the computed PFTPs in horses was confirmed. However, the flow

volume loop generated by this method possessed a long plateau phase in the middle of the flow

indicating that airflow limitation and high resistance in the system were likely caused by the

nasotracheal tube. The experimental apparatus developed for this study created a system to

directly measure airflow generated by the forced expiration maneuver, and to eliminate the

limitation of previously described systems.

Obj ectives in thi s study include:

* To develop an electro-mechanical system with the capability of directly measuring the
airflow generated by FE in horses and with minimal airflow resistance artifact.

* To investigate the effect of negative pressure applied to the airways of horses and to
determine a suitable negative pressure for inducing reproducible FE measurements.

* To compare the diagnostic value of the PFTPs computed from the FE maneuver, and with
a histamine response curve from HB generated by respiratory inductance plethysmography
/ pneumotachography, to BALf cytology.

Methods

Subject

Twenty-four mature Thoroughbred horses with no evidence of cardiovascular, respiratory

or musculoskeletal problems by physical exam were used in this study. The horses were

assembled into groups of two to four and kept in paddocks with shelters. Physical fitness was









maintained with eight minutes of exercise on a treadmill three times per week. The exercise

included two minutes of warm-up trotting, four minutes of cantering, and two minutes of cool-

down trotting. The horses were fed twice daily with commercial concentrate. Good quality hay

and water were available ad libidum. None of the horses had any hi story of respiratory problems

and received no medications during the two weeks prior to the experiment. Protocol for animal

use was approved by the University of Florida Institutional Animal Care and Use Committee

(Permit A-130).

Study Design

The airway hyper-responsiveness of the horses was determined by challenge with

histamine diphosphate (HD). The histamine response curve was generated using inductance

pl ethy sm ograp hy/pn eum ota chography. Th e te st wa s rep eated twi ce at there e to fi ve week i nterval

to determine the repeatability of the test. After each HB, BALf cytology of each horse was

evaluated. Five weeks after the last HB, a pulmonary function test was performed in these horses

with rapid partial FE maneuver. The results of the histamine response curve from HB, BALf

cytology, and PFTPs derived from rapid partial FE maneuver were compared, and their

correlations were investigated.

Histamine Bronchoprovocation and Respiratory Inductance Plethysmography and
Pneomotachography (Open PlethTM)

Histamine bronchoprovovation and respiratory inductance plethysmography and

pneumotachography were conducted indoors in a well-ventilated airconditioned room. Two

horses were brought into the room at a time to minimize separation anxiety. Horses subj ected to

HB test were physically restrained in a stall, and the HB was performed on one horse at a time.

After a brief clinical examination, the horse was chemically restrained by intravenous

administration of 0.75 mg/kg of xylazine hydrochloride into the left external jugular vein. Five









minutes after tranquilization, the external nares of the horse were cleaned with damp-gauze.

Pulmonary function was determined by respiratory inductance plethysmography and

pneumotachography (Open PlethTAI, Ambulatory Monitoring, Inc, NY) before and after exposure

to HD at various concentrations using the following protocol. After calibration of the inductance

pl ethy sm ography/p neum ota chograp hy, th e test wa s p erforme d a ccordi ng to the m ethod provi de d

by the manufacturer:186

* Baseline data on pulmonary function were obtained following two minutes of a normal
saline nebulization to the airway by the equine aero mask tube nebulizer. Nebulization was
generated by the ProneboUltrall compressor nebulizer system with the Pari LCo Plus
Reusable Nebulizer (Pari VA USA). The solution was nebulized at a rate of 0. 148+0.018
ml/min. Pulmonary function data were recorded for two minutes immediately after the
nebulization. Collected data included inductance plethysmography and pneumotachgraphy.
These data served as a baseline.

* Histamine bronchoprovocation was performed by nebulization ofHD, as described by
Hoffman et al.ls Briefly, histamine diphosphate (HD) (1VP Biomedicals #100343) was
diluted in sterile normal saline solution (Baxter #NDC 0338-0048-04) to a final
concentrations of 2.0, 4.0, 8.0, 16.0, and 32.0 mg/ml. Histamine bronchoprovocation was
begun with nebulization of 2 mg/ml HD for 2 minutes and immediately followed by 2
minutes of data collection. The procedure was then repeated by substituting 2.0 mg/ml of
HD with 4.0, 8.0, 16.0, and 32.0 mg/ml of HD, sequentially.

* Alteration in flow rate after each concentration ofHD nebulization was plotted against
histamine concentration using FlowmetricTM Software. The concentration of histamine that
caused a 3 5% increase in delta flow (PC3 5 delta flow) WaS computed based on the histamine
dose response curve. Thi s PC3 5 delta flov i S also referred to as the concentration of hi stamine
that results in a 35% reduction in dynamic compliance of the airway. The degree of airway
hyper-sensitivity was categorized according to the manufacturer' s protocol (Table 3-1).

Bronchoalveolar Lavage.

Broncho-alveolar lavage (BAL) was performed within 1 hour after HB. Briefly, the horse

was chemically sedated with intravenous administration of 0.75 mg/kg of xylazine

hydrochloride. After 5 -10 minutes, the external opening of the right nasal cavity was cleaned

with damped gauze, and a 3 -meter long fiberoptic endoscope with a biopsy channel (Pentax EG-

2901XL) was inserted into the ventral nasal meatus of the right nasal cavity until reaching the









nasopharynx. The tip of the endoscope was further advanced into the distal trachea. The

epithelial lining of the trachea and bronchi were then desensitized by an infusion of 10 ml of 1 %

lidocaine hydrochloride. After desensitization, the endoscope was introduced into the left lung

lobe until the tip of the endoscope was wedged in small bronchi. One hundred ml of 3 7oC sterile

normal saline was infused into the small bronchi via the biopsy channel of the endoscope. The

fluid was immediately withdrawn via vacuum generated by pulling the plunger of the 60 ml

syringe. The lavage procedure was repeated two times to yield a total volume of fluid instillation

of300 ml. The recovered BAL fluid (BALf) was pooled, and the recovery volume was measured

with a 250 ml sterile cylinder. The BALf was filtrated through a double layer of sterile gauze

prior to being transferred into a sterile glass container. The physical appearance of BALf was

noted, and the BALf sample was kept on ice until analysis. The total number of nucleated cells in

the fresh BALf sample was determined by manual counting with a hemocytometer. The counting

was done in 4 large squares in the corners of 2 counting chambers. An average of the total

number of cells in 2 chambers was calculated. Number of cells in 1 ml was computed by the

following formula:

Number of cells in 1 ml = Average of number of cells x 2500 (3-1)

Two BALf cytological slides were prepared with Cytospin (Shandon). Briefly, 2-3 drops

of BALf were transferred into the Cyto-funnel, which was mounted on the microscopic slide that

was pre-loaded in a Cytospin rack. The slides were spun for 5 minutes at 100 RPM. The slides

were air-dried and stained with Dip Quick stain (Jorgensen Laboratories Inc., CO). BALf

cytology was determined under the light microscope. Two hundred nucleated-cells were

evaluated and counted with a cell counter. All counts were made by the same person.









Percentage of recovered tracheo-bronchial epithelial lining fluid (ELF) was determined

by urea dilution.18819 Urea is a small molecule that can diffuse across the mucus membrane

freely. In normal physiological circumstances, urea concentration in plasma and tracheo-

bronchial ELF is in equilibrium and equal in concentration. This fact has been confirmed in

sheep by a direct measurement of pulmonary ELF urea concentration.191 Therefore, if the BALf

sample is being withdrawn immediately after instillation, the dilution of ELF in the BALf sample

can be determined by measuring the urea concentrations of BALf and plasma samples

simultaneously. Urea concentrations in plasma and BALf samples were analyzed by quantitative

enzymatic colorimetric assay with Stanbio Enzymatic Urea Nitrogen (BUN) Procedure No. 2050

(Stanbio Laboratory, Texas). The assay was performed according to the manufacturer' s protocol.

Briefly, urea in the sample is hydrolyzed by the enzyme urease to yield ammonia and carbon

dioxide. The ammonium ions then react with a mixture of salicylate, sodium nitroprusside and

hydrochlorite to yield a blue-green chromophore. The intensity of the chromophore is evaluated

by reading an absorbance at 600 nm. The intensity of the color is proportional to the urea

concentration in the sample. Concentration of urea in the samples was calculated based on an

absorbance of the standard.

Rapid Partial Forced Expiration Maneuver

Unlike humans, it is impossible to control by coaching frequency and depth of active

voluntary breathing patterns of animals. However, it is feasible to mimic the breathing pattern of

an animal. The airways can be inflated with air at a controlled pressure to total lung capacity

(TLC) to mimic inspiration. Normal expiration, on the other hand, is caused by the force of

elastic recoil properties of pulmonary tissues, chest wall, and relaxation of the diaphragm. Forced

expiratory maneuver can be accomplished by emptying an airtight, sealed airway with a suitable

negative pressure system. Negative pressure causes more emptying of the air from the lung than










the force of elastic recoil and muscle contraction. As a result, the functional reserve volume of

the lung is reduced. With this information, a system that is capable of performing a rapid partial

forced expiration maneuver in horses was designed. The system is constructed with five maj or

electro/mechanical components.

* Negative pressure generator and vacuum reservoir.
* System for artificial inspiration.
* Airflow measurement apparatus.
* Airflow direction control system.
* Data acquisition system.

Negative pressure generator and vacuum reservoir

The negative pressure was generated by a vacuum pump (#2667-V108 Gast Imfg Corp)

driven by an industrial motor (#6K702BA Dayton). The vacuum was stored in a custom-made

850 liters reservoir. The negative pressure reservoir was made of 4 steel tubes, connected in

parallel by a 1" NPT PVC pipe. Each steel tube was 184.5 cm long by 38.5 cm internal diameter.

A single manual relay switch was used to turn the vacuum pump on and off. The level of

negative pressure was controlled with a 1" NPT manually actuated ball valve, which was

installed between the vacuum pump and the negative pressure reservoir. When the valve was

open, atmospheric air passes through the exhaust port and counters the negative pressure. To

create the negative pressure, the valve was closed and the vacuum pump was turned on. Negative

pressure in the vacuum reservoir was monitored by a pressure transducer (DP103, Validyne)

directly connected to the negative pressure reservoir by a rigid polyethylene tube (Parker

Parflex), (1/4" NPT external diameter and a 0.04" thick wall). The pressure transducer was

calibrated with a U-tube mercury manometer (#1223, Dwyer). Signals generated by the pressure

transducer were transferred to the data acquisition hardware and were recorded in a portable










computer by the data acquisition software. Details about data acquisition are described in the

section on the data acquisition system.

System for artificial inspiration

To mimic inspiration, an airtight sealed airway was pressurized with atmospheric air.

Because the airway was manually inflated, the following concerns were addressed.

* To mimic an inspiration pattern of the animal, the airways must be slowly inflated to its
TLC.

* A person performing artificial inspiration must be able to monitor the inflated airway
continuously and be able to quickly adjust the airway pressure as needed.

* Artificial inspiration must overcome the animal's physiological respiratory drive.

* Because atmospheric air is used to inflate the airways, introduction of air-born particles
into the airway must be prevented.

Based on these concerns, a 1300O-watt detachable blower of Shop-Vac (#723 0997, Shop-

Vac Corporation, Williamsport, PA) that is capable of generating a maximum airflow greater

than 3000 standard liter per minute (SLPM) was used. This blower was also used in an airflow

calibration process of the laminar flow element (LFE). The method of LFE calibration is

described in the airflow measurement system.

To address the first concern, the airflow rate generated by the blower was controlled. A

variable transformer capable of transforming 120 volts of alternating current (AC) to 0-140 volts

AC (Powerstat) (Figure 3-1) was used to control electrical voltage current to the blower. The

amount of airflow generated by the blower was positively correlated to the amount of voltage

supplied by the variable transformer. Gradually increasing the electrical current to the blower

slowly increased the amount of the airflow, and gently inflated the airway.

To address the second concern, pressure in the airways was monitored visually by an

analog pressure gauge (#LPG1, Dwyer) mounted in the system manifold near the horse. A









manually actuated 2" NPT PVC valve was installed in the manifold to serve as a pressure relief

valve to prevent over-inflation of the airway. Airway pressure was also measured by a pressure

transducer (DP45 Validyne) that connected to the manifold close to the junction of the

endotracheal tube with 1/4" NPT external diameter and a 0.04" thick wall of rigid polyethylene

tube (Parker Parflex). The pressure transducer was calibrated with a U-tube water manometer

(#1223 Dwyer). Airway pressure signals originated from the pressure transducer were transferred

to the data acquisition system and recorded by data acquisition software.

Normal respiration is controlled by the autonomic nervous system, which responds to

signals from chemoreceptors in the medulla oblongata (a change of pH) and the carotid and

aortic bodies (a change in partial pressure of oxygen and carbon dioxide in blood).192 The

respiratory center was over-ruled by hyperventilation at a rate of 25-30 times per minute,

approximately twice the normal resting respiratory rate and induction of hyperoxia and

hypocapnea. Decreasing pCO2 and increasing pO2 in the blood resulted in an increase in the

blood pH and suppressed the respiratory center.

The airway inflation method in the system was designed to bypass the upper respiratory

tract, and was specifically measure biomechanical properties of the lower airways. More

specifically, the system excluded airflow resistance caused by the anatomical structure of the

upper airways. However, because the artificial inspiration had bypassed all of the anatomical and

physiological mechanisms in the upper respiratory tract required for trapping air-born particles,

the air used for insulation was filtrated.

Airflow measurement apparatus

Airflow generated by rapid partial FE was measured using differential pressure (AP)

generated across the laminar flow element (LFE). In an ideal laminar flow, fluid molecules move









in parallel along the length of the flow path, and without turbulence. The differential pressure

drop can be measured across the LFE. Airflow measurement in this system was based on the

physics ofHagen-Poiseuille' s equation, which quantifies the relationship between pressure drop

and flow of fluid as:193

AP = 8rLQ/ xn r 4 (3 -2)

The formula can be re-written as:

Q = |P1 P2 2 74 / 8yL (3-3)

When: Q = Volumetric flow rate.
P1 = Static pressure at the inlet.
P2 = Static pressure at the outlet.
x= Mathematical constant (approximately 3.141592654).
r = Radius of the pipe.
r = (eta) absolute viscosity of the fluid.
L = Length of the pipe.

Since 2n, r and L are constant in our apparatus, the equation can be rewritten as:

Q = K(AP/p?) (3 -4)

In this case K is a constant factor determined by the geometry of the flow restriction. The

equation shows a linear relationship between volumetric flow rate (Q), differential pressure (AP),

and fluid viscosity (r) in a simple form.

To measure the AP generated by airflow, LFE (50MC2 4" ID, Meriam) was installed

according to the company' s installation recommendation. Briefly, a 4" NPT of 40" straight PVC

pipe and a 4" NPT of 20" straight PVC pipe were installed upstream and downstream of the

LFE, respectively. Upstream, the pipe was linearly connected to the manifold used for artificial

inspiration. Differential pressure across the LFE was measured by a pressure transducer (DP45,

Validyne) connected to the laminar flow element with 1.5 meters of rigid polyethylene tubes

(Parker Parflex) having a 1/4" NPT external diameter and a 0.04" thick wall. The pressure









transducer was calibrated with a water manometer. Differential pressure signals generated by

airflow were transferred to the data acquisition system and recorded by data acquisition software.

An additional inline filter made of two layers of 1.5 mm2 HylOn mesh was installed

between the artificial inspiration component and LFE to trap dirt and secretion prior to reaching

the LFE.

Differential pressure across LFE generated by airflow was calibrated with a NIST

traceable mass flow element (MFE) (8104-1416 FM, Matheson) prior to each experiment. In the

calibration process, LFE and the connected PCV pipe were temporally disconnected from the

whole apparatus. The air blower and the MFE were connected to the entry port and exit port of

the LFE, respectively. All of the connecting points were sealed with electrical tape to prevent air

leakage. To obtain calibration data, an air blower (under control of a variable transformer) was

used to generate airflow through the LFE at different flow rates. Airflow rate data from MFE

direct readings and AP data generated by a particular airflow were recorded. The calibration

process was repeated at the end of the experiment each day.

Temperature of airflow in the system was measured by a fast response thermocouple

probe (SRTC-TT-T-40-36, Omega) installed proximally to the LFE. The thermocouple probe

was connected to the meter (DP-41B, Omega) with a direct digital readout for temperature value

and a capability for generating a digital signal output. The temperature digital signal output was

transferred to the signal interface prior to being connected to the analog digital converter.

Pressure differential and temperature data were recorded with data acquisition software.

Airflow direction control system

Airway pressure and airflow direction in the system were controlled by manually

actuated PVC ball valves. As mentioned earlier in the system for artificial inspiration, the










inspiratory pressure was continuously monitored during airway inflation, and its pressure was

controlled by a 2" NPT manually actuated PVC ball valve. An inline 2" NPT manually actuated

PVC ball valve installed between the airflow measurement system and the negative pressure

reservoir was used for separating the negative pressure reservoir from the airflow measurement

component of the system. This valve was also used to control the rapid partial FE maneuver. To

induce expiration, the valve was quickly opened to expose a fully inflated airway to the negative

pressure reservoir. Difference in pressure between these two compartments generated airflow

through the airflow measurement component.

Data Acquisition system

Pressure and temperature data were acquired and recorded by Windaq Pro+ software

(Windaq). The software was kindly provided by Professor James H. Jones, Department of

Surgical and Radiological Science, University of California, Davis, for research cooperation on

equine exercise physiology. Briefly, signals from the pressure transducers were conditioned with

high-gain carrier demodulators (CD-19A, Validyne) installed in a 10-channel-module case

(MC 1-10, Validyne) prior to being relayed to a signal interface (DP250, Windaq). Analog

signals of temperature were relayed directly to the signal interface. All analog signals gathered at

the signal interface were transferred to the analog to digital converter (DP702 USB, Windaq).

Data were acquired and recorded by the Windaq Pro+ software at a frequency of 1000 Hz with a

laptop computer. Diagram of system set up for RP-FE is shown in Figure 3-2.

Animal Preparation for rapid partial forced expiration.maneuver.

The horses were chemically restrained with intravenous administration of detomidine

hydrochloride (Domosedan, Pfizer) at a dosage of 20-30 pg/kg. Following administration, horses

were left undisturbed for 10 minutes. Body temperature was obtained with a rectal mercury

thermometer. The oral cavity was then thoroughly rinsed with a water hose to remove saliva and









food materials. A mouth gag made from an 8" x 1 1/2" NPT PVC pipe (Figure 3-3) was inserted

between the lower and upper incisors. One-third of the cuffed end of a 2.6 cm internal diameter

endotracheal (ET) tube was lubricated with sterile water-soluble lubricant (KY Jelly, Johnson &

Johnson). The ET tube was passed through the mouth with the animal's head held in a straight

and fully extended position. Once the ET tube was in the trachea, it was advanced to the mid-

cervical trachea and the cuff of the ET tube was inflated with 80-100 ml of air. A fiberoptic

endoscope, which was preinstalled with a homemade three-way connecter, was inserted into the

ET tube until the tip of the endoscope was proximal to the tracheal bifurcation. The homemade

three-way connecter was then connected to the ET tube and to the manifold of the artificial

inspiration system. All of the connections were sealed with electrical tape to prevent leakage of

air.

Once the ET tube was connected to the RP-FE apparatus, the airway and lung were

artificially inflated with the air blower under the control of the variable transformer. The airway

was gradually inflated to a pressure of25-30 cm H20, and the variable transformer was turned

off. The lung and airway deflated under the force of the lung and chest wall elastic recoil

properties until the airway pressure dropped to near zero when the airway pressure relief valve

was opened. Artificial inspiration was maintained at a rate of25-30 times/minute.

Induction of Rapid Partial Forced Expiration

Prior to initiation of RP-FE, the negative pressure in the reservoir was adjusted to a

desired level, and the airway was concurrently inflated to its TLC, at an airway pressure of30

cm H20. At thi s airway pressure, the pressure relief valve and the valve connecting the artificial

inspiration manifold with the blower were closed and the valve located downstream of the

airflow measuring system was quickly opened to expose the inflated airway to negative pressure.

The difference in pressure between the vacuum reservoir and the airway emptied the air from the










airway, which mimicked voluntary forced expiration. Once the airway was emptied, the FE

controlling valve was closed and the valve connecting the artificial inspiration manifold and the

blower was opened, and artificial inspiration was resumed.

Rapid partial forced expiration maneuver was conducted with a negative pressure at 25,

50, 75, 100, 150, 200, and 250 Torr. After the last maneuver, the RP-FE system was

disconnected from the ET tube, the cuff of the ET tube was deflated, and the ET tube removed

from the horse.

Calculation of the Pulmonary Function Test Parameters

Reset the calibrated pressure differential of LFE with pressure/temperature corrected flow
rate

Airflow rate data from the NIST traceable MFE and AP data generated from the airflow,

were used to confirm a linear relationship between the flow rate and differential pressure.

Maximum airflow rate and its pressure differential from MFE/LFE calibration were used to

calculate pressure and temperature-corrected airflow rates. The original airflow rate from MFE

(at the standard condition: 210C, 760 Torr, and dry) was corrected with barometric pressure (BP)

and body temperature (BT) to reflect airflow rate of the expired air from the horse during RP-FE

maneuver. Barometric pressure data were obtained from Gainesville Regional Airport weather

station, available online, National Weather Service

(http://weather.noaa.gov/weather/current/KNVhtml). The BP- and BT- corrected airflow rate

was used to reset the high calibration of the AP of the LFE at the same MFE data point, and a AP

of LFE at no flow was reset to zero. After resetting the high and the low calibrations of LFE' s

pressure differential with a BT/BP-corrected flow rate, the LFE data represent the airflow rate

generated by the RP-FE maneuver. These data were used for computation of PFTPs.










Calculation of expiratory volume from airflow data

Volume of airflow generated by the RP-FE was obtained by integration of the area under

the curve for the relationship between airflow rate and time. Volume integration was computed

with the Advanced CODAS add-on module for the Windaq Pro+ data acquisition software. The

derivative of the integrated volume calculated by the Advance CODAS was used to determine

the beginning of the RP-FE.

Determination of pulmonary function test parameters

After computing the integrated volume and calculating its derivative, the PFTPs were

computed or obtained either with Windaq waveform browser software or EXCEL. Pulmonary

function test parameters of interest include forced vital capacity (FVC), peak expiratory flow

(PEF), FEV0.5, FEV0.75, FEV1, FEV1.5, FEV2, FEV2.5, FEV3, forced vital capacity (FVC),

and FEV1/FVC ratio. Airflow rates at 25, 50, and 75 % of FVC (1VEF25%, 50%, and 75%) were

also computed.

Determination of suitable negative pressure for the rapid partial forced expiration
maneuver

During the FE maneuver, negative pressures at 25, 50, 75, 100, 150, 200, and 250 Torr

were tested for their capacity to empty air from the airway. The impact of negative pressures on

lower airway mucosa was determined by endoscopic examination during the RP-FE maneuver.

Statistical Analysis

Histamine concentrations causing PC3 5 delta flow Were categorized from 1 to 4 according to

the PC3 5 delta flow interpretation protocol (Table 3 -1). Agreement between the 1st and 2nd HB tests

of the degree of airway hyper-sensitivity (severe, moderate, mild, and none) was investigated

using the Friedman test and Spearman correlation. HB results were then categorized into two

categories (horses with hyper-sensitive airways and horses without), and agreement between the









1st and 2nd HB tests for identifying airway hyper-sensitivity was investigated using the Friedman

test and Spearman correlation. Broncho-alveolar lavage fluid cytology data from the 1st and 2nd

HB were compared using Wilcoxon Signed Rank test, and the correlation between the two tests

was investigated using Spearman correlation.

Linear relationships between volumetric flow rate (Q) and differential pressure (AP) of

the LFE pressure transducer obtained before RP-FE and after RP-FE were evaluated using

regression analysis. Pulmonary function test parameters derived from RP-FE at 25, 50, 75, 100,

125, 150, 200, 250 Torr were presented in mean+SD. Reproducibility of PF TPs was investigated

using Wilcoxon Signed Rank test. Analysis of PFTPs reproducibility was conducted on PFTPs

data derived from RP-FE at 150, 200, and 250 Torr.

Data of horses for which the 1st HB test result agreed with the 2nd HB test (hyper-

sensitive or non hyper-sensitive) were selected for further analysis. After categorizing the horses

into hyper-sensitive and non-hyper-sensitive groups, BALf cytology and PFTPs were recorded as

meantSD. Differences in BALf cytology of horses with sensitive and horses with non-sensitive

airways were compared using Mann-Whitney U test. Pulmonary function test parameters of

horses with sensitive airways were compared to those with non-sensitive airways using Mann-

Whitney U test. P value I 0.05 was considered significant.

Results

Subj ect

Twenty-four horses used in this study included six mares and 18 geldings. Mean+SD and

range of age and body weight were 6.811.7 years (range = 2.5 -9) and 529141 kg (range = 412-

599). When the horses were tested for HB and RP-FE, they were clinically normal.









Histamine Bronchoprovocation and Respiratory Inductance Plethysmography and
Pneumotachography (Open PlethTM)

The test of airway hyper-sensitivity by the Open PlethTIL System is a non-invasive

technique for measuring the response of the airway to hi stamine. The technique also i s called

histamine bronchoprovocation (HB). Most of the horses tolerated the test procedures well. Only

one horse in this study developed extreme anxiety and restlessness after a facemask was put on,

and an additional administration of xylazine hydrochloride (100 mg) was required. The test

procedure took 35 -50 minutes from the first inj section of chemical restraint. Xylaxine

hydrochloride administration at a dosage of 0.75 mg/kg produced a short period of moderate to

strong sedation, which lasted less than 20 minutes. Most of the horses became restless when the

sedative effect subsided. Therefore, to immobilize the horses throughout the test, two or three

doses of xylazine hydrochloride were administered. During HB, most of the horses developed

clinical signs of dyspnea and respiratory noise originating from the upper airways. There was

also an increase in nasal secretion in some horses after HB. Histamine concentrations causing a

35% decrease in airway dynamic compliance (PC35 delta flow) fTOm the 1st and the 2nd HB test are

presented in Table 3 -2, and descriptive statistics are presented in Table 3 -3.

In the 1st HB test, 18 horses were classified as having airway hyper-sensitivity

(interpretation = 1-3), and 6 horses were classified as having normal airways (interpretation =

4). In the 2nd test 1 1 horses were classified as having airway hyper-sensitivity, and 13 horses

were classified as having normal airways. Fifteen horses produced the same results in the 1st and

the 2nd HB test, and 9 horses did not. Among these 15 horses, 10 possessed hyper- sensitivity

airways, and 5 possessed normal airways.

Airway hyper-sensitivity (severe, moderate, mild, and normal) in the 1st and the 2nd HB

tests did not differ significantly (p-value = 0. 11), and results between the tests were not









significantly correlated (R = 0.3 83, p-value = 0.07). When only the presence of airway hyper-

sensitivity was considered (horses with airway hyper-sensitivity and horses without), the results

of the tests differed significantly (p-value = 0.02) and they were not significantly correlated (R =

0.338, p-value = 0.11) (Table 3 -4).

Broncho-alveolar Lavage

MeanfSD percentage of BALf rcvr was 57f16 and ranged from 22 to 81i.

Measurement of recovered BALf did not include the foamy part of the BALf. Physical

appearances of all samples were colorless and slightly turbid. There were no visible flakes of

mucus in the BALf. MeanfSD percentage of epithelial lining fluid in BALf determined by urea

concentration was 1.52f0.73, and ranged from 0.3 to 3.6. Cytology results compared between

the 1st HB and the 2nd HB tests were not significantly different. MeanfSD BALf cytology results

of the 1st HB test and the 2nd HB test and their statistical comparison are presented in Table 3 -5.

To determine the relationship between the airway hyper-sensitivity and BALf cytology,

data from subjects with the same results in the 1st and 2nd HB tests were used, and the degree of

hyper-sensitivity was not considered. After excluding subj ects with different results in the 1st and

2nd HB tests, 10 horses had hyper-sensitive airway and 5 horses had normal airways. BALf

cytology data from horses with hyper-sensitive airways were not significantly different from

those of horses with normal airway s. However, the percentages of BALf neutrophils in horses

with hyper-sensitive airways and horses with normal airways approached significance different

in both tests (Table 3-6). Flow-volume loops derived from average flow data and volume data of

horses with normal airways and horses with hyper-sensitive airway were not different (Figure 3-

4).










Rapid Partial Forced Expiration (RP-FE) Maneuver

The linear relationship between volumetric flow rate (Q) and differential pressure (AP) of

the LFE pressure transducer was evaluated using regression analysis. The relationship of the

airflow rate data and their AP was linear (Figure 3-5). The simple linear regression models of the

relationship between airflow rates measured by MFE and their AP, before and after RP-FE, were

Y = (-0.05 605 5) + (0.091 098)X and Y = (-0.043 768) + (0.090272)X, respectively, with X =

MFE airflow and Y = AP. When X = 10 to 150, differences in Y between these models were not

greater than 0.8%. The maximum number of 150 was tested because RP-FE was not expected to

produce peak airflow greater than 150 liters/second.

Negative pressure at 200 Torr emptied air from the airways without causing visible

damage to the lower airways. Lower negative pressures incompletely emptied the airways.

Integrated volumes from airflow data were positively correlated with the amount of negative

pressure. Negative pressure at 250 Torr produced the highest FVC and caused visible airway

mucosal damage, which was characterized by sub-mucosal petechial and echimotic hemorrhage.

There was no evidence of vi sible intra-luminal hemorrhage in any horse after the RP-FE.

After being chemically restrained with an intravenous administration of 20-30 Clg/kg

detomidine hydrochloride, endotracheal (ET) tube intubation was successful in all but one horse.

This one horse was 2.5 years old, was the youngest of the subj ects and was excluded from RP-

FE study. Endotracheal tube intubation induced coughs in some horses. The cough subsided

once the ET tube was in place. Artificial inspiration caused expansion of the thoraco-abdominal

wall, which in turn collapsed when the air blower was turned off. There were no major signs of

discomfort during artificial inspiration or RP-FE. Negative pressure at 100, 150, 200, and 250

Torr emptied the airways which could be visualized either by endoscope in the trachea or by









contraction of the thoraco-abdominal wall when the airways were exposed to the negative

pressures. A negative pressure at 250 Torr caused the greatest collapse of the trachea and the

greatest contraction of the thoraco-abdominal wall, but caused endoscopically visible diffused

submucosal petechial and ecchymotic hemorrhage at the distal end of the trachea and in maj or

bronchi. Raw data for RP-FE from 4 horses were not used in the PFTPs calculation due to an

erroneous setting of the LFE pressure transducer, which caused an error in the recording of

pressure differential signals generated by the airflow. Figure 3 -6 illustrates the data acquisition

window of Windaq data acquisition software. Pulmonary function test parameters derived from

lower negative pressures were less than the PFTPs derived from the higher negative pressures;

i.e., PFTPs25 < PFTPs50 < PFTPsl00 < PFTPsl25 < PFTPsl50 < PFTP200 < PFTP250 (Table

3-7). Figures 3-7 and 3-8 demonstrate an example of comparison in expiratory flow rate and

flow-volume loop generated by different negative pressure.

Mean+SD of PFTPs from the 1st and the 2nd RP-FE maneuvers at 150, 200, and 250 Torr

are presented in Table 3-8. The PFTPs of the 1st and 2nd maneuvers were not significantly

different when RP-FE was performed at 150 or 200 Torr. However, PFTPs derived from the 1st

and 2nd maneuvers at 250 Torr were significantly different (Table 3 -9). Example of FV loops

form the 1st and the 2nd RP-FE maneuvers at 150, 200, and 250 Torr are presented in Figure 3-9.

These results demonstrated that PFTPs derived from RP-FE of 150 and 200 Torr were

repeatable, while the PFTPs of 250 Torr were less repeatable.

The potential correlation between the percentage of BALf neutrophils and the PFTPs was

investigated. Data on five horses with the highest percentage of BALf neutrophils were

compared with data on five horses with the lowest percentage of BALf neutrophils. Horses used

in this comparison were selected if their percentage of BALf neutrophils was consistent in both









the 1st and the 2nd BALf collections. Data on horses with highest and lowest percentages of BALf

neutrophils were compared to correlate BALf neutrophil with PFTPs alteration.

Percentages of B ALf neutrophils of these two groups of horses were signifi cantly

different (Table 3 -10), in view of the previously mentioned reasons. Pulmonary function test

parameters derived from these selected populations were not significantly different at a p-value

of 0.05. Statistics of FEV0.5, FEV0.75, PEF, and MEF25% approached 0. 1 (Table 3 -1 1).

Despite the lack of statistical significance between these two groups, their FV loops generated

from average flow data and volume data did differ (Figure 3-10).

Discussion

Histamine is a vasoactive amine substance. It is synthesized from amino acid histidine by

decarboxylation reaction. The reaction is catalyzed by the enzyme histidine decarboxylase.194

Once synthesized, it is mostly stored in granules of mast cells or basophils. Histamine plays

important role in the local immune responses and controls normal functions of the

gastrointestinal system.195,196 Non-mast cell-associated histamine can be found in other tissues,

for example, in the central nervous system where it acts as a neurotransmitter. 197 An increase in

the number of mast cells and hi stamine concentration in BALf has been demonstrated in human

asthma.198 Once released, histamine binds to histamine receptors on the bronchial smooth muscle

cells and causes contraction, which leads to bronchoconstriction. Histamine also activates the

histamine receptors (H4) on the surface of the mast cells, which intensifies the release of

hi stamine and regulates the mast cell immune response.199 As a result, the effects of hi stamine

are augmented.

Lower airway inflammatory diseases caused by non-infectious agents in horses include

IAD, RAO, and SPAOPD. They are characterized by chronic inflammation of the lower airways

and are thought to associate with TH1 and TH2 cytokines dys-regulation.86,200 Numbers of









neutrophils in BALf increased in affected horses." An increase in the number of mast cells also

has been demonstrated in some studies, but the results were inconsistent. 171,200,201 Elevation in

the number of pulmonary tissue mast cells after being challenged with moldy hay suggested that

mast cells may play an important role in RAO pathogenesis and may be involved in pulmonary

tissue remodeling in chronic RAO.202 Histamine, the mast cell product, is thought to be involved

in airway hyper-sensitivity, which is commonly seen in inflammatory diseases of lower airways

in both humans and animals.17,s In human medicine, the degree of airway hyper-sensitivity to

exogenous histamine has been used extensively in the diagnosis of respiratory diseases such as

bronchial asthma and COPD.17

Histamine challenge in veterinary medicine is modified from histamine bronchoprocation

(HB) in humans. Hi stamine bronchoprocation i s a non-invasive technique of measuring airway

hyper-sensitivity. The test is commonly performed in human patients when either obstructive

(disease affecting airways that carry gas into and out of the lung such as asthma and COPD) or

restrictive (disease affecting pulmonary tissue such as neoplasia and fibrosis) pathologies of the

pulmonary ti issues are suspected.182 Exposure of the affected airway s to hi stamine causes

bronchoconstriction and an increase in airway resistance.

In this study, horses without clinical signs of respiratory problems responded to HB

inconsi stently. Horses with a high percentage of BALf neutrophils were more likely to have

hyper-sensitive airways. However, correlations between percentages of BALf neutrophils and

airway hyper-sensitivity were not significant. The difference in HB results between tests in this

study agreed with those of a previous study, suggesting a high variation in individual response to

HB.184 Results from the previous study suggested that the HB response in normal horses and

horses with low-grade lung disease (with no clinical signs of respiratory tract distress) were not









significantly different. However, horses with severe pulmonary disease required a significantly

lower histamine concentration to cause 35% reduction in lung dynamic compliance.184

The cause of individual variation in HB response is not known. However, a possible

explanation is a nonspecific response of upper airways to histamine, especially when a facemask

was used during HB test. A previous report suggested that upper airway resistance in normal

horses is approximately 50% of the total pulmonary resistance.203 Abnormalities of either

pharyngeal or laryngeal functions, such as displacement of soft palate and laryngeal hemiplegia,

increase resi stance of airflow in the upper airway s.204-206 Temporary malfunction of these

structures can be found in some horses following the administration of chemical restraints. This

may be due partly to the effect of generalized muscle relaxation, which resulted in a relaxation of

pharyngeal and laryngeal muscle tone.207

Xylazine hydrochloride being used in this study during HB is an ot-2 adrenoceptor

agonist. It produces bradycardia and alters rhythm of cardiac contraction.208 After administration,

it initially produced hypertension followed by a prolonged hypotension, decrease in cardiac

output, and respiratory depression.208 Xylazine hydrochloride caused the head carriage position

to be less than 1800 (when 1800 was defined as a position when both head and heck are parallel

to the horizontal plane).209 Thi s resulted in an increase in the resi stance in both the upper and

lower airways. However, when the head and neck were re-positioned to >1800, the previous

increase in airway resistance from low head carriage was restored to values seen prior to the

beginning of sedation.210 During HB in this study, the head and neck positions were maintained

close to the horizontal plane by using a support at the mandible.

Histamine not only induces the contraction of the smooth muscles of the airways, but also

causes vasodilatation of blood vessels in the airways. Vasodilatation leads to edema of the









respiratory mucosa and increases mucus production in both upper and lower airways.211 These

results may contribute to an increase in non-specific resistance of the upper airways that

potentially increases total resistance of the respiratory tracts. A pharmacological effect of

histamine on mucus production was observed in this study; that is, the vast maj ority of the horses

had an increase in their nasal secretion during and after exposed to histamine.

Histamine causes immediate bronchoconstriction in humans, which is different from

bronchoconstriction naturally occurring in equine lower airway inflammatory diseases (IAD,

RAO, and SPAOPD). After challenge with known environmental inciting agents,

bronchoconstriction slowly developed over a few hours, and the resolution of clinical signs was

delayed after the agents were removed.' These results suggested that bronchoconstriction in

horses affected by lower airway inflammation may depend on an unknown intermediate

substrate, which might be chemical substance release from tissue resident inflammatory cells,

vascular endothelium, or inflammatory cells present in bronchioalveolar fluid." Among these

cells, alveolar macrophages and neutrophils are the most studied. Besides phagocytic activity,

alveolar macrophages release pro-inflammatory cytokines in response to external stimuli. Results

from an in vitro study demonstrated that activation of alveolar macrophages and monocytes with

LPS and fungal antigen increased their TNF-a production.144 Tumor necrosis factor alpha can

activate macrophages, endothelial cells, and other immune cells to produce more inflammatory

cytokines, which amplify the inflammatory reactions. Neutrophils in BALf are recruited from

pulmonary circulation in response to TNF-u.212 The migration of neutrophils across endothelium

requires chemotactic cytokine, including interleukin-8 (IL-8). Interleukin-8 can be produced by

endothelial cells, cells from broncho-alveolar lavage, and bronchial epithelium. 177,200,213 An

increase in IL-8 concentration in BALf following an exposure to hay dust that was accompanied









by an increase in the number of neutrophils in BALf, indicated that IL-8 plays an important role

in pathogenesis of lower airway inflammatory diseases in horses.214

Results from this study suggested that the HB test can be used to reinforce diagnosis of

equine lower airway inflammatory diseases, but interpretation of HB test results obtained from

clinically normal horses should be done with great care. Information derived from an HB test

should be used together with results from other diagnostic information, including BALf

cytology, clinical signs, history of illness, and response to therapy.

Forced expiration maneuver is another test of pulmonary function modified from human

medicine. In human medicine, the test is the most common diagnostic procedure performed in

respiratory clinics. Derived parameters reflect the biomechanical characteristics of pulmonary

parenchyma and air conductive tissues. The test is voluntary and non-invasive in humans.

Quality of the test depends on subj ect cooperation and ability of the respiratory diagnostician to

coach the patient to breath correctly.18 Results from the test are repeatable.183

Forced expiration in horses requires additional maneuvers and is more invasive than that

in humans. A design for a system that is capable of intervening inspiration and expiration is

necessary since coaching on breathing in horses is not possible. In this study, the inspiration

maneuver was accomplished by inflating the airways with atmospheric air at a positive pressure

3 0-45 cm H20 at the rate of approximately 25-30 breaths/minute. At this pressure and rate,

artificial inspiration suppressed the physiological respiratory drive of the test subj ects. The

suppression might be due partly to hypocapnea and increased partial pressure of blood oxygen

from hyperventilation. This, in turn, reduced the respiratory drive governed by the autonomic

nervous system. Whether artificial ventilation in this study affected the blood gas parameters is

unknown and is worth further investigation.









The RP-FE maneuver in this study lasted 7-10 minutes. However, duration of the

maneuver did not reflect the total time required for conducting the test since this experiment

included the evaluation of RP-FE generated by various negative pressures. Less time will be

needed if a single negative pressure is used for the maneuver in the future.

Other problems in this RP-FE set up included control of airway pressure at TLC prior to

an induction of RP-FE. Determination of TLC in this study was based on airway pressure at the

end of artificial inspiration, which was expected to 30 cm H20. Airway pressure data suggested

that the inspiratory manifold set up in this study was insufficient to produce repeatable airway

pressure at TLC. This might due partly to leakage of air through the connection sites in the

manifolds. Manually adjusting the airway pressure also contributed to high variation in airway

pressure at TLC. Leakage of air in the manifold upstream of the LFE not only affected the ability

to control the airway pressure, but also produced airflow signals on LFE pressure transducer

during RP-FE when airways were already emptied. The flow signals caused by air leakage could

interfere with PFTPs calculations.

Pulmonary function test parameters from this study were different from values reported

by a previous study.m7 The differences in PFTPs may be caused by differences in manifold

designs, in the negative pressures used to empty airways, or in methods of measuring airflow.

Regardless of the total length of the manifold used in the two studies, the maj or factor

that contributed to airflow resistance was the diameter of the manifold. The internal diameter

(ID) was determined by the selection of an endotracheal tube (2.6 cm, this study) versus

nasotracheal (2.2 cm, previous study). Intubation possessed an advantage over the facemask in

obtaining PFTPs, since the tube bypassed the upper respiratory structures, which potentially

contributed to a non-specific resistance to airflow. Poiseuille' s equation demonstrates the









relationship between flow resistance and geometric restriction of flow.193 Resistance is inversely

related to the fourth power of the radius:

R = 8yL/xR4 (3-5)

When R = Resistance.
r = Fluid viscosity.
L = length of pipe.
R = Radius.

Despite seemingly a minor difference in the internal diameter between these two designs,

resistance caused by 2.2 cm ID is twice that of 2.6 cm ID. Relationship of the radius to the

volumetric flow rate also is demonstrated by a variation of Poiseuille' s equation; the amount of

the volumetric flow rate is positively related to the fourth power of the radius.

Q = (APxR4)/8pL (3 -6)

When Q = Volumetric flow rate.
AP = Differential pressure.

Pulmonary function test parameters derived from different negative pressures indicated

that PEF and FVC values were positively correlated with the amount of negative pressure used to

generate RP-FE. Negative pressure at 200 Torr was selected in this study because it emptied the

air from airways without causing observable damage to the tracheal mucosa. The PEF and FVC

values derived from 200 Torr in this study were greater than the previously reported values,

when FE was induced by 161.8 Torr (-220 cm H20). In this RP-FE experiment, airflow data

were obtained from a direct measurement of AP generated by airflow. The AP data were

calibrated with NIST-traceable lVFE. Additional calibration with a known-volume inj section

through the LFE should be considered in future experiments. By comparing the integrated

volume (computed from IVFE calibration) to a known volume inj ected through LFE, accuracy of

measurements can be determined.









Even though subj ects used in this study were clinically normal, BALf cytology of some

horses possessed a high percentage of BALf neutrophils. In inflamed airways, neutrophils are

recruited during an active disease episode, and an increase in their numbers in BALf has been

used as an indication of inflammation of the lower airways. Previously reported values for

percentages of B ALf neutrophils in normal horses vari ed among studi es.176,215 However,

numbers greater than 5% in the samples have been used to suggest the presence of an

inflammation within the lower respiratory tract.201,216 MeanfSD percentages of BALf neutrophils

from the 1st and 2nd BALf collections in this study were 9.0f4 and 7.7f5, respectively. These

results agreed with previously reported values in clinically normal horses (6.812.7215 and

1 1.518.9176). Despite no clinical signs of respiratory problems, a high percentage of BALf

neutrophils might indicate an ongoing inflammatory reaction in the pulmonary tissues. Flow-

volume loop derived from horses with low percentages of BALf neutrophils (< 6%) was larger

than that from horses with a high percentage of BALf neutrophils (>9%). These results suggested

a possible compromise in the pulmonary function when inflammation is present in the lower

airways. However, this study could not confidently identify sub-clinical lower airway

inflammation in the selected population. Future investigation ofPFTPs derived from clinically

active cases of lower airway inflammatory diseases will be highly beneficial.









































Figure 3-1. Variable transformer.




























~I


Component symbols

Negative pressure reservoir m Temoerature meter
Air blower laSignal conditioner

Variable transformer Signal interface

8 ~Analor! Dressure rrause Analog to digital converter
4 O Laminar flow element Riaid volveroovlene tube
Filter Thermocouoles
Pressure transducer Connecting. cable
Manually actuated PVC valve Electrical lower cord
Computer
Figure 3 -2. Diagram of device setup to perform rapid partial forced expiration maneuver in
horses and component symbols.







































Figure 3 -3. Mouth gag made from an 8" x 1 1/2" NPT PVC pipe. Hand guard made from black
rubber curry comb.































0 20 40 60 80 100 120

Flow rate (liters/second)


- Normal airway


- Hyper-sensitive airway


Figure 3 -4. Flow-volume loops derived from average flow data and volume data of horses with
normal airways and horses with hyper-sensitive airway.


10 20 30 40 50


Direct reading ofMFE airflow (liters/second)


- Before RP-FE


- After RP-FE


Figure 3 -5. Relationship of airflow rates measured by NIST-calibrated mass flow element
(MFE) and differential pressure generated before and after rapid partial forced
expiration (RP-FE).













File Edit View Search Scaling Transform XV Qptions blelp
250 00


1= mHg















-152 14
4=4 cmH2


70 000 -


[iBL

-5 000
1800




-50 .0
MID 62.5700 SEC(TBF) 2.2200 SEC(TM) 78.2 %EOF T: .2000 SEC/DIV

Figure 3 -6. Example of data acquisition window of Windaq Pro+ software. Data from pressure
transducer connecting to the negative pressure reservoir, the laminar flow element,
and the airway were recorded in channels 1, 2 and 4, respectively. Channel 3 was set
for recording the temperature of air in the inspiratory manifold. Channel 5 is the
forced expiratory volume calculated from integration of airflow data as a function of
forced expiratory time. Channel 6 represents the derivative of the forced expiratory
volume.






























111















- 25
- 50
75
100
- 125
- 150
- 200
- 250


0 0.5 1 1.5 2 2.5 3

Forced expiratory time (second)

Figure 3-7. Example of forced expiratory flow rates generated by different negative pressures.


- 25
- 50
75
100
- 125
- 150
- 200
- 250


0 20 40 60 80 100 120 140 16

Forced expiratory airflow rate (liters/second)

Figure 3-8. Example of flow-volume loops generated by different negative pressures.














70

~60

S50

o40

'~30

8 20


10


- 1st 150
- 2nd 150
- 1st 200
- 2nd 200
1st 250
- 2nd 250


0 50 100 150

Forced expiratory airflow (liters/second)

Figure 3-9. Example of flow-volume loops generated by negative pressures at 150, 200, and 250
Torr.


70.00

60.00

50.00

40.00

30.00


20.00

10.00

0.00
0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00


Airflow (liters/second)


- Horses with low % BALf-Neu


- Horses with high % BALf-Neu


Figure 3-10. Flow-volume loops derived from average flow data and volume data
low percentages of BALf neutrophils and horses with high percentages
neutrophils.


of horses with
of BALf










Table 3 -1 Interpretation of histamine concentrations that cause 3 5% increase in delta flow
(modified from operation manual of Open PlethTM System).
Histamine concentration that causes
PC3 5 delta flow Interpretation of airway hyper-sensitivity/ category
0-2 mg/ml Severe / 1
2 4 mg/ml Moderate/ 2
4 8 mg/ml Mild/ 3
8 >32 mg/ml Normal / 4
(Source:.Operation manual of Open Pleth System. Ambulatory Monitoring, Inc, 2006.1s)

Table 3 -2. Histamine concentration causing PC 3 5 delta flow and their interpretations from the 1st
and the 2nd HB tests.
PC 3 5 delta flow of Interpretation of PC 3 5 delta flow of Interpretation of
Horse the 1st test the 1st test* the 2nd test the 2nd test*
H14 0.88 1 1.62 1
H2# > 32.00 4 15.00 4
H3 + 0.92 1 7.38 3
H4# 12.87 4 9.00 4
H5 2.17 2 24.55 4
H6 + 1.05 1 1.85 1
H7 1.33 1 21.05 4
H8 + 1.00 1 1.50 1
H9 4 5.23 3 1.20 1
H10 + 1.25 1 0.18 1


Hl le 3.41 2 1.97 1
H112 2.35 2 27.02 4
H13# > 32.00 4 21.94 4
H114# > 32.00 4 21.94 4
H15 + 1.62 1 1.41 1
H16+ 3.35 2 1.91 1
H117 0.49 1 14.38 4
H18 1.56 1 29.33 4
H119 6.94 3 9.00 4
H20 6.53 3 12.00 4
H21 3.58 2 8.51 4
H22# 13.00 4 > 32.00 4
H23 + 0.29 1 2.34 2
H24 8.14 4 0.44 1
* = Interpretation of the PC 3 5 delta flow results (1= severe airway hyper-sensitivity, 2 = moderate
airway hyper-sensitivity, 3 = mild airway hyper-sensitivity, and 4 = normal airway). Method of
interpretation is explained in Table 3-1. HB histamine bronchoprovocation, + horse with
airway hyper-sensitivity regardless of severity, and the result of the 1st test agreed with the 2nd
test, # = horse with normal airway, and the result of the 1 st test agreed with the 2nd test.










Table 3 -3. Descriptive statistics of the degree of airway hyper-sensitivity based on results from
the 1st and 2nd tests of hi stamine bronchoprovocation.
Degree of airway Frequency of the 1st Frequency of the 2nd
hyper- sensitivity test Percent test Percent
1 10 41.7 9 37.5
2 5 20.8 1 4.2
3 3 12.5 1 4.2
4 6 25.0 13 54.2
Total 24 100.0 24 100.0
= Interpretation of the PC 3 5 delta flow results (1= severe airway hyper-sensitivity, 2 = moderate
airway hyper-sensitivity, 3 = mild airway hyper-sensitivity, and 4 = normal airway). Method of
interpretation is explained in Table 3-1.

Table 3 -4. P-values from comparisons of the HB results between the 1st and 2nd tests and their
correlations.
Degree of airway hyper-sensitivity Result based on presence or absence of
(categorized from 1-4) airways hyper-sensitivity
Comparison* Correlation coefficient** Comparison* Correlation coefficient**
0.11 0.383 0.02 0.338
(p-value = 0.07) (p-value = 0. 11)
* = Friedman test, ** = Spearman' s rho.

Table 3 -5. MeantSD of BALf cytology results of the 1st and 2nd HB test and p-value of
Wilcoxon Signed Rank test and Correlations between the tests.
Stati stics TNC (x106 cell/ml) Mac (%) Lym (%) Neu (%) Mast (%)
MeanitSD 1st test 29.41 39.96 49.14 9.03 1.82


~9.16 17.69 16.92 +4.17
2nd test 32.12 39.06 50.39 7.79
~13.50 18.89 17.89 15.22
P-value 0.49 0.29 0.33 0.14
Correlation coefficient# **0.768 *0.479 0.400 *0.473


1.27
2.32
:1.52
0.25
0.375


TNC total nucleated cell, Mac macrophage, Lym lymphocyte, Neu neutrophil, Mast
mast cell, # Spearman' s rho statistic, ** = correlation is significant at the 0.01 level, -
correlation is significant at the 0.05 level.












2nd test


BALf = Broncho alveolar lavage fluid, TNC = total nucleated cell, Mac = macrophage, Lym =
lymphocyte, Neu = neutrophil, Mast = mast cell.


Table 3-6. MeantSD and Mann-Whitney U statistics of BALf cytology from horses with hyper-


sensitive and normal airways.
1st test


BALf cytology
parameters
TNC
(x106 cell/ml)
Mac (%)

Lym (%)

Neu (%)

Mast (%)


Hyper-
sensitive
28.24
~12.31
38.48
~8.55
49.35
~5.79
10.33
~4.71
1.85
~1.83


Hyper-
P-value sensitive
0.95 36.11
116.86
0.59 34.78
110.50
0.61 52.58
18.10
0.19 10.18
16.69
1.00 2.15
11.84


Normal
30.89
15.24
38.45
16.52
51.85
16.75
8.20
11.96
1.45
10.57


Normal
28.52
16.49
44.40
15.89
47.1
15.79
6.15
+2.03
1.55
10.44


P-value
0.37

0.13

0.27

0.14

0.83





partial forced expiration, FEVx


Table 3-7. Mean SD of PFTPs derived from RP-FE at negative pressures of 25, 50, 75, 100,
125, 150, 200, and 250 Torr (n=19).


Negative pressure
100 12
23.45 27.6r
10.96 10.71
29.36 34.4(
11.13 10.91
33.80 39.4~
11.28 1.
38.48 42.7(
+3.09 12.9:
39.15 43.1I
13.46 12.9
39.25 43.3:
13.45 12.8
39.25 43.3~
13.45 12.8:
39.25 43.3~
13.45 12.8:
0.60 0.6~
10.05 10.0
0.75 0.81
10.06 10.0
0.87 0.9
10.05 10.0
78.06 90.01
+3.00 12.4
RP-FE = rapid p~


r)
150*
34.45
11.18
43.99
11.55
48.94
13.36
50.70
+4.34
49.49
16.77
51.04
+4.38
51.07
+4.38
51.07
+4.38
0.68
10.04
0.86
10.05
0.96
10.02
108.15
13.48


(Tor
5
8
9
6
6
4
9
9
3
8
1
2
3
4
2
4
2
4
4
0
4
1
4
8
5


PFTPs
FVEO.5

FVEO.75

FEV1.0

FEV1.5

FEV2 .0

FEV2.5

FEV3 .0

FVC

FEV0.5S/FVC

FEV0 .75/FVC

FEV1/FVC

PEF


25
9.98
~0.88
12.71
~1.09
14.79
~1.31
17.55
~1.71
18.61
~2.11
18.95
~2.35
19.01
~2.33
19.01
~2.33
0.53
~0.03
0.67
~0.03
0.78
~0.03
32.53
~3.87


50
14.12
11.13
17.98
11.49
20.95
11.80
24.93
12.49
26.53
+3.14
26.84
13.37
26.88
13.43
26.88
13.43
0.53
10.04
0.67
10.05
0.78
10.05
47.39
14.60


75
18.69
10.92
23.54
11.11
27.32
11.32
32.15
12.26
33.47
+3.06
33.77
13.18
33.86
13.21
33.86
13.21
0.55
10.04
0.70
10.05
0.81
10.05
62.62
13.27


200*
43.57
11.09
55.44
11.67
60.49
13.42
62.00
+4.01
62.28
+4.01
62.45
+4.06
62.47
+4.05
62.47
+4.05
0.70
10.03
0.89
10.04
0.97
10.01
133.64
13.44


250*
52.44
11.21
66.75
11.95
71.66
13.31
73.13
13.7
73.38
13.74
73.44
13.76
73.44
13.77
73.44
13.77
0.72
10.03
0.91
10.02
0.98
10.01
157.17
13.65


forced vital capacity, and PEF = peak expiratory


,
=


PFTPs = pulmonary function test parameters
forced expiratory volume at x second, FVC
flow, = average value of2 maneuvers.










Table 3-8. Mean fSD of PFTPs derived from the 1st and the 2nd RP-FE, when 150, 200, and 250
Torr were used to induced RP-FE.


150 Torr


200 Torr


250 Torr
1st 2nd
52.68 51.89
f1.30 f1.00
67.22 65.72
f1.80 f2.10
72.32 70.25
f3.10 f3.80
73.82 71.65
f3.40 f4.30
74.06 71.91
f3.50 f4.30
74.11 71.97
f3.50 f4.40
74.12 71.98
f3.50 f4.30
74.12 71.98
f3.50 f4.40
0.71 0.72
f0.03 f0.04
0.91 0.91
f0.02 f0.03
0.98 0.98
f0.01 f0.01
158.00 155.57
f3.70 f2.90


2nd
34.60
f1.10
44.00
f1.60
49.07
f3.50
50.91
f4.70
51.14
f4.70
51.21
14.70
51.21
f4.70
51.21
14.70
0.68
f0.05
0.86
f0.05
0.96
f0.02
108.51
f3.60


2nd
43.54
f1.10
55.47
f1.80
60.53
f3.50
62.03
f4.00
62.31
f4.00
62.55
f4.10
62.57
f4.10
62.57
f4.10
0.70
f0.03
0.89
f0.04
0.97
f0.01
133.58
f3.40


1st
34.56
f1.10
43.97
f1.60
48.82
f3.44
50.48
f4.40
50.77
f4.40
50.88
14.40
50.93
f4.40
50.93
14.40
0.68
f0.04
0.87
f0.04
0.96
f0.02
108.00
f3.60


st
43.60
f1.10
55.41
f1.70
60.46
f3.50
61.96
f4.10
62.25
f4.10
62.35
f4.10
62.34
f4.10
62.37
f4.10
0.70
f0.04
0.89
f0.03
0.97
f0.01
133.98
f3.50


PFTP
FVEO.5

FVEO.75

FEV1

FEV1.5

FEV2

FEV2.5

FEV3

FVC

FEV0.5S/FVC

FEV0 .75/FVC

FEV1/FVC

PEF


PFTP = pulmonary function test parameter. RP-FE
expiration. FEV = forced expiratory volume, FVC
expiratory flow.


= rapid partial forced expiration. FE = forced
forced vital capacity. PEF = peak










Table 3-9. Wilcoxon Signed Rank test statistics (p-value) of pulmonary function test parameters
(PFTPs) from the 1st and the 2nd RP-FE maneuver at 150, 200, and 250 Torr.
Vacuum FEV FEV FEV FEV FEV FEV FEV
(Torr) 0.5 0.75 1.0 1.5 2.0 2.5 3.0 FVC PEF
150 1.00 0.89 0.94 0.87 1.00 0.90 0.75 0.75 0.19
200 0.54 0.84 0.81 0.81 0.75 0.94 0.98 0.98 0.29
250 0.02 <0.01 0.01 0.02 0.02 0.02 0.02 0.02 <0.01
P-value less than 0.05 indicated that the PFTPs from the 1s and the 2n RP-FE maneuvers were
significantly different, RP-FE = rapid partial forced expiration. FEV = forced expiratory volume,
FVC = forced vital capacity, and PEF = peak expiratory flow.


Table 3 -10. MeantSD of percentage of BALf neutrophils of horses with low % Neu and horses
with high % Neu, and Mann-Whitney U test statistics.
Test Horse with high % Neu (n=5) Horse with low % Neu (n=5) P-value
1st 14.0512.73 4.8011.64 <;0.01
2nd 14.9516.93 5.8510.70 <;0.01










Table 3 -11i. MeantSD of PFTPs of horses with low percentage of BALf neutrophils and horses
with high percentage of BALf neutrophil and Mann-Whitney U test stati stics.
Horse with high % Neu Horse with low%/ Neu
PF TPs (n= 5) (n= 5) P-valu e
FEV0.5 42.804 43.917 0.11
~0.802 11.345
FEV0.75 54.302 56.131 0.15
~1.643 11.725
FEV1.0 59.325 61.625 0.55
~3.749 13.522
FEV1.5 60.916 63.138 0.55
~4.275 +4.113
FEV2.0 61.263 63.390 0.55
~4.248 +4.111
FEV2.5 61.67 63.458 0.55
~4.621 +4.059
FEV3.0 61.686 63.476 0.55
~4.610 +4.037
F VC 61.686 63.476 0.55
~4.610 +4.037
PEF 13 1.226 134.828 0.15
~1.894 14.415
MEF25% 130.982 134.576 0.10
~1.957 14.364
MEF50% 98.636 99.674 1.00
~6.624 11.836
MEF75% 54.320 53.956 1.00
~6.176 12.427
FEV0.5S/F VC 0.697 0.693 1.00
~0.048 10.024
FEV0.75/F VC 0.8 82 0.8 84 0.95
~0.047 10.031
FEV1.0/F VC 0.962 0.971 0.22
~0.0 15 +0.007
PFTPs = pulmonary function test parameters, BAL = broncho-alveolar lavage, FEVx = forced
expiratory volume at x second, FVC = forced vital capacity, PEF = peak expiratory flow,
MEFx% = forced expiratory flow rate when x% of FVC was exhaled, FEVx/FVC = ratio
between forced expiratory volume at x second to the forced vital capacity.









CHAPTER 4
EFFECTS OF ELECTROACUPUNCTURE ON PULMONARY FUNCTION AND IMMUNE
RESPONSE IN HORSES

Introduction and Background

The respiratory system plays an important role in equine athletic performance. Unlike the

cardiovascular and musculoskeletal system, competence of the lung and respiratory tracts are not

changed by exercise training programs. The respiratory system has been postulated to be the

maj or factor limiting the maximal performance of the horse.217,218 Abnormalities of the

respiratory system, which decrease gas transport capacity of the airways, diminish alveolar gas

exchange, or both, reduce athletic performance capacity.21

Chronic lower airway inflammatory disease is a naturally occurring respiratory disorder

in horses.220 Three forms of this disorder have been described based on the age of affected horses

and seasonal occurrences, including inflammatory airway disease (IAD), recurrent airway

obstruction (RAO), and summer pasture associated obstructive pulmonary disease (SPAOPD).221

Inflammatory airway disease is diagnosed in young training racehorses, while the other two

diseases are commonly diagnosed in older horses, and usually associated with poor stable

management. Recurrent airway obstruction occurs primarily in horses being kept in stables and

in the winter months, and is also called heaves.220 Summer pasture associated obstructive

pulmonary disease occurs primarily in horses being kept in pastures, and the clinical signs are

more severe in the summer season.222 These diseases cause chronic inflammation of the lower

airways. They produce non-specific clinical signs and produce similar broncho-alveolar lavage

fluid (BALf) cytology, suggesting that they might partly share a common disease mechanism.223

Several etiologies have been suggested to cause inflammation of the lower airways,

including hay dust and fungal spores in the stable environment.224,225 Laan et al.144 demonstrated

an increase in production of tumor necrosis factor alpha (TNF-oc) and interleukin-1 beta (IL-1P)









by alveolar macrophages following in vitro challenge with a solution of hyphae and conidia

prepared from Aspergillus fumiga~tus. The role of a fungal antigen in pathogenesis of lower

airways diseases has been also demonstrated in an in vivo study. After challenging the airways of

RAO-susceptible horses with A. fumigatus antigen, the numbers of total nucleated cells and

neutrophils in BALf increased.226 The challenge also increased the mRNA expression of TNF-ot,

IL-1 and IL-8 in alveolar macrophages. These inciting antigens caused immunological responses

at both local and systemic levels. An increase in BALflgA specific to M~icrosporum faeni and A.

fumigatus in RAO-affected horses has been demonstrated.227 There was also a significant

increase in serum IgE specific to Aspergillus antigen in RAO-affected horses.228

The role of endotoxin in pathogenesis of lower airway inflammatory diseases has also

been been shown. Nebulization of bacterial lipopolysaccharide (LPS) solutions into the airways

induced neutrophil infiltration in a dose-dependent maner.229 However, LPS nebulization alone

did not cause clinical signs of respiratory problems in either normal horses or in RAO-affected

horses. Fungal antigen such as Aspergillus jitmigatus has been suggested to play an important

role in equine lower airway inflammatory diseases.144 Further study demonstrated that the

increase in the numbers of neutrophils in airways was significantly reduced when the airways

were challenged with a LPS-depleted fungal antigen. These results suggested that endotoxins

present in the antigens that play an important role in pulmonary inflammation associated with

RAO and other lower airway inflammatory diseases in vivo.230

Until recently, there was no single diagnostic procedure that could be used to accurately

distinguish among these diseases. Currently diagnosis relies on case history, clinical signs, BALf

cytology, and response to treatment.170 The ability to definitively diagnose these diseases at their

early stage is important. However, early diagnosis is difficult due to a lack of specific clinical









manifestations and, more importantly, the affected horses may not show clinical signs of

respiratory problems until the amount of pulmonary tissue affected is greater than the functional

reserve. It is believed that most affected horses are undiagnosed because of the sub-clinical

nature of the diseases. Several progressive diagnostic methods have been developed in attempts

to identify early stages of these diseases, including direct measuring of intra-pleural pressure,

histamine bronchoprovocation, and forced expiration.172-175

Treatments of chronic lower airway inflammatory diseases are complex due to an

incomplete understanding of the causes of these diseases and their pathogenesis. Conventional

therapy requires long-term medication and an adjustment of the housing environment to remove

probable inciting causes.86 Significant improvement in clinical signs may be seen in some cases

after making changes in the animal's environment. Bronchodilator, mucolytic, and anti-

inflammatory agents alone or in combination together are normally prescribed in order to control

the clinical signs of dyspnea and cough.170 Improvement in the clinical signs does not guarantee

complete resolution of the disease. Horses with a history of previous lower airway inflammatory

disease are more likely to experience a recurrence of clinical signs when inciting factors are re-

introduced. Failure or delay in delivery of proper treatment at early stages of these diseases may

lead to chronic irreversible changes in the histopathological structures of the small airways and

pulmonary parenchyma.86,231 An increase in airway resistance and decrease in pulmonary

compliance and gas exchange capacity are common consequences of these changes.

Besides the standard medical management discussed above, alternative treatments such

as acupuncture (AC), electroacupuncture (EA), and Chinese herbs have been used as adjunctive

therapieS.109,232 They are intended to decrease the dosage requirements of bronchodilators and

anti-inflammatory agents and improve the quality of life of animals suffering from chronic









respiratory diseases.233 In the United States, the integration of AC and EA into conventional

veterinary practice is new when compared to their practice in China. They were not widely

practiced outside of China until 1974 when the International Veterinary Acupuncture Society

was founded.8 In contrast to the use of AC or EA to induce analgesia, modern research data on

the use of AC and EA for treating respiratory diseases in horses is limited. Until recently, the

benefit of AC and EA in treating equine respiratory problems was inconclusive. Previous

research demonstrated an improvement in pulmonary function in RAO affected horses following

a single AC treatment, but that was not statistically significant. After treatment, respiratory rate

and pulmonary resistance decreased, while the maximal change in pleural pressure, dynamic

compliance, and tidal volume increased. These improvements lasted less than 24 hours.88 In rats

with bronchial asthma induced by ovalbulmin, EA at GV-14, BL-13, Fei-shu, Ding-chuan, LU-

1, CV-17, ST-36, and SP-6 has been shown to significantly reduce lung inflammation relative to

a sham treatment.97 Peri-bronchial and peri-vascular infiltrations of inflammatory cells were

significantly reduced in the EA group. Electroacupuncture treatment also significantly reduced

the percentage ofpolymorphonuclear cells in BALf.97

Application of AC/EA for treating chronic respiratory problems is also supported by the

study conducted by the World Health Organization (WHO). The report reviewing data from

controlled clinical trials by WHO in 2003 suggested that AC possesses therapeutic benefits for

allergic rhinitis and bronchial asthma.9 More recent scientific investigations also supported this

conclu si on.87,234,235 Previ ou s research in humans demonstrated an immediate bronchodilating

effect after 30O minutes of AC treatment in patients suffering from asthma.87 In this study,

patients who received AC treatment had their forced expiratory volume in the first second

(FEV1) increased by 1 1%, while the increase of FEV1 in the sham group was improved less than









1%. The benefit oflaser-AC combined with pro-biotic supplementation as an adjunctive therapy

over conventional treatment in children with intermittent asthma has also been reported.234 In this

study, acupoints were chosen according to traditional Chinese medicine (TCM) diagnostic

methods (questioning, palpation, tongue and pulse diagnosis). Prescription of acupoints for each

patient was based on TCM, and a maximum of 16 acupoints were chosen from a preformulated

list. Selected acupoints were stimulated with laser light for 20 seconds without skin contact.

Study patients were treated with laser-AC once a week for 10 treatments in conjunction with

seven weeks of pro-biotic supplementation, and controls given only conventional therapy. At the

end of the study, peak flow variability (PFV) of patients in the treatment group was significantly

decreased; PFV is one of the pulmonary function test parameters used to measure hyper-

sensitivity of bronchi. Moreover, patients in the treatment group had a less severe respiratory

tract infections compared to patients in the control group.234

Information on immune modulatory properties of AC/EA was reviewed in Chapter 2. In

Chapter 2 the evidence presented suggested that EA at GV-14, LI-4, and LI-10 once a day for

three consecutive days suppressed in vitro TNF-a production from stimulated whole blood. In

contrast, the degree of suppression caused by AC was not significantly different compared to the

TNF-a concentration prior to AC. The anti-inflammatory effect of EA was thought to be

mediated by modulation of mononuclear leukocytes mediated innate immune response. This in

vitro anti-inflammation action ofEA was demonstrated using antigens that have been proported

to cause lower airway inflammatory disease, including fungal antigens and bacterial endotoxins.

In Chapter 3, a method of performing the rapid partial forced expiration (RP-FE) in

horses using a direct measurement of the pressure differential (AP) generated by airflow was

demonstrated. One obj ective of the study was to identify airway obstructions that might be









caused by sub-clinical inflammation of the lower airway in horses. This was done by comparing

BALf cytology to the degree of airway hyper-sensitivity [using the histamine

bronchoprovocation (HB) test] and the pulmonary function test parameters (PFTPs) derived from

the RP-FE. When the test of airway hyper-sensitivity was repeated within 2 months, horses

showed individual variation in the degree of airway hyper-sensitivity. However, the test can be

used to identify horses with airway hyper-sensitivity when the degree of hyper-sensitivity is not

considered. There was no significant correlation between airway hyper-sensitivity and the

percentage of BALf neutrophils, with a p-value greater than 0. 1.

Flow-volume (FV) loops generated from airflow data and volume data from the RP-FE

maneuver (Chapter 3) in clinically normal horses demonstrated the variability in airflow

limitation from horse to horse. Flow-volume loops of some horses possessed a scoop-out

characteristic after the peak expiratory flow, and some did not. The scoop-out characteristic is a

result of a rapid decrease of airflow due to a narrowing of the small airway. Even though FV

loop from horses (n=5) with a high percentage of BALf neutrophils (> 9%) was smaller than that

of the horses (n=5) with a low percentage of BALf neutrophils (< 7%), the difference in PTFTs,

including forced expiratory volume at 0.5, 0.75 second (FEV0.5, FEV0.75), peak expiratory flow

(PEF), forced expiratory flow rate when 25% of FEV has been expired (MEF25%), and ratio of

forced expiratory volume at 1 second to the forced vital capacity (FEV1.0/FVC) between these

two groups were not significant at a 95% confidence level. However, the p-value for MEF25%

was less than 0.1, and the p-values for FEV0.5, FEV0.75, and PEF were close to 0.1.

Although results from the experiment in Chapter 3 suggested that both HB and RP-FE

tests might benefit diagnosis of lower airway inflammatory diseases, they could not confidently

be used to identify horses with possible sub-clinical lower airway inflammation. Some horses









enrolled in the Chapter 3 experiment possessed persistent high percentages of BALf neutrophils.

Even though the FV loop derived from average airflow data and volume data of these horses was

smaller than that derived from data from horses with low percentages of BALf neutrophils, the

difference in PFTPs between these two groups was not significant at a 95% confidence level

To test the effects of EA on biomechanical properties of lower airways and lung tissues,

the RP-FE maneuver is more suitable than HB with facemask for the following reasons.

* Rapid partial forced expiration using endotracheal tube (ET) intubation through the mouth
eliminates non-specific airway resistance originating in the upper respiratory tracts.
Therefore, derived PFTPs are thought to truly reflect mechanical properties of lower
airways. Intubation with a large ET tube also is superior to that using a smaller tube
because the larger tube markedly reduces airflow resistance created by the system
manifold.

* Pulmonary function test parameters and FV loop computed from the airflow data and
volume data from the RP-FE maneuver are reliable. This factor is critical since the study
measured effects of treatment that may cause only subtle changes in pulmonary function.

The obj ective of experiment was to evaluate the effects of EA and sham EA at acupoints

regime mimicking acupoints commonly used for managing equine respiratory problems on the

PFTPs, anti-inflammatory activity, and circulating immunoglobulins.

Materials and Methods

Animals

Seventeen horses that had been previously used in the study of Chapter 3 were used in

this study. Three horses were unable to enroll in this study. Additional three horses were

recruited from the herd to yield a total numbers of 20. These 20 horses were regrouped and

assigned for individual treatment. Horses were kept outdoors in paddocks in groups of two or

four. This pattern was used because of space limitations. Groups of two or four individuals also

have been found to be the most practical for subj ect handling. Groups were randomly arranged,

and horses were numbered from 1 to 20. Horses with odd numbers were treated with EA, and









horses with even numbers were treated with sham EA. Horses were grouped according to their

housing prior to assigning numbers to avoid separation anxiety that usually occurs among horses

when at least one in their group is taken away. All horses of each group were brought into the

experimental room at the same time. At the end of the last treatment, a period of 3 weeks wash

out was provided prior to the crossover experiment. The horses had no clinical signs of

respiratory disease during the past 3 months. Protocol for animal use was approved by the

University of Florida Institutional Animal Care and Use Committee (Permit A-
130).Electroacupuncture

The regime of therapeutic acupoints in this study was formulated according to previous

research data and equine clinical acupuncture practice. Acupoints selected in this study

mimicked the acupoint regime recommended for treating horses with RAO and other chronic

lower airway inflammatory problems. Therefore, BL-13, Ding-chuan, Fei-men, Fei-pan, Fei-

shu, CV-22, and GV-14 were stimulated.78,97 Thirty-two gauge, 2- or 3-inch long disposable

stainless steel acupuncture needles (Kingli, China) were used. Details about needle size and

direction of needle insertion for each acupoint are listed in Table 4-1. Needle insertion was

performed by two veterinarians who were certified in equine acupuncture practice. Information

about locations of acupoints was reviewed and the methods of needle insertion were rehearsed by

the acupuncturists prior to the beginning of the experiment.

Acupoints were bilaterally connected and stimulated with an electrostimulator (Pantheon

Research), except GV-14 and CV-22 were connected to one another (Figure 4-1). Acupoints

were stimulated with 20 Hz for 10 minutes and then with 200 Hz for 10 minutes. The amplitude

of stimulation was adjusted to the point at which slight muscle contraction was observed or to a

level that the animal could comfortably tolerate. The frequency, duration, and amplitude of EA

stimulation were based on the guidelines published by the Council of Acupuncture and Oriental









Medicine Association and previous research.236-238 However, when possible, the amplitude was

maximally adjusted to 4 mA (but not greater than this level). Animals were treated once a day for

seven consecutive days followed by two treatments per week for another two and a half weeks.

Each horse received total 12 treatments. The first seven consecutive days of treatment was

designed to investigate acute aggressive effect of EA, while the subsequent less frequent

treatment was carried out to investigate effects of chronic treatment. Sham EA was performed

using the same procedure except that the needles were taped to acupoints using adhesive tape

(duct tape) without skin penetration (Figure 4-2). The sham EA mimicked animal handling

procedures during the experiment, including needle placement, electrical wiring, and animal

restraint.

Data and Sample Collection

Blood, plasma, and serum samples were collected one week prior to the beginning of the

experiment (Pr), 20-24 hours after the 7th treatment (Po), and 24-48 hours after the 12th treatment

(Ps). Samples collected prior to the beginning of the treatment are referred to as the pre-treatment

samples. All samples were collected from the left external jugular vein. Twenty-five ml of

whole blood were collected in a 35-ml syringe containing sodium heparin (NDC 0641-2470-41).

The final concentration of sodium heparin was 10 units/ml. This sample was used for in vitro

whole blood stimulation. Additional blood samples were collected with partially evacuated blood

collection tube containing sodium heparin (10 ml), no anti-coagulant (10 ml), and EDTA (7 ml)

vacutainer tubes (BD Vacutainero). Plasma or serum was harvested from each of these tubes.

The samples collected in the EDTA containing tubes were submitted to the clinical pathology

diagnostic laboratory of the College of Veterinary Medicine at University of Florida for

complete blood counts.









Bronchoalveolar lavage fluid (BALf) samples were collected one week prior to the

experiment (Pr) and 24-48 hours after the 10Oth treatment (Po). The percentage of epithelial lining

fluid (ELF) in the BALf samples was determined based on the urea dilution method. Details of

the method explained in Chapter 3. The percentage of ELF in BALF was used to calculate the

total nucleated cells count relative to 100% ELF (ELF-corrected TNC) and concentrations of

immunoglobulins relative to 100% ELF (ELF-corrected BLAf immunoglobulins). The RP-FE

test was performed prior to the beginning of the experiment (Pr), 20-24 hours after the 7th

treatment (Po), and 24-48 hours after the 12th treatment (Ps). Collection of the Po sample of

BALf on the same day when RP-FE was performed was not desirable, since the procedures may

affect the PFTPs.

Rapid Partial Forced Expiration Maneuver

The rapid partial forced expiration (RP-FE) maneuver was performed with the apparatus

modified from the system described in Chapter 3. During the previous operation of this

apparatus, some technical limitations were encountered.

* Reproducibility of airway pressure at total lung capacity (TLC).

* False differential pressure signals in the laminar flow element (LFE) caused by artificial
inspiration.

* Residual signals from the pressure transducer at the end of RP-FE when the airway was
empty.

* Requirement of additional personnel to control the vacuum level of the negative pressure
reservoir and the initiation of RP-FE.

During the RP-FE maneuver, the TLC of the lung was determined when the airway

pressure at the end of the artificial inspiration reached 30 cm H20. Variability of TLC airway

pressure in the previous operation was due partly to a leak in the air direction control system









manifold, lack of an appropriate pressure relief valve, and manual dependence on controlling

airflow and initiating the RP-FE maneuver.

To improve reproducibility of the airway pressure at TLC, a pressure regulator (EB5NL6,

EquiliBar) (Figure 4-3) was installed on the manifold of the artificial inspiration system. The

operation of this pressure regulator required an air supply at a set reference pressure. The set

reference pressure was maintained by a set-point regulator (type 41, Belloframe) (Figure 4-3)

connected to an air compressor (Campbell Hausfeld). A manually actuated ball valve controlling

the air from the air blower in the artificial inspiration system was replaced with a 2.5" NPT

solenoid valve (8215A90, AscoRedhat) (Figure 4-4).

False pressure differential signals in the laminar flow element (LFE) caused by artificial

inspiration were prevented by isolation of the LFE from the artificial respiration manifold and

the negative pressure reservoir by installation of two 2.5" NPT solenoid valves (82 1 5a90,

AscoRedhat). One was installed upstream to the LFE, and the other was installed downstream.

This eliminated pressure fluctuation in the LFE caused by artificial respiration. These two

solenoid valves were controlled with a single relay switch for simultaneous operation. Opening

these valves initiated the RP-FE maneuver.

The vacuum level in the negative pressure reservoir was maintained by a vacuum switch

(9016 GAW1, Square D) and a 1/4" NPT solenoid valve (8016G, AscoRedhat). Briefly, the

vacuum switch was used to inactivate the vacuum pump when the negative pressure of the

vacuum reservoir reached 215 Torr, and to actuate the vacuum pump when the vacuum dropped

to 130 Torr. The vacuum switch alone could not repeatedly reproduce an accurate negative

pressure. Therefore, the negative pressure range (215-130 Torr) was preset. Final fine adjustment

to lower the vacuum was accomplished by activation of the solenoid valve. This also allowed









the device operator to synchronize inflation of the lung to TLC while adjusting the vacuum of the

negative pressure reservoir to a value very close to 200 Torr for the RP-FE maneuver. Two-

hundred Torr of vacuum was chosen based on the previous experiment, which indicated that this

amount of vacuum completely emptied the airway without causing visible damage to the tracheal

mucosal.

In this experiment, an 865 watts/1500VA battery backup and power surge protector

(BR1500, APC) was installed and connected to the module case of the signal conditioner (MC 1-

10), temperature meter (DP41-B-A-C24-TC), analog to digital converter (DI720), and a laptop

computer. The system setup is illustrated in Figure 4-5. Symbols of the device component are

illustrated in Figure 4-6.

Airflow calibration of LFE was based on airflow values determined by the mass flow

element (MFE). This MFE was routinely calibrated and was National Institute of standards and

technology (NIST) traceable. Setup of LFE calibration is shown in Figure 4-7. In this

experiment, the accuracy of MFE calibration was tested by comparing integrated airflow signals

generated by pulling a known volume of air through the pipe connected downstream of the

calibrated LFE. A 12.829-liter syringe was used to test the integrated airflow signals (Figure 4-

8). By comparing the integrated volume derived from the AP data to the known volume of the

syringe, a correction factor for calibration was computed.

Meanf SD volume of air from integrated airflow signals generated by pulling 12.829-liter

of air through the calibrated LFE was 13.63 856f0.023401 liters. This result indicated that the

volume from integration was greater than the actual volume of the syringe being inj ected by

6.31%. This means that the volume integration result of 13.639 liters was, in fact, 12.829 liters.









Therefore, the calculated correction factor for MFE/LFE is 1.063105 (13.639/12.829). This

correction factor was used in the calculation of calibrated volumes.

Arterial Blood Gas Analysis During RP-FE

After chemical restraint, a 1.25-inch 21 G arterial catheter (SurflashThi, Terumo) was

inserted in the left transverse facial artery on eight horses. Briefly, hairs over the left transverse

facial artery were shaved, and the skin was cleaned and disinfected with a surgical scrub. The

catheter, which was pre-flushed with sterile heparinized saline (10 units/ml), was inserted into

the transverse facial artery. A 1-foot extension with a three-ways stop valve, which was pre-filled

with sterile heparinized saline, was connected to the catheter. The catheter hub was secured in

place with superglue, and the catheter was flushed with 3 -5 ml sterile heparinized saline. An

arterial blood sample was collected with a 3 -ml syringe, which was pre-flushed with heparin. To

obtain an arterial blood sample, 5 ml of blood and saline in the extension set was withdrawn and

discarded, and 1 ml of arterial blood was withdrawn with the prepared syringe. The syringe with

the arterial blood sample was capped and put on ice. The extension cord was flushed with 10 ml

sterile heparinized saline. Arterial blood samples were analyzed with a CG8+ cartridge and i-

STAT blood gas analyzer (Abbott Laboratories) within 10 minutes.

Calculation of the Pulmonary Function Test Parameters

The linear relationship between airflow rate data from a direct reading of NIST-traceable

MFE and AP data from LFE calibrations was tested using regression analysis. Maximum flow

rate from NIST-traceable MFE and the corresponding AP from the calibration were used for the

calculation of pressure- and temperature-corrected airflow rates. The original airflow rate from

MFE (at the standard condition: 210C, 760 Torr, and dry) was corrected with barometric pressure

(BP) and body temperature (BT) to reflect the airflow rate of the expired air from the horse









during the RP-FE maneuver. Barometric pressure data were obtained from the weather station of

the Department of Physics at the University of Florida, Gainesville. Data were available online at

http://www. phy s.ufl.edu/weather/. Pressure- and temperature-corrected airflow rate was further

adjusted with a correction factor obtained from the syringe calibration. This adjusted rate was

used for resetting the high calibration of the AP of the LFE at the same MFE data point, and AP

of LFE at no flow was reset to zero. After resetting the high and low calibrations of LFE AP, the

LFE data represent the airflow rate generated by the RP-FE maneuver. These airflow data were

used for computation of PFTPs as described in Chapter 3.

BALf Collection and Preparation

The procedure used for BALf collection was performed as described in Chapter 3.

Methods used for evaluations of BALf cytology were similar to those explained in Chapter 3.

The percentage of the epithelial lining fluid (ELF) recovered in BALfwa determined with the

urea dilution principle. The method for the urea assay was explained in Chapter 3.

The BALf sample from each horse was collected in a sterile glass bottle and kept on ice

until processed. Broncho-alveolar lavage cells were isolated from BALf by centrifugation at 600

RCF at 40C for 15 minutes. Supernatant from BALf was aliquoted and stored in -800C for

further analysis.

Tumor Necrosis Factor Alpha (TNF-a) Production of Whole Blood and TNF-a Assay

The anti-inflammatory activity of EA was indirectly determined by in vitro TNF-a

production of whole blood after stimulation with selected immunological stimulants.

Heparinized-blood stimulation and TNF-a assay were performed as described in the Chapter 2.

Culture extract ofA. Jiemigatus (CE), which was used in Chapter 2, was not used alone in this









study due to low response. Moreover, the results from Chapter 2 showed that whole blood from

some horses may not produce TNF-oc after being stimulated with CE for 6 hours.

Immunoglobulin Assay

Determination of circulating immunoglobulin isotypes was used as an indicator of

humoral immunity. Concentrations of immunoglobulin isotypes (IgA, IgM, IgGa, IgGb, and

IgG(T)), in plasma and BALf supernatant, were determined with horse immunoglobulin ELISA

Quantitation Kits (Bethyl Laboratories, Inc. Texas #E70-116, #E70-114, #E70-124, #E70-127,

and #E70-105). Assays were performed according to the protocols provided by the manufacturer

as described in Chapter 2.

Cortisol Assay

Quantitative analysis of cortisol was performed in serum samples with a commercial

cortisol ELISA test kit (Endocrine Technologies). The assay followed the manufacturer' s

protocol. Briefly, 50 Cll of standards, controls, and samples were added to designated wells in

duplicate. One hundred Cll of the Cortisol Enzyme Conjugate solution was added to each well,

except wells that were designated as controls. After incubating the plate at 370C for 1 hour,

excess standards, controls, and samples were removed from the plate by inverted snapping.

Residual fluid was then removed from the plate by firmly tapping the plate on a clean absorbent

paper towel. Each well was washed three times with 300 Cll of washing solution, and residual

washing solution was removed from the plate by firmly tapping the plate on a clean absorbent

paper towel. After washing, 100 Cll of TMB substrate solution was added to each well, and the

plate was incubated at room temperature for 20 minutes. After incubation, the peroxidase

reaction of the TMB substrate was terminated by adding 50 Cll of a stop solution to each well.

The stop solution was added in the same sequence as the TMB substrate. The plate was










evaluated with a microtiter plate reader at a wavelength of 450 nm. The concentrations of

cortisol in serum samples were calculated based on a four-parameter logistic curve-fit generated

by KC4 software.

Statistical Analysis

Linear relationships between volumetric flow rate (Q) and differential pressure (AP) of

the LFE pressure transducer obtained before RP-FE and after RP-FE were evaluated using

regression analysis.

Homogeneity of data from pre-EA and pre-sham treatment groups for hematological

parameters, BALf cytological parameters, concentrations of plasma immunoglobulins,

concentrations of BALf immunoglobulins, concentrations of TNF-a from an in vitro whole

blood stimulation, PFTPs from RP-FE, and concentrations of serum cortisol were initially tested

using Student' s t-test for independent samples.

Effects of treatment (EA, sham), sampling time (Pr, Po, Ps), trial (1st, 2nd), horse (1 -20),

and sequence (1, 2) on dependent variables were tested using a general linear model. A variable

called sequence was created for testing effect of treatment order. Horses receiving EA treatment

first were assigned as sequence 1. Horses receiving sham treatment first were assigned as

sequence 2. The dependent variables included hematological parameters, BALf cytological

parameters, concentrations of plasma immunoglobulins, concentrations of ELF-corrected BALf

immunoglobulins, concentrations of TNF-a from an in vitro whole blood stimulation, PFTPs

from RP-FE, and concentrations of serum cortisol. Main effects and their interactions were

tested using the Type III sum of squares test. Sequence nested in horse was treated as a random

factor.









Differences among sampling times (Pr, Po, Ps) within dependent variables were

compared using the multiple t-tests with Bonferroni correction. The Bonferoni correction is used

to keep the total chance of erroneously reporting a difference below alpha = 0.05. Differences in

BALf cytological parameters and in concentrations of BALf immunoglobulin i sotypes between

Pr and Po were compared using paired t-test. Statistical analysis was performed using SPSS 17

software for Windows. Data is reported as meanfstandard error of mean (meanfSE). P-value I

0.05 was used for determining significance.

Results

Subj ects

Horses used in this study included 19 geldings and one mare. MeansfSD of the age and

body weight of all horses were 8f1.1 years and 5 51137 kg. Means SD of age and body weight

of horses receiving odd number assignments were 8f0.6 years and 5411f41 kg. MeansfSD of age

and body weight of horses receiving even number assignments were 8f1.3 years and 560f32 kg.

During the study period, one even-numbered horse (H4) was treated for hoof abscess, and one

odd-numbered horse (H11) was treated for acute abdomen. Multiple dosages ofNSAIDs were

administered for treatments of these diseases and data derived from these horses were not

included in statistical analyses.

EA and Sham Treatments

Reactions of subj ects to the EA and the sham treatments were observed. Reactions to

sham treatment ranged from no reaction to agitation, restlessness, and trying to remove the

needle. Reactions to EA ranged from no reaction to severe agitation. One horse in the odd-

numbered group did not accept the EA procedure during the 2nd EA session, and EA was

discontinued on this horse. Experimental data from this horse for the EA trial were not included









in statistical analyses. Animal reactions to EA were more prominent than those to the sham

treatment. A summary of reactions to EA and to sham treatment is presented in Table 4-2.

Reactions to needles were greatest at GV-14, Ding-chuan, and BL-13. There was no reaction at

CV-22 in either EA or sham treatments.

Complete Blood Count and Other Hematological Parameters

Mean values of complete blood counts and other hematological parameters of the pre-

treatment samples of the EA and the sham groups were not significantly different; p-values from

independent t-tests white blood cell counts (Wbc) (0.45), percentage of neutrophils (Neu) (0.84),

percentage of lymphocytes (Lym) (0.63), percentage of monocytes (Mono) (0.40), percentage of

eosinophils (Eos) (0.33), percentage of basophils (Baso) (0.86), red blood cell counts (Rbc)

(0.53), hemoglobin (Hb) (0.45), hematocrit (Hct) (0.46), mean corpuscular volume (Mcy) (0.87),

mean corpuscular hemoglobin (Mch) (0.76), mean corpuscular hemoglobin concentration

(Mchc) (0.96), cellular hemoglobin concentration (Chcm) (0.55), and corpuscular hemoglobin

content (Ch) (0.72).

P-values from the Type III sum of squares test of the main effects and their interactions

for the white blood cell parameters are given in Table 4-3. Wbc, mean percentages of Neu, Lym,

Mono, Eos, and Baso of all samples were within normal limits throughout the study (Table 4-4).

Pairwise comparisons between Pr, Po, and Ps samples of all the white blood cell indices

in the EA group were not significantly different (Table 4-5). In the sham group, even though the

Wbc of Po was significantly different from that of the Ps sample (p-value = 0.02), Wbc from

both Po and Ps samples were within the normal reference values (Table 4-4).

P-values from the Type III sum of squares test of the main effects and their interactions

for the red blood cell indices are given in Table 4-6. Mean+SE of the red blood cell indices are










given in Table 4-7. P-values of pairwise comparisons between Pr, Po, and Ps samples of all the

red blood cell indices in the EA and the sham groups are given in Table 4-8. Even though some

pairwise comparisons of Rbc, Hct, Hb, Mcy, and Ch in the EA and the sham group were

significantly different, mean values of these red blood cell parameters were within the normal

reference values. The mean values of Mche and Chcm were in a high normal range or greater

than the normal reference values throughout the experiment.

Hemoglobin (Hb) values from pre-treatment samples from 6 of 17 horses in the EA group

and 7 of 19 horses in the sham group were greater than normal reference values (11.2-16.2

mg/dL), and ranged from 16.3-18.5 and 16.3-18.7 mg/dL, respectively. The high Hb values in

these horses corresponded to their hematocrit (Hct) values, which were in the upper limit of the

normal reference values (3 0-43%) or greater. Both Hb and Hct of all horses were within normal

limits in samples collected after the 7th and 12th treatments. An increase in Hb in pre-treatment

samples may result from excitation caused by initial handling. The excitement activates the

sympathetic nervous system, which could cause a spleenic contraction and released stored red

blood cells.

Broncho-alveolar Lavage and BALf Cytology

Meanf SD percentages of BALf recovered by using a fiberoptic endoscope and

percentages of ELF in BALf determined by urea dilution technique are shown in Table 4-9.

Meanf SD percentage of overall BALf recovered was 75f5.5%, and percentages ranged from 60

to 85%. Measurements of recovered BALf did not include the foamy part of the BALf. The

physical appearance of all samples was clear and slightly turbid. Based on simple observation,

there were no visible mucus flakes. Mean values of the BALf cytology indices of the EA and the

sham groups were not significantly different; p-values from independent t-tests the ELF-









corrected TNC (0.39), percentage of macrophages (Mac) (0.68), Lym (0.67), Neu (0.87), Mast

(0.63), and Eos (0.35).

P-values from the Type III sum of squares test of the main effects and their interactions

for the BALf cytological parameters are given in Table 4-10. MeanfSE values of the ELF-

corrected TNC, Mac, Neu, Mast, and Eos in BALf are given in Table 4-11i.

Within-treatment comparisons of ELF-corrected TNC, Mac, Lym, and Eos of pre-

treatment and post-10th treatment samples were not significantly different (Table 4-12). The

mean percentages of Neu in post-10Oth treatment samples of the EA group was significantly

higher than that of the pre-treatment samples (p-value = 0.04). The mean percentages of Neu in

post-10Oth treatment samples of the sham group also increased, but the increase was not significant

(p-value = 0.06). The mean value of Mast in post-10Oth treatment samples of the EA and sham

groups significantly decreased (p-value of the EA group = 0.05, and p-value of the sham group =

0.01).

Immunoglobulins

Mean concentrations of immunoglobulin isotypes in pre-treatment samples of the EA and

the sham groups were not significantly different based on independent t-tests IgA (0.3), IgM

(0.7), IgGa (0.7), IgGb (0.7), and IgG(T) (0.8).

P-values from the Type III sum of squares test of the main effects and their interactions

for plasma immunoglobulin isotypes are given in Table 4-13. MeanfSE concentrations of

plasma immunoglobulin isotypes are given in Table 4-14. Pairwise comparison between Pr, Po,

and Ps samples of all plasma immunoglobulin isotypes are given in Table 4-15.

Even though p-values of the treatment effect for all immunoglobulin isotypes were

significant, results of pairwise comparisons between Pr, Po, and Ps samples of all









immunoglobulin isotypes indicated that the significantly different might be due partly to

individual subj ect difference.

Mean concentrations ofimmunoglobulin isotypes in the ELF-corrected BALf of pre-

treatment samples of the EA and the sham group were not significantly different; p-values IgA

(0.5), IgM (0.4), IgGa (0.7), IgGb (0.9), and IgG(T) (0.9).

P-values from the Type III sum of squares test of the main effects and their interactions

for concentrations of IgA, IgM, IgGa, IgGb, and IgGt in the ELF-corrected BALf are given in

Table 4-16. Mean+SE concentrations of ELF-corrected BALf immunoglobulins are given in

Table 4 -17. Within-group compari sons of ELF -corrected B ALf immunoglobulin i sotypes of Pr-

and Po-samples in the EA and the sham groups were not significantly different (Table 4-18).

TNF-cl Production of Whole Blood Stimulation

After in vitro incubation of whole blood, TNF-a production with no stimulant added was

less than 0.08 x103 pg/ml. There was no detectable TNF-a by ELISA assay when only PBS was

added. Mean concentrations of TNF-a production from whole blood stimulated with LPS, Zym,

CA, CA+CE, CA+LPS, CE+LPS, and CA+CE+LPS of the pre-treatment samples of the EA and

the sham were not significantly different based on independent t-tests; p-values LPS (0.3), Zym

(0.4), CA (0.9), CA+CE (0.9), CA+LPS (0.6), CE+LPS (0.6), and CA+CE+LPS (0.7).

P-values from the Type III sum of squares test of the main effects and their interactions

for the TNF-a production from stimulated whole blood are given in Table 4-19.

Mean+SE concentrations of TNF-a production from whole blood when stimulated with

each stimulant are given in Table 4-20. EA generally suppressed TNF-a production when the

whole blood was stimulated with all stimulants. The significant of suppressions were greatest

between pre-treatment and post-7th treatment samples, and were greater than those of between









pre-treatment and post-12th treatment samples (Table 4-21). Suppression of TNF-a production

from whole blood by EA treatment of post-7th treatment and post-1 2th treatment samples when

the whole blood was stimulated with LPS, CA, CA+CE, CA+LPS, CE+LPS, and CA+CE+LPS

were not significantly different (Table 4-21).

In the EA group, TNF-a production of whole blood stimulated with Zym in the pre-

treatment samples did not significantly differ from that of post-7th and post 12th-treatment

samples (Table 4-21). However, the decrease from the post-7th treatment samples to post-12th

treatment samples was significant (p-value 5 0.01). This was due partly to large differences in

individual responses to Zym. The individual variation in response to Zym can be seen from the

large standard error of the mean TNF-a concentration derived from this stimulant (Table 4-20).

In the sham treatment group, mean concentrations of TNF-a production from whole

blood samples stimulated with Zym, CA, CA+CE, CA+LPS, CE+LPS, and CA+CE+LPS were

not significantly different between sampling times (Table 4-21). When LPS was used as a

stimulant alone, TNF-a production from whole blood samples collected after the 12th treatment

significantly increased when compared to those of pre-treatment and post-7th treatment samples

(Table 4-21). Moreover, when CA was used as stimulant alone, TNF-a production from whole

blood samples collected after the 12th sham treatment significantly increased when compared to

the post-7th treatment sample, but was not significantly different when compared to the pre-

treatment samples.

Rapid Partial Forced Expiration Maneuver

The relationship of the airflow rate to the pressure differential (AP) was linear. Examples

of linearity of the airflow rate to AP before RP-FE and after RP-FE are shown in Figure 4-9.









After being chemically restrained with an intravenous administration of 20-30 Clg/kg

detomidine hydrochloride, an endotracheal (ET) tube was successfully inserted in each horse.

Endotracheal tube intubation induced coughs in some horses. Coughing subsided once the ET

tube was in place. Artificial inspiration caused expansion of the thoraco-abdominal wall, which

resolved when the air blower was turned off. There were no maj or signs of discomfort during

artificial inspiration or the RP-FE maneuver.

Mean values of PFTPs of the pre-treatment samples of the EA and the sham groups were

not significantly different; p-values from independent t-tests FEV0.5 (0.88), FEV0.75 (0.78),

FEV1.0 (0.69), FEV1.5 (0.98), FEV2.0 (0.72), FEV2.5 (0.71), FEV3 .0 (0.71), FVC (0.71), PEF

(0.72), MEF25% (0.79), MEF50% (0.5 1), MEF75% (0.48), FEV0.5/FVC (0.75), FEV0.75/FVC

(0.54), FEV1.0/FVC (0.44), FEV1.5/FVC (0.29), and FEV2.0/FVC (0.52). P-values from the

Type III sum of squares test of the main effects and their interactions for PFTPs are given in

Table 4-22 and Table 4-23.

Mean+ SE values of PFTPs are given in Table 4 -24 and Table 4 -25. P-values of pairwi se

comparisons between Pr, Po, and Ps data of PFTPs in the EA and the sham groups are given in

Table 4-26 and Table 4-27.

In the EA treatment group, FEV0.75 and FEV1.0 after the 7th treatment were

significantly greater than those in the pre-treatment. When pre-treatment PFTPs were compared

with those obtained after the 12th treatment, FEV0.75, FEV1.0, FEV1.5, PEF, MEF 25%, MEF

50%, and MFE 75% significantly increased. Moreover, FEV1.0, FEV1.5, PEF, and MEF 25%

obtained after the 12th treatment were significantly greater than those obtained after the 7th

treatment (Tables 4-24 and Table 4-25).









In the sham group, when pre-treatment PFTPs were compared with those obtained after

the 7th treatment, FEV1.0, PEF, and MEF 25% significantly increased. FEV0.5, FEV0.75,

FEV1.0, FEV1.5, FEV2.0, FEV2.5, FEV3.0, FVC, PEF, and MEF 50% obtained after 12th

treatment were significantly greater than pre-treatment PFTPs. Moreover, FEV0.75, FEV1.0,

FEV1.5, and PEF obtained after the 12th treatment were significantly greater than values

obtained after the 7th treatment (Tables 4-24 and 4-25).

Arterial Blood Gas Analysis During RP-FE

Results of arterial blood pH, pO2, pCO2, and concentrations of HCO3 tOn pre- and post-

RP-FE, and 1, 2, and 5 minutes after RP-FE was initiated) were obtained from 8 horses, are

presented in Figures 4-10 to 4-13. When artificial inspiration was initiated, the arterial blood pH

and oxygen partial pressure (pO2) inCreaSed, and the concentration of HCO3 and carbon dioxide

partial pressure (pCO2) decreased. The results indicated that hyperventilation of the airways

suppressed the normal respiratory drive of the horses by increasing blood oxygenation. Low

arterial concentration of HCO3 and pCO2 Suggested hypocapnea. Together with an increase in

the arterial blood pH, results from blood gas analysis indicated that artificial ventilation during

RP-FE induced respiratory alkalosis.

Serum Cortisol

Pre-treatment serum cortisol concentrations in the EA and sham groups were not

significantly different (p-value = 0.85). P-values from the Type III sum of squares test of the

main effects and their interactions for serum cortisol are given in Table 4-28. Mean+SE

concentrations of serum cortisol are given in Table 4-29. Pairwise comparisons of serum cortisol

from the EA and the sham groups were not significantly different. P-values from pairwise

comparisons of Pr-Po, Pr-Ps, and Po-Ps of the EA group were 0.7 1, 1.00, and 0.5 1. P-values









from pairwise comparisons, of Pr-Po, Pr-Ps, and Po-Ps of the sham group were 0.50, 1.00, and

0.79.

Discussion

Seventeen of 24 horses that were used in the study detailed in Chapter 3 also were used in

this study. Three additional horses were recruited from the research herd to yield a total of 20.

None of the horses that had been used in the Chapter 3 experiment possessed significantly

different PFTPs from one another, suggesting that these horses have normal pulmonary

functions. Thi s may be one of the maj or factors explaining why there were no effects of EA on

BALf cytology and PFTPs in thi s study.

To investigate effects of EA on the immune system and pulmonary function, multiple

acupoints were used. Twelve acupoints used in this study were chosen from acupoints

recommended for treating equine chronic respiratory disorders.78 All acupoints are located on the

cranial part of the body or on the lateral thoracic wall. Cutaneous sensations originating from

GV-14 and Ding-chuan are likely transmitted to the central nervous system via the cutaneous

branch of the local spinal nerves.239 Cutaneous sensations from BL-13 and Fei-men are likely

transmitted via the dorsal branch of the local thoracic nerve. Cutaneous sensation from Fei-pan

is likely transmitted via both dorsal branch of the local thoracic nerve and the intercostobrachial

nerve. Cutaneous sensation from Fei-shu is likely transmitted via the intercostal nerve.

Cutaneous sensation originating from CV-22 area is likely transmitted via the ventral branch of

the 6th COTVICal HOTVe and cranial branch of the supraclavicular nerve.169,239

Piercing an acupoint with an acupuncture needle not only generates a nerve signal in the

skin, but also activates sensory receptors residing in the subcutaneous tissue and muscles located

under the acupoint. When the selected twelve acupoints are stimulated, likely nerve of activation

include facial, intercostobrachial, cutaneous branch of the local spinal nerve, and dorsal branches









of local spinal nerve. 169,239-241 Details of acupoint locations, cutaneous innervations, muscles

involved and their innervations are given in Table 4-30.

It is obvious that inserting an acupuncture needle through the skin at an acupoint activates

both mechanoreceptors and pain receptors in the tissues. Mechanoreceptors in the skin and

subcutaneous tissues include Meissner' s corpuscles, Pacinian corpuscles, and Merkel' s disks.242

Activation of these receptors generates sensory signals, which are transmitted to the spinal cord

via AP axon. These receptors have a low threshold of activation. Receptors for pain sensation, or

nociceptors, are free nerve endings that are associated with either C or AS axon. They have a

high threshold of activation and transmit pain, temperature, and crude touch sensation.242

Electroacupuncture in this study seemed to generate both mechanical and pain sensations.

Some horses showed no reaction to EA, while some horses were agitated by the procedure. The

reactions to EA ranged from mild skin twitching to severe agitation. Chemical restraint with a

short-acting sedative during needle placement can be used, but this practice was not considered

in this experiment. However, when additional physical restraint was required, either a lip chain

or shoulder twitch was used.

Numbers of Wbc and Rbc of all subj ects were within normal reference values throughout

the study. Results from the differential counts of white blood cells from individual horses were

within normal reference values throughout the study. Horses used in this study were handled

regularly (3-5 times a week) and were assumed to be accustomed to the research facilities.

However, physiological excitation caused by initial handling for taking pre-treatment blood

samples was observed. Hemoglobin concentrations of pre-treatment samples from 6 of 17 horses

in the EA group and 7 of 19 horses in the sham group were greater than normal reference values

(1 1.2-16.2 g/dL), and ranged from 1 6.3 -18.5 and 16.3-18.7, respectively. High values of Hb in









these horses coincided with their high Hct values, which were in the upper limit of the normal

reference values (30-43%) or greater. Both Hb and Hct of these horses were within normal

limits after the 7th treatment and after the 12th treatment. It is assumed that the high Hb and Hct

values of the pre-treatment samples in this study resulted from excitation caused by initial

handling. The excitement activates the sympathetic nervous system, which could cause spleenic

contraction and release of stored red blood cells into circulation.

Mean Mche and Chcm of the EA and sham groups were in high normal or greater than

the reference values throughout the experiment. High Mche and Chcm might be caused by

hemolysis of red blood cells or by the clumping of red blood cells, which led to an under

estimate of Rbc numbers. Blood samples in EDTA vacutainer tube for hematology in this study

were stored on ice prior to being submitted to the laboratory. In humans with cold agglutinin (an

auto-immune disease that caused hemolytic anemia or AIHA), the red blood cells agglutinate and

cause high Mche and Chcm when blood temperature is colder than 370C. In veterinary medicine,

cold agglutinin has been described most often in dogs and horses. The condition is idiopathic and

may be secondary to chronic infections, other auto-immune disorders, or neoplastic

diseases.243,244 Whether this condition present in horses being used in this study was not

investigated, but is not likely. Another possibility is rouleaux formation, a physiologic

phenomenon in which red blood cells of horses stack together forming a line when the blood

sample is still. Rouleaux formation disappears when the blood sample is mixed thoroughly.

Percentages of BALf neutrophils in pre-treatment samples of the EA and sham groups in

the first trial ranged from 0.5-6.5%. This level of percentages in BALf neutrophils has been

reported as characteristic of BALf cytology of normal horses.215 The results suggested that

horses used in this study did not have pulmonary disorders. Percentages of neutrophils were










significantly higher in BALf samples that were collected after the 10th treatment, which was 8-10

days after the second RP-FE maneuver. Also, percentages of BALf neutrophils in pre-treatment

samples from the second trial were greater than those of the first trial. These results together

suggested that RP-FE induced pulmonary inflammation, which was carried over to the second

trial.

Collecting BALf samples and performing the RP-FE maneuver at the same time was

avoided during the initial study design. A period of 8-10 days was first thought to be enough for

the pulmonary tissues to recover from RP-FE-induced inflammation. However, the results

indicated that inflammation in the airways and pulmonary tissues caused by RP-FE lasted longer

than the period first hypothesized. Despite RP-FE-induced inflammation in the pulmonary

tissues, the PFTPs were not compromised.

According to the amount of TNF-a produced from whole blood stimulation, LPS was

more potent than Zym and CA. There was a synergistic effect when CA or CE was individually

or both added to LPS for the stimulation. Electroacupuncture, but not the sham treatment,

significantly suppressed in vitro TNF-a production from whole blood when stimulated with LPS,

CA+CE, CA+LPS, CE+LPS, and CA+CE+LPS.

In the EA treatment group, mean TNF-a production when whole blood was stimulated

with Zym alone decreased over time (Figure 4-14), and it is possible that EA progressively

suppressed TNF-a production. However, the suppression was not shown to be statistically

significant due to a large variation in individual response.

Tumor necrosis factor alpha is an inflammatory cytokine. It is an important component of

the early response in the innate immune system. It is thought to be involved in the pathogenesis

of chronic lower airway inflammation in horses.144 Suppression of TNF-a production from









immune cells after EA treatment, as demonstrated when the whole blood was stimulated with

LPS, CA+CE, CA+LPS, CE+LPS, and CA+CE+LPS, may partly play a role in how EA exerts

an anti-inflammatory action.

In RP-FE maneuver, after the ET tube was connected to the FE-system, the RP-FE

maneuver for each horse took less than five minutes. When the ET tube was connected to the FE-

system, the artificial inspiration was initiated. Inspiration was accomplished by inflation of the

airway to a pressure of 30 cm H20. Expiration was driven by passive mechanism, which is based

on the elastic recoil properties of pulmonary tissues and thoracic walls. Artificial respiration at

the rate of 25-30 breaths/minute suppressed the physiological respiratory drive of the tested

subj ects, and allowed the RP-FE operator to override the normal respiratory rate of the horse.

Results from the blood gas analysis suggested that artificial inspiration during RP-FE increased

blood pH and pO2, while decreasing pCO2 and HCO3, which indicated respiratory alkalosis.

Pulmonary function test parameters from this study were different from values reported

by a previous study. 17 The causes of differences in PFTPs were discussed in detail in Chapter 3,

and included differences in manifold designs, in the negative pressures used to empty airways,

and in methods of measuring airflow. Mean FVC from both the EA and sham groups in this

experiment were greater than the TLC value suggested for equine species (55 liters).192 Possible

explanations for high FVC include decreased density of expired air during RP-FE-maneuver.

This may have happened when gas in the airways was exposed to the negative pressure. In

normal expiration, gas in the lungs and airways is compressed due to the contraction of the

thoracic walls by expiratory muscle action. Expiration i s a result of an increase in intra-pleural

pressure, which also increases airway pressure. When airway pressure is greater than

atmospheric pressure, air in the lungs and airways is expired. Artificial expiration in RP-FE









maneuver is caused by pulling air out from the airways and lungs. At TLC (artificially inflating

airways to 30 cm H20), gas in the airways was compressed. When the compressed gas in the

airways and lungs was exposed to the negative pressure, the gas moved and expanded at the

same time.

Another possible explanation for high FVC is that the RP-FE maneuver might have

completely emptied air from the airways and lungs, including air in the physiological dead

spaces. In this study, an individual RP-FE maneuver for each horse was composed of 3-5

artificial forced expirations. Forced vital capacity, PEF and flow-volume loop of the first

artificial forced expiration in every maneuver were always greater than those of the subsequent

artificial forced expirations (Figures 4-15 and 4-16). It is possible that some parts of the alveoli

and airways were collapsed after the first artificial forced expiration, and were not inflated by the

artificial inspiration. Because of the elastic properties of airways and alveoli, which are located

in a collapsible thoracic cavity, it might be possible to completely empty air from the airways

and lungs. At the end of the artificial forced expiration, the thoracic and abdominal cavities were

severely contracted.

This study also investigated the effects of treatments (EA and sham) and RP-FE on serum

cortisol concentration. The hormone cortisol is released mainly from the adrenal cortex and has

been used as an indicator of stress response in various species, including the horse. Average

concentrations of serum cortisol from horses in the EA and sham groups in this experiment were

6.6 and 6.9 Clg/dL, respectively. These values were less than the reference values ( 13 Clg/dlL)

for normal horses.245 Serum cortisol concentrations among three sampling times (pre-, after the

7th-, and after the 12th treatments) were not significantly different. These results suggested that

neither treatments (EA and sham) nor RP-FE maneuvers in this study induced change in cortisol.









In summary, the results of this study suggest that EA provides anti-inflammatory effect as

demonstrated by a suppression of TNF-oc production of stimulated whole blood. These effects

may help modulate inflammatory response present in several equine diseases, including chronic

respiratory diseases. Therefore, we believe that EA treatment at GV-14, CV-22, BL-13, Ding-

chuan2, Fei-men, Fei-pan, and Fei-shu has merit in the treatment of inflammatory diseases in

horses, but requires further study to be fully validated.












































Figure 4-1. Electroacupuncture.


















































rlgure 4-2. Ynam electroacupuncture.







































Figure 4-3. A pressure regulator and a set point regulator installed on the manifold of the
artificial inspiration system. Two analog pressure gauges, G1 and G2 were used for
monitoring pressure in the manifold of inspiratory system and pressure of the set
point regulator.





































Figure 4-4. Solenoid valve used for controlling the air from the air blower in the artificial
inspiration system and for isolating the LFE from the artificial respiration manifold
and the negative pressure reservoir.































1
- I


5

t'


I MM I


Figure 4-5. Diagram of the rapid partial forced expiration apparatus.











156










L






8




I









O


B


Negative pressure reservoir

Air blower

Variable transformer

Analog pressure gauge

Laminar flow element

Filter

Pressure transducer

Manual actuated PVC valve

Computer

Solenoid valve

Airway pressure relive valve

Vacuum Pump


Temperature meter

Signal conditioner

Signal interface


Analog digital converter

Rigid polypopylene tube

Thermocouples

Connecting cable


Electrical power cord

Manual relay switch

Electrical power source

Vacuum switch


Figure 4-6. Component symbols.


o-+


1~

1IIA

II~






































Figure 4-7. Apparatus setup for laminar flow element (LFE) calibration using NIST-traceable
mass flow element (MFE). The LFE, MFE, and air blower were linearly connected
and air was sucked out through the MFE. Yellow arrows indicate direction of airflow.





































Figure 4-8. The 12.829-liter syringe used for testing an accuracy of integrated airflow volume
compare with a 60 ml disposable syringe.














x5







"2

1
w
F4
c]


0 10 20 30 40 50 60

Direct reading ofMFE airflow (liters/second)
Before RP-FE After RP-FE

Figure 4-9. Linear relationship of airflow rate to AP before RP-FE and after RP-FE. Lines were
plotted using calibration data from RP-FE on July 26, 2008. After the initial
calibration, RP-FE was performed in 4 horses.


7.55


7.5


7.45


7.4


7.35


Pre RP-FE


Post RP-FE


Time (minute)


Figure 4-10. Mean+SD arterial
horses).


blood pH during RP-FE maneuver (data were obtained from 8















r 45
O
O
o
S35


Pre RP-FE


1 2 5


Post RP-FE


Time (minute)

Figure 4-11. MeaniSD arterial blood pCO2 during RP-FE maneuver (data were obtained from 8
horses).





S130

r 110

S90


70


Pre RP-FE


1 2 5


Post RP-FE


Time (minute)


Figure 4-12. MeaniSD arterial blood pO2 during RP-FE maneuver (data were obtained from 8
horses).














~28


O

o

2 24


Pre RP-FE


1 2 5


Post RP-FE


Time (minute)

Figure 4-13. Mean+SD arterial blood HCO3 during RP-FE maneuver (data were obtained from 8
horses).


5000


4500
4000
3500
3000
2500
2000
1500
1000
500
0


Po
Sampling time


Figure 4-14. Mean+SD TNF-oc production in electroacupuncture (EA) and sham-EA (sham)
groups when whole blood was stimulated with Zymosan. Pr = pre-treatment, Po
after the 7th treatment, Ps after the 12th treatment.


5 EA
SSham
















S60
2nd FE

I~l /-3rd FE
S40 I 4th FE


S20




0 0.5 1 1.5 2 2.5 3
Forced expiratory time (second)


Figure 4-15. Airflow rates from five artificial forced expirations (FE) during rapid partial forced
expiration maneuvers on one horse in 20 July 2008.


70



9 50 -t 1 st FE
li -2ndFE
> 40
~ 3rd EF
30 -4th FE

S20 _-5hF
0 0


20 40 60 80


Forced expiratory airflow rate (liters/second)

Figure 4-16. Flow-volume loops from five artificial forced expirations (FE) during rapid partial
forced expiration maneuvers on one horse in 20 July 2008. Data from same horse as
in Figure 5-15.













Needle insertion
method
Perpendicular to
skin


Acupoint
GV-14


CV-22


BL-13





Fei-men



Fei-pan


Fei-shu






Ding-chu


2 Perpendicular to
skin

3 Perpendicular to
skin




2 Caudoventrally


2 Cranioventrally


2 Ventromedially
and parallel to
thoracic wall




2 Perpendicular to
skin


Cun = unit of measurement in TCVM. Length of cun i s relative to the body size of the animal,
and the width of scapula from the cranial to caudal border is 3 cun.246 (Sources: Xie H,
Yamagiwa K. Equine Classical Acupoints In: Xie H,Preast V, eds. Xie's veterinary acupuncture.
1st ed. 2007; pages 89-127, and Xie H, Trevisanello L. Equine Transpositional Acupoints In: Xie
H,Preast V, eds. Xie's veterinary acupuncture. 2007; pages 27-87.78,135)


Table 4-1. Anatomical location of acupoints, their Western medical indication, needle size and


method of insertion.


Location
C7-T1 dorsal midline
cranial to depression of
wither
Ventral midline at
depression cranial to
sternum
Caudal edge of scapular
cartilage (8th
intercostals space) 3
cun lateral to dorsal
midline
1/3 distance from top of
scapular along cranial
border
1/3 distance from top of
scapular along caudal
border
9th intercostals space on
line connecting
shoulder and
coxofemoral joint


an2 0.5 cun lateral to GV-14


Needle
size
(inch)
2


Indication in Western
medicine
Fever, cough, heaves,
and immune
stimulation
Cough, heaves, and
asthma

Cough, heaves, and
asthma




Upper respiratory
problem, cough,
and asthma
Lower airway and
lung disease

Upper airway
infection, cough,
heaves, asthma,
bronchiti s,
pneumonia, flu,
and COPD
Cough and asthma













Degree of reaction
Study Treatment A B C D E Total
it s1trial EA 5 1 1 2 1 10


Table 4-2. Numbers of horses categorized by degree of reaction to EA and to sham treatments in
the 1 st and 2nd trials prior to eliminating the horse that did not accept EA treatment
and horses receiving NS AIDs.


Shana 7 2 1 0 0 10
2nd trial EA 6 1 0 2 1 10
Shana 8 1 1 0 0 10
A = no reaction, B = mild skin twitching, C = moderate skin twitching, D = intermittent forceful
skin twitching, E = continuous forceful skin twitching and restlessness, EA = electroacupuncture,
Sham = sham EA.

Table 4-3. P-values from the Type III sum of squares test on main effects and their interactions
for white blood cell indices.
Factor Wbc Neu Lym Mono Eos Baso
Treatment 0.91 0.92 0.50 0.10 0.68 0.95
Sampling time 0.19 0.93 0.84 0.58 0.37 0.29
Trial 0.25 0.55 0.74 0.94 0.43 0.84
Sequence 0.13 0.75 0.70 0.87 0.81 0.24
Treatments sampling 0.16 0.93 0.89 0.29 0.39 0.61
time
Horse sequence 0.02 5 0.01 5 0.01 0.11 5 0.01 0.64
* = interaction between factors, Wbc = white blood cell counts, Neu = percentage of neutrophils,
Lym = percentage of lymphocytes, Mono = percentage of monocytes, Eos = percentage of
eosinophils, Baso = percentage of basophils.










Table 4-4. MeantSE white blood cell indices, by treatment and sampling time. Normal
reference values in parentheses.


Wbc
x103cell/pL
(5.5-11.0)
8.77
~0.28
8.78
~0.28
8.65
~0.28
9.07
~0.26
8.20
~0.26
8.93
~0.26


Sampling
Tx time
EA Pr


Po


Ps


Sham Pr


Po


Ps


Neu%
(28.0-82.8)
61.9


61.9


61.8


61.6


61.2


62.3


Lym% h
(19.8-58.9) (1
29.0


28.9


29.0


27.9


28.3


28.9


Ps 0.05, Pr = pre
= treatment, EA =


Mlono%
.4-10.5)
4.6
10.4
3.9
10.4
4.5
10.4
5.1
10.4
5.3
10.4
4.4
10.4
-treatment,


Eos% Baso%
(0-8.7) (0-2.0)
3.9 0.4
10.5 1.
4.6 0.3
10.5 1.
4.1 0.3
10.5 1.
4.8 0.4
10.5 1.
4.5 0.4
10.5 10.0
3.8 0.2
10.5 10.0
Po after the 7th


+ p-value of comparison between Po and
treatment, Ps after the 12th treatment, Tx


electroacupuncture, Sham sham
lymphocyte, Mono = monocyte,


EA, Wbc = white blood cell count, Neu = neutrophil, Lym
Eos = eosinophil, Baso = basophil.










Table 4-5. P-values from pairwise comparisons of white blood cell indices, by treatment and
sampling time.


Tx Pairwise
EA Pr-Po
Pr-Ps
Po-Ps
Sham Pr-Po
Pr-Ps
Po-Ps
Pr = pre-treatment, Po = after
= electroacupuncture, Sham =


Wbc


Neu


Lym
1.00
1.00
1.00
1.00
1.00
1.00
after the


Mono
0.51
1.00
1.00
1.00
0.99
0.90
12h treatment,


Eos


Baso


0.48 1.00
0.62 1.00
0.72 1.00
1.00 1.00
0.71 0.76
0.65 0.30
Tx = treatment, EA
= neutrophil, Lym


1.00 1.00
1.00 1.00
1.00 1.00
0.06 1.00
1.00 1.00
0.02 1.00
the 7t treatment, Ps
Ssham EA, Wbc = wl


hite blood cell count, Neu


lymphocyte, Mono = monocyte, Eos = eosinophil, Baso = basophil.


Table 4-6. P-values from the Type III sum of squares test on main effects and their interactions
for red blood cell indices.
Factor Rbc Hb Hct Mcy Mch Mche Chcm Ch


0.68
5 0.01
0.02


0.69
5 0.01
0.15


0.47
5 0.01
<0.01


0.74
5 0.01
<0.01


0.30
5 0.01
0.76


0.93
5 0.01
0.03


0.68
5 0.01
0.06


Treatment

Sampling time
Trial


0.61
5 0.01
<0.01


Sequence 0.83 0.76 0.82 0.45 0.51 0.71 0.44 0.49
Treatments sampling 0.33 0.43 0.49 0.13 0.27 0.35 0.07 0.77
time
Horse sequence 5 0.01 5 0.01 5 0.01 5 0.01 5 0.01 0.64 5 0.01 5 0.01
* = interaction between factors, Rbc = red blood cell count, Hct = hematocrit, Hb = hemoglobin,
Mcy = mean corpuscular volume, Mch = mean corpuscular hemoglobin, Mche = mean
corpuscular hemoglobin concentration, Chcm = cellular hemoglobin concentration, and Ch =
corpuscular hemoglobin content.





Table 4-7. MeantSE red blood cell indices, by treatment and sampling time. Reference values in


parentheses.
Rbc
(6.7- Hct
Sampling 10.0x106 (30.0-
Tx time cell/CIL) 43.0%)
EA Pr 9.28 39.6
+0.21 10.9
Po 8.90 37.6
+0.21 10.9
Ps 8.71 36.9
+0.21 10.9
Sham Pr *#9.49 *#40.4
+0.20 10.8
Po 8.69 36.9
+0.20 10.8
Ps 8.93 37.7
+0.20 10.8


Hb
(11.2-
16.2
g/dL)
#15.3
10.3
14.7
10.3
14.1
10.3
*#15.6
10.3
14.4
10.3
14.4
10.3


Mche
(36.4-
38.8
g/dL)
*38.7
10.4
+39.1
10.4
38.4
10.4
*38.7
10.4
+39.2
10.4
37.3
10.4


Chcm
(34.9-
37.6
g/dL)
38.6


38.6

30.1

38.7



38.3


38.9


Mch
(14.0-
18.7 pg)
#16.5
10.2
+16.5
10.2
16.2
10.2
*#16.4
10.2
+16.6
10.2
16.2
10.2


Ch
(13.7-
17.9 pg)
*16.5
10.2
16.4
10.2
16.5
10.2
*16.5
10.2
+16.4
10.2
16.5
10.2


Mcy
(37.5-
50.0 L)
*#42.7
10.5
42.3
10.5
42.3
10.5
42.5
10.5
42.4
10.5
42.3
10.5


* = p-value of comparison between Pr and Po: 0.05, # = p -value of comparison between Pr and
Ps I 0.05, + p-value of comparison between Po and Pse 0.05, Pr = pre -treatment, Po after
the 7th treatment, Ps after the 12th treatment, Tx treatment, EA = electroacupuncture, Sham =
sham EA, Rbc = red blood cell count, Hct = hematocrit, Hb = hemoglobin, Mcy = mean
corpuscular volume, Mch = mean corpuscular hemoglobin, Mche = mean corpuscular


hemoglobin concentration,
hemoglobin content.


Chcm = cellular hemoglobin concentration, and Ch = corpuscular










Table 4-8. P-values from pairwise comparisons of the red blood cell indices, by treatment and
sampling time.
Tx Pairwise Rbc Hct Hb Mcy Mch Mche Chcm Ch
EA Pr-Po 0.20 0.08 0.22 <0.01 1.00 0.03 1.00 <0.01
Pr-Ps 0.12 0.07 0.04 0.02 0.03 0.27 1.00 1.00
Po-Ps 1.00 1.00 0.61 1.00 <0.01 <0.01 0.60 0.15
Sham Pr-Po <0.01 <0.01 <0.01 1.00 <0.01 <0.01 1.00 0.02
Pr-Ps 0.03 0.02 <0.01 0.27 <0.01 0.13 0.10 1.00
Po-Ps 0.54 0.78 1.00 0.18 <0.01 <0.01 0.12 0.02
Pr = pre-treatment, Po = after the 7t treatment, Ps = after the 12h treatment, Tx = treatment, EA
= electroacupuncture, Sham = sham EA, Rbc = red blood cell counts, Hct = hematocrit, Hb =
hemoglobin, Mcy = mean corpuscular volume, Mch = mean corpuscular hemoglobin, Mche =
mean corpuscular hemoglobin concentration, Chcm = cellular hemoglobin concentration, and Ch
= corpuscular hemoglobin content.

Table 4-9. MeantSD and range (in parenthesis) of percentages of recovered BALf and
percentages of ELF in BALf samples determined by urea dilution technique.
Percentage of BALf
Tx Sampling time recovered Percentage of epithelial lining fluid
EA Pr 76.315.3 (85.0-65.3) 1.310.4 (2.1-0.6)
Po 75.415.0 (83 .3-63.3) 1.310.5 (2.2-0.6)
Sham Pr 75.616.9 (83 .3-60.0) 1.210.5 (2.4-0.6)
Po 72.214.1 (80.0-64.0) 1.210.3 (1.9-0.9)
Pr = pre-treatment, Po = after the 10th treatment, Tx treatment. EA = electroacupuncture, Sham
= sham EA, BALf = broncho-alveolar lavage fluid, ELF = epithelial lining fluid.










Table 4-10. P-values from the Type III sum of squares test on main effects and their interactions
for broncho-alveolar lavage fluid cytological parameters.
Factor ELF-corrected TNC Mac Lym Neu Mas Eos
Treatment 0.59 0.38 0.36 0.59 0.85 0.26
Sampling time 0.43 0.55 0.23 I 0.01 5 0.01 0.89
Trial <0.01 0.28 0.10 <0.01 0.85 0.67
Sequence 0.04 0.30 0.90 0.75 0.21 0.33
Treatments 0.45 0.35 0.73 0.78 0.72 0.47
sampling time
Horse sequence 0.18 0.69 5 0.01 5 0.01 5 0.01 5 0.01
* = interaction between factors, ELF = epithelial lining fluid, TNC = total nucleated cell count,
Mac = macrophage, lym = lymphocyte, Neu = neutrophil, Mast = mast cell, and Eos =
eosinophil .

Table 4-11. MeantSE ELF-corrected TNC and differential counts of BALf cells, by treatment
and sampling time.
Sampling ELF-corrected TNC
Tx time (x107 cell/ml) Mac (%) Lym (%) Neu (%) Mast (%) Eos (%)
EA Pr 2.84 41.0 52.3 *4.5 *1.8 0.3
~0.27 17.8 115 0.6 10.2 1.0
Po 2.81 51.7 54.4 6.1 1.0 0.5
~0.27 17.8 115 0.6 10.2 1.0
Sham Pr 3.17 40.7 51.2 4.7 *1.7 0.8
~0.25 17.2 113 0.6 10.2 10.2
Po 2.77 38.5 52.3 6.6 1.0 0.6
~0.25 17.2 113 0.6 10.2 1.0
*=p-value of difference between Pr and Per 0.05, Pr = pre -ramnP=atrte1t
treatment, Tx = treatment, EA = electroacupuncture, Sham = sham EA, BALf = broncho-alveolar
lavage fluid, ELF = epithelial lining fluid, TNC = total nucleated cell count, Mac = macrophage,
lym = lymphocyte, Neu = neutrophil, Mast = mast cell, and Eos = eosinophil.










Table 4-12. P-values from paired samples t-test of BALf cytological parameters by treatment.
Tx Pairwi se ELF-corrected TNC Mac Lym Neu Mast Eos
EA Pr-Po 0.97 0.47 0.39 0.04 0.05 0.73
Sham Pr-Po 0.31 0.27 0.50 0.06 0.01 0.50
Pr = pre-treatment, Po = after the 10Oth treatment, Tx = treatment, EA = electroacupuncture,
Sham = sham EA, BALf= broncho-alveolar lavage fluid, ELF = epithelial lining fluid, TNC =
total nucleated cell count, Mac = macrophage, lym = lymphocyte, Neu = neutrophil, Mast = mast
cell, and Eos = eosinophil.

Table 4-13. P-values from the Type III sum of squares test on the main effects and their
interactions for plasma concentration of immunoglobulin isotypes.
Factor IgA IgM IgGa IgGb IgG(T)
Treatment <0.01 <0.01 0.03 <0.01 <0.01
Sampling time 0.13 0.76 I 0.01 5 0.01 0.55
Trial 0.06 0.03 <0.01 0.03 0.98
Sequence 0.37 0.54 0.86 0.82 0.88
Treatments sampling time 0.74 0.97 0.65 0.15 0.66
Sequence horse 5 0.01 5 0.01 0.05 5 0.01 5 0.01
* = interaction between factors.

Table 4-14. MeantSE concentrations of plasma immunoglobulin isotypes (x105 ng/ml), by,
treatment and sampling time.
Tx Sampling time IgA IgM IgGa IgGb IgG(T)
EA Po 12.91 5.59 24.24 48.38 42.22
~2.46 10.56 11.59 12.76 16.18
Pr 13.47 5.62 25.51 50.70 42.69
~2.46 10.56 11.59 12.76 16.18
Ps 15.80 5.76 28.94 39.62 43.26
~2.46 10.56 11.59 12.76 16.18
Sham Po 17.85 7.99 28.27 56.56 63.62
2.43 10.54 11.52 12.67 +6.01
Pr 18.69 8.09 27.53 53.41 60.81
~2.43 10.54 11.52 12.67 +6.01
Ps 19.49 8.35 34.09 49.31 68.35
~2.43 10.54 11.52 12.67 +6.01
Pr = pre-treatment, Po = after the 7t treatment, Ps = after the 12h treatment, Tx = treatment, EA
= electroacupuncture, Sham = sham EA.























Tx = treatment, EA = electroacupuncture, Sham = sham electroacupuncture, Pr = pre-treatment,
Po = after the 7th treatment, Ps after the 12th treatment.

Table 4-16. P-values from the Type III sum of squares test on main effects and their interactions
for concentration of immunoglobulin isotypes in ELF-corrected BALf.
Factor IgA IgM IgGa IgGb IgG(T)
Treatment 0.84 0.45 0.26 0.71 0.71
Sampling time 0.96 0.94 0.53 0.32 0.25
Trial 0.21 0.44 0.46 0.31 0.12
Sequence 0.36 0.97 0.44 0.96 0.89
Treatments sampling time 0.21 0.83 0.37 0.27 0.42
Horse sequence 0.22 0.43 0.88 0.52 0.31
*= interaction between factors.

Table 4-17. MeanfSE concentrations of immunoglobulin isotypes in ELF-corrected BALf (x10S
ng/ml), by treatment and sampling time.
Tx Sampling time IgA IgM IgGa IgGb IgG(T)
EA Po 24.12 20.32 9.87 18.92 11.57
~2.96 12.80 11.09 12.41 11.47
Pr 20.58 23.61 8.05 13.87 8.82
~2.96 12.80 11.09 12.41 11.47
Sham Po 20.32 0.31 7.71 14.42 10.20
~2.80 10.06 11.03 12.28 11.39
Pr 23.61 0.31 7.97 14.59 9.68
~2.80 10.06 11.03 12.28 11.39
Pr = pre-treatment, Po = after the 10Oth treatment, Tx = treatment, EA = electroacupuncture,


Table 4-15. P-values from pairwise comparisons of concentration of plasma immunoglobulin


isotypes, by treatment and sampling time.
Tx Pairwi se IgA IgM
EA Pr-Po 0.95 1.00
Pr-Ps 0.07 1.00
Po-Ps <0.01 0.34
Sham Pr-Po 1.00 1.00
Pr-Ps <0.01 1.00
Po-Ps < 001 0 19


IgGa
1.00
0.29
0.32
1.00
0.06
0 07


IgGb
1.00
<0.01
<0.01
1.00
0.45
< 001


IgG(T)
1.00
0.03
<0.01
0.12
0.02
0 99


I j


I j


Sham = sham EA, BALf= broncho-alveolar lavage fluid, ELF = epithelial lining fluid.










Table 4-18. P-values from pairwise comparisons for concentrations of immunoglobulin isotypes
in ELF-corrected BALf.
Tx Sampling time IgA IgM IgGa IgGb IgG(T)
EA Pr-Po 0.93 0.37 0.86 0.52 0.52
Sham Pr-Po 0.67 0.86 0.68 0.72 0.55
Tx treatment, EA = electroacupuncture, Sham = sham EA, Pr = pre-treatment, Po = after the
10th treatment.

Table 4-19. P-values from the Type III sum of squares test on main effects and their interactions
for TNF-oc production from stimulated whole blood by stimulants.
Stimulants
CA+
CA+ CA+ CE+ CE+
Factor None PBS LPS Zym CA CE LPS LPS LPS
Treatment 0.08 -0.04 0.08 <0.01 <0.01 <0.01 <0.01 <0.01
Sampling time 0.18 -0.11 0.05 0.02 0.15 5 0.01 0.02 0.02
Trial 0.31 -0.05 0.95 0.26 0.12 0.06 0.29 0.14
Sequence 0.34 -0.66 0.53 0.82 0.63 0.38 0.20 0.62
Treatments 0.25 -0.03 0.03 <0.01 0.02 <0.01 <0.01 <0.01
sampling time
Horse sequence 0.43 0.01 5 0.01 < 0.01 5 0.01 5 0.01 < 0.01 5 0.01
* = interaction between factors, None = no stimulant added, PB S = phosphate buffer saline, LPS
= lipopolysaccharide, Zym = zymosan, CA = cellular antigen from A. fumigatus, CE = culture
extract of A.fumigatus.










Table 4-20. MeantSE TNF-oc concentrations in whole blood after stimulation,
sampling time. Concentrations are x103 pg/ml.
Stimulants


by treatment and


CA+
CE +
LPS
*#5.78
10.30
4.60
10.30
5.03
10.30
5.69
10.29
5.87
10.29


Sampling
Tx time
EA Pr


Po


Ps


Sham Pr


Po


CA +
None PBS LPS Zym CA CE


CA +
LPS
*#5.59
10.27
4.24
10.27
4.69
10.27
5.45
10.26
5.36
10.26


CE +
LPS
*#5.32
10.26
4.24
10.26
4.44
10.26
5.17
10.25
5.12
10.25


0.00 0.00 *5.10
~0.01 10.00 10.29


1.66 *4.12
10.36 10.21


*4.23
10.23
3.58
10.23
3.73
10.23
4.22
10.22
4.30
10.22


0.04
~0.01
0.01
~0.01
0.00
~0.01
0.00
~0.01


0.00
10.00
0.00
10.00
0.00
10.00
0.00
10.00


4.82
10.29
4.94
10.29
#4.90
10.28
5.11
10.28


+0.56
10.36
0.22
10.36
1.13
10.35
1.67
10.35


3.38
10.21
3.64
10.21
4.10
10.21
+4.09
10.21


Ps 0.00 0.00 5.71 0.97 4.65 4.76 5.88 5.79 6.37
~0.01 10.00 10.28 10.35 10.21 10.22 10.26 10.25 10.29
* = p-value of comparison between Pr and Po I 0.05, # = p-value of comparison between Pr and
Ps I 0.05, + p-value of comparison between Po and Pse 0.05, Tx = treatment, EA =
electroacupuncture, Sham sham EA, Pr pre-treatment, Po after the 7th treatment, Ps = after
the 12th treatment, None no stimulant added, PB S phosphate buffer saline, LPS =
lipopolysaccharide, Zym = zymosan, CA = cellular antigen from A. fumigatus, CE = culture
extract of A.fumigatus.










Table 4-21. P-values from pairwise comparisons of TNF-oc concentrations in whole blood after
stimulation, by treatment.
Stimulants


CA+
CE +
LPS
<0.01
0.03
0.54
0.21
0.07
0.28


CA+
CE
<0.01
0.20
0.57
1.00
0.29
0.26


CA+
LPS
<0.01
<0.01
0.58
1.00
0.53
0.28


CE +
LPS
<0.01
<0.01
1.00
1.00
0.17
0.11


Tx Pairwise
EA Pr-Po
Pr-Ps
Po-Ps
Sham Pr-Po
Pr-Ps
Po-Ps


PBS LPS
-<0.01
-0.073
-1.00
-0.10
-0.05
-0.21


CA
<0.01
0.06
0.79
1.00
0.10
0.04


None
0.53
1.00
0.99
0.99
0.52
1.00


Zym
0.29
0.12
<0.01
0.87
0.79
0.75


Tx treatment, EA = electroacupuncture, Sham = sham EA, Pr pre-treatment, Po after the
7th treatment, Ps = after the 12th treatment, None = no stimulant added, PB S phosphate buffer
saline, LPS = lipopolysaccharide, Zym = zymosan, CA = cellular antigen from A. fumigatus, CE
= culture extract ofA. fumigatus.










Table 4-22. P-values from the Type III sum of squares test on the main effects and their
interactions for FEVx, FVC, and PEF.
FEV FEV FEV FEV FEV FEV FEV
Factor 0.5 0.75 1.0 1.5 2.0 2.5 3.0 FVC PEF
Treatment 0.79 0.79 0.86 0.786 0.79 0.82 0.83 0.83 0.84
Sampling time 0.03 5 0.01 5 0.01 5 0.01 5 0.01 5 0.01 5 0.01 5 0.01 5 0.01
Trial <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01
Sequence 0.98 0.76 0.52 0.61 0.77 0.75 0.73 0.73 0.53
Treatments 0.99 0.96 0.68 0.86 0.95 0.95 0.95 0.95 0.57
sampling time
Horse 0.89 0.89 0.76 <0.01 <0.01 <0.01 <0.01 <0.01 0.68
sequence
* = interaction between factors, FEVx = forced expiratory volume at x seconds, FVC = forced
vital capacity, PEF = peak expiratory flow.

Table 4-23. P-values from the Type III sum of squares test on the main effects and their
interaction for IVEFx% and FEVx/FVC ratio.
1VEF IVEF IVEF FEV0.5/ FEV0.75 FEV1.0/ FEV1.5/ FEV2.0/
Factor 25% 50% 75% FVC /FVC FVC FVC FVC
Treatment 0.47 0.87 0.56 0.76 0.71 0.74 0.96 0.48
Sampling time 0.19 5 0.01 0.03 0.35 0.16 0.26 0.96 0.23
Trial <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 0.45 <0.01
Sequence 0.71 0.93 0.87 0.78 0.80 0.83 0.94 0.26
Treatments 0.49 0.37 0.23 0.99 0.93 0.71 0.35 0.28
sampling time
Horse 0.72 <0.01 <0.01 0.24 <0.01 <0.01 <0.01 <0.01
sequence
* = interaction between factors, MEF% = forced expiratory flow rate after x% of forced vital
capacity has been expired, FEVx/FVC = ratio of forced expiratory volume at x seconds/forced
vital capacity.










Table 4-24. Mean SE FEVx, FVC, and PEF obtained by the rapid partial forced expiration
maneuver, by treatment and sampling time.


Sampling
Tx time
EA Pr


Po

Ps


Sham Pr

Po


Ps


FEV
0.5
10.34
~0.21
10.72
~0.21
10.96
~0.21
#10.40
~0.19
10.81
~0.19
10.98
~0.19


FEV
0.75
*#29.73
10.2
30.49
10.26
31.24
10.26
#29.89
10.24
+30.53
10.24
31.19
10.24


FEV FEV
1.0 1.5
*#45.61 #62.29
10.23 10.42
+46.59 +63.15
10.23 10.42
47.66 64.14
10.23 10.42
*#45.86 #62.26
10.21 10.39
+46.56 +62.93
10.21 10.39
47.46 63.81
10.21 10.39


FEV
2.0
65.19
10.66
65.88
10.66
66.66
10.66
#64.76
10.60
65.48
10.60
66.56
10.60


FEV
2.5
65.46
10.66
66.15
10.66
66.94
10.66
#65.00
10.61
65.78
10.61
66.86
10.61


FEV
3.0
65.76
10.67
66.45
10.67
67.26
10.67
#65.29
10.61
66.12
10.61
67.17
10.61


FVC
65.76
10.67
66.45
10.67
67.26
10.67
#65.29
10.61
66.12
10.61
67.17
10.61


PEF
#80.16
10.35
+81.80
10.35
83.91
10.35
*#80.61
10.33
+81.59
10.33
83.61
10.33


* = p-value of comparison between Pr and Po< 0.05, # = p -value of comparison between Pr and
Ps I 0.05, + p-value of comparison between Po and Pse 0.05, Tx = treatment, EA =
electroacupuncture, Sham sham EA, Pr pre-treatment, Po after the 7th treatment, Ps after
thel2th treatment, FEVx forced expiratory volume at x seconds (liters), FVC = forced vital
capacity (liters), PEF = peak expiratory flow (liters/second).










Table 4-25. MeantSE MEFx% and FEVx/FVC ratio obtained by the rapid partial forced
expiration maneuver, by treatment and sampling time.
Sampling MEF MEF MEF FEV0.5/ FEV0.75 FEV1.0/ FEV1.5/ FEV2.0/
Tx time 25% 50% 75% FVC /FVC FVC FVC FVC
EA Pr #78.39 #71.75 #46.52 0.15 0.45 0.695 0.94 0.99
~1.11 10.55 10.78 10.00 10.01 10.01 10.01 10.00
Po +79.92 73.41 47.47 0.16 0.46 0.70 0.95 0.99
~1.11 10.55 10.78 10.00 10.01 10.01 10.01 10.00
Ps 81.61 75.50 49.20 0.16 0.46 0.70 0.95 0.99
~1.11 10.55 10.78 10.00 10.01 10.01 10.01 10.00
Sham Pr *78.73 #72.57 47.30 0.15 0.45 0.70 0.95 0.99
~1.02 10.50 10.71 10.00 10.01 10.01 10.01 10.00
Po 80.00 73.42 47.57 0.16 0.46 0.70 0.95 0.99
~1.02 10.50 10.71 10.00 10.01 10.01 10.01 10.00
Ps 79.46 74.81 47.87 0.16 0.46 0.70 0.95 0.99
~1.02 10.50 10.71 10.00 10.01 10.01 10.01 10.00
* = p-value of comparison between Pr and Po I 0.05, # = p-value of comparison between Pr and
Ps I 0.05, + p-value of comparison between Po and Pse 0.05, Tx = treatment, EA =
electroacupuncture, Sham sham EA, Pr pre-treatment, Po = after the 7th treatment, Ps after
the 12th treatment, MEFx% = forced expiratory flow rate after x% of forced vital capacity has
been expired (liters/second), FEVx/FVC = ratio of forced expiratory volume at x seconds/forced
vital capacity.















































7th treatment, Ps = after the 12th treatment, MEF% = forced expiratory flow rate after x% of
forced vital capacity has been expired (liters/second), FEVx/FVC = ratio of forced expiratory
volume at x seconds/forced vital capacity.


Table 4-26. P-values from pairwise comparisons of the FEVx, FVC, and PEF obtained by the
rapid partial forced expiration maneuver.


FEV FEV FEV FEV FEV
0.5 0.75 0.1 1.5 2.0
0.53 0.05 0.03 0.51 1.00
0.13 <0.01 <0.01 <0.01 0.14
1.00 0.35 0.05 <0.01 0.39
0.78 0.32 0.03 0.12 0.46
0.02 <0.01 <0.01 <0.01 0.02
0.55 0.04 <0.01 0.02 0.07
= electroacupuncture, Sham = sham EA, Pr


FEV
2.5
1.00
0.14
0.39
0.38
0.02
0.07


FEV
3.0
1.00
0.13
0.39
0.33
0.02
0.07


FVC PEF


Tx Pairwise
EA Pr-Po
Pr-Ps
Po-Ps
Sham Pr-Po
Pr-Ps
Po-Ps
Tx treatment, EA
7th treatment, Ps = a
FVC = forced vital (


1.00
0.13
0.39
0.33
0.02
0.07


0.19
<0.01
<0.01
<0.01
<0.01
<0.01


after the


pre-treatment, Po


afterr thel2th treatment, FEVx = forced expiratory volume at x seconds (liters),
capacity (liters), PEF = peak expiratory flow (liters/second).


Table 4-27. P-values from pairwise comparisons of the MEFx% and FEVx/FVC ratio obtained


by the rapid partial forced expiration maneuver
MEF MEF MEF FEV0.5/
rwi se 25% 50% 75% FVC
Po 0.27 0.06 0.57 1.00
Ps <0.01 <0.01 0.02 0.98
Ps <0.01 0.19 0.47 1.00
Po <0.01 0.08 0.68 1.00


0.15
1.00
sham E


FEV0.75
/FVC
0.91
0.46
1.00
1.00
0.14
1.00
A. Pr = prI


FEV1.0/
FVC
0.86
0.39
1.00
1.00
0.96
1.00


FEV1.~
FVC
1.00
0.73
1.00
1.00
1.00
0.98


5/ FEV2.0/
FVC
1.00
1.00
1.00
0.12
0.19
1.00
after the


Ps
Ps
nent, EA


Tx Pai
EA Pr-
Pr-
Po-
Sham Pr-
Pr-
Po-
Tx treaty


1.00 <0.01 1.00
1.00 0.21 1.00
=electroacupuncture, Sham


,


I


-e-treatment, Po










Table 4-28. P-values from the Type III sum of squares test on main effects and their interactions
for serum cortisol concentration.
Factor (s) P-value
Treatments 0.93
Sampling time 0.17
Trial 0.29
Sequence 0.09
Treatments 0.53
sampling time
Sequence 0.00
horse
* = interaction between factors.


Table 4-29. Mean+SE


Tx Sampling time
EA Pr

Po

Ps

Sham Pr

Po


concentrations of serum cortisol, by treatment and sampling time.
Concentration of serum
cortisol (Clg/dl)
6.271
~0.687
5.408
~0.687
6.847
~0.687
6.992
~0.639
5.564
~0.639


Ps 6.111
~0.639
Tx treatment, EA electroacupuncture, Sham sham EA.Pr pre-treatment, Po = after the 7th
treatment, Ps = after the 12th treatment, P-values of pariwise comparisons; Pr-Po, Pr-Ps, and Po-
Ps in the EA group were 0.71, 1.01, and 0.51i, respectively. P-values of pairwise comparisons; Pr-
Po, Pr-Ps, and Po-Ps in the sham group were 0.50, 1.00, and 0.79, respectively.










Table 4-30. Details of cutaneous and muscle innervations at acupoints used in this study.
Acupoint locations are described in Table 4-1.


Acupoint Cutaneous innervations (N = nerve)
GV-14 Dorsal branch of 7h cervical N. and
1 st thoraci c N.
CV-22 Ventral cutaneous branch of 6th
cervical spinal N and cranial branch
of supraclavicular N.
BL-13 Dorsal branch of thoracic N.









Fei-men Ventral cutaneous branch of C 6th
cervical spinal N.




Fei-pan Lateral cutaneous branch of 2nd and 3rd
thoracic N (component of
intercostobrachial N) and lateral
cutaneous branch of 4th thoracic N.
Fei-shu Intercostal N.


Muscles/innervations (N = nerve)


Cutaneous coli/Ramus coli of facial N.


Cutaneous trunci/Lateral thoracic and
intercostobrachial N.
Trapezius thoracic/Dorsal branch of
accessory N.
Rhomb oi deu s thoraci s/Medi oventral
branch of local thoracic N.
Latissimus dorsi/Thoracodorsal N.
Longismus thoracis/Dorsal branch of
local spinal N.
Trapezius cervicis/Dorsal branch of
accessory N.
Subclavius/Cranial pectoral N.
Longissimus cervicis/Dorsal branch of
local spinal N.
Cutaneous trunci/Lateral thoracic and
intercostobrachial N.
Triceps (long head)/Radial N.

Cutaneous trunci/Lateral thoracic and
intercostobrachial N.
Intercostal muscle/Intercostal N. (from
ventral branch of Thoracic N.).
Trapezius cervicis/Dorsal branch of
accessory N.


Ding-
chuan


Dorsal branch of local cervical and
thoracic spinal N.


Rhomb oi deu s cervi ci s/Medi oventral
branch of cervical N.
Splenius/Dorsal branch of local spinal N.
and dorsal branch of accessory nerve.
(Sources; Hackett MS, Sack WO, Simmons MA, et al. Forelimb In: Hackett MS,Sack WO, eds.
Rooney's guide to the dissection of the horse. 7th ed. 2001;page 1 12-152., Budras K-D, Sack
WO, Roick S, et al. Selected body systems in tabular form In: Budras K-D, Sack WO, Roick S, et
al., eds. Anatomy of the horse : an illustrated text. 3rd ed. 2001;page 8 1-102., Fleming P.
Transpositional equine acupuncture atlas In: Schoen AM, ed. Veterinary acupuncture : ancient
art to modern medicine. 2n ed. 2001; page 393-431., and Blythe LL, Kitchell RL.
Electrophysiologic studies of the thoracic limb of the horse. Am J Vet Res 1 982;43;pagel5 11 -
1524.169,239-241)









CHAPTER 5
SUMMARY AND CONCLUSIONS

Introduction

Cardiopulmonary fitness is an important factor determining athletic performance of

horses. Diseases and abnormalities that alter the normal airflow and gas exchange of the lung

may originate from the upper or lower airway s.231 Di orders of the upper airway that increase

resistance and limit the normal airflow include recurrent laryngeal hemiplegia, pharyngeal

lymphoid hyperplasia, dorsal displacement of the soft palate, nasopharyngeal collapse, sub-

epiglottic cyst, and entrapment of the epiglottis.

Abnormalities of the lower airways affect horses at all ages, of all breeds, and in

physiological stages. Severity of the diseases depends on several factors including the age of the

animal, immunological status, and the cause of the disease. Important diseases that cause non-

septic inflammation of the lower airways include inflammatory airway disease (IAD), heaves or

recurrent airway obstruction (RAO) and summer pasture associated obstructive pulmonary

disease (SPAOPD).17 Horses affected by theses diseases show clinical signs of dry cough,

serious nasal discharge, increased expiratory effort, flaring of nostrils, and exercise intolerance.222

Horses that are being kept for competition, sports and recreational purposes are unable to

participate in their routine training program when affected by these conditions and a decrease in

performance capability is seen. Opportunities to develop other pulmonary disease-associated

complications such as pleuropneumonia, pleuritis, emphysema and fibrosis are increased if

appropriate treatment is delayed and may lead to an incomplete recovery. Irreversible changes in

the histological structure and severe compromise of the normal respiratory physiological

function can lead to the termination of an animal' s athletic career or, in severe cases, euthanasia.









Until recently, there has been no single diagnostic procedure that can be used to

accurately distinguish these diseases and, more importantly, to diagnose these diseases at the

earliest stage. Current diagnosis relies on case history, clinical signs, BALf cytology, and

response to treatments. Ability to recognize these diseases at an early stage is important.

However, it is difficult due to a lack of specific clinical manifestations. Several diagnostic

methods have been developed to identify early disease stages in affected horses, including direct

measurement of intra-pleural pressure, histamine broncho-provocation, and forced expiration.172-



Histamine Bronchoprovocation as a Test for Equine Airways Hyper-sensitivity

Histamine bronchoprovocation (HB), also known as histamine challenge, has been used

to determine the degree of airway hyper-sensitivity. The hyper-sensitive airway is though to be a

sequel to chronic inflammation of the lung and to be ref lective of the severity of airway disease.

Using HB result alone as a diagnostic tool for respiratory disease is questionable.

Bronchoconstriction in lower airway inflammatory diseases in horses develops over a few hours

after exposure to an irritant, and the improvement of clinical signs is delayed following the

causes are removed." The bronchoconstriction effect of HB is of much shorter duration.

Horses without clinical signs of respiratory problems responded to HB inconsistently as

was demonstrated in Chapter 3. Horses with a high percentage of BALf neutrophils were more

likely to have hyper-sensitive airways. However, the correlation between percentage of BALf

neutrophils and airway hyper-sensitivity was not significant. Variation in HB test results for

individuals, when the test was repeated, agreed with the finding of previous research which

suggested a high level of inconsistency in individual response to HB.184,185 Variation in

individual responses to HB also has been demonstrated in clinically normal foals.ls

Concentrations of hi stamine causing a decline in lung dynamic compliance by 3 5% in these foal s









ranged from 1 mg/ml to more than 8 mg/ml. Results of previous research also has suggested that

the HB response in normal horses and horses with low-grade lung disease (with no clinically

evident signs of respiratory tract disease) were not significantly different. However, horses with

severe pulmonary disease required a lower histamine concentration to cause a 35% decline in the

lung dynamic compliance.184

The cause of variability in individual response to HB is unknown. However, it may be

caused by the non-specific response of the upper airway to histamine, especially when a

facemask was used to conduct the HB test. Histamine not only induces the contraction of the

smooth muscles of the airways, but also causes vasodilatation of blood vessels in the airways.

Vasodilatation leads to edema of the respiratory mucosa and increased mucus production in the

upper and lower airways.211 These effects may contribute to an increase in a resistance of the

upper airways that potentially increases total resistance of the respiratory tracts. A

pharmacological effect of histamine on mucus production was observed in the experiment of

Chapter 3; that is, the vast maj ority of the horses had increased nasal secretion during and after

exposure to histamine.

Using a facemask for delivering histamine is another factor that may affect HB test

results. The nebulization rate of hi stamine solution depends on the pressure and type of nebulizer

being used in the test. With the facemask, the exact amount of histamine reaching the lower

airway is unknown. The quantity of histamine reaching the lower airways likely depends on

respiratory frequency and depth of breathing.

Results in Chapter 3 suggested that the HB test can be used to determine airway hyper-

sensitivity and may be used to support the diagnosis of equine lower airway inflammatory

diseases. However, interpretation of HB test results obtained from clinically normal horses










requires great care. Results from the HB test should be used in conjunction with other diagnostic

information (BALf cytology, clinical signs, history of illness, and response to therapy) for a

more reliable diagnosis.

Rapid Partial Forced Expiration for Testing Equine Pulmonary Function

Rapid partial forced expiration (RP-FE) is another pulmonary function test modified from

the forced expiration test in human medicine. The technique is novel in veterinary medicine and

not widely practiced. The test requires additional maneuvers and is more invasive than that in

humans. A design for a system that is capable of intervening inspiration and expiration is

necessary since coaching of breathing in horses to obtain maximal breth excursions is not

possible. To perform RP-FE test in horses, an airtightsealed airway of the horse was first inflated

with atmospheric air at a controlled pressure to total lung capacity (TLC). Then the airway was

exposed to a negative pressure reservoir. The difference in pressure between the airway and

negative pressure reservoir moved air out of the lung. Negative pressure causes emptying of the

air from the lung beyond the effect of elastic recoil properties of the pulmonary tissues and the

chest wall. The functional reserve volume of the lung is also emptied, mimicking forced

expiration.

Pulmonary function test parameters (PFTPs) from RP-FE test results in Chapters 3 and 4

differed from values reported by a previous study.m' The differences in PFTPs may be caused by

differences in manifold designs, in the negative pressures used to empty airways, or in the

methods of measuring airflow.

Regardless of the total length of the manifold used in the two studies, the maj or factor

that contributed to airflow resistance was the diameter of the manifold. The internal diameter

(ID) was determined by the selection of an endotracheal tube (2.6 cm, study in Chapter 3 and 4)

versus a nasotracheal tube (2.2 cm, previous study). Relationship of the radius to the volumetric









flow rate can be demonstrated by Poiseuille' s law,193 which states that the amount of the

volumetric flow rate is positively related to the fourth power of the radius (Equation 3 -6).

Results in Chapter 3 demonstrated that PEF and FVC values were positively correlated

with the amount of negative pressure used to generate RP-FE. Negative pressure at 200 Torr was

used for RP-FE in Chapter 4 because the studies cited in Chapter 3 suggested that it eventually

emptied the air from airways without causing observable damage to the tracheal mucosa. The

PEF and FVC values derived from 200 Torr used for inducing RP-FE in Chapters 3 and 4 were

greater than the previously reported values, when FE was induced by a vacuum at 161.8 Torr (-

220 cm H20).17

Forced expiratory airflow during the RP-FE maneuver in Chapters 3 and 4 was measured

directly via the pressure differential generated by the maneuver. This method of measurement

differed from that in previous research in which the airflow rate and expiratory volume were

indirectly calculated from an immediate change in negative pressure in a vacuum reservoir. The

AP generated by airflow in RP-FE was measured by the MFE-calibrated laminar flow element.

The MFE used in the calibration was regularly tested for its accuracy and was NIST (National

institute of standards and technology) traceable.

From Poiseuille' s law, in the laminar flow condition the volumetric flow rate is linearly

related to the drop in AP measured across the flow tube.193 The Equation 3-6 can be re-written as

Equation 3-3. When factors determined by the geometry of the flow restriction are reduced to the

K constant, Equation 3-3 can be rewritten as Equation 3-4. This equation shows a linear

relationship between volumetric flow rate (Q), differential pressure (AP), and fluid viscosity (r)

in a simple form. This simple linear relationship between Q and AP was confirmed by pre- and

post-RP-FE calibrations in Chapters 3 and 4.









In Chapter 4, the accuracy of mass flow element (MFE) calibration was tested by

comparing integrated airflow signals generated by pulling a known volume of air through the

pipe connected downstream of the calibrated laminar flow element (LFE). A 12.829-liter syringe

was used to test the integration of airflow signals. The results indicated that the volume from

integration was greater than the actual volume of the syringe being inj ected by 6.3 1%. This

means that the volume integration result of 13.639 liters was, in fact, 12.829 liters. Therefore, the

calculated correction factor for MFE/LFE was 1.063 105 (13.63 9/12.829). This correction factor

was used in the calculation of calibrated volumes in Chapter 4. These results suggested that a

carefully calibrated LFE is suitable for measuring airflow during RP-FE. Measurement of forced

expiratory airflow by LFE in the RP-FE relied on the same principle as that of a

pneumotachograph.

Commercially available pneumotachographs are superior to an ordinary LFE in that they

are electrically heated. This prevents condensation of moisture from the expired air inside the

pneumotachograph. The condensation might have occurred in the LFE of the RP-FE apparatus

during the experiments. Laminar flow element used in Chapters 3 and 4 was 4 inches in internal

diameter and was nearly four times greater than that of the endotracheal tube (1.024 inches),

which was a bottleneck of airflow during the RP-FE maneuver. It can be assumed that minor

condensation in the LFE would not significantly alter the resistance of the apparatus.

The increase in the percentage of neutrophils in the bronchoalveolar lavage fluid (BALf)

after RP-FE testing suggested that RP-FE induced airway inflammation. Percentages of

neutrophils were significantly higher in BALf samples collected after the 10th treatment, which

was 8-10 days after the second RP-FE maneuver. Also, percentages of BALf neutrophils in pre-

treatment samples from the second trial were greater than those in the first trial. These results










together suggested that RP-FE induced pulmonary inflammation, which was carried over to the

second trial. The magnitude of RP-FE induced airway inflammation observed might be due

partly to an accumulation of inflammatory reaction when the horses were scheduled for re-

testing too soon. A period of 8-10 days was first thought to be enough time for the pulmonary

tissues to recover from RP-FE-induced inflammation. However, the increase in the percentages

of neutrophils in BALf suggested that the pulmonary tissue had not completely recovered, and

that inflammation in the airways and pulmonary tissues caused by RP-FE lasted longer than the

period first hypothesized. There were no significantly differences in PFTPs between pre- and

post-RP-FE observed, even in the face of RP-FE-induced inflammation in the pulmonary tissues.

Previous research demonstrated that when airways of rats were ventilated with an end

expiratory pressure of 0 cm H20 and with a peak inspiratory pressure of 45 cm H20 for 20

minutes, lung tissues were damaged as indicated by edema of the pulmonary tissues and an

increase in percentages of neutrophils in BALf.247 COncentrations of inflammatory cytokines in

BALf, including macrophage inflammatory protein-2, IL-1P, heat shock protein-70, and matrix

metalloproteinase were also increased.

Negative pressure-induced pulmonary injury has been documented in humans subjected

to general anesthesia.248,249 It is usually caused by attempted ventilations against an acute upper

airway obstruction in the perioperative period. Negative intrathoracic pressure develops during

such respiratory efforts.250 This results in accumulation of fluid in the pulmonary tissue.251 The

mechanism and extent of pulmonary tissue injury induced by RP-FE in horses have never been

tested.

Artificial inspiration and artificial forced expiration during the RP-FE maneuver caused a

rapid swing in intrapleural pressure. A continuous abrupt change in intrapleural pressure has









been observed in horses during strenuous exercise, and has been thought to be one of the factors

contributing to exercise-induced pulmonary hemorrhage.252 In the future, the RP-FE apparatus

may be used in research in thi s field.

Partial collapse of the small airways and alveoli that might be caused by artificial forced

expiration is potentially a problem of the RP-FE maneuver, as demonstrated by the fact that

forced vital capacity (FVC), peak expiratory flow (PEF), and flow-volume loop of the first

artificial forced expiration were always greater than those of the subsequent artificial forced

expirations (Figures 9 and 10 in Chapter 4). Artificial inspiration during RP-FE maneuvers in

Chapters 3 and 4 was adequate to overcome the physiological drive of normal respiration, but a

thorough ventilation of all alveoli could not be ensured, especially after the first artificial forced

expiration in each RP-FE test. In the future, temporarily disconnecting the RP-FE apparatus from

the endotracheal tube after each artificial forced expiration cycle prior to performing the

subsequent RP-FE cycles, may allow the horse to regain physiological control of respiration.

Physiologically, inspiration is initiated by expansion of the thoracic cavity and a decrease in

intrapleural pressure, unlike artificial inspiration. Artificial inspiration was initiated by positive

pressure generated from inflation of the airways. Allowing the horse to spontaneously breath

unassisted may improve ventilation of the collapsed small airways and alveoli.

Testing equine pulmonary function with RP-FE is an emerging technique for measuring

biomechanical properties of the lung. There is no single standard method for obtaining PFTPs,

and reference values for horses are lacking. RP-FE maneuver explained in Chapters 3 and 4, and

its PFTPs provided more information on the pulmonary function in clinically normal

thoroughbred horses. Additional PFTPs from RP-FE in horses of different breeds, sex, age, body









weight, and with known pulmonary diseases, will be highly beneficial for comparisons of these

parameters in horses in general.

Acupuncture and Electroacupuncture Efects on Equine Immune Response and Pulmonary
Function

Acupuncture and electro-acupuncture (AC/EA) combined with Chinese herbs have been

used as adjunctive therapies for treating equine chronic respiratory disorders.78,232 They are

intended to decrease the dosage requirements of bronchodilators and anti-inflammatory agents

and improve the quality of life of horses suffering from chronic respiratory diseases.233

Therapeutic results obtained from humans and laboratory animals suggested that AC/EA possess

therapeutic benefits for respiratory problems.97,253-255

Acupuncture and EA applications in the respiratory system require stimulation of

multiple acupoints. The points selected in the treatment strategy are intended to replenish the

normal lung function, to reduce cough, to alleviate clinical signs of heaves and dyspnea, and to

improve immune function.'" Selected acupoints can be stimulated with an acupuncture needle,

with low voltage electricity, or with an inj section of a mild irritant sterile solution such as normal

saline or a solution of water-soluble vitamins. Stimulation of acupoints with a laser also has been

used.234 Frequently used acupoints and their functions (in traditional Chinese veterinary

medicine) are listed in Table 5-1.78,135,256

In respiratory disorders, the affected animals develop clinical signs of respiratory

discomfort due to reduced airway ventilation, impaired gas exchange capacity, increased

production and accumulation of airway secretions and increased inflammatory reaction in the

lower airway.86 Alleviation of one or more of these disease mechanisms may reduce the severity

of the clinical signs. Acupuncture treatment in humans suffering from asthma has been shown to

improve the pulmonary function and significantly improved the quality of life of patients.87,25









However, a more scientifical based explanation of how AC and EA benefit treatment of

respiratory diseases is still needed. By extrapolating from recent scientific research on AC/EA in

animal models of disease, it can be proposed that AC/EA benefit the respiratory system by

several mechanisms, including:

* Improved mucociliary action of the airway epithelium.
* Reduced airway and pulmonary tissue inflammation.
* Activation of cholinergic anti-inflammation.
* Alteration of immune responses.
* Modulation of autonomic nervous system.
* Alteration in the peripheral sensory input from inflamed pulmonary tissues.
* Other mechanisms (as yet to be fully defined).

Improved Mucociliary Action of the Airway Epithelium

An increase in mucus production is a consequence of airway inflammation. Normally,

mucus is immediately removed by epithelial mucociliary action. Disruption of the mucociliary

clearance occurs in response to chronic airway inflammation and is thought to be caused by

neutrophil-derived elastase.98 The increased production and accumulation of mucus decreases

airway diameter and increase total airway resistance. Until recently, no scientific evidence

directly demonstrated the effect of AC or EA on mammalian mucociliary clearance. However,

Tai et al.99 demonstrated that EA at acupoints LU-1 and CV-22 significantly increased the rate of

tracheal mucociliary transport in quail compared to a control group.99 Moreover, EA at these

acupoints significantly reversed the decrease in mucociliary transport caused by the

admini strati on of human neutrophil-derived elastase.

Several mediators have been shown to regulate the rate of mucociliary clearance. These

included endogenous nitric oxide (NO) and substance-P.257,258 However, whether these

substances are involved in AC/EA-mediated mucociliary clearance needs further investigation.









Reduced Airway and Pulmonary Tissue Inflammation

Analgesic and anti-inflammatory effects of AC and EA are well documented in both

somatic and visceral tissues.60,89 Mechanisms associated with these analgesic and anti-

inflammatory effects have been linked to a release of endogenous opioid substances in the

central nervous system (CNS) during AC/EA treatment.28 These endogenous substances activate

the opioid receptors in the CNS tissues, such as the substantial gelatinosa of the spinal cord and

the periaqueductal grey of the midbrain, and produce analgesia.9293 This endogenous opioid-

dependent anti-nociception has been shown to be modulated via the mu-opioid receptor.93

Immunocytes such as macrophages, monocytes, and polymorphonuclear cells possess

opioid receptors on their cell surfaces where mu-opioid receptors predominate.94 Once activated,

the receptor induces an anti-inflammatory response via the down regulation of the transcription

factor, nuclear factor kappa-B (NF-rB).95 The NF-KB down regulation has been demonstrated to

reduce the mRNA expression of other inflammatory cytokines including tumor necrosis factor

alpha (TNF-oc), I-1P, IL-6, nitric oxide synthase (iNOS), and metalloproteinase.96 Carneiro et

al.97 demonstrated that EA reduced the inflammatory cell infiltration in the peribronchial tissue

and in the pulmonary perivascular spaces in rats with ovalbumin-induced bronchial asthma.97

Moreover, the number of total nucleated cells and the percentages of neutrophilic and

eosinophilic leukocytes in bronchoalveolar fluid (BALf) were significantly decreased when

compared to control and sham EA groups. In this study, the EA was performed on acupoints GV-

14, BL-13, LU-1, CV-17, ST-36, SP-6, and Ding-chuan which mimicked the acupoints used to

treat human asthma.









Activation of Cholinergic Anti-inflammation

The vagus nerve is a maj or parasympathetic nerve that contains afferent and efferent

nerve fibers for both somatic and visceral tissues.79,80 Acethylcholine is a major neurotransmitter

in both preganglionic and postganglionic parasympathetic nerve synapses. The cholinergic

influence on immunological functions has been demonstrated in laboratory animals.8 Electrical

stimulation of the vagus nerve suppressed the in vivo release of TNF-oc and inhibited

lipopolysaccharide (LPS)-induced endotoxic shock. In this experiment, the authors also

demonstrated attenuation in release of IL-1P, IL-6, and IL-18, but not IL-10, by acethylcholine in

LPS-stimulated human macrophage culture. This cholinergic dependent anti-inflammatory

pathway suppresses the non-specific, innate immune response and may explain how AC/EA

works in treating other diseases affecting visceral organs.

The direct effect of AC/EA on the cholinergic-associated anti-inflammatory response in

visceral organs has never been demonstrated. However, the effect of EA on the cholinergic anti-

mnflammatory response of somatic tissue has been shown in rats with collagen-induced arthritis."

In this model, EA at ST-36 showed significant analgesic and anti-inflammatory properties. These

analgesic and anti-inflammatory activities of EA were suppressed when the muscarinic

cholinergic receptor antagonist was co-administered with the EA. This suggests that EA at ST-36

may activate a local cholinergic anti-inflammatory mechanism preventing an inflammatory

reaction and thus reducing pain. It is also possible that endogenous opioids and cholinergic

pathways work together to create the anti-inflammatory action associated with AC/EA.

Alteration of Immune Responses

When treating chronic respiratory diseases with AC/EA in clinical practice, acupoints

that benefit the immune system are routinely stimulated in addition to acupoints that have









specific indications for respiratory problems. Acupoints that are normally used for this purpose

include LI-4, LI-11, ST-36 and GV-14. 138,139,259,260

Tian et al.76 Studied the anti-inflammatory benefit ofEA stimulation at ST-36 in rats with

induced colitis.76 He reported a significant decrease in circulating TNF-a and a down regulation

of mRNA coding for TNF-a expression in inflamed colonic tissue. Electroacupuncture treatment

in Chapters 2 and 4 generally suppressed TNF-a production in whole blood cultures. The

suppression of TNF-a production in whole blood cultures when LPS alone was added was more

significant (Chapter 4). The difference in suppression was variable possibly due partly to a

different in EA treatment protocol. In Chapter 4, more acupoints were stimulated during the

treatment, and the assay for TNF-a production in whole blood cultures were performed after the

7th and 12th EA treatments (versus after the 3rd EA in Chapter 2). Electroacupuncture in Chapters

2 and 4 involved GV-14 stimulation. This may suggest that GV-14 plays an important role in

modulating immune response. Further investigation on thi s acupoint will be beneficial.

Electroacupuncture at ST-25 and CV-6 significantly reduced I-1P, IL-6, and TNF-a

secretion by monocytes during experimentally induced colitis in rats and reversed the decrease in

apoptosi s rate of peripherally circulating neutrophils.75 Prolonged apoptosi s of neutrophils i s

thought to b e a re sult of the presence of pro-inflammatory-tri ggeri ng sub stances. All eviati on i n

the suppression of neutrophil apoptosis may be due to the reduction of inflammation of local

tissue and a decrease in the production of inflammatory cytokines.7

Electroacupuncture may possess an activity similar to that of direct electrical stimulation

of the vagus nerve, thereby possibly also stimulating the cholinergic anti-inflammatory pathway,

inhibiting macrophage activation, and decreasing the production of TNF-u, I-1P, IL-6, and IL-









18.261 A down regulation of TNF-oc released by AC/EA also has been demonstrated in other

experimental models.76,262,263

Modulation of Autonomic Nervous Systems

Equine airway smooth muscle and lymphoid tissue are innervated by both sympathetic

and parasympathetic nervous systems.264 Alterations of the activity in both systems have been

demonstrated following AC treatment.265 It has been proposed that the spinal cord and the 10th

cranial nerve are essential to relay AC/EA sensory signals to higher relay centers such as the

brain stem and the hypothalamus.266 All the afferent signals converge at these relay centers prior

to sending a signal to the somatosensory cortex.

Bradycardia following AC/EA is commonly seen in both clinical practice and laboratory

experiments demonstrating the parasympathomimetic and sympatholytic properties of

AC/EA.267,268 Electroacupuncture at ST-36 decreased the excitability of the cardiovascular

system manifested by bradycardia, which may be associated with sympathetic inhibition and

modification of the central baroreflex arch.269 Modulation of the sympathetic response also has

been reported in a study of AC performed at PC-6.270 Chan et al.271 TepOrted that AC can be used

to treat post-traumatic sympathetic dystrophy in humans with a 70% improvement in clinical

signs.271 In another study, EA at LI-11 and LI-4 produced moderate hypoalgesia in humans

concurrent with by a significant increase in muscle sympathetic nerve activity.272

Several studies indicate that AC/EA possess an immediate mild to moderate

bronchodilator effect. A clinical study of human asthma found that AC at LU-7, LI-4, PC-6, ST-

40, LI-11, and PC-3 for 15 minutes induced bronchodilation manifested by an increase in forced

expiratory volume in the first second (FEV1).8 Improvement in pulmonary function parameters

(pleural pressure, tidal volume, minute ventilation, peak inspiratory flow and peak expiratory









flow) in RAO-affected horses also has been demonstrated after a single AC treatment.88 In this

previous study, the authors concluded that the improvements were due to animal handling.

Electroacupuncture treatment in Chapter 4 did not cause a change in PFTPs specific to the EA

treatment. Results suggested that in clinically normal horses, EA has no effect on PFTPs. Horses

airways are in the normal state likely maximally dilated. Any bronchodilatory effect is probably

mechanically impossible in normal horses.

Alteration in the Peripheral Sensory Input From Inflamed Pulmonary Tissues

Although the non-myelinated nerve fibers (C fibers) have never been described in the

equine airway, they can be identified in several animal species.273,274 Therefore, it can be

assumed that this type of nerve fiber is also present in equine species. It is a vagally mediated

non-myelinated nerve fiber and functions as a polymodal receptor, activated by tissue damage,

edema, or inflammatory mediators." Irritations caused by inhaled particles or mediators

released by airway resident inflammatory cells activate this nerve fiber. Activation also leads to

airway hyper-responsiveness, a classical clinical manifestation of lower airway diseases.

Attenuation of nociceptive signal conduction by C fibers has been hypothesized as the

main analgesic mechanism of AC/EA.29 This is also known as the Gate Control Theory,

proposed by Melzack and Wall. In this theory, sensory input from AC conducted by the AP

myelinated nerve fiber reaches the spinal cord at a faster speed than does the nociceptive signal

that travels through the non-myelinated nerve fiber. When the sensory signal reaches the

substantial gelatinosa of the spinal cord, it stimulates the inhibitory interneuron and prevents

subsequent conduction of the slow nociceptive signal transmitted by the non-myelinated C fiber

and AS fiber. Based on this theory, ascending non-nociceptive signals originating from AC/EA









may modulate the nociceptive input from C fibers from the airway at the level of the spinal cord

and alleviate airway hyper-responsiveness.

Other Mechanisms

Positive therapeutic effects following sham AC/EA in human subj ects suggest a

psychogenic influence and a placebo effect.276 Whether a similar placebo effect is present in

other animals is questionable. Animals do not expect a positive effect treatment unless the

investigator predictably rewards the animal following the treatment. Subj ective parameters that

require human assessment of an animal, such as appetite and degree of pain, are unavoidably

affected by human bias. Also, it should be kept in mind that handling during the experiment

potentially plays a maj or role in producing stress, which is frequently demonstrated by elevation

of the endogenous corticosteroid concentrations. In Chapter 4, mean concentrations of serum

cortisol in the EA and the sham groups were not significantly changed throughout the study, and

the concentrations of serum cortisol of all samples were within normal reference values. These

results at least suggested that neither treatments (EA and sham) nor RP-FE maneuvers in thi s

study induced stress when serum cortisol concentration was used as a stress indicator.

Even though the thoracic pain associated with respiratory disease has never been studied

in horses, it is thought to be present in horses affected by chronic respiratory diseases.27

Immediate relief in respiratory discomfort following AC/EA could be a result of a non-specific

nociceptive inhibition by a diffuse noxious inhibitory control mechanism (DNIC). In DNIC,

noxious stimulation, including AC needle insertion, may evoke analgesia by a non-specific

attenuation of the afferent pain sensation.27 Alleviation of respiratory discomfort following

AC/EA at acupoints located around the thoracic region, including BL-13, Fei-men, Fei-pan, and

Ding-chuan2, may be the result of thi s mechani sm.










Summary

Histamine bronchoprovocation test can be used to reinforce diagnosis of equine lower

airway inflammatory diseases, but interpretation of HB test results obtained from clinically

normal horses should be done with great care. Information derived from the HB test should be

used together with results from other diagnostic information, including BALf cytology, clinical

signs, history of illness, and response to therapy for determining the severity of the equine lower

airway diseases. Forced expiration test in horses is an emerging technique of measuring

biomechanical properties of the lung. Standard methods for obtaining PFTPs and reference

values for horses are still lacking. Rapid partial forced expiration maneuver and its PFTPs

provided more information on the pulmonary function in horses. Rapid partial forced expiration

possesses great potential for diagnosis of lower airway diseases. Additional data from RP-FE in

horses of different breeds, sex, age, body weight, and in horses with known lung disease are

likely to be highly informative.

Electroacupuncture was more effective than AC in modulation of innate immune

response of the horses tested in these studies. This anti-inflammatory action was likely governed

by modulation of a cellular component of the innate immune system by altering the production

of inflammatory cytokine upstream of the inflammatory cascade. Effects of EA on pulmonary

biomechanics are still unclear and further study in horses with clinical signs of respiratory

disease is needed.











Table 5-1.


Acupoints commonly used to treat chronic respiratory diseases of horses.


Acupoint Location
BL-13 At caudal edge of scapular cartilage
(8th intercostal space), 3 cun from
dorsal midline.
BL-23 At 2nd Lumbar intercostal space (L2-
L3), 3 cun from dorsal midline.
CV-17 On ventral midline at level of 4th
intercostals space (caudal border
of elbow).
CV-22 On ventral midline in depression just
cranial to manubrium of sternum.
LU-9 On medial side of carpus at junction
of radius and first row of carpal
bone, at level of accessory carpal
bone.
Ding- 0.5 cun lateral to midline at level of
chuan2 GV14 (on dorsal midline between
C7-T 1).
Fei-pan On the caudal edge of the scapula,
1/3 from upper border.
Fei-men On cranial edge of scapula, 1/3 from
upper border.
LI-4 At level of upper one third of cannon
bone in a depression between 2nd
and 3rd metacarpal bone.
LI-11 In depression cranial to elbow on
lateral aspect, in transverse cubital
crease cranial to lateral epicondyle
of humerus.
ST-36 3 cun distal to ST-35 and 0.5 cum
lateral to the cranial aspect of the
tibial crest over the cranial tibial
mu scle.


Attributes and Indications
Lung Back Shu Association point to
strengthen lung.

Kidney Back Shu Association point to
strengthen kidney.
Cough, dyspnea.


Cough, dyspnea.

Chronic cough, asthma, and heaves.



Chronic lung problems, cough, asthma,
and dyspnea.

Lower airway problems, heaves and
cough.
Upper airway disease

Immune deficiency.


Immune deficiency



General weakness.




Cough, heaves, and immune modulation.


GV-14


Dorsal midline between dorsal
spinous processes C7-T1.


(Sources: Xie H, Yamagiwa K. Equine Classical Acupoints In: Xie H, Preast V, eds. Xie's
veterinary acupuncture. 1 st ed. 2007; pages 89-127, Xie H, Trevisanello L. Equine
Transpositional Acupoints In: Xie H,Preast V, eds. Xie's veterinary acupuncture. 2007; pages 27-
87, and Xie HS, Preast, V. Appendix D: Acupuncture Point Locations In: Xie HS, Preast, V., ed.
Traditional Chinese Veterinary Medicine. 2002; pages 559-581.78,135,256)









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BIOGRAPHICAL SKETCH

Weerapongse Tangj itj aroen received his degree in Doctor of Veterinary Medicine from

Kasetsart University, Bangkok, Thailand, in 1997 with first class honors. After practicing in

equine medicine for two and a half years, he returned to academia by j oining the Faculty of

Veterinary Medicine, Chiang Mai University, which is located in the north of Thailand. At

Chiang Mai University, he lectured in the fields of equine medicine and surgery as well as

supervised veterinary students during their clinical rotation and clinical clerkships. In 2004 he

came to the University of Florida to pursue graduate study. At the University of Florida, he

worked in the Equine Performance Laboratory under the academic supervi sion of Professor Dr.

Patrick T. Colahan. His dissertation focused on investigating the effects of acupuncture and

electroacupuncture on immune responses and pulmonary functions in horses. After completing

his Ph.D., he will return to Chiang Mai University.


220





PAGE 1

1 EFFECTS OF ACUPUNCTURE AND ELECTROACUPUNCTURE ON IMMUNE RESPONSES AND PULMONARY FUNCTIONS IN HORSES By TANGJITJAROEN WEERAPONGSE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULF ILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009

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2 2009 Tangjitjaroen Weerapongse

PAGE 3

3 To my Mum and my Dad To all who nurtured my academic interest and have made thi s mile stone possible

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4 ACKNOWLEDGMENTS Completing the experiments and writing this dissertation has been both academically challenging and rewarding. Without the inspiration, support, encouragement, and guidance of the following people, this dissertation would have never have been completed. I wish to express my deepest gratitude to the following. At the very first, I gratefully acknowledge the Royal Thai Government for financial support of my graduate study. Thank you to Dr. Chamnan Trinarong, Chair of t he Equine Clinic at the Faculty of Veterinary Medicine, Chiang Mai University, for giving me an extraordinary opportunity to pursue overseas graduate study. I wish to deeply thank Professor Dr. Patrick T. Colahan for accepting me as his graduate student. H is academic and professional commitment inspired and motivated me. Without his patient academic guidance and support, this dissertation could not have been completed. I sincerely thank Dr. Huisheng Xie and his family for inspiration, support, and steady en couragement. His excellence in the traditional Chinese veterinary medical profession and teaching skills are extraordinary. Through his kindheartedness, I shared a small part of his wisdom, and I am grateful. I also sincerely thank Dr. David J. Hurley for providing a foundation and guidance in immunological assay. The importance of his mentoring in the immunological aspect of my study made his importance to the completion of my dissertation second to no one. I thank Dr. Richard D. Johnson for motivating me to achieve high academic standards, and to have given me the opportunity to learn equine anatomy in his class. A thorough knowledge of anatomy has served as a foundation for every aspect of my studies. I thank Dr. Daiqing Liao for accepting the invitation to join my committee in the last period of my program. Even though I did not have many opportunities to interact with him, I

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5 greatly appreciate his taking on a responsibility that added to his already full time academic work. I thank Professor Dr. James H. Jones, Chair of the Department of Surgical and Radiological Science at the School of Veterinary Medicine, UC Davis, for his guidance and excellent technical support in helping me build the forced expiration device. It is a marvel for me, who has no engi neering background, to have built this machine. His guidance and input in the engineering aspect of the forced expiration device made his contribution to the completion of my dissertation second to no one. I thank Dr. Steve Giguere for his guidance in the field of equine lower airway inflammatory disease, and allowing me to used the inductance plethysmography and pneumotachograpgy machine and other facilities of the Internal Medicine Clinic in my research. I also sincerely thank Brett Rice, Stacie Atria, Je nnifer A. Claflin, Ted Broome, and other members of the staff of the Equine Performance Laboratory at the University of Florida. The assistance I have received from them was invaluable. They were essential to every research project I accomplished. I gratef ully thank my family; Dad and Mum for nourishing my life and for encouraging me to achieve this academic work, and my sisters and brother who cared for my Dad and Mum while I am studied overseas. Last but not least, I sincerely thank everyone who contribut ed to my research but whom I have forgotten to mention in this acknowledgement.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................................... 4 LIST OF TABLES .............................................................................................................................. 10 LIST OF FIGURES ............................................................................................................................ 14 LIST OF ABBREVIATIONS ............................................................................................................ 16 CHAPTER 1 HISTORY OF TRADITIONAL CHINE SE VETERINARY MEDICINE AND A REVIEW OF MODERN SCIENTIFIC RESEARCH ON EQUINE ACUPUNCTURE ....... 23 Introduction ................................................................................................................................. 23 History of Traditiona l Chinese Medicine and Traditional Chinese Veterinary Medicine ........................................................................................................................... 23 Origin of Traditional Chinese Veterinary Medicine and Equine Acupuncture Research ............................................................................................................................ 26 Equine Acupuncture Research ................................................................................................... 29 Research in the Field of Analgesia and Musculoskeletal Pain ......................................... 29 Acupuncture for Managing Ocular Problems .................................................................... 34 Acupuncture Research in Gastrointestinal Disorders ........................................................ 36 Acupuncture Research in Respiratory Disor ders ............................................................... 39 Acupuncture Research in Other Medical Problems .......................................................... 41 AC Research in Reproductive System ............................................................................... 42 Research in Diagnostic Potential of Acupoints and Meridians ........................................ 46 Summary ...................................................................................................................................... 48 2 MODULATION OF IMMUNOLOGICAL RESPONSE BY ACUPUNCTURE AND ELECTROACUPUNCTURE AT LI 4, LI 11, AND GV14 IN CLINICALLY NORMAL HORSES. .................................................................................................................. 51 Introduction ................................................................................................................................. 51 Methods ....................................................................................................................................... 52 Animal .................................................................................................................................. 52 Acupuncture and Electroacupuncture ................................................................................. 53 Sour ce of Fungal Antigens .................................................................................................. 53 Leukocyte Separation .......................................................................................................... 54 Reactive Oxygen Species Generation of Neutrophil ......................................................... 55 Heparinized -blood Stimulation ........................................................................................... 56 Equine Tumor Necrosis Factor Alpha (TNF ) Assay ..................................................... 57 Immunoglobulin Assay ....................................................................................................... 58 Statistical Analysis ............................................................................................................... 59

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7 Results .......................................................................................................................................... 59 Animals ................................................................................................................................ 59 Acupuncture and Electroacupuncture Procedures ............................................................. 59 Homogeneity of Samples Before Electroacupuncture and Acupuncture ......................... 60 Plasma Immunoglobulins .................................................................................................... 60 Reactive Oxygen Species Generation of Neutrophil ......................................................... 61 Heparinized -blood Stimulation and Equine Tumor Necrosis Factor Alpha (TNF ) Assay ................................................................................................................................. 62 Discussion .................................................................................................................................... 63 3 COMPARISON OF INDUCTANC E PLETHYSMOGRAPHY AND PNEUMOTACHOGRAPHY AND THE RAPID PARTIAL FORCED EXPIRATION MANEUVER FOR DIAGNOSIS OF EQUINE LOWER AIRWAY INFLAMMATORY DISEASE. .................................................................................................................................... 77 Introduction ................................................................................................................................. 77 Methods ....................................................................................................................................... 80 Subject .................................................................................................................................. 80 Study Design ........................................................................................................................ 81 Histamine Bronchoprovocation and Respiratory Inductance Plethysmography and Pneomotachography (Open PlethTM) .............................................................................. 81 Bronchoalveolar Lavage. .................................................................................................... 82 Rapid Partial Forced Expiration Maneuver ........................................................................ 84 Negative pressure generator and vacuum reservoir ................................................... 85 System f or artificial inspiration ................................................................................... 86 Airflow measurement apparatus .................................................................................. 87 Airflow direction control system ................................................................................. 89 Data Acquisition system .............................................................................................. 90 Animal Preparation for rapid partial forced expiration.maneuver. ................................... 90 Induction of Rapid Partial Forced Expiration .................................................................... 91 Calculation of the Pulmonary Function Test Parameters .................................................. 92 Reset the calibrated p ressure differential of LFE with pressure/temperature corrected flow rate .................................................................................................... 92 Calculation of expiratory volume from airflow data ................................................. 93 Determination of pulmonary function test parameters .............................................. 93 Determination of suitable negative pressure for the rapid partial forced expiration maneuver ................................................................................................. 93 Statistical Analysis ............................................................................................................... 93 Results .......................................................................................................................................... 94 Subject .................................................................................................................................. 94 Histamine Bronchoprovocation and Respiratory Inductance Plethysmography and Pneumotachography (Open PlethTM) .............................................................................. 95 Broncho alveolar Lavage .................................................................................................... 96 Rapid Partial Forced Expiration (RP -FE) Maneuver ........................................................ 97 Discussion .................................................................................................................................... 99

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8 4 EFFECTS OF ELECTROACUPUNCTURE ON PULMONAR Y FUNCTION AND IMMUNE RESPONSE IN HORSES ...................................................................................... 121 Introduction and Background ................................................................................................... 121 Materials and Methods .............................................................................................................. 127 Animals .............................................................................................................................. 127 Electroacupuncture ............................................................................................................ 128 Data and Sample Collection .............................................................................................. 129 Rapid Partial Forced Expiration Maneuver ...................................................................... 130 Arterial Blood Gas Analysis During RP -FE .................................................................... 133 Calculation of the Pulmonary Function Test Parameters ................................................ 1 33 BALf Collection and Preparation ..................................................................................... 134 Tumor Necrosis Factor Alpha (TN F ) Production of Whole Blood and TNF Assay ............................................................................................................................... 134 Immunoglobulin Assay ..................................................................................................... 135 Cortisol Assay .................................................................................................................... 135 Statistical Analysis ............................................................................................................. 136 Results ........................................................................................................................................ 137 Subject ................................................................................................................................ 137 EA and Sham Treatments .................................................................................................. 137 Complete Blood Count and Other Hematological Parameters ....................................... 138 Broncho alveolar Lavage and BALf Cytology ................................................................ 139 Immunoglobulins ............................................................................................................... 140 TNF Production of Whole Blood Stimulation ............................................................. 141 Rapid Partial Forced Expiration Maneuver ...................................................................... 142 Arterial Blood Gas Analysis During RP -FE .................................................................... 144 Serum Cortiso l ................................................................................................................... 144 Discussion .................................................................................................................................. 145 5 SUMMARY AND CONCLUSIONS ...................................................................................... 182 Introduction ............................................................................................................................... 182 Histamine Bronchoprovocation as a Test for Equine Airways Hyper -sensitivity ................ 183 Rapid Partial Forced Expiration for Testing Equine Pulmonary Function............................ 185 Acupuncture and Electroacupuncture Efects on Equine Immune Response and Pulmonary Function .............................................................................................................. 190 Imp roved Mucociliary Action of the Airway Epithelium ............................................... 191 Reduced Airway and Pulmonary Tissue Inflammation .................................................. 192 Activation of Choline rgic Anti inflammation ................................................................. 193 Alteration of Immune Responses ...................................................................................... 193 Modulation of Autonomic Nervous Systems ................................................................... 195 Alteration in the Peripheral Sensory Input From Inflamed Pulmonary Tissues ............ 196 Other Mechanisms ............................................................................................................. 197 Summary .................................................................................................................................... 198

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9 LIST OF REFERENCES ................................................................................................................. 200 BIOGRAPHICAL SKETCH ........................................................................................................... 220

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10 LIST OF TABLES Table page 1 1 Treatment efficacy of AC and its related technique in equine thoracolumbar pain. ......... 50 1 2 Treatment efficacy of AC and its related techniques in equine lameness originating from joint problems. ............................................................................................................... 50 2 1 Anatomical location of acupoints and treatment indications. ............................................. 70 2 2 Demographic information on horses in the EA and AC groups. ........................................ 70 2 3 MeanSE concentration of immunoglobulin isotypes (x105 ng/ml) from pre treatment samples of EA and AC groups. ............................................................................ 70 2 4 MeanSE neutrophil -ROS response ratio from pre treatment samples of EA and AC groups. ..................................................................................................................................... 71 2 5 MeanSE TNF co ncentrations (pg/ml) from pre -treatment samples of EA and AC groups. ..................................................................................................................................... 72 2 6 Mann Whitney Rank Sum test statistics between -group comparisons of pre -treatment samples in EA and AC groups. ............................................................................................. 73 2 7 Mean SE immunoglobulin isotype concentrations (x105 ng/ml) of pre and post EA and AC treatments, and test statistics of within -group comparisons. ................................. 73 2 8 Mean SD neutrophil ROS response ratios of pre and post EA and AC treatments, and test statistics of within -group comparisons. .................................................................. 74 2 9 Mean SD TNF concentration ( in pg/ml) of pre and post EA and AC treatments, and test statistics of within -group comparisons. .................................................................. 75 2 10 Mann Whitney Rank Sum test statistics for between -group comparisons of post treatment data in EA and AC groups. ................................................................................... 76 2 11 Cutaneous and muscle innervations of acupoints being stimulated. ................................... 76 3 1 Interpretation of his tamine concentrations that cause 35% increase in delta flow (modified from operation manual of Open PlethTM system). ............................................ 114 3 2 Histamine concentration causing PC 35 delta flow and their interpre tations from the 1st and the 2nd HB tests. ........................................................................................................... 114 3 3 Descriptive statistics of the degree of airway hyper -sensitivity based on results from the 1st and 2nd tests of histamine bronchoprovocation. ...................................................... 115

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11 3 4 P values from comparisons of the HB results between the 1st and 2nd tests and their correlations. .......................................................................................................................... 115 3 5 MeanSD of BA Lf cytology results of the 1st and 2nd HB test and p value of Wilcoxon Signed Rank test and Correlations between the tests. ...................................... 115 3 6 MeanSD and Mann -Whitney U statistics of BALf cytology from horses with hyper sensitive and normal airways. .............................................................................................. 116 3 7 MeanSD of PFTPs derived from RP FE at negative pressures of 25, 50, 75, 100, 125, 150, 200, and 250 Torr. ............................................................................................... 117 3 8 Mean SD of PFTPs derived from the 1st and the 2nd RP FE, when 150, 200, and 250 Torr were used to induced RP -FE. ...................................................................................... 118 3 9 Wilcoxon Signed Rank test s tatistics (p -value) of pulmonary function test parameters (PFTPs) from the 1st and the 2nd RP -FE maneuver at 150, 200, and 250 Torr. ............... 119 3 10 MeanSD of percentage of BALf neutrophils of horse s with low % Neu and horses with high % Neu, and MannWhitney U test statistics. ..................................................... 119 3 11 MeanSD of PFTPs of horses with low percentage of BALf neutrophils and horses with high percentage of BALf neutrophil and MannWhitney U test statistics. .............. 120 4 1 Anatomical location of acupoints, their Western medical indication, needle size and method of insertion. ............................................................................................................. 164 4 2 Numbers of horses categorized by degree of reaction to EA and to sham treatments in the 1st and 2nd trials prior to eliminating the horse that did not accept EA treatment and horses receiving NSAIDs. ............................................................................ 165 4 3 P values from the Type III sum of squares test on main effects and their interactions for white blood cell indices. ................................................................................................ 165 4 4 MeanSE white blood cell indices, by treatment and sampling time. Normal reference values in parentheses. .......................................................................................... 166 4 5 P values from pairwise comparisons of white blood cell indices, by treatment and samp ling time. ...................................................................................................................... 167 4 6 P values from the Type III sum of squares test on main effects and their interactions for red blood cell indices. .................................................................................................... 167 4 7 MeanSE red blood cell indices, by treatment and sampling time. Reference values in parentheses. ...................................................................................................................... 168 4 8 P values from pairwise comparisons of the red blood cell indices, by treat ment and sampling time. ...................................................................................................................... 169

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12 4 9 MeanSD and range (in parenthesis) of percentages of recovered BALf and percentages of ELF in BALf samples determined by urea dilution technique. ............... 169 4 10 P values from the Type III sum of squares test on main effects and their interactions for broncho alveolar lavage fluid cytological parameters. ................................................ 170 4 11 MeanSE ELF -corrected TNC and differential counts of BALf cells, by treatment and sampling time. ............................................................................................................... 170 4 12 P values from paired samples t test of BALf cytological parameters by treatment. ....... 171 4 13 P values from the Type III sum of squares test on the main effects and their interactions for plasma concentration of immunoglobulin isotypes. ................................ 171 4 14 MeanSE concentrations of plasma immunoglobulin isotypes (x105 ng/ml), by, treatment and sampling time. .............................................................................................. 171 4 15 P values from pairwise comparis ons of concentration of plasma immunoglobulin isotypes, by treatment and sampling time. .......................................................................... 172 4 16 P values from the Type III sum of squares test on main effects and their interactions for conce ntration of immunoglobulin isotypes in ELF corrected BALf. ......................... 172 4 17 MeanSE concentrations of immunoglobulin isotypes in ELF -corrected BALf (x105 ng/ml), by treatment and sampling time. ............................................................................ 172 4 18 P values from pairwise comparisons for concentrations of immunoglobulin isotypes in ELF corrected BALf. ....................................................................................................... 173 4 19 P values fro m the Type III sum of squares test on main effects and their interactions for TNF production from stimulated whole blood by stimulants. ................................ 173 4 20 MeanSE TNF concentrations in whole blood a fter stimulation, by treatment and sampling time.. ..................................................................................................................... 174 4 21 P values from pairwise comparisons of TNF concentrations in whole blood after stimulation, by treatment. .................................................................................................... 175 4 22 P values from the Type III sum of squares test on the main effects and their interactions for FEVx, FVC, and PEF. ............................................................................... 176 4 23 P values from the Type III sum of squares test on the main effects and their interaction for MEFx% and FEVx/FVC ratio. ................................................................... 176 4 24 MeanSE FEVx, FVC, and PEF obtained by the rapid partial forced expiration maneuver, by treatment and sampling time. ....................................................................... 177

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13 4 25 MeanSE MEFx% and FEVx/FVC ratio obtained by the rapid partial forced expiration maneuver, by treatment and sampling time. ..................................................... 178 4 26 P values from pairwise comparisons of the FEVx, FVC, and PEF obtained by the rapid partial forced expiration maneuver. ........................................................................... 179 4 27 P values from pairw ise comparisons of the MEFx% and FEVx/FVC ratio obtained by the rapid partial forced expiration maneuver. ............................................................... 179 4 28 P values from the Type III sum of squares test on main effects and their inte ractions for serum cortisol concentration. ......................................................................................... 180 4 29 Mean SE concentrations of serum cortisol, by treatment and sampling time. ................ 180 4 30 Details of cutaneous and muscle innervations at acupoints used in this study.. .............. 181 5 1 Acupoints commonly used to treat chronic respiratory diseases of horses. ..................... 199

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14 LIST OF FIGURES Figure page 3 1 Variable transformer. ........................................................................................................... 107 3 2 Diagram of device setup to perform rapid pa rtial forced expiration maneuver in horses and component symbols. .......................................................................................... 108 3 3 Mouth gag made from an 8 x 1 1/2 NPT PVC pipe. Hand guard made from black rubber curry comb. ............................................................................................................... 109 3 4 Flow -volume loops derived from average flow data and volume data of horses with normal airways and horses with hyper -sensitive airway. .................................................. 110 3 5 Relationship of airflow rates measured by NIST calibrated mass flow element (MFE) and differential pressure generated before and after rapid partial forced expiration (RP -FE). .............................................................................................................. 110 3 6 Example of data acquisition window of Windaq Pro+ software. ...................................... 111 3 7 Example of forced expiratory flow rates generated by different negative pressures. ...... 112 3 8 Example of flow -volume loops generated by different negative pressures. ..................... 112 3 9 Example of flow -volume loops generated by negative pressures at 150, 200, and 250 Torr. ....................................................................................................................................... 113 3 10 Flow -volume loops derived from average flow data and volume data of horses with low percentages of BALf neutrophils and horses with high percentages of BALf neutrophils. ........................................................................................................................... 113 4 1 Electroacupuncture. .............................................................................................................. 152 4 2 Sham electroacupuncture. .................................................................................................... 153 4 3 A pressur e regulator and a set point regulator installed on the manifold of the artificial inspiration system. ................................................................................................ 154 4 4 Solenoid valve used for controlling the air from the air blower in the artificial inspiration system and for isolating the LFE from the artificial respiration manifold and the negative pressure reservoir. .................................................................................... 155 4 5 Diagram of the rapid partial forced expiration apparatus. ................................................. 156 4 6 Component symbols. ............................................................................................................ 157 4 7 Apparatus setup for laminar flow element (LFE) calibration using NIST traceable mass flow elemen t (MFE). .................................................................................................. 158

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15 4 8 The 12.829liter syringe used for testing an accuracy of integrated airflow volume compare with a 60 ml disposable syringe. .......................................................................... 159 4 9 Linear relationship of airflow rate to P before RP FE and after RP FE. ....................... 160 4 10 MeanSD arterial blood pH during RP -FE maneuver (data were obtained from 8 horses). .................................................................................................................................. 160 4 11 MeanSD arterial blood pCO2 during RP FE maneuver (data were obtained from 8 horses). .................................................................................................................................. 161 4 12 MeanSD arterial blood pO2 dur ing RP -FE maneuver (data were obtained from 8 horses). .................................................................................................................................. 161 4 13 MeanSD arterial blood HCO3 during RP -FE maneuver (data were obtained from 8 horses). .................................................................................................................................. 162 4 14 Mean SD TNF production in electroacupuncture (EA) and sham -EA (sham) groups when whole blood was stimulated with Zymosan. ................................................ 162 4 15 Airflow rates from five arti ficial forced expirations (FE) during rapid partial forced expiration maneuvers on one horse in 20 July 2008. ......................................................... 163 4 16 Flow -volume loops from five artificial forced expirations (FE) during rap id partial forced expiration maneuvers on one horse in 20 July 2008.. ............................................ 163

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16 LIST OF ABBREVIATION S C Degree Celsius Fluid viscosity Mathematical constant (approximately 3.141592654) P Pressure differential ABTS 2,2' -Azi no -bis(3 Ethylbenzthiazoline 6 -Sulfonic Acid) AC Acupuncture ACTH Adrenocorticotropic hormone AFU Arbitrary fluorescent units AID Airway inflammatory disease AquA Aquaacupuncture AurA Auricular acupuncture AVMA American Veterinary Medical Association AVMA The American Veterinary Medical Association BAL Broncho alveolar lavage BALf Broncho alveolar lavage fluid B.C.E. Before current era BL Bladder meridian BP Barometric pressure BSA Bovine serum albumin BT Body temperature CA Aspergillus fumigatus cellular antigen system C.E. Current era CE Aspergillus fumigatus culture filtrate antigen CFA Complete Freunds adjuvant

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17 cm Centimeter cm H2O Centimeter of water CNS Central nervous system COPD Chronic obstructive pulmonary disease CRH Corticoid releasing hormone CSF Cerebrospinal fluid CPT Cutaneous pain threshold CV Conception vessel meridian DNIC Diffuse noxious inhibitory control EA Electroacupuncture EHV1 Equine herpes virus type 1 ELISA Enzyme -linked immunosorbent assay ELF Epithelial lining fluid ET Endotrac heal FE Forced expiration FEV Forced expiratory volume FEVx Forced expiratory volume at x seconds (such as FEV0.5, FEV1.0) FSH Follicle stimulating hormone FVC Forced vital capacity FV loop Flow volume loop GB Gall bladder meridian GDNF Glial cell line -der ived neurotrophil factor GnRH Gonadotrophin releasing hormone GV Governing vessel meridian HA Hemoacupuncture

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18 HB Histamine bronchoprovocation HD Histamine diphosphate HPOA Hypothalamic -pituitary ovarian axis HWRl Hoof withdrawal reflex latency Hz Hertz ID Internal diameter IVAS International Veterinary acupuncture Society IL 10 Interleukin 10 IL 18 Interleukin 18 IL 1B Interleukin 1 beta IL 6 Interleukin 6 INOS Nitric oxide synthase IVAS International Veterinary Acupuncture Society kg Kilogram KID Kidney me ridian L Length of the pipe LAC Laser acupuncture LAurA Laser auricular acupuncture LFE Laminar flow element LH Lutnizing hormone LI Large intestine meridian LIV Liver meridian LPS Lipopolysaccharide LU Lung meridian mA Milliampere

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19 MEFx% Expiratory flow r ate when x% of forced vital capacity has been expired MFE Mass flow element mg Milligram ml Milliliter mm Millimeter mRNA Messenger ribonucleic acid NAA National Acupuncture association NF B Nuclear factor kappa B NIST National institute of standard tech nique nm Nanometer NPT National pipe thread NSAIDs Non -steroidal antiinflammatory drugs NSC Neuronal stem cell PBS Phosphate buffered saline PC Pericardium meridian PC35 delta flow Concentration of histamine that results in a 35% reduction in dynamic com pliance of the airway pCO2 Partial pressure of carbon dioxide PEF Peak expiratory flow PFTPs Pulmonary function test parameters PMA Phorbal 12 -myristate 13 acetate pO2 Partial pressure of oxygen PVC Polyvinylchloride Q Volumetric flow rate r Radius RAO Rec urrent airway obstruction

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20 RFC Relative centrifugal force ROS Reactive oxygen species RP FE Rapid partial forced expiration RPM Round per minute SI Small intestine meridian SLPM Standard liter per minute SP Spleen meridian SPAOPD Summer pasture associated obstructive pulmonary disese ST Stomach meridian TCM Traditional Chinese medicine TCVM Traditional veterinary medicine TH Triple heater meridian TLC Total lung capacity TMB 3,3',5,5' tetramethylbenzidine TNF Tumor necrosis factor alpha WHO World Health O rganization Zym Zymosan

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21 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EFFECTS OF ACUPUNCTURE AND ELECTROACUPUNCTURE ON IMMUNE RESPONSES AND PULMONARY FUNCTIONS IN HORSES By Tangjitjaroen Weerapongse August 2009 Chair: Patrick T. Colahan Major: Veterinary Medical Sciences Using acupuncture (AC) and electroacupuncture (EA) as alternative therapies to conventional equine medical p ractice is increasing worldwide. The benefit of these therapies for treating chronic musculoskeletal disorders such as pain in the thoracolumbar area is well documented and seems to be superior to that of conventional treatment alone. Acupuncture and EA al so are used for treating other medical problems such as gastrointestinal, ophthalmic, and respiratory disorders. However, modern scientific evidence supporting their use for treating these diseases in horses is limited. This study investigates the effects of AC and EA on immune responses and pulmonary functions of Thoroughbred horses. These two topics were chosen because a healthy pulmonary system is vital to improving athletic performance, and inflammation of the lower airways occurs commonly in horses. Se veral forms of inflammation of the lower airways have been described, including inflammatory airway disease (IAD), recurrent airway obstruction (RAO), and summer pasture associated obstructive pulmonary disease (SPAOPD). These diseases are thought to be ca used by dysregulation of immune responses, and investigation of the effects of AC an EA on the immune system might help explain how AC and EA contributes to treatment of these diseases.

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22 The initial investigation compared effects of AC and EA at acupoints LI 4, LI 11, and GV14 on immune functions. Results indicated that only EA significantly induced anti inflammation as demonstrated by in vitro suppression of TNF production in antigen -stimulated whole blood. Electroacupuncture at acupoints commonly used for treating equine chronic respiratory diseases (GV 14, CV 22, BL 13, Ding -chuan, Fei -men Fei -pan, and Fei -shu ) produced similar results. This in vitro anti inflammation was likely governed by modulation of a cellular component of the innate immune syste m by altering the production of inflammatory cytokine upstream of the inflammatory cascade. Effects of EA on pulmonary functions were investigated using the rapid partial forced expiration maneuver. Results showed that EA produced no significant change in pulmonary functions in clinically normal horses. The rapid partial forced expiration maneuver is an emerging technique for measuring biomechanical properties of the lung. Additional studies using this technique in horses of different breeds, ages, body w eights, and horses with known lung disease are needed.

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23 CHAPTER 1 HISTORY OF TRADITION AL CHINESE VETERINAR Y MEDICINE AND A REVIEW OF MODERN SCIENTIFIC RE SEARCH ON EQUINE ACU PUNCTURE Introduction History of Traditional Chinese Medicine and Traditional Chin ese Veterinary Medicine Acupuncture (AC), a branch of traditional Chinese medicine (TCM), has been practiced worldwide for thousands of year. Its clinical benefits have been demonstrated in both human and veterinary medicines. It is clear that AC originate d in ancient China, as the earliest document of AC has been found as part of archeological explorations of very early Imperial sites within modern China. These materials include the most important TCM manuscript Huangdi Neijing. which also is known as The Inner Canon of Huangdi or Emperor Huangs Inner Canon. Huangdi Neijing contains two texts presented in a question and answer format between Huangdi (Emperor Huang) and six of his medical ministers.1 The first text, Suwen (basic question), contains the theoretical foundation of TCM and a method of diagnosing diseases. The second text, Lingshu (spiritual pivot), explains AC treatment methods. Joseph Needham (19001995) and Lu Gwei Djen (19041991), highly respected scholars in TCM, believed that Suwen was composed during the 2nd to 4th centuries B.C.E.2 The version of Huangdi Neijing Suwen used today is called Chong Guang Bu Zhu Huangdi Neijing Suwen (Huangdi Neijing Suwen; Again Broadly Corrected [and] Annotated), and is derived from the Imperial Editorial Office of the Song Dynasty (960 1279 C.E), and is based on a revision of Wang Bings Suwen manuscript (762 C.E ). 3 The Inner Canon of Huangdi has played an important role in the modernization of TCM, including traditional Chinese veterinary medicine (TCVM). It has served as a primary reference for the foundations and doctrine s of both TCM and TCVM.

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24 Over time, AC has spread to other Asian countries giving rise to new forms of AC techniques such as Hari (meridian therapy) and Sujok (hand and foot acupuncture) of Japan and Korea respectively. Even so, the practice of AC was almos t abandoned in China during the early 20th century. This was due in large part to the Boxer Protocols, a treaty signed on 7 September 1901 between the Qing Empire of China and the Eight Nation Alliance (Russia, United States, and several European countries ), and imposed upon China subsequent to the failed of Boxer Rebellion in 1900. 4 This formal agreement mandated the Chinese Imperial government of the Qing Dynasty to transform and adopt Western ideas. This also cause to a declining in the imperial power of the Qing Dynasty, which later overthrown by the revolution in 1911. Following the revolution, traditional medicine and AC were blamed for the ir antiquity, and their practice almost was restricted in 1914. Despite traditional medicine was not totally banned, by the mid 1949, the number of TCM doctors trained in China declined to 270,000, while the number of Western trained doctors increased to m ore than 1.7 million. 5 Then, Mao Zedong, the leader of the Peoples Republic of China from 1949 until his death in 1976, lead a campaigned to revitalize TCM and to promote an integration of TCM into Western medicine.6 It is not obvious why Mao chose to promote an integration of TCM and Western medicine rather than continuing the development of Western medicine alone in China. However, many historians believe that conventional medical care was not widely available during the time of his nationwide campaign due to socioeconomic conditions. Further, TCM seemed to be the only affordable medical care that could accommodate the large Chinese population. After the civil war, Mao played a n important role in revitalizing TCM and AC by encouraging the Chinese medical community to study and incorporate TCM into mainstream medical care. Promoting

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25 TCM in China was not problematic since the basic concept of TCM was deeply rooted in Chinese cultu re. Although European physicians have known about AC since at least the17th century, it was not until the late 20th century that American physicians were motivated to consider its use. During U.S. President Richard Nixons diplomatic trip to China in 1971 James Reston, a reporter for the New York Times traveling with Nixon, received AC treatment after undergoing an emergency appendectomy. After returning to the United States, Reston wrote about his experience with post -operative pain relief following AC treatment.7 Because of his story, the American medical community became interested in AC and initiated serious scientific investigations of its clinical benefits In 1974, in cooperation with the National Acupuncture Association (NAA), the International Veterinary Acupuncture Society (IVAS) was founded in the United States. The organization promotes the practice of veterinary AC and encourages addition of AC into modern veterinary medical practices.8 In response to a worldwide increase in AC practice, the World Health Organization (WHO) hosted the Nation al Symposia of Acupuncture and Moxibustion and Acupuncture Anesthesia in 1979 in Peking, China. Acupuncturists from various countries were invited to identify conditions that they believed might benefit from AC treatment. The symposium concluded that 43 di seases could be treated by AC.9 As most of the published data were derived from cases reports in this symposium. Therefore, the creditability of this body of data has been questioned. However, subsequent new data from controlled clinical trials, that were reviewed by WHO in 2003 confirmed the clinical benefits of AC.9 This review classified the benefits of AC for treating diseases into four groups. Diseases, symptoms or conditions for which the effectiveness of AC treatment has been proven through controlled trials.

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26 Diseases, symptoms or conditions for which the therapeutic effect of AC has been shown, but for which further proof is needed. Diseases, symptoms or conditions for which there is only one controlled trial reporting some therapeutic effect, but for which AC is an alternative because conventional med icine provides no or little benefit. Diseases, symptoms or conditions for which AC may be tried when the practitioner has special modern medical knowledge and adequate monitoring equipment. Origin of Traditional Chinese Veterinary Medicine and Equine Acupuncture Research How TCVM developed is unclear. However, it can be postulated that it may have originated from experimental AC treatments in horses and other farm animals. These animals played important role as family asset in ancient Chinese cultures as f ood sources than did companion animals like dogs and cats, and for transportation. Horses were particularly important and widely used in ancient Chinese warfare. Their importance is demonstrated by the inclusion terra -cotta army in the tomb of emperor Qin -shi of a four -horse war chariot, a soldier leading a horse, and a stable boy. Equine acupuncture has played an important role in Chinese civilization since ancient times. Bole s Cannon of Veterinary Medicine (659621 B.C.), written by Sun-yang during Spri ng and Autumn periods (770 476 B.C.) of the Eastern Zhou Dynasty (770 221 B.C.) demonstrated that TCVM extends well back in ancient China.10 In his text, Sun-yang describes diseases of livestock and horses, and provides some of the ther apeutic foundations used in modern practice, including methods for AC treatment for illnesses in horses. Unfortunately, the text contains no explanation as to how the AC system was discovered or how TCVM developed, and no date has been established for the foundation of ancient equine medical practices. However, TCVM shares many similarities with TCM, and it is likely that TCVM was derived from TCM. The application of traditional Chinese medical doctrines ( Qi Yin/Yang, Wu Xing or Five Elements, Eight

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27 Princi ples, ZangFu or organ physiology, and meridians), in historical philosophical frameworks in TCVM is similar to those of in TCM. The discovery of equine acupoints is thought to have been accidental. For example, a chronic illness of a horse may have disapp eared after an injury at a certain spot on the body was treated with a sharp object (e.g., an arrow, spear, or sword). If this same treatment seemed to have a beneficial effect in several horses, over time it likely inspired ancient Chinese veterinarians and horsemen to conclude that a specific point, even remote to the site of injury, could be used to treat disease. With the spread of evidence -based knowledge from the scientific revolution, philosophybased and experiential knowledge has been replaced by newer concepts. That modern evidence based information derived from controlled experiments have replaced the understanding based on philosophy and simple historical observation as a basis for practice. Medicine is one of the fields that has been most affe cted by this transformation. Despite this recent trend, the practice of TCVM is still based mostly on traditional doctrines and principles. To become fully integrated into modern medical practice, TCVM needs scientific evaluation to determine if it possess es real clinical benefits. Further study will also be required to fully explain the physiological mechanisms.involved in mediating those clinical benefits. Most current research is investigating the effects of AC practices on the nervous system and humoral substances via an assessment of the level of endogenous opioids and substance -P relative to treatment. Scientifically elucidating how AC works is necessary before TCVM can be widely integrated into modern veterinary practice. A better understanding of the basis of AC will facilitate development of more specific and more effective therapeutic strategies.

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28 Even though AC and herbal medicine have been used for treating animals in China for more than 1,000 years, their application to treat animal diseases in mo dern veterinary practice is relatively new in the United States and Europe. Early research and reports of clinical benefits were published in Chinese and were associated with cases being treated in Chinese academic institutions. These publications were generally available only in China and usually were often overlooked by Western veterinary medicine. Early scientific investigations in dogs and horses by Westerntrained veterinarians provided promising results for AC, especially in chronic musculoskeletal pa in such as equine back pain.11 These scientific investigation brought AC to the attention of conventional practitioners. They noted that various AC techniques, often in combination with herbal medicine, were useful for treating chronic diseases that respond poorly to conventional medical management. The clinical benefits of AC have impressed many conventional veterinary practitioners who struggled when treating chronic diseases that respond poorly to conventi onal medical treatment. This experience has produced a strong incentive for veterinarians to pursue AC training and incorporate TCVM into their practice. In the United States, it has been estimated that approximately 2,000 veterinarians are certified as eq uine or small animal acupuncturist. Currently, veterinary AC training in the United States is offered by three major institutions, Chi Institute of Traditional Chinese Veterinary Medicine, International Veterinary Acupuncture Society (IVAS), and the Univer sity of Colorado. It is predicted that the demand for veterinary AC and other forms of complementary medicine will steadily increase due to the increasing popularity among pet owners of holistic medicine and natural products. The American Veterinary Medica l Association (AVMA) has provided a guideline for complementary and alternative veterinary medicine and has stated that:

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29 Veterinary acupuncture and acutherapy are considered an integral part of veterinary medicine. These techniques should be regarded as s urgical and /or medical procedures under state veterinary practice acts. It is recommended that educational programs be undertaken by veterinarians before they are considered competent to practice veterinary acupuncture.12 Acupuncture was first introduced to modern vete rinary medicine as an alternative therapeutic option for chronic pain management. Its therapeutic benefit for treating chronic musculoskeletal pain has been shown to be superior to that of the conventional therapeutic regime. Neurohormonal explanations of the therapeutic mechanisms of AC provide an understanding into how AC works and promote the integration of AC into general practice. A rapid increase in the use of TCVM to treat animals has resulted in several veterinary colleges around the world integrati ng AC and other types of complementary medicine into their curricula. The increase in popularity of AC and related modalities is facilitated by increased numbers of veterinary acupuncturists, now estimated at about 2,000 in the United States, The popularit y of AC is also buoyed by a positive public perception of complementary medical practices seen as natural or holisticalternative to Western medicine. However, the academic and scientific veterinary community is split over the perceived benefits of AC. Th ere exists a disparity, although narrowing, between the experience based medicine touted by acupuncturists and the evidence-based medicine required for widespread acceptance by the veterinary community. In this review, a critical analysis of available lite rature has been attempted to highlight areas of experimental and clinical research in the horse. Due to limited research available on horses, relevant studies on humans and other species are included. Equine Acupuncture Research Research in the Field of An algesia and Musculoskeletal Pain Standard medical management for chronic musculoskeletal pain, including lameness, back pain, laminitis, and navicular disease, includes long term prescription of non -steroidal anti -

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30 inflammatory drugs (NSAIDs), symptomatic t reatment, and supportive therapy. In diagnoses carrying a poor prognosis, such as chronic laminitis, most of the affected horses will be subjected to euthanasia. This may be due to a lack of improvement in clinical signs after a long period of treatment or a decision of the owner to terminate the treatment due to economic constraints. Even with a better prognosis, intensive nursing care until the animal is able to survive independently often is essential. Hospitalization care usually is followed by a long p eriod of rest, which sometimes may last a year or more. Consequently, any forms of adjunctive therapy that reduces the hospitalization period or improves clinical signs are beneficial. Acupuncture and EA may be a suitable modality for managing pain, reduci ng inflammation and, ultimately, improving the outcome in horses with these diseases.1315 Chronic back pain is one of the most common musculoskeletal problems in horses and is a leading cause of poor performance. D iagnosis is based on the performance history and a finding of hyperalgesia or hypersensitivity in the thoracolumbar area during digital palpation. Due to a lack of other specific clinical signs, the condition can be overlooked or incorrectly diagnosed. Wit hout adequate treatment, the horse may suffer from chronic pain.14 AC and EA are highly recommended for treating equine chronic back pain, as confirmed by recent investigations and reports.14,16 19 Application of AC and EA for back pain was also strongly supported by clinical studies in people.20,21 Several methods of treatment have been employed, with no statistical differences in results among needle stimulation, electrical stimulation, acupoint injection (saline or injectable vitamin B12), and low power infrared stimulation.22,23 Acupoints for treatment of back pain include Bai-hui back -associated acupoints along the Bladder meridian, Hua -tuo jia ji, Shen-shu Shen-peng, and Shen-jiao .24,25 With a success rate of the AC treatment and related techniques was 98% (Table 1 1).

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31 Local analgesia has been produced after EA with 4080 Hz stimulation at the Yan -chi acupoint. After treatment, the local pain threshold was increased 1.7 and 1.4 times for horses and mules, respectively.26 These results suggest that the analgesic effect is derived from diffuse noxious inhibitory control (DNIC), in which neuronal signals originating from noxious stimulation in the ir receptive fields are inhibited by other pain sensations.27 The local increase in pain threshold demonstrated an immediate segmental analgesic benefit fr om the treatment. Bilateral EA and AC at BL 18, 23, 25, and 28 on horses significantly increased skin temperature and the cutaneous pain threshold (CPT) over the lumbar area when measured by the radiant heat -evoked skin twitch reflex.28 Electroacupuncture produced a greater increase in CPT at all times measured ( 30, 60, 90, and 120 minutes after treatment) than did AC, and the increase in CPT of the AC group was greater than that in the control group. The researchers also demonstrated that the skin temperature and CPT of the EA group reached their maximum level at 30 minutes post treatment. Moreover, the -endorphin concentration of the spinal cord cerebrospinal fluid (CSF) collected from the spinal cord was increased at 30, 60, 90, and 120 minutes post treatment. These increases in CSF -endorphin concentrations i n the EA group were significantly greater than the concentrations prior to the treatment for all post treatment measurements. Acupuncture treatment also increased the CSF -endorphin at all the sampling points, but reached its highest concentration at 60 m inutes post treatment. However, the AC induced increases in -endorphin were not statistically significant. In the EA group, the concentration of -endorphin was highest at 120 minutes after treatment. The plasma endorphin concentration in horses that re ceived EA and AC tended to increase, but with no significant difference from the concentration prior to the treatment observed28 In this study, the skin temperature and CPT reached their maximum value sooner after t reatment than the

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32 maximum concentration CSF -endorphin were reached. These results suggest that in addition to the opioid -mediated analgesic pathway, AC and EA also induces an immediate effect that alters a local neuronal circuit, leading to an immediate increase in skin temperature and CPT. The best known mechanisms proposed to explain the rebound inhibition of neuronal transmission in AC and EA are the Gate Control Theory proposed by Melzack and Wall and the DNIC described by Le Bars.27,29,30 Electroacupuncture alters hoof withdrawal reflex latency (HWRL).18 HWRL is defined as the duration between the initiation of radiant heat lamp illumination and retraction of the hoof. In this study, either two or four acupoints, in combination, and selected from among Bai-hui SI 9, San-yang -lou Qian -chan-wan and Qian -Jiu increased HWRL regardless of the frequency of the electrical current being used when, compared to a negative control treatment (2 ml of a single saline injection subcutaneously). However, HWRL increases were not great er than a positive control treatment (2 ml of 0.5% bupivacaine hydrochloride injection subcutaneously). Electroacupuncture applied at SI 9, San-yang-lou Qian -chan -wan and Qian -jiu at 80 120 Hz caused a greater increase in HWRL than EA at 20 Hz alone. These results suggested that EA at 80120 Hz is more effective at reliving pain than EA at 20 Hz, and that dynorphins, rather that mu acting -endorphins, are responsible for the observed local analgesia.18 A clinical response after treatment of equine facial nerve paralysis with AC and EA also has been reported. This f inding is consistent with the purported success of AC and EA in neural preservation and regeneration. The overall success rate was greater than 95%.31 35 Other nerve related disorders for which AC and related treatm ent techniques may be used as an alternative therapy include paralysis of the supra -scapular nerve and recurrent laryngeal nerve paralysis. Studies on rats demonstrated that local EA stimulation of the lumbar muscles increased blood

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33 supply of the sciatic nerve more than 50%, while EA at the lumbar nerve root and at the pudendal nerve increased local blood flow by 100%.36 Similar clinical effectiveness of AC and EA for treating dogs with hind limb weakness or paralysis has been shown.37 40 The causes of canine hind limb weakness and paralysis are usually related to a focal disruption at some levels of the spinal cord. Electroacupuncture at GV 1, GV 2, GV6, and GV 9 has been shown to improve survival rate of neuronal stem cells (NSC) being transplanted into a fully transected spinal cord in rats, and increased their migration distance toward the caud al part of the transected spinal cord when compared to the non -treatment group.41 Electrical stimulation of the peripheral nerves has been shown to increase proliferation and differentiation of neural progenitor ste m cells.42 It also promoted re -myelination and neuronal repair. The neuronal plasticity following EA may be partly due to a modulation of factors involved in gene transcription, including c Fos and c -Jun.43 Studies of cats with dorsal rhizotomy at the lumbosacral region of the spinal cord demonstrated that EA significantly increased the neurotrophin 3 and the expression of endogenous nerve growth factor at both protein and mRNA levels.44 These data strongly suggest that AC and EA may also augment the treatment of a variety of nerve relat ed disorders. Arthritis is a major cause of lameness in performance horses. In degenerative osteoarthritis, AC may not reverse pathological changes within synovial structures, but it provides prolonged symptomatic relief.45 Research in China indicated that AC, EA, and their related treatment techniques (hemoacupuncture and aquapuncture) can effectively treat joint associated lameness originating from the forelimb and general arthritis in horses.46 49 Details of this research are shown in Table 1 2. A review of 10 randomized, controlled trials in humans, which included 1,456 participants, demonstrated that AC is an effective treatment for pain and

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34 physical dysfunction associated with osteoarthritis of the knee.50 It also significantly improved the quality of life compared to that of patients who received only conventional treatment.51 Other smaller clinical trials for this clinical problem also demonstrated the same positive result.5256 Pa in relief has been thought to depend on either opioids or nonopioids analgesic mechanisms.57 Electroacupuncture has been shown to suppress the expression of the glial cell line derived neurotrophic factor (GDNF) immunoreactivity of cells from the dermal and subcutaneous tissues of the Complete Freunds adjuvant (CF A) induced arthritis rat.58 Studies demonstrated that EA not only significantly suppressed nocicepive behavior, but also inhibited spinal microglia activation induced by intra articular injection of CFA.59 Suppression of spinal microglia activation by EA also resulted in a down regu lation of the inflammatory cytokines in the spinal cord. This finding suggested that EA might have an important anti -neuroinflammatory effect. In addition to the therapeutic mechanisms discussed above, activation of the muscarinic cholinergic receptors and serotonergic receptors also has been demonstrated.60 Acupuncture for Managing Ocular Problems In human medicine, AC and EA have been used to treat ocular pain, dry eye syndrome, ocular hypertension, and nerve assoc iated ocular disorders.6166 Their use for ophthalmic treatment in conventional equine medicine is an emerging field. In combination with NSAIDs, they effectively alleviate ocular inflammation and ocular pain. Resul ts from clinical practice and laboratory animal experiments are promising. However, scientific data for horses are limited. Most of the data were obtained from case reports, clinical trials in humans, and research conducted on laboratory animals such as do gs and rabbits. Thirty minutes of AC at Tai -yang, BL 2, and TH 23 significantly increased tear production in rabbits as measured by the Schirmers Test.67 Subsequent histological exa mination of the lacrimal gland indicated that an immediate increase in tear production after AC was due to an increase in the secretory activity of

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35 the gland. Moreover, after ten consecutive days of AC, tear production remained greater than the value obtai ned prior to the beginning of AC treatment, and the histological structure of the gland indicated an increase in glandular synthesis and secretory activities.67 Increase in the intra ocular pressure is a sign of glaucoma, a leading cause of ocular pain. Previous research strongly indicated that AC and EA at locations distant from the eyes and selected according to TCVM principles, reduced intraocular pressure in several animal species. A single session of AC at LIV 4, LIV 3, and GB 37 significantly reduced intraocular pressure in healthy dogs.68 A similar study in rabbits has been conducted using a different acupoint; an hour of EA at GB 30 reduced intraocular pressure, systemic blood pressure, and aqueous humor flow rate.69 These reductions were concurrent with a significant increase in endorphin in the aqueous humor, while the concentrations of the sympathetic neurotransmitter, norepinephrine, and dopamine were decreased. Moreover, the effect of intraocular hypotension from a single EA treatment lasted longer than 9 hours. This intraocular hypotensi ve effect could be prevented by a pre treatment with an opioid antagonist. An involvement of sympathetic innervation in the EA -induced intra -ocular hypotension has been demonstrated in rabits. 70 In this model the cervical sympathetic trunk and superior cervical ganglion were surgically excised. After sympathetic de nervation, the EA -induced intraocular hypotension was significantly reduced. Further study indicated that this EA induced intraocular hypotension was associated with activation of -opioid receptors in the intraocular tissues, which require intact sympathe tic innervation.70 It is clear that the mechanisms of AC and EA in treating ocular disorders depend on the autonomic nervous system and the endogenous opioids. However, an involvement of the rebound analgesia pathway such as the Gate Control Theory or DNIC is highly feasible. Both tactile and

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36 pain sensations arisin g from ocular and surrounding tissues are relayed at the trigeminal ganglion and transmitted to the ventral posterior medial nucleus of the thalamus via the trigeminothalmic tract before being relayed to the primary somatosensory cortex. Sensations generat ed by AC and EA are transmitted via the A nerve fibers to the spinal cord and the trigeminal ganglion at a faster rate than those of pain sensation, transmitted via the C nerve fibers. Activation of the inhibitory interneurons leads to an inhibition of pa in sensation transmitted by the C nerve fibers.71 Even though it is unknown how AC and EA suppress ocular inflammation, it is possible that they reduce intraocular pressure and pain, thereby constraining inflammation and providing for ocular tissue healing. However, the increase in -endorphin in the aqueous humor after AC suggests that this endogenous opioid substance might play an important role in how AC reduces inflammation of the eyes. Based on available research data, it seems that AC and EA at acupoints located around the eyes such as BL 2, Tai -yang and TH 23 are suitable for treating ocular pain and stimulating tear production, while AC and EA at a cupoints remote from the eyes such as GB 30, GB 36, LIV 3, and LIV 4 are suitable for controlling the fluid dynamics of aqueous humor. Acupuncture Research in Gastrointestinal Disorders Disorders of the gastrointestinal tract that can be treated by AC and EA include indigestion, diarrhea, and colic. It is generally accepted that AC and EA are not recommended in cases that require surgical intervention such as intussusception and torsion of the large intestine. Previous research suggested that therapeutic ef fects of AC and EA for treating colic may be complex and may not be explained by a single mechanism. Bilateral application of EA at BL 21, BL 25, BL 27, ST 36, and Bai-hui has been shown to increase the rectal pain threshold in a controlled rectal distensi on model.72 Although the increase in pain threshold was not as great as

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37 with butorphanol, the result suggested that EA at those acupoints could be used to suppress pain originating from the large intestine. Chronic diarrhea and chronic inflamm atory bowel diseases are other disorders for which AC and EA therapeutic efficacy have been frequently investigated. GV 1 is the most important single acupoint for treating diarrhea in humans and animals. The mechanism of GV 1 AC for stopping diarrhea is u nclear. GV 1 AC has been shown to stop diarrhea as well as significantly reduce colonic motility and inflammation in rats with experimentally induced colitis.73 Pre treatment of these rats with naloxone, an opioid a ntagonist, prior to the GV 1 AC application abolished the AC effect. This result suggested that the anti -diarrhea and anti -colitis action of GV1 may be due partly to the endogenous opioid anti inflammation pathway.73 In humans suffering from hemorrhoids, EA at GV 1 reduced pain sensations during defecation.74 The analgesic effect was as potent as the standard conventional treatment evaluated with the pain visual analog scale. Local analges ia following EA at GV 1 may be explained by the Gate control theory and DNIC mechanism. In TCVM practice, LI 4 and ST 36 also are commonly used in conjunction with GV 1 to treat diseases associated with the gastrointestinal system. Research has demonstrate d that AC and EA possesse therapeutic effects via a down regulation of the production of inflammatory cytokines such as IL1 IL 6 and TNF .75 Electroacupuncture at ST 36 has been shown to decrease plasma TNF co ncentration and down regulate its mRNA expression in the colonic tissue of rats with experimentally induced colitis.76 Moreover, EA at LI 4 and ST 36 significantly reduced the macroscopic lesions caused by colitis and significantly reduced myeoperoxidase activity of the inflamed colonic tissues. In this study, the researchers demonstrated that the anti -inflammatory activity of EA on LI 4 and ST 36 was suppressed by the

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38 administration of a adrenoceptor antagonist.76 Therefore, EA at these acupoints may activate the sympathetic anti -inflammatory pathway, mediated through adrenoceptors.77 Mi jiao -gan, an acupoint, can be translated as vagosympathetic trunk. This acupoint is recommended for treating poor appetite, diarrhea, and indigestion.78 The point is located at the junction of the cranial and middle one -third of the neck and dorsal to the jugular vein. The vagus nerve is located in this area, and is a major parasympathetic nerve that contains both afferent and efferent nerve fibers for both somatic an d visceral tissues.79,80 Acetylcholine is a major neurotransmitter in both preganglionic and postganglionic parasympathetic nerve synapses. This acupoint is a good example of an AC application that is supported by a related anatomical structure. Direct electrical stimulation of the vagus nerve attenuates the release of TNF and prevents LPS induced endotoxic shock.81 Moreover, acetylcholine attenuates the in vitro release of IL 1 IL 6, and IL 18, but not the anti inflammatory cytokine, IL 10, in LPS -stimulated human macrophage culture. This cholinergic -dependent anti -i nflammatory pathway suppressed the non-specific innate immune response and may explain how AC and EA function in treating diseases of the gastrointestinal system and other visceral organs. A study of the visceral analgesic effects of EA at Guan-yuan-shu (BL 21) on horses with colic, experimentally induced by duodenal balloon distension, did not demonstrate an improvement in the clinical signs of colic.82 In contrast, early clinical practice in China indicated that AC at Jiang ya can be used effectively to control clinical signs of colic.83,84 Moreover, data from a case report indicated that AC and EA may possess positive clinical benefits in treating chronic recurrent colic.85 To confirm a therapeutic benefit of AC and EA, additional studies with modern research design are necessary. Until then, AC and EA may not be a treat ment of choice

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39 when colic horses are admitted to veterinarians, since the vast majority of the cases are acute, often emergencies, and a delay in diagnosis and treatment may negatively affect their prognosis. Acupuncture Research in Respiratory Disorders Chronic respiratory diseases such as recurrent airway obstruction (RAO) and summer pasture associated chronic obstructive pulmonary disease (SPAOPD) are chronic debilitating respiratory disorders. Conventional treatment includes improving the stable environment to eliminate potential inciting causes and symptomatic therapy.86 Long term medical management is required in most cases. When the inciting cause is re -introduced, clinical signs of a respiratory problem reappear. Be sides the conventional medical managements, AC, EA, and Chinese herbs have been used as adjunctive therapies to control clinical signs. They are intended to decrease the dosage requirements of bronchodilators and anti -inflammatory agents, and improve the quality of life of animals suffering from chronic respiratory diseases. Several studies indicated that AC and EA induce immediate mild to moderate bronchodilator effects. A clinical study in human asthma found that AC at LU 7, LI 4, PC 6, ST 40, LI 11, and PC 3 for 15 minutes improved the forced expiratory volume in the first second (FEV1).87 Improvement in pulmonary function parameters (transpleural pressure, tidal volume, minute ventilation, peak inspiratory flow, and peak expiratory flow, in RAO affected horses also has been demonstrated after a single AC treatment.88 However the increases were not statistically significant, and the researchers concluded that the improvements in those pulmonary function parameters were due partly to animal handling. Analgesic and anti inflammatory effects of AC and EA are well recognized in both somatic and visceral tissues.60,89 Major acupoints that are indicated for these purposes include LI 4, IL10, and ST 36. 60 90 91 These acupoints are commonly included in the treatment regime of equine respiratory disorders. Their analgesic and anti inflammation mechanisms have been

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40 linked to a release of endogenous opioid substances in the central nervous system (CNS).28 These endogenous substances activate opioid receptors in CNS tissues, such as the substantia gelatinosa of the spinal cord and the periaqueductal grey of the midbrain, and produce analgesia.92 93 Immunocytes such as macrophages, monocytes, and polymorphonuclear cells also possess opioid receptors predominately -opioid receptors on their cell surfaces.94 Once activated, the receptor induces an anti -inflammatory response via the down regulation of the transcription factor, nuclear factor kappa B (NF B).95 NF B down regulation has been demonstrated to reduce mRNA expression of other inflammatory cytokines, including TNF IL 1 IL 6, nitric oxide synthase (iNOS), and metalloproteinase.96 Carneiro et al.97 demonstrated that, in rats with ovalbumin -induced bronchial asthma, EA reduces the inflammatory cell infiltration in the peribronchial tissue and in the pulmonary perivascular spac es.97 Moreover, the number of total nucleated cells and the percentages of neutrophil and eosinophil leukocytes in bronchoalveolar fluid (BALf) are significantly decreased compared to control and sham EA groups. In this study, EA was performed on acupoints mimicking the treatment regime for human asthma, including GV 14, BL 13, LU 1, CV 17, ST 36, SP 6, and Ding -chuan. An increase in mucus production is a common consequence of airway inflammation. Until recently no s cientific evidence has directly demonstrated the effect of AC or EA on mammalian mucociliary clearance. Disruption of normal mucocilliary clearance is a consequence of chronic airway inflammation and was hypothesized to be caused by neutrophil derived elas tase.98 The increased production and accumulation of mucus decreases airway caliber and increases total airway resistance. Tai et al.99 demonstrated that EA at acupoints LU 1 and CV 22 significantly increased the rate of tracheal mucociliary transport in treated quails

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41 compared to a control group.99 Moreover, EA at these acupoints significantly reversed the decrease in mucociliary trans port caused by the administration of human neutrophil derived elastase. Acupuncture Research in Other Medical Problems In combination with appropriate Chinese herbal formulas, AC and EA are safe for long term medical management for geriatric animals, beha vioral problems, Cushings disease, and anhydrosis. Unfortunately, the scientific data on the therapeutic effects of AC and related techniques on these diseases are limited. A positive clinical response of horses (N=4) suffering from anhydrosis to AC has been demonstrated.100 A retrospective study of 24 horses affected by anhydrosis and treated with T CVM indicated that using a combination of AC, EA, and a Chinese herbal formula (New Xiang Ru San) was an effective therapeutic approach.101 In this study, ow ners of 25 horses were interviewed by telephone and asked to grade improvement in clinical signs after their horses were treated with TCVM. The improvement grading system was developed by the authors and was based on a visual analog scale. Thirty -six perce nt of the clients reported complete recovery, 32% reported significant improvement, 28% reported slight improvement, and 4% reported no improvement. The positive clinical benefit may be due partly to AC/EA or supplementation with New Xiang Ru San, or a com bination of the treatments. Although it is uncertain as to which therapeutic modality is more beneficial for the treatment of anhydrosis, the finding is significant since conventional medicine provides no therapeutic benefit for affected horses. Electroacu puncture significantly increased blood cortisol concentrations of horses when compared to sham treatment.102 This increase was hypothesized to be mediated through activation of the hypothalamic pituitary adrenal axis causin g a release of -endorphin and

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42 ACTH.102 Cortisol or endogenous glucocorticoid, is produced by the adrenal cortex. It is an important steroid hormone, which regulates the immune response, glucose metabolism, and amino acid mobilization. Electroacupuncture at Er jian or Qun -hui acupoints has been used to produce analgesia for surgery in horses (n=62).103 The degree of analgesia was adequate to allow a veterinary surgeon to perform a standing operation.103 Another study in dogs demonstrated that EA at ST 36 and GB 34 produced greater analgesic effect than EA performed at ST 36 and SP 6. In dogs in which analgesia has been induced, it was feasible to perform a simple laparotomy.104 Using AC an d EA analgesic to control pain during operations on horses seems far from realistic. Since horses respond unpredictably and violently to nociceptive stimuli, and without appropriate and dependable control of anesthetic depth, a life threatening injury to t he surgeon, damage to the surgical facility and equipment, or injury to the patient may occur. AC Research in Reproductive System Application of AC and EA for treating disorders of the reproductive system is another field in which the positive clinical res ponse rate is significant. Among all reproductive system problems, infertility is the most common disorder treated by AC. Research in humans has demonstrated that AC is an effective therapy for women with infertility. Following in vitro fertilization and e mbryo transfer, AC has been used to improve the pregnancy rate.105107 Causes of equine infertility may be simply categorized as due to infection or noninfection. Common infectious agents that can cause infertility include viruses, bacteria, and fungi. Non -infectious causes range from anatomical defects and lameness to stress -induced infertility. In some cases the problem may be multi -factorial. Examples of common noninfectious causes of equine infertility include hormonal imbalance, excess uterine fluid, endometrial cysts, and lameness.

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43 The cause of infertility requires careful diagnosis before an appropriate treatment, whether conventional or integrative, is implemented. In TCVM, mare reproductive function is rela ted to the Chinese parenchymatous visceral organs Kidney and Liver. It also depends on the Conception vessel and the Penetrating channel. Therefore, acupoints commonly stimulated for treating mare infertility include Bai-hui Yan -chi BL 23, Shen-shu Shen-peng KID 3, KID 7, GV 3, GV 4, CV 4, CV 6, and LIV 3.108,109 A TCVM pattern diagnosis provides the core of any acupuncture treatment. In Thoroughbred mares with a history of excess uterine fluid and/or uterine pooling that did not respond to the conventional treatment, adjunctive AC treatment significantly reduced uterine fluid accumulation as determined by transrectal ultrasonography.110 A high percentage (81%) of this group of mares was later able to get pregnant.110 Another report using EA for the same purpose has reported a significant increase in uterine tone within 24 hours after the treatment.111 The decrease in the uterine fluid accumulation may be due pa rtly to an improvement in local circulation and the uterine blood flow mediated by central inhibition of sympathetic nerve activity.112 Additional studies in other animals also support the benefit of AC and EA for treating infertility. A small -sample study of anestrous sows indicated that AC at Baihui and Wei -ken could be used to induce estrous within 14 days after the treatment, but not in a sham AC group when needles were used to stimulate Chian-feng and Chou-shu acupoints. The induction of estrous in sows using AC was superior to treatment with gonadotrophin releasing hormone (GnRH) alone.113 A similar study in repeat -breeder cows that failed to respond with GnRH treatment and failed to become pregnant after more than three inseminations showed the same positive result.114 In this study aquapuncture with 10 ml and 5 ml of 50% glucose solution were

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44 injected into Bai-hui and Shen-peng acupoints, respectively. The authors reported that most of the cows exhibited estrous signs within 14 days and were artificially inseminated. Later pregnancy diagnosis by rectal palpation revealed that 66% of the treated cows were pregnant. However, the percentage of cows that carried their pregnancy to a full term and successfully calved was 44%.114 A lower rate of successful calving may be due partly to the fact that the repeat -breeder cows are likely to be predisposed to other stress factors and disorders such as metabolic acidosis and hot climate, which may contribut e to a loss of their pregnancy. A positive clinical benefit of EA also has been shown in cows with ovarian cysts.115 Aquapuncture at Bai-hui with prostaglandin induces luteolysis of the corpus luteum in mares.116 The luteolytic effect and the abilit y to induce estrous cycle were as effectively as intramuscullar administration of prostaglandin at the recommended dosage. Bai-hui aquapuncture with micro-dose of prostaglandin was still effective, and caused less systemic adverse effects than prostaglandi n alone at the recommended dosage.116 However, a later study refuted these claims.117 Further scientific scrutiny on this topic i s required. Electroacupuncture at appropriate acupoints reversed abnormal functions of the hypothalamic -pituitary -ovarian axis (HPOA).118 A series of AC treatments at BL 18, BL 23, CV 3, CV 4, and SP 6 in women with ovulatory dysfunction has been shown to improve the ovulation rate over 80%.119 Hormonal profiles also showed that AC and EA were capable of adjusting the level of the follicle stimulating hormone (FSH), lutinizing hormone (LH), estrogen, and progesterone to their normal physiological concentrations.119,120 A study on rats demonstrated that EA at CV 4 increased the immunohistochemical reactivity for GnRH in the medial pre op tic area, the arcuate nucleus, and the nucleus periventricularis of the hypothalamus.121 Ovariectomized rats possess abnormal reproductive

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45 profiles and have been used as an experimental model to study abnormalities of reproductive hormones. Based on this experimental model, EA has been shown to restore the function of the HPOA.122 Electroacupuncture significantly increased the number of GnRH positive neurons in the hypothalamus of ovariectomize d compared to both ovariectomized and normal rats.123 GnRH positive neurons in the nucleus paraventricularis also have been found to co -localize with the corticoid releasing hormone (CRH) -positive neurons identified by immunofluorescent double labelling histochemistry and laser confocal scanning microscopy.123 This data suggested that AC and EA may modulate the hypothalamic pituitary gonadal axis by altering the synthesis and secretion of regulatory hormones GnRH and CRH from the hypothalamus, and modulate the reproductive hormones and homeostasis. Release of GnRH after EA also has been show in rabbits.124 Even though the benefits of AC and EA have been confirmed in clinical practice, their mechanisms remain unclear. Sensory signals generated by AC and EA may modulate either neuroendocrine control of the hypothalamic -pituitary -gonadal axi s or modulate the peripheral autonomic neural control of the reproductive tract, or both. A more thorough understanding of their mechanisms will allow the practitioner to specify the action of each acupoint and improve the effectiveness of the treatment. A positive clinical benefit of AC and EA in the treatment of male infertility has been demonstrated in humans. Acupuncture decreased the number of defective spermatozoa and improved their motility.125127 Improvement in acrosome ultrastructure, nuclear shape, axonemal pattern and shape, and accessory fibers of sperm organelles also has been shown.125 Acupuncture also increased sperm count per ejaculate, especially in subjects with a history of genital tract

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46 inflammation.128 Even though the results in humans seem promising, a placebo effect was not ruled out. Resea rch in Diagnostic Potential of Acupoints and Meridians In TCVM, acupuncturists can use Ah -shi point to diagnose animal disorders. Ah -shi point is also known as trigger point and as myofascial pain point. Trigger points are defined as locations of pain duri ng pressure and palpation. These locations may be objectively detected by pressure algometry, pressure threshold measurement, magnetic resonance, thermography, and history of illness. The associated acupuncture principle states that where there is a pain, there is an acupoint. Therefore, occurrence of a trigger point may be at the same location as an ordinary acupoint or at a non-specific location on the body surface. Hypersensitivity upon palpation at trigger point may indicate underlying local tissue inflammation or a specific pathology of a certain tissue. The presence of a trigger point in horses has been demonstrated by using sensitivities within the cleidobrachialis muscle. Horses with chronic musculoskeletal pain possessed an objective sign of spont aneous electrical activity, spike activity and local twitch response at the myofascial trigger point location.129 McCorm ick demonstrated a relationship between five diagnostic trigger points and the cause of a fore limb lameness.130 In his study, acupoints LI 18, SI 16, TH 1 5, BL 42, and BL 14 were used to represent channel diagnosis for the Large intestine, Small intestine, Triple heater, Lung, and Pericardium channels, respectively. Hypersensitivity of these acupoints has been proposed to be a result of imbalance in the cha nnel they represent. The pathways of these channels are located in the fore limb, and their imbalances are thought to be caused by pathology in the fore limb. The study found that the evidence of hypersensitivity among these trigger points was significantl y greater in heel lameness and laminitis, but not lameness originating from sub -solar regions. Pericardium channel imbalance

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47 caused by heel lameness was significantly greater than channel imbalance caused by laminitis.130 The result also showed that pain originating in the toe region is highly correlated with the imbalance of the Lung and the Large intestine channels. Subsequent intra articular anesthesia of the distal interphalangeal joint abolished the Lung and the Large intestine channel imbalance in all of these horses. Moreover, lameness and response to intra articular therapy of the proximal interphalangeal joint (fetlock joint) may be evaluated by a se nsitivity of SI 16, TH 15, BL 14, LI 18 and BL 42.131 The sensitivity of these acupoints was demonstrated in 54% of horses (176 of 327 horses) in which the clinical signs and medical history suggest that the cause of lameness originates from the fetlock joint. Sensitivity of LI 18 and BL 42 was detected in all 176 horses. Sensitivity of SI 16, TH 15, and BL 14 w as also increased, but not consistently among these horses. Subsequent intra articular injection therapy reduced the sensitivity of those acupoints in 54% of these horses, and 63% of them became sound. Horses with no change in their sensitivity to acupoint s or still unsound after intra articular therapy, were later demonstrated to have concomitant sources of pain, such as interphalangeal joint, carpal joint, and pain originating from hind limbs.131 These results demonstrate the presence of trigger points in fetlock pain and the diagnostic value of those trigger points. Therefore, it is suggested that the hypersensitive reaction at LI 18 and BL 42 may be caused by pathology within the distal interphalangeal joint. The benefit and specificity of BL 18, BL 19, BL 36 Xie -qi and Shen-shu acupoints for diagnosing hind limb lameness caused by tarsal joint and metatarso phalangeal joint also have been demonstrated.132 Using sensitivity of acupoints for diagnosing equine herpes virus type 1 (EHV1) and equine protoz oal myelitis also has been proposed. However, results from standard diagnosis did not confirm the researchers supposition.133,134

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48 Tenderness and neurologic inflammation at GV 1 also has been demonstrated in rats w ith induced colonic inflammation.73 GV 1 is an important acupoint for treating diarrhea, bloody defecation, constipation, and perineal problems.135 Summary Current research has confirmed certain therapeutic benefits of AC and EA. In musculoskeletal disorders, the success rate of the treatment was nearly 100% for thoracolumbar pain. For this type of disorder, AC, EA, aquapuncture, or laser AC can be used with no significant difference in their therapeutic efficacy. For treating nerve associated disorders, EA seems to be the best method, and electrical stimulation has been shown to be or beneficial in the promotion of nerve repair, re -myelination, and neuronal plasticity. Little modern research on equine arthritis has been conducted, and clinical application of AC and EA to treat equine arthritis is still based on empirical and personal experience. How ever, results from human research seem promising, and research on their therapeutic effects for treating equine arthritis may be worthwhile. For ophthalmic treatment, local acupoints around the eyes are appropriate for ocular pain and inflammation. Possibl e therapeutic mechanisms include opioid analgesia and anti inflammation. Using AC and EA for treating an increased intra ocular pressure also is promising. The therapeutic mechanism is thought to be opioid and sympathetic innervation dependent. In the fie ld of reproductive disorders, AC and EA therapeutic effects are thought to be mediated by the ability of AC and EA to restore balance of the hypothalamic pituitary gonadal axis and to promote uterine tone by the central inhibition of the sympathetic nerve activity. Currently available research data were obtained from studies on humans. Interpretation of results should be done with care, since the positive results of treatments on humans may by due to the placebo effect. Additional studies in animals may eli minate this confounding positive

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49 psychological effect, and demonstrate the true effects of AC and EA. To date, scientific scrutiny of the mechanisms of AC and EA for treating reproductive problems in horses has been limited to studies on mares, and is abse nt for stallions. Acupuncture and EA provide mild to moderate analgesia and are not the anesthetic methods of choice when surgical intervention is required. Because the vast majority of colic cases are acute and rapidly progress, delay in diagnosis and tre atment may affect their prognosis. Therefore, all cases of colic presented to veterinarians are likely to be treated first with conventional medicine, and the clinical benefits of AC and EA are rarely investigated. However, for chronic gastrointestinal pro blems, treatment efficacy for both AC and EA has been demonstrated. Treatment benefit was thought to be due party to an activation of the cholinergic anti inflammatory pathway, leading to a down regulation of inflammatory cytokines such as IL1 IL 6 and TNF In respiratory disorders, EA might be beneficial for treating RAO. Results from laboratory animals suggested that the possible treatment mechanisms include anti inflammation, bronchodilation, and increased mucociliary clearance. Anhidrosis is anothe r medical problem for which AC and EA is useful. This is important because TCVM seems to be a treatment of choice. The only other effective treatment is relocation of the affected horses to a more temperate environment. Finally, diagnostic and therapeutic values of trigger points have been confirmed and, for some, an increase in sensitivity is highly positively correlated with a suspected disorder. Even though previous research supports using AC and EA for veterinary medicine, more carefully controlled rese arch in every aspect of TCVM for the horse is necessary.

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50 Table 1 1. Treatment efficacy of AC and its related technique in equine thoracolumbar pain. Investigator. AC Technique (s) Total cases Number of cases with positive response to treatment Number of cases not response to treatment Klide 1984 14 AC 15 13 2 Petermann 2002 16 AC+AurA+ LAurA+LAC 512 502 10 Xie 2005 17 AquA+ Herbal medicine *4 4 0 Rungsri(Kulchaiwat) 2009 19 EA **16 16 0 Total 547 535 12 AC = acupuncture, AurA = auricular acupuncture, LAurA = laser auricular acupuncture, LAC = laser acupuncture, EA = electroacupuncture, = Effectiveness of EA treatment on these horses was compared to the treatment efficacy of oral administration with phenylbutazone and normal saline in others 11 horses, ** = Effectiveness of EA treatment on these horses was compared to the treatment efficacy of sham EA in others 7 horses. Table 1 2. Treatment efficacy o f AC and its related techniques in equine lameness originating from joint problems. Location. Reference AC technique Total cases Sound Improve Failure All four limbs. 46 EA 198 174 21 3 Forelimb. 47 AquA+HA 85 83 2 0 Forelimb or hindlimb. 48 AquA+Herbal medicine 161 136 15 10 Rhumatism. 49 EA 153 143 0 10 EA = electroacupuncture, AquA = aquapuncture, HA = hemoacupuncture.

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51 CHAPTER 2 MODULATION OF IMMUNOLOGICAL RESPONSE BY ACUPUNCTURE AND ELECTROACUPUNCTURE AT LI 4, LI 11, AND GV14 IN CLINICALLY NOR MAL HORSES. Introduction Application of alternative medicine, such as traditional Chinese veterinary medicine (TCVM), in treating diseases caused by disorders of the immune system is both an ancient and a novel, at least in Western cultures, approach to veterinary medicine. Acupuncture (AC), a part of TCVM, was developed in ancient Chinese culture and has been practiced in China for more than a thousand years. With a long history of clinical benefits, AC is now be ing integrated into Western veterinary medicine, and is being adopted as an alternative therapy for a number of diseases. Interestingly, AC has changed only slightly since the ancient Chinese texts described the methods for treating illness. Electroacupun cture (EA) is a form of treatment that was derived from and remains closely related to AC. It was developed by an integration of knowledge about electrophysiology of body tissues that was merged with the classical practices of AC. In human medicine, both AC and EA have been used as adjunctive therapies for several diseases, including asthma, human immuno deficiency virus (HIV), allergic disease, and disorders of the immune system.87,136,137 A common pathophysiologic aspect of each of these diseases is a dysregulation of immunological function. Under TCVM treatment principles, several acupoints, including LI 11, ST 36, LI 4, and GV 14, have been used for improving health and modulating the immune system.135,137 Multiple AC treatments at LI 11, GV 14, SP 10, and ST 36 have been shown to significantly decrease the number of peripheral circulating leukocytes (including lymphocytes) in healthy humans.138 It is possible that the alterations in these hematological parameters were due to movements of immune cells between cell storage compartments.138 Modulation of the cellular

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52 immune response was demonstr ated in AC performed at LI 11 twice a week for a total of eight treatments.139 After the fourth treatment, the increase in reactive oxygen species (ROS) production by neutrophils was greater in the AC group than in the placebo group. In human medicine, ulcerative colitis is another chronic inflammation for which AC treatment possessed a positive clinical benefit. According to the randomized single -blind study, AC performed at the acupoints selected according to the traditional Chinese medical diagnosis significantly reduced the colonic activity index and improved the quality of life of the patients.140 Further study in the rat indicated that the mechanism of the treatment may be partly associated with a down regulation of colonic tumor necrosis factor alpha (TNF ) mRNA expression.76 In veterinary medicine the LI 4, LI 11, and GV 14 acupoints are commonly included in treatment protocols of several aliments, including fever, immunodeficiency, and dental pain.135 This study compares the effects of AC and EA on circulating immunoglobulins, ROS production by circulating neutrophils after in vitro stimulation, and TNF production in stimulated whole blood cultures in vitro, as evidence of in vivo immune modulation. Methods Animal Twenty -four mature Thoroughbred horses with no history of illness or hospitalization during the past month were randomly assigned for A C (n=12) and EA (n=12) treatments. All horses were kept in paddocks in groups of 2 or 4. Shelter was available in each paddock. The horses were fed twice a day with balanced commercial feed. Clean water and good quality hay were available ad libidum. Numbe rs from 1 2 and 14 were randomly assigned to the horses in the groups of 2 and 4 horses, respectively. Horses with odd numbers were assigned to EA treatment. Horses with even numbers were assigned to AC treatment. Protocol for animal use

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53 was approved by t he University of Florida Institutional Animal Care and Use Committee (Permit A 130). Acupuncture and Electroacupuncture Acupoints used in our study were LI 4, LI 11, and GV 14. These acupoints were selected because, according to the TCVM, they are recommen ded for tonifying Qi (defined as the life energy that nourishes and propels physiological activities of living organisms). LI 4 is commonly used in treating inflammation of the front leg and is known to possess analgesic properties. LI 11 is an immune -stim ulation acupoint. According to acupuncture treatment protocol, GV 14 is recommended for treating fever and excessive sweat and other disorders that are diagnosed as excess Heat pattern in TCVM.135 Detailed descriptions of each acupoint and TCVM indication are shown in Table 2 1. A 0.35 mm diameter x 50 mm disposable sterile stainless steel acupuncture needle (Kingli, China) was used at LI 11 and GV 14 acupoints. A 0.35 mm x 25 mm acupuncture needle was used at LI 4. For EA, acupoints were stimulated with an electrical stimulator (Pantheon Research). Intensity and frequency were set at 3 5 mA of 15 Hz for 10 minutes and immediately followed by 3 5 mA 200 Hz for 10 minutes. The needles were secured in place with superglue. Horses assigned to the AC were treated the same way except needles were not connected to the electrical stimulator, and needles were left in each acupoint for 20 minutes. A fter AC and EA, needles were gently removed. Electroacupuncture and AC were performed once a day for 3 consecutive days. Source of Fungal Antigens Aspergillus fumigatus antigens used in this study were obtained in the form of sterile commercial products av ailable for immunological testing in humans and animals (Greer source materials). The manufacturer prepared the cellular antigen (CA) from a defatted dry powder of

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54 A. fumigatus, ATCC strain 1022, obtained from a 20-day static culture (#XPM3X1A5). Fungal ce llular antigen was extracted using 0.01M ammonium bicarbonate at 1:20 weight/volume. The extract was then concentrated, and dialyzed with an AmiconTM filtration/dialysis system. Finally the fluid was passed through a 0.2 m sterile filter and was lyophiliz ed. Culture filtrate antigen (CE), (#XPM3 -F16 50.0) was obtained from a 20 -day old static culture medium. The culture fluid was concentrated and dialyzed with an AmiconTM filtration/filtration system. The fluid was then passed through a 0.2 m sterile filt er and was lyophilized. Protein content of each antigen was measured using the Bradford protein assay. Leukocyte Separation Sixty ml of peripheral blood was collected from the left external jugular vein into a pre heparinized sterile syringe. Sodium hepar in (Abraxis Pharmaceutical) was used as an anticoagulant and its final concentration was 10 unit/ml. Polymorphonuclear leukocytes were isolated from leukocyte rich plasma prepared by the method that has been previously described by Sun et al.141 To obtain leukocyte rich plasma, the heparinized blood was allowed to settle for 1520 minutes. Neutrophils were separated from mononuclear cells by the single step gradient method using lymphocyte separation medium, density 1.077 g/cm3 (Mediatech #25 072CV).142 Leukocyte rich plasma from the blood sample was then carefully layered over 10 ml of leukocyte separation medium in a 60 ml sterile conical centrifuge tube (Fisher #0644318). The mononuclear cells were separated from the polymorphonuclear leukocytes and contaminated red blood cells by centrifugation at 800 RCF for 30 minutes and 21 C After centrifugation, the polymorphonuclear leukocytes were recovered from the cell pellet. Contaminated red blood cells were removed by hypotonic lysis with 20 ml of sterile distilled deionized water (Mediatech #25055CV) and vortexed for 30 seconds. The osmolarity of the cell suspension was immediately

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55 restored to isotonic condition by adding 20 ml of sterile double strength phosphate buffer saline (2 x PBS). Cell suspensions were then adjusted to 45 ml with 1 x sterile PBS for washing (Mediatech #21 040CV). The tubes were centrifuged at 800 RCF at 21C for 10 minutes. In total, the neutrophil s were washed three times with 40 ml of sterile 1 x PBS and centrifuged at 800 RCF for 5 minutes at 21C Before the final wash, the cell concentration was determined by counting with a hemocytometer, and cell viability was determined by Trypan blue exclus ion. After the final wash, concentration of polymorphonuclear leukocytes was adjusted to 3 x106 cell/ml with media containing 10% fetal bovine serum (FBS, Hyclone #SH30070), 1% L glutamine (Mediatech #25005CL), 2% sodium pyruvate (Mediatech #25 000CL), and 0.1% gentamicin sulfate (Mediatech #30 005CR) in RPMI 1640 without phenol red and L gultamine (Mediatech #17 105CV). Reactive Oxygen Species Generation of Neutrophil The assessment of equine neutrophil ROS generation was modified from the method des cribed by Donovan et al.143 Briefly, 100 l of neutrophil suspension in media or of media alone were added to each well of a 96 -well black opaque flat bottom plate (Costar #3915). Solutions of stimulants were prepar ed in the media, including purified E coli 0111:B4 lipopolysaccharide (LPS) (InvivoGen # tlrl -eblps), zymosan (Zym) (Molecular Probes # Z2849), Aspergillus fumigatus CA, A. fumigatus CE, and phorbal 12-myristate 13 acetate (PMA) (Biomol #PE 160). To each designated well, in triplicate, were added 10 l of media alone, 106 M PMA, LPS (10, 1, 0.1 g/ml), Zym (10 g/ml), CA (10 g/ml), or CE (10 g/ml), and 10 l of 100 M of DHR 123 (Invitrogen #D632). Lipopolysaccharide, CA, and CE were used because they h ave been suspected of being involved in the pathogenesis of equine acute abdomen, laminitis, and

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56 recurrent airway obstruction.144 146 Zym is a standard fungal antigen, which is routinely used in general immunologica l testing. After adding DHR 123, the plate was shaken with a plate shaker (MS1 Minishaker) for 30 seconds and incubated at 37 C in 5% CO2 for 3 hours in the dark. Each plate was evaluated by using a fluorescent plate reader (Bioteck) with 485 nm excitation and 538 nm emission to monitor conversion of the dye from the non-fluorescent to the fluorescent form in arbitrary fluorescent units (AFU). Phorbal 12 myristate 13 acetate was used to generate the maximum stimulation of ROS production and was used as a positive control for each sample. The ROS production of neutrophils in response to each stimulant was determined from a calculation of the response ratio using the following formula: ROS response ratio = AFU of stimulated cells/AFU of non-stimulated cells (2 1) Heparinized blood Stimulation A 1440 l of heparinized blood was transferred into 10 sterile pyrogen-free microfuge tubes (Fisher #05 408129). Sterile solutions of each stimulant, including LPS, Zym, CA, and CE, at concentrations of 1 g/ml (10X), 2 g/ml (20X), and 3 g/ml (30X), were prepared with 1 x PBS. These concentrations were used for single, double, and triple stimulations, respectively. For a single stimulation, 160 l of 1 g/ml of each stimulant was added to the microfuge tube. For double stimulations, 80 l of 2 g/ml of 2 combinations among LPS, CA, and CE were added. For triple stimulations, 53.3 l of 3 g/ml of LPS, CA, and CE were added to the tube. Microfuge tubes were incubated in a 150 rounds/minute shaker for 6 hours at 37 C. At the end of incubation, the microfuge tubes were placed in ice for 5 minutes prior to centrifugation at 14000 RCF for 1.5 minutes. Two 250 l aliquots of plasma were collected in microfuge tubes and stored in 20 C for TNF analysis.

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5 7 Equine Tumor Necrosis Factor Alpha (TNF ) Assay Tumor necrosis factor alpha in plasma collected from stimulated -heparinized blood was analyzed with ELISA (protocol was kindly provided by Dr. David J. Hurley, College of Veterinary Medicine, University of Georgia). Briefly, 100 l of 3 g/ml of anti -equine TNF polyclonal antibody (Endogen #PETNFAI) in a carbonate bicarbonate coating buffer at pH 9.7 (Sigma Aldrich #C 3041) was added to the 96 -well plate (Nunc ImmunoTM Modules #469949). The plate was incubated overnight at 4 C. After coating, the coating buffer was removed, and 200 l of blocking buffer containing 1% BSA (Sigma Aldrich #B4287) in PBS was added. The plate was then incubated at room temperature for one hour. After incubation, each well was washed three times with 2 50 l of washing buffer containing 0.05% Tween 20 (FisherBiotech #BP337500) in PBS. A series of two-fold dilutions of equine recombinant TNF (ErTNF ) standard (from 10000 pg/ml to 39.05 pg/ml) was prepared with diluent containing 1% BSA and 0.05% Tween 20 in PBS. Plasma was diluted 1:5 with the diluent. Then 100 l of each concentration of standard and diluted plasma was added to a designated well, in duplicate, and the plate was incubated at 37 C for two hours. After incubation, each well was washed three times with 250 l of washing buffer. After washing, 100 l of equine TNF biotin labeled polyclonal antibody (Endogen #PETNFABI) at a concentration of 0.9 g/ml was added to each well and the plate was incubated for 90 minutes at 37C. After incubation, the washing step was repeated three times and 100 l of 1:5000 of AvidinHPR (BD Bioscience Pharmingen #554058) was added to each well. The plate was incubated at 37C in the dark for one hour. After incubation, the washing step was repeated five times, and 100 l of ABTS peroxidase substrate system for HRP (KPL #50 6200) was added to each well. The plate was then incubated for 30 minutes at room temperature. The plate was evaluated with a universal microplate reader

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58 (Elx800, Bio Tek) at a wavelength o f 405 nm. TNF concentration in the plasma was calculated based on a four -parameter logistic curve -fit generated by KC4 software. Immunoglobulin Assay Plasma immunoglobulins (IgA, IgM, IgGa, IgGb, and IgG(T)) were determined with horse immunoglobulin ELIS A Quantitation Kits (Bethyl laboratories, inc. Texas #E70 116, #E70114, #E70124, #E70127, and #E70105). Assays were performed according to the protocols provided by the manufacturer. Briefly, Nunc MaxiSorp C bottom well modules in a 96 well frame were coated with 100 l of goat anti horse immunoglubumin diluted in carbonate bicarbonate coating buffer (Sigma #C3041). After 1 hr of incubation, the buffer was removed and each well was washed three times with 200 l of washing buffer (Sigma #T9039). After w ashing, 200 l of postcoat solution (T6789) was added to each well and the plate was incubated for 30 minutes. After incubation, the washing step was repeated three times. Dilutions of immunoglobulin standard were prepared from the standard serum according to the protocol. Plasma samples were diluted with sample/conjugate diluent. A suitable dilution of sample was predetermined with equine plasma with the same assay. 100 l of each dilution of standard and diluted plasma was added to a designated well in du plicate. The plate was incubated for one hour at room temperature. After incubation, the washing step was repeated three times, and 100 l of diluted goat anti horse Ig HRP conjugate was added to each well. The plate was then incubated for 1 hour. After i ncubation, the excess HRP conjugate was removed, and the washing step was repeated five times. After washing, 100 l of TMB peroxidase substrate was added to the each well, and the plate was incubated for 15 minutes. The peroxidase reaction was stopped by adding 100 l of 2M of H2SO4 (Fisher #SA818500) to each well. The plate was evaluated with a universal microplate reader at a wavelength of 450 nm. The concentration of immunoglobulin in

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59 the sample was calculated based on a four -parameter logistic curve -f it generated by KC4 software. Statistical Analysis Immunoglobulin concentrations, ROS generation ratios, and TNF concentrations were first evaluated by box plot. Outlier and extreme outlier data in each data set were excluded from further statistical ana lysis. Differences between preEA and pre -AC treatment groups were compared using Mann-Whitney Rank Sum test. Differences between pre and post -within -group treatments were compared using Wilcoxon Signed Rank test. Differences between post -EA and post -AC t reatment groups were compared using Mann Whitney Rank Sum test. Because of presumed genetic differences and variation in housing environments, a p value for determining significance. Statistical analysis was performed with SPSS.17 for Window s. Results Animals Twenty -one geldings and 3 mares were included in this study. The EA group contained 12 geldings, while the AC group contained 9 geldings and 3 mares. MeanSD age of subjects in EA and AC groups were 8.21.1 years (ranged from 6 10) and 7.42.1 years (ranged from 310), respectively. MeanSD body weight of subjects in EA and AC groups were 54635 kg (ranged from 490590) and 53946 kg (ranged from 473615), respectively. Demographic information on horses in the EA and AC groups is presente d in Table 2 2. Acupuncture and Electroacupuncture Procedures With physical restraint by a halter and leading rope held by a handler, most of the horses accepted the AC and EA procedures. One horse in the AC group showed signs of mild discomfort, including restlessness, and continuously contracted its cutaneous muscles at the first AC treatment. The signs of discomfort decreased during the second and the third AC treatments.

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60 Two horses in the EA group showed signs of severe discomfort when acupuncture needles were stimulated with electricity. The signs of discomfort included restlessness and twitching of the cutaneous muscles. However, EA was completed on all horses without additional physical restraint. Rhythmic muscle contraction during the low frequency stimulation was not observed in our study. Homogeneity of Samples Before Electroacupuncture and Acupuncture Mean SE concentrations of immunoglobulins, ROS ratios generated by circulating neutrophils, and TNF production of whole blood in pre -treatment sa mples of EA and AC groups are given in Tables 2 3 to 2 5. Concentrations of IgA, IgM, and IgGb between EA and AC groups before treatment were quite uniform. Reactive oxygen species ratios generated by circulating neutrophils following stimulation with PMA, LPS, Zym, CA, and CE between EA and AC groups before treatment were also essentially the same. Tumor necrosis factor alpha production of whole blood following LPS, Zym, CA, CE, and CA+CE stimulations between EA and AC groups before treatment were not sign ificantly different. However, concentrations of IgGa, IgG(T), and the TNF production of whole blood stimulated with CA+LPS, CE+LPS, and CA+CE+LPS were significantly different in pre treatment samples of the two groups of horses (Table 2 6). Plasma Immuno globulins After three treatments of AC and EA, plasma concentrations of the five immunoglobulin isotypes measured in this experiment were not significantly different from the levels prior to treatment (Table 2 7). Between groups comparisons of post -treatment samples of EA and sham groups were not significantly different (Table 2 10).

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61 Reactive Oxygen Species Generation of Neutrophil The viability of isolated neutrophils was greater than 96% in all samples prior to the final wash. The assessment of ROS was done using a fixed number of neutrophils recovered from blood samples (300,000 cells per sample); thus, it represents a measure of an average per cell response rather than a direct measure of the change in the total circulating ROS production capacity of the horse. The change in the number and fraction of circulating neutrophils would be required to generate an assessment of the total impact in the horse. Mean SD ROS ratios generated by each stimulant and test statistics from within-group comparisons are presented in Table 2 8. Phorbal 12 -myristate 13 acetate generated the greatest ROS production in all samples. Electroacupuncture treatment induced a significantly increased neutrophil ROS response when the cells were stimulated with PMA. Phorbal 12-myris tate 13 acetate induces ROS production by maximal intracellular simulation of enzymatic action, primarily through complete activation of protein kinase C at the concentration tested. However, EA treatment significantly reduced ROS production by neutrophil s when the cells were stimulated with 1 g/ml of LPS, but not with lower concentrations (0.1 and 0.01 g/ml). Lipopolysaccharide induces ROS through TLR4 mediated signaling and its associated inflammatory activation. Neutrophil ROS response ratios generated by neutrophils when the cells were stimulated with Zym, CA, and CE were not affected by EA. Acupuncture treatment did not affect the production of ROS by any of the stimulants utilized in these trials (Table 2 8). Between -group comparisons of post treatment samples of EA and sham groups were not significantly different (Table 2 10).

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62 Heparinized blood Stimulation and Equine Tumor Necrosis Factor Alpha (TNF ) Assay Tumor necrosis factor alpha production in whole blood cultures is a measure of the capacity of all the cells in a fixed volume sample of blood to mount a specific inflammatory response under conditions directly simulating blood in the circulation. The TNF production response is impacted by both the circulating numbers and fraction of monocytes (the cells primarily responsible for TNF production) and their fraction of circulating cells. After in vitro incubation of whole blood with only PBS added, TNF production in the blood samples obtained from two horses receiving AC treatment were greate r than 500 pg/ml. The high concentrations of TNF in the absence of an inducing stimulant may indicate contamination of the blood samples. Therefore, data derived from these horses were excluded from the statistical analyses. For all horses, production o f TNF was significantly greater in whole blood cultures stimulated with LPS, Zym, or CA than for cultures containing only PBS. However, CE proved to be an ineffective stimulant for TNF production in this whole blood culture system. Thus, the data from CE stimulated whole blood cultures were excluded from within-group analysis. Electroacupuncture treatment significantly reduced TNF production in response to Zym, CA, CA+CE, CA+LPS, CE+LPS, and CA+CE+LPS (Table 2 9). However, AC treatment did not signi ficantly reduce TNF production with any stimulants used (Table 2 9). TNF production by whole blood culture containing Zym, CA+LPS, CE+LPS, and CA+CE+LPS of horses receiving EA treatment was significantly lower than that receiving AC treatment; p values were 0.03, 0.06, <0.01, and 0.06, respectively. Moreover, there were no significantly differences in TNF production by whole blood culture containing LPS, CA, CE, and CA+CE after EA and AC treatment (Table 2 10).

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63 Discussion Acupoints that possess immune stimulatory properties include LI 4, LI 11, ST 36, GB 39, SP 6, GV 14, BL 11, BL 20, BL 23, BL 24, BL 25, BL 27, BL 28, and CV 12. 137 Our study investigated the immune modulatory effe cts of AC and EA when stimulations were performed at LI 4, LI 11, and GV 14. These acupoints have been demonstrated to possess anti inflammatory and analgesic properties in rats.147 They are located on the cranial p art of the body including the front limbs. Their locations offer a safe and easy approach for the veterinary acupuncturist to insert an acupuncture needle. LI 4, also known as He -gu, is one of the most studied acupoints in humans and laboratory animals. I t has been used for reducing pain and alleviating inflammation originating in the face and jaw regions.148,149 An increase in the neural activity of the spinal cord at the 6th cervical vertebra to the 1st thoracic v ertebra after AC at LI 4 indicated that sensory input originating from AC is partly involved in the analgesic and therapeutic mechanisms of this acupoint.150 During acupoint stimula tion, cutaneous and somatic sensory inputs were transmitted to the spinal cord. Sensory inputs generated from LI 4 and LI 11 were transmitted through the brachial plexus, while the sensory input originating from GV 14 was transmitted to the spinal cord via the dorsal cutaneous branches of the dorsal branches of the cervical and thoracic nerves. Details of the cutaneous and somatic sensory innervations are shown in Table 2 11. Electroacupuncture at LI 4 has been shown to increase the functional magnetic resonance imaging (fMRI) signal in several parts of the brain including, the pre -central gyrus, postcentral gyrus, and putamen, and insula, while AC decreased the fMRI signal in the posterior cingulate, superior temporal gyrus, putamen, and insula.151 These results suggested that EA and AC recruited different neural networks for their treatment mechanisms.151

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64 GV14, also know as Da -zhui is recommended in TCVM for treating diseas e patterns caused by Excess Heat, including fever, skin rash, seizure, sweat, and other abnormalities such as cough and cervical pain.135 Recent research showed that a series of AC at GV 14, LI 11, SP 10, and ST 36 decreased the total number of peripherally circulating leukocytes and the number of lymphocytes.138 Acupuncture has been shown to decrea se the level of serum IgM in humans suffering from Behcets disease.152 The disease is characterized by a chronic inflammation of blood vessels throughout the body. This inflammation is likely caused by an anti -endothelial antibody (i.e., IgM). Patients receiving AC treatment also benefited from a lower recurrence rate in clinical signs than did patients receiving conventional treatment. However, how AC reduced serum IgM was unknown.152 Decreases in serum IgM and increases in serum IgG after AC also have been demonstrated in human asthma.153 AC at CV 3, GV 4, GV 14, GV 20, SP 6, SP 9, LI 4, LI 11, ST 30, ST 36, KI 3, KI 5, TH 5, LIV 2, B L 28, BL 31, BL 43, and PC 6 for 12 treatments over a period of one month has been shown to be beneficial in the treatment of chronic pelvic inflammatory disease in women.154 After the last treatment, serum IgM was decreased, while total serum globulin was increased. The benefit of AC to immune modulation was demonstrated in stress related immune suppression in competing athletes. Daily AC at LI 4, ST 36, ST 6, and LU -6 for 15 minutes has been shown to inhibit exercise -induced decrease of salivary IgA, and exerciseinduced increase of salivary cortisol.155 After the treatments, subjects reported high sc ores for their mental wellness. Acupuncture inhibition of exercise induced alteration of immunological and endocrinological parameters in this study was positively correlated with psychological parameters. Therefore, the results were potentially due to pla cebo effects. Electroacupuncture and AC treatment regimes in our study did not alter the levels of circulating immunoglobulin.

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65 This may be due to the fact that immunoglobulin is a product of the secondary immune response, and a longer period of time for th e immunoglobulinproducing cells to adapt to the immunological stimulation might be required. Phorbal 12 -myristate 13 acetate caused maximum increase in ROS production by mimicking the action of diacylglycerol which activates protein kinase C (PKC), the m ajor cellular phorbal ester receptor.156,157 Lipopolysaccharide induces ROS production through the binding of the Toll like receptor 4 (TLR4) receptor and its associated signaling cascade. Electroacupuncture signifi cantly increased the neutrophil ROS response when cells were stimulated by PMA, but significantly decreased the response when cells were stimulated with LPS at a concentration of 1 g/ml. Phorbal 12 -myristate 13 acetate induces maximal ROS responses in hor se neutrophils, essentially representing the total enzymatic capacity of cells to produce ROS. In contrast, LPS induces ROS production based on extracellular signaling and is limited by the level of TLR4 receptor expression, and the capacity of neutrophils to amplify TLR4 mediated signaling. Thus, the enhancement of PMA induced ROS and reduction in maximal LPS induced ROS may represent a down -modulation in the level of proinflammatory receptors (such as TLR4), but an increase in the general enzyme levels needed to produce ROS. This suggests that a complex set of regulatory processes in the function of neutrophils are induced by EA, but the same regulatory level is not reached by simple AC stimulation. However, there is no research evidence demonstrating up regulation of PKC by either EA or AC. Previous research in rats showed that EA stimulation at ST 36, GV 20, Yin-tang, and ST 40 could reverse an over -expression of PKA and PKC in the hippocampus.158 Neutrophil -ROS responses to other stimulants did not differ relative to EA treatment. Each was due to signaling through different families of cell surface receptors. The response to

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66 zymosan is regulated by several receptors and the lack of significant change in the overa ll response may have been due to a shift in the expression of individual receptors that gave an equivalent balance in the response. The responses to both the fungal antigens utilized were rather weak, so a larger enhancement or depression of the response w ould have been needed to measure a significant level of change. The EA induced increase in ROS production of neutrophils when stimulated with PMA corresponded to the results of earlier research, which demonstrated an increase in reactive oxygen burst (RB) activity of neutrophils following a series of AC treatment.139 In the previous research, RB of neutrophils was induced by either priming with initial incubation with recombinant TNF followed by receptor stimulati on with N -formyl -methionyl leucyl phenylalanine (FMLP), FMLP activation alone, or by phagocytosis of E. coli. N -formyl methionyl -leucyl phenylalanine is derived from bacterial protein degradation.159 Receptors for FMLP can be found on the surface of granulocytes.160 Binding of FMLP receptors activates phospholipase C and causes increase production of diacylglecerol and inositol triphosphate. These secondary messengers later activate the PKC, which is also activated by P MA. Lipopolysaccharide activates the TLR4 present on the cell surface, which activates MAP kinase (Erk) and nuclear factor B (NF B), which in turn, control transcription of target genes.161 Decrease in ROS production by neutrophils after the cells were stimulated with LPS suggested that EA might possess an anti inflammatory effect. However, its mechanism is still inconclusive. Previous research on the effects of EA at ST 36 in experimentally induced cutaneous anaphylaxis in mice demonstrated that EA inhibited cutaneous anaphylaxis and inhibited the release of IL 6.162 The anti anaphylactic activity of EA was associated with a

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67 reduction in DNA binding activity of NF B, as determined by Western blot analysis and transcription factor enzyme-linked immunoassay. In our assessment of TNF production in whole blood cultures we found that LPS was the most potent stimulant. Lipopolysaccharide was more potent than CA, CE, or Zym. Culture filtrate antigen of A. fumigatus was the weakest stimulant of TNF production in whole blood cultures. TNF production induced by Zym was i nconsistent, and only two horses from each group responded to Zym. The cause of inconsistent responses to Zym is unknown, but may be due partly to individual subject differences in expression of the family of receptors regulating Zym response. There was ev idence of a synergistic effect when CA or CE was individually, or if both were added to LPS. Electroacupuncture generally suppressed TNF production in whole blood cultures, except when LPS was added alone. Results of cultures with multiple stimulants com bined with LPS (i.e. CA+LPS, CE+LPS, and CA+CE+LPS) indicated that EA generally suppressed TNF production. In the AC group, TNF production in whole blood cultures was not significantly different from the baseline samples prior to treatment. Tumor necro sis factor alpha is an inflammatory cytokine. It is an important component of the early response of the innate immune system. Therefore, suppression of TNF release from immune cells, as demonstrated in this study, may partly play a role in how EA exert i ts anti inflammatory action. Until recently, it has not been clear how AC and EA modulate immune functions. Previous research in electrical stimulation of the vagus nerve has been shown to attenuate endotoxin-induced shock in rats. This modulation was char acterized by an inhibition of TNF synthesis in the liver and a reduction of the TNF concentration in plasma.81 The inhibition of endotoxic shock was suggested to be partly due to the ability of EA to restore the hepatic

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68 metabolism of rats subjected to LPS -induced shock. The restoration of hepatic metabolism was characterized by the ability of the hepatocytes to maintain their glycogen storage level and the activity of the enzyme sorbital dehydrogenase (SDH), gluc ose 6 -phosphate, and ATPase.163 The vagus nerve is a major part of the cholinergic nervous system and contains both afferent and efferent nerve fibers for both somatic and visceral tissues.79,80 The cholinergic efferent signals have been demonstrated to cause vasodilatation in visceral tissues and alter the contractility of visceral smooth muscle, including intestines and uterus.164,165 Electrical stimulation of the efferent vagus nerve attenuated macrophage -dependent intestinal inflammation in the post -operative ileus in mice.166 This attenuation has been suggested to be due in part to the activ ation of the alpha 7 nicotinic acetylcholine receptors on the macrophage. Activation of endothelium nicotinic acetylcholine receptors also has been shown to reduce the expression of the adhesion molecule and chemokine release.167 Therefore, cholinergic dependent anti inflammation may be due partly to an inhibition of inflammatory cell migration to the site of tissue damage, and down regulation of the production of pro-inflammatory cytokines that cause inflammation. T his finding may be used to explain how EA reduces inflammation in diseases of the visceral organs.76 Whether AC possesses the same therapeutic mechanism or not is still inconclusive and needs further investigation. Electroacupuncture and acupuncture in our study did not produce immunological modulation that was fully comparable with those of previous studies. In these studies, EA treatment, but not AC treatment, significantly modulated neutrophil production of ROS induced with either PMA or LPS, and modulated TNF production by blood cells stimulated wi th LPS, both alone and in combination with other stimuli tested. This could be because the horses used in our study were healthy. Thus, they might not gain as much benefit from either EA or AC

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69 treatment as sick people or animals. According to one of the TCVM treatment principles, the AC and EA treatment objective is to cure an imbalance of the health status. Healthy horses can be assumed to able to maintain their health. Thus, the treatment should do neither harm, nor provide significant benefit to them. Moreover, in a real clinical TCVM treatment, multiple EA or AC treatments are performed over a long period of time at a pre-defined interval, such as weekly treatment for five to ten consecutive weeks. The clinical AC/EA treatment also differs from our AC/ EA treatment in terms of the numbers of acupoints being stimulated, and how each acupoint was selected. However, our study suggested that short term aggressive stimulation at LI 4. LI 11, and GV 14 has a potential impact on immunological function. Despite the lack of a clear impact on neutrophil ROS response to EA and AC treatments, both EA and AC reduced the level of TNF production in whole blood culture after exposure to individual and combinations of microbial stimulants. Our results also suggest that the immunomodulatory effect of EA and AC may be partly due to a modulation of the innate immune response mediated primarily by monocytes, and that EA is more effective than AC.

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70 Table 2 1. Anatomical location of acupoints and treatment indications. Acupoi nts Anatomical location Treatment indications LI 4 Distal and medial to base of second metacarpus. Facial paralysis, dental pain, sore throat, anhydrosis, fever, and immunodeficiency. LI 11 In depression cranial to elbow in the transverse cubital crease. Fever, dental pain, uveitis, sore throat, seizure, abdominal pain, diarrhea, and paralysis of front limb. GV 14 Dorsal midline at depression in cervicothoracic vertebral space (C7 T1) Fever, cough, heaves, cervical stiffness, skin rash, and seizure. (So urce: Xie H, Trevisanello L. Equine Transpositional Acupoints In: Xie H,Preast V, eds. Xie's veterinary acupuncture. 2007; page 27 87.135) Table 2 2. Demographic information on horses in the EA and AC groups. Group Gelding Mare Total Age in year (mean SD) Age range (years) EA 12 0 12 8.2 1.1 6 10 AC 9 3 12 7.4 2.1 3 10 EA = electroacupuncture, AC = acupuncture. Table 2 3. Me anSE concentration of immunoglobulin isotypes (x105 ng/ml) from pre -treatment samples of EA and AC groups. Ig isotype Group EA (x10 5 ng/ml) AC (x10 5 ng/ml) IgA 10.761 0.877 11.410 1.312 IgM 6.773 0.701 7.326 1.120 IgGa 33.603 2.183 28.701 2.088 IgGb 55.046 3.489 57.810 4.057 IgG(T) 46.295 2.228 39.125 2.938 EA = electroacupuncture, AC = acupuncture.

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71 Table 2 4. MeanSE neutrophil ROS response ratio from pre -treatment samples of EA and AC groups. Stimulant Group EA AC PMA 20.60 0.80 2 0.89 1.03 LPS (1.0 g/ml) 1.52 0.13 1.69 0.15 LPS (0.1 g/ml) 1.33 0.08 1.54 0.13 LPS (0.01 g/ml) 1.26 0.06 1.40 0.08 Zym 1.79 0.13 1.95 0.09 CA 1.19 0.06 1.34 0.11 CE 1.10 0.03 1.16 0.02 ROS = reactive oxygen species, EA = electroacup uncture, AC = acupuncture, PMA = phorbol 12myristate 13 acetate, LPS = lipopolysaccharide, Zym = zymosan, CA = cellular antigen of A. fumigatus CE = culture extract antigen from static growth of A. fumigatus

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72 Table 2 5. MeanSE TNFconcentrations (pg /ml) from pre treatment samples of EA and AC groups. Stimulant Group EA AC None 0 0 0 0 PBS 0 0 0 0 LPS 3893 576 5647 915 Zym 530 214 1029 264 CA 4487 891 4492 464 CE 0 0 0 0 CA+CE 4309 737 5715 938 CA+LPS 4801 668 7004 963 CE+LP S 3759 344 6595 896 CA+CE+LPS 5282 605 8008 1064 EA = electroacupuncture, AC = acupuncture, PMA = phorbol 12-myristate 13 acetate, LPS = lipopolysaccharide, Zym = zymosan, CA = cellular antigen of A. fumigatus CE = culture extract antigen from stati c growth of A. fumigatus

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73 Table 2 6. Mann -Whitney Rank Sum test statistics between -group comparisons of pre -treatment samples in EA and AC groups. Ig isotype P value ROS of neutrophils by stimulant P value TNF production of whole blood stimulation by stimulant P value IgA 0.843 PMA 0.608 None 1.000 IgM 0.977 LPS 1.0 0.468 PBS 1.000 IgGa *0.089 LPS 0.1 0.401 LPS 0.148 IgGb 0.843 LPS 0.01 0.217 Zym 0.180 IgG(T) *0.089 Zym 0.316 CA 0.624 CA 0.562 CE 1.000 CE 0.169 CA+CE 0.219 CA+LPS *0.093 CE+LPS *0.006 CA+CE+LPS *0.059 ROS = reactive oxygen species, PMA = phorbol 12 myristate 13 acetate, LPS = lipopolysaccharide, Zym = zymosan, CA = cellular antigen of A. fumigatus CE = culture extract antigen from static growth of A. fumigatus # = outlier and extreme outlier data determined by box -plot were excluded from the analysis, = significant difference was accepted at p 0.1. Table 2 7. Mean SE immunoglobulin isotype concentrations (x105 ng/ml) of pre and post EA and AC treatment s, and test statistics of within -group comparisons. Ig isotype Treatment EA (12 AC (12 Pre (x10 5 ng/ml) Post (x10 5 ng/ml) P value Pre (x10 5 ng/ml) Post (x10 5 ng/ml) P value IgA 10.761 0.877 10.820 0.966 0.57 11.410 1.312 11.020 1.185 0.38 IgM 6.773 0.701 6.654 0.699 0.52 7.326 1.120 7.2 058 1.141 0.42 IgGa 33.603 2.183 32.845 2.041 0.20 28.701 2.088 28.345 2.105 0.47 IgGb 55.046 3.489 54.596 3.482 0.47 57.810 4.057 58.868 4.945 0.83 IgG(T) 46.295 2.228 46.657 2.129 0.69 39.125 2.938 40.597 3.172 0.69 Ig = immunoglobulin isotype, EA = electroacupuncture, AC = acupuncture.

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74 Table 2 8. Mean SD neutrophil ROS response ratios of pre and post EA and AC treatments, and test statistics of within -group comparisons. Stimulant Treatment EA (12 AC (12 Pre Post P value Pre Post P value PMA 20.60 0.80 23.19 1.13 <0.01 20.89 1.03 22.10 1.34 0.55 LPS1.0 ( g/ml) 1.52 0.13 1.23 0.03 0.05 1.69 0.15 1.47 0.12 0.19 LPS0.1 ( g/ml) 1.33 0.08 1.25 0.06 0.43 1.54 0.1 3 1.38 0.09 0.19 LPS0.01 ( g/ml) 1.26 0.06 1.33 0.13 0.58 1.40 0.08 1.27 0.06 0.27 Zym 1.79 0.13 2.11 0.06 0.15 1.95 0.09 2.06 0.08 0.27 CA 1.19 0.06 1.16 0.02 0.72 1.34 0.11 1.17 0.04 0.19 CE 1.10 0.03 1.15 0.02 0.23 1.16 0.02 1.15 0. 03 0.57 EA = electroacupuncture, AC = acupuncture, PMA = phorbol 12-myristate 13 acetate, LPS = lipopolysaccharide, Zym = zymosan, CA = cellular antigen of A. fumigatus CE = culture extract antigen from static growth of A. fumigatus

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75 Table 2 9. Mean SD TNF concentration (in pg/ml) of pre and post EA and AC treatments, and test statistics of within -group comparisons. Stimulant Treatment EA (12 AC ( 10 Pre Post P value Pre Post P value None 0 0 0 0 1.00 0 0 0 0 1.00 PBS 0 0 0 0 1.00 0 0 0 0 1.00 LPS 3893 576 3834 611 0.97 5647 915 5014 643 0.19 Zym 530 214 14 14 0.06 1029 264 706 261 0.54 CA 4487 891 2675 713 0.06 4492 464 4292 783 0.64 CE 0 0 41 33 1.00 0 0 0 0 1.00 CA+CE 4309 737 2889 62 0.07 571 5 938 4594 908 0.30 CA+LPS 4801 668 3646 468 0.06 7004 963 5821 766 0.49 CE+LPS 3759 344 2851 319 0.08 6595 896 5578 735 0.49 CA+CE+LPS 5282 605 4125 693 0.03 8008 1064 5873 661 0.20 EA = electroacupuncture, AC = acupuncture, PMA = phorbo l 12 -myristate 13 acetate, LPS = lipopolysaccharide, Zym = zymosan, CA = cellular antigen of A. fumigatus CE = culture extract antigen from static growth of A. fumigatus

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76 Table 2 10. Mann-Whitney Rank Sum test statistics for between -group comparisons of post treatment data in EA and AC groups. Ig isotype P value ROS of neutrophils by stimulant P value TNF production of whole blood stimulation by stimulant P value IgA 0.79 PMA 0.56 None 1.00 IgM 1.00 LPS 1.0 0.19 PBS 1.00 IgGa 0.114 LPS 0.1 0.30 LPS 0.16 IgGb 0.608 LPS 0.01 0.65 Zym *0.03 IgG(T) 0.123 Zym 0.59 CA 0.12 CA 0.84 CE 0.57 CE 0.97 CA+CE 0.19 CA+LPS *0.06 CE+LPS *<0.01 CA+CE+LPS *0.06 ROS = reactive oxygen species, PMA = phorbol 12 myristate 13 acetate, LPS = lipopoly saccharide, Zym = zymosan, CA = cellular antigen of A. fumigatus CE = culture extract antigen from static growth of A. fumigatus # = outlier data determined by box plot were excluded from the analysis, = significant difference was accepted at p 0.1. Table 2 11. Cutaneous and muscle innervations of acupoints being stimulated. Acupoint Cutaneous nerve Muscle / innervation LI 4 Medial cutaneous antebracheal of the musculocutaneous nerve None / medial palmarmetacarpal LI 11 Cranial cutaneous antebrac hial, lateral cutaneous antebrachial nerve of the superficial branch of the radial nerve, and lateral cutaneous branch of 2nd thoracic nerve (component of the intercostobrachial nerve) Triceps (lateral head) / radial Brachialis / musculocutaneous and radi al GV 14 Dorsal branch of local cervical and local thoracic spinal. (Source: Budras K D, Sack WO, Rck S, et al. Anatomy of the horse : an illustrated text. 4th ed. Hannover: Schltersche, 2003. Blythe LL, Kitchell RL. Electrophysiologic studies of the thoracic limb of the horse. Am J Vet Res 1982;43:15111524 168,169)

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77 CHAPTER 3 COMPARISON OF INDUCT ANCE PLETHYSMOGRAPHY AND PNEUMOTACHOGRAPHY AND THE RAPID PARTIAL FORCED EXPIRATION MANEUVER FOR DIAGNOS IS OF EQUINE LOWER AIRWAY INFLAMM ATORY DISEASE. Introduction Chronic lower airway inflammatory disease is one of the most common causes of poor performance in horses. These include inflammatory airway disease (IAD), recurrent airway obstruction (RAO) or heaves, and sum mer pasture associated obstructive pulmonary disease (SPAOPD). Inflammatory airway disease is diagnosed in young training racehorses, while the other two diseases are commonly diagnosed in older horses and usually associated with poor stable management.170 It is still unclear whether horses that have been diagnosed for IAD at on early age may progress to RAO or SPAOPD. Horses that are being kept in a poorly ventilated or dusty environment are at a higher risk of developing the diseases. Affected horses show signs of chronic coughing, dyspnea, exercise intolerance, and poor performance. There are also increases in respiratory rate, nasal discharge, and neutrophils in broncho alv eolar lavage fluid (BALf).86 Inflammatory airway disease, RAO, and SPAOPD are thought to share many similarities in their disease pathogenesis. In chronic long -standing cases, alteration of the pulmonary and airway histolo gical structures in response to the persistent exposure of a causative antigen results in increased airway resistance and a decreased gas exchange capacity of the lung.171 Until recently, there has been no single diagnostic procedure that can be used to accurately distinguish these diseases. Current diag nosis is based on case history, clinical signs, broncho alveolar fluid (BALf) cytology, and response to the treatments. The ability to recognize these diseases at an early stage is important. However, this is difficult due to the lack of specific clinical manifestations and, more importantly, affected horses may not show clinical signs of respiratory problems until the amount of pulmonary tissue affected is greater than the functional

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78 reserve. For these reasons, many affected horses are undiagnosed due to t he sub-clinical nature of these diseases. Several progressive diagnostic methods have been developed in attempting to identify early stages of the disease, including intra -pleural pressure, histamine bronchoprovocation, and forced expiration.172175 Airway hyper responsiveness is thought to be a sequel to chronic airway inflammation. Inflammation of airways increases mucus secretion and accumulation in the airways.176 It also increases the respiratory resistance caused by contraction of the airway smooth muscles; this is triggered by inflammatory cytokines being released locally by infiltrated leukocytes. Airway hyper responsiveness can be provoked by exposing the affected airw ays to a known irritant or by an administration of exogenous histamine.144,171,177 In human medicine, testing a physiological response of the airway with histamine is called histamine bronchoprovocation (HB) or hist amine challenge. Together with the pulmonary function test, HB has been used in the diagnosis and determination of the severity of chronic respiratory problems like bronchial asthma and chronic obstructive pulmonary disease (COPD).178 Comparison of a series of HB tests over time has been suggested to be useful for objectively determining improvement in clinical signs and response to therapy. The effects of HB were determined by pulmonary function testing, conducted immediately after HB. The most commonly performed test in human respiratory clinics is forced expiration (FE).179 Computed parameters derived from the test are collectively called pulmonary function test parameters (PFTPs). Commonly computed PFTPs of FE include forced expiratory volume in x second (FEVx), forced expiratory volume (FEV) or forced vital capacity (FVC), peak expiratory flow (PEF), and forced expira tory volume in 1 second/FEV ratio (FEV1/FEV). The most commonly reported FEVx is the FEV1. Expiratory flow rates at 25, 50, and 75% of

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79 FVC has been expired (MEF25%, MEF50%, and MEF75%) also can be computed.179 These PFTPs reflect biomechanical characteristics of the airways and pulmonary tissues, incl uding lung volume, airway resistance, and lung compliance. Deviation of these parameters from reference values can be used as indicator of pathology in pulmonary tissues. Pulmonary disease produces alteration in PFTPs at the end of expiration. When the ai rflow rate and expiratory volume are plotted, flow volume (FV) loops are generated. Pulmonary pathology affects both PFTPs and the FV loop. The FV loop of a patient suffering from obstructive pulmonary disease demonstrates a scoop out characteristic after peak flow. This scooped out characteristic indicates decreased airflow rate caused by narrowing of the small airways, is a common pathological change found in lower airway inflammatory diseases.180,181 The forced expiration test procedure in humans is self -induced and voluntary. It is accomplished with coaching by respiratory therapist or diagnostician. The test procedure is explained to the patient before the recording is made. In some instances, a patient may be asked to practice the test before recording of the data is attempted. The quality of the test depends on good coaching and the cooporation of the patient. Explaining the details of the test to the patient, and emphasizing that the reliability of the test depends exhaling as rapidly and as fully as possible is critical.182 Forced expiration is considered a superior method of testing pulmonary function. It is reliable and consistent.183 In equine medicine, HB also has been described.174,184,185 By comparing respiratory function following administration of various concentrations of histamine to the airways, a histamine response curve h as been generated. Results suggested that the technique provides a non invasive alternative diagnostic technique for clinical practices.174

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80 In equine practices, the measurement of FE and computation of the associat ed PFTPs have been described.175 The maneuver has been accomplis hed by exposing an airtight -sealed airway and maximally inflating the lung to its total lung capacity (TLC) to a negative pressure. A 90 cm long by 22 mm internal diameter nasotracheal tube with inflatable cuff was used to connect the lower airways with the negative pressure reservoir. Airflow and respiratory volume were indirectly computed by measuring the instantaneous change in pressure in a vacuum reservoir. The flow volume (FV) loop thus derived was diagnostic for obstructive pathologies of airways.175 Reproducibility of the computed PFTPs in horses was confirmed. However, the flow volume loop generated by this method possessed a long plateau phase in the middle of the flow indicating that airflow limitation and high resistance in the system were likely caused by the nasotracheal tube. The experimental apparatus dev eloped for this study created a system to directly measure airflow generated by the forced expiration maneuver, and to eliminate the limitation of previously described systems. Objectives in this study include: To develop an electro -mechanical system with the capability of directly measuring the airflow generated by FE in horses and with minimal airflow resistance artifact. To investigate the effect of negative pressure applied to the airways of horses and to determine a suitable negative pressure for induc ing reproducible FE measurements. To compare the diagnostic value of the PFTPs computed from the FE maneuver, and with a histamine response curve from HB generated by respiratory inductance plethysmography / pneumotachography, to BALf cytology. Methods Subject Twenty -four mature Thoroughbred horses with no evidence of cardiovascular, respiratory or musculoskeletal problems by physical exam were used in this study. The horses were assembled into groups of two to four and kept in paddocks with shelters. Physi cal fitness was

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81 maintained with eight minutes of exercise on a treadmill three times per week. The exercise included two minutes of warm up trotting, four minutes of cantering, and two minutes of cool down trotting. The horses were fed twice daily with com mercial concentrate. Good quality hay and water were available ad libidum None of the horses had any history of respiratory problems and received no medications during the two weeks prior to the experiment. Protocol for animal use was approved by the University of Florida Institutional Animal Care and Use Committee (Permit A 130). Study Design The airway hyper responsiveness of the horses was determined by challenge with histamine diphosphate (HD). The histamine response curve was generated using inductan ce plethysmography/pneumotachography. The test was repeated twice at three to five week interval to determine the repeatability of the test. After each HB, BALf cytology of each horse was evaluated. Five weeks after the last HB, a pulmonary function test w as performed in these horses with rapid partial FE maneuver. The results of the histamine response curve from HB, BALf cytology, and PFTPs derived from rapid partial FE maneuver were compared, and their correlations were investigated. Histamine Bronchoprov ocation and Respiratory Inductance Plethysmography and Pneomotachography (Open PlethTM) Histamine bronchoprovovation and respiratory inductance plethysmography and pneumotachography were conducted indoors in a well -ventilated airconditioned room. Two horse s were brought into the room at a time to minimize separation anxiety. Horses subjected to HB test were physically restrained in a stall, and the HB was performed on one horse at a time. After a brief clinical examination, the horse was chemically restrain ed by intravenous administration of 0.75 mg/kg of xylazine hydrochloride into the left external jugular vein. Five

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82 minutes after tranquilization, the external nares of the horse were cleaned with damp -gauze. Pulmonary function was determined by respiratory inductance plethysmography and pneumotachography (Open PlethTM, Ambulatory Monitoring, Inc, NY) before and after exposure to HD at various concentrations using the following protocol. After calibration of the inductance plethysmography/pneumotachography, the test was performed according to the method provided by the manufacturer:186 Baseline data on pulmonary function were obtained following two minutes of a normal saline nebulization to the airway by the equine aero mask tube nebulizer. Nebulization was generated by the PronebUltraII compressor nebulizer system with the Pari LC Plus Reusable Nebulizer (Pari VA USA). The solution was nebulized at a rate of 0.148 0.018 ml/min. Pulmonary function data were recorded for two minutes immediately after the nebulization. Collected data included inductance plethysmography and pneumotachgraphy. These data served as a baseline. His tamine bronchoprovocation was performed by nebulization of HD, as described by Hoffman et al.187 Briefly, histamine diphosphate (HD) (MP Biomedicals #100343) was diluted in sterile normal saline s olution (Baxter #NDC 0338004804) to a final concentrations of 2.0, 4.0, 8.0, 16.0, and 32.0 mg/ml. Histamine bronchoprovocation was begun with nebulization of 2 mg/ml HD for 2 minutes and immediately followed by 2 minutes of data collection. The procedur e was then repeated by substituting 2.0 mg/ml of HD with 4.0, 8.0, 16.0, and 32.0 mg/ml of HD, sequentially. Alteration in flow rate after each concentration of HD nebulization was plotted against histamine concentration using FlowmetricTM software. The co ncentration of histamine that caused a 35% increase in delta flow (PC35 delta flow) was computed based on the histamine dose response curve. This PC35 delta flow is also referred to as the concentration of histamine that results in a 35% reduction in dynam ic compliance of the airway. The degree of airway hyper -sensitivity was categorized according to the manufacturers protocol (Table 3 1). Bronchoalveolar Lavage. Broncho alveolar lavage (BAL) was performed within 1 hour after HB. Briefly, the horse was che mically sedated with intravenous administration of 0.75 mg/kg of xylazine hydrochloride. After 5 10 minutes, the external opening of the right nasal cavity was cleaned with damped gauze, and a 3 -meter long fiberoptic endoscope with a biopsy channel (Pentax EG 2901XL) was inserted into the ventral nasal meatus of the right nasal cavity until reaching the

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83 nasopharynx. The tip of the endoscope was further advanced into the distal trachea. The epithelial lining of the trachea and bronchi were then desensitized by an infusion of 10 ml of 1% lidocaine hydrochloride. After desensitization, the endoscope was introduced into the left lung lobe until the tip of the endoscope was wedged in small bronchi. One hundred ml of 37C sterile normal saline was infused into the small bronchi via the biopsy channel of the endoscope. The fluid was immediately withdrawn via vacuum generated by pulling the plunger of the 60 ml syringe. The lavage procedure was repeated two times to yield a total volume of fluid instillation of 300 m l. The recovered BAL fluid (BALf) was pooled, and the recovery volume was measured with a 250 ml sterile cylinder. The BALf was filtrated through a double layer of sterile gauze prior to being transferred into a sterile glass container. The physical appear ance of BALf was noted, and the BALf sample was kept on ice until analysis. The total number of nucleated cells in the fresh BALf sample was determined by manual counting with a hemocytometer. The counting was done in 4 large squares in the corners of 2 co unting chambers. An average of the total number of cells in 2 chambers was calculated. Number of cells in 1 ml was computed by the following formula: Number of cells in 1 ml = Average of number of cells x 2500 (3 1) Two BALf cytological slides were pre pared with Cytospin (Shandon). Briefly, 2 3 drops of BALf were transferred into the Cyto -funnel, which was mounted on the microscopic slide that was pre loaded in a Cytospin rack. The slides were spun for 5 minutes at 100 RPM. The slides were air -dried and stained with Dip Quick stain (Jorgensen Laboratories Inc., CO). BALf cytology was determined under the light microscope. Two hundred nucleated-cells were evaluated and counted with a cell counter. All counts were made by the same person.

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84 Percentage of re covered tracheo -bronchial epithelial lining fluid (ELF) was determined by urea dilution.188190 Urea is a small molecule that can diffuse across the mucus membrane freely. In normal physiological circumstances, urea concentration in plasma and tracheo bronchial ELF is in equilibrium and equal in concentration. This fact has been confirmed in sheep by a direct measurement of pulmonary ELF urea concentration.191 Therefore if the BALf sample is being withdrawn immediately after instillation, the dilution of ELF in the BALf sample can be determined by measuring the urea concentrations of BALf and plasma samples simultaneously. Urea concentrations in plasma and BALf samples were analyzed by quantitative enzymatic colorimetric assay with Stanbio Enzymatic Urea Nitrogen (BUN) Procedure No. 2050 (Stanbio Laboratory, Texas). The assay was performed according to the manufacturers protocol. Briefly, urea in the sample is hydrolyze d by the enzyme urease to yield ammonia and carbon dioxide. The ammonium ions then react with a mixture of salicylate, sodium nitroprusside and hydrochlorite to yield a blue green chromophore. The intensity of the chromophore is evaluated by reading an abs orbance at 600 nm. The intensity of the color is proportional to the urea concentration in the sample. Concentration of urea in the samples was calculated based on an absorbance of the standard. Rapid Partial Forced Expiration Maneuver Unlike humans, it is impossible to control by coaching frequency and depth of active voluntary breathing patterns of animals. However, it is feasible to mimic the breathing pattern of an animal. The airways can be inflated with air at a controlled pressure to total lung capac ity (TLC) to mimic inspiration. Normal expiration, on the other hand, is caused by the force of elastic recoil properties of pulmonary tissues, chest wall, and relaxation of the diaphragm. Forced expiratory maneuver can be accomplished by emptying an airti ght, sealed airway with a suitable negative pressure system. Negative pressure causes more emptying of the air from the lung than

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85 the force of elastic recoil and muscle contraction. As a result, the functional reserve volume of the lung is reduced. With this information, a system that is capable of performing a rapid partial forced expiration maneuver in horses was designed. The system is constructed with five major electro/mechanical components. Negative pressure generator and vacuum reservoir. System for artificial inspiration. Airflow measurement apparatus. Airflow direction control system. Data acquisition system. Negative pressure generator and vacuum reservoir The negative pressure was generated by a vacuum pump (#2667V108 Gast Imfg Corp) driven by a n industrial motor (#6K702BA Dayton). The vacuum was stored in a custom -made 850 liters reservoir. The negative pressure reservoir was made of 4 steel tubes, connected in parallel by a 1 NPT PVC pipe. Each steel tube was 184.5 cm long by 38.5 cm internal diameter. A single manual relay switch was used to turn the vacuum pump on and off. The level of negative pressure was controlled with a 1 NPT manually actuated ball valve, which was installed between the vacuum pump and the negative pressure reservoir. When the valve was open, atmospheric air passes through the exhaust port and counters the negative pressure. To create the negative pressure, the valve was closed and the vacuum pump was turned on. Negative pressure in the vacuum reservoir was monitored by a pressure transducer (DP103, Validyne) directly connected to the negative pressure reservoir by a rigid polyethylene tube (Parker Parflex), (1/4 NPT external diameter and a 0.04 thick wall). The pressure transducer was calibrated with a U -tube mercury manometer (#1223, Dwyer). Signals generated by the pressure transducer were transferred to the data acquisition hardware and were recorded in a portable

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86 computer by the data acquisition software. Details about data acquisition are described in the section on the data acquisition system. System for artificial inspiration To mimic inspiration, an airtight sealed airway was pressurized with atmospheric air. Because the airway was manually inflated, the following concerns were addressed. To mimic an inspirat ion pattern of the animal, the airways must be slowly inflated to its TLC. A person performing artificial inspiration must be able to monitor the inflated airway continuously and be able to quickly adjust the airway pressure as needed. Artificial inspiration must overcome the animals physiological respiratory drive. Because atmospheric air is used to inflate the airways, introduction of air born particles into the airway must be prevented. Based on these concerns, a 1300-watt detachable blower of Shop -Vac (#7230997, ShopVac Corporation, Williamsport, PA) that is capable of generating a maximum airflow greater than 3000 standard liter per minute (SLPM) was used. This blower was also used in an airflow calibration process of the laminar flow element (LFE). T he method of LFE calibration is described in the airflow measurement system. To address the first concern, the airflow rate generated by the blower was controlled. A variable transformer capable of transforming 120 volts of alternating current (AC) to 0 1 40 volts AC (Powerstat) (Figure 3 1) was used to control electrical voltage current to the blower. The amount of airflow generated by the blower was positively correlated to the amount of voltage supplied by the variable transformer. Gradually increasing t he electrical current to the blower slowly increased the amount of the airflow, and gently inflated the airway. To address the second concern, pressure in the airways was monitored visually by an analog pressure gauge (#LPG1, Dwyer) mounted in the system manifold near the horse. A

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87 manually actuated 2 NPT PVC valve was installed in the manifold to serve as a pressure relief valve to prevent over -inflation of the airway. Airway pressure was also measured by a pressure transducer (DP45 Validyne) that connect ed to the manifold close to the junction of the endotracheal tube with 1/4 NPT external diameter and a 0.04 thick wall of rigid polyethylene tube (Parker Parflex). The pressure transducer was calibrated with a U -tube water manometer (#1223 Dwyer). Airway pressure signals originated from the pressure transducer were transferred to the data acquisition system and recorded by data acquisition software. Normal respiration is controlled by the autonomic nervous system, which responds to signals from chemorecep tors in the medulla oblongata (a change of pH) and the carotid and aortic bodies (a change in partial pressure of oxygen and carbon dioxide in blood).192 The respiratory center was over ruled by hyperventilation at a rate of 2530 times per minute, approximately twice the normal resting respiratory rate and induction of hyperoxia and hypocapnea. Decreasing pCO2 and increasing pO2 in the blood resulted in an increase in the blood pH and suppressed the respiratory center. The airway inflati on method in the system was designed to bypass the upper respiratory tract, and was specifically measure biomechanical properties of the lower airways. More specifically, the system excluded airflow resistance caused by the anatomical structure of the upp er airways. However, because the artificial inspiration had bypassed all of the anatomical and physiological mechanisms in the upper respiratory tract required for trapping air -born particles, the air used for insuflation was filtrated. Airflow measuremen t apparatus Airflow generated by rapid partial FE was measured using differential pressure ( P) generated across the laminar flow element (LFE). In an ideal laminar flow, fluid molecules move

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88 in parallel along the length of the flow path, and without turbulence. The differential pressure drop can be measured across the LFE. Airflow measurement in this system was based on the physics of Hagen-Poiseuilles equation, which quantifies the relationship between pressure drop and flow of fluid as:193 P = 8 4 (3 2) The formula can be re -written as: Q = |P1 P24 / 8 L (3 3) When: Q = Volumetric flow rate. P1 = Static pressure at the inlet. P2 = Static pressure at the outlet. = Mathematical constant (approximately 3.14 1592654). r = Radius of the pipe. = (eta) absolute viscosity of the fluid. L = Length of the pipe. Q = K( P/ ) (3 4) In this case K is a constant factor deter mined by the geometry of the flow restriction. The equation shows a linear relationship between volumetric flow rate (Q), differential pressure ( P), and fluid viscosity ( ) in a simple form. To measure the P generated by airflow, LFE (50MC2 4 ID, Meria m) was installed according to the companys installation recommendation. Briefly, a 4 NPT of 40 straight PVC pipe and a 4 NPT of 20 straight PVC pipe were installed upstream and downstream of the LFE, respectively. Upstream, the pipe was linearly conne cted to the manifold used for artificial inspiration. Differential pressure across the LFE was measured by a pressure transducer (DP45, Validyne) connected to the laminar flow element with 1.5 meters of rigid polyethylene tubes (Parker Parflex) having a 1/ 4 NPT external diameter and a 0.04 thick wall. The pressure

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89 transducer was calibrated with a water manometer. Differential pressure signals generated by airflow were transferred to the data acquisition system and recorded by data acquisition software. A n additional inline filter made of two layers of 1.5 mm2 nylon mesh was installed between the artificial inspiration component and LFE to trap dirt and secretion prior to reaching the LFE. Differential pressure across LFE generated by airflow was calibrate d with a NIST traceable mass flow element (MFE) (8104 1416 FM, Matheson) prior to each experiment. In the calibration process, LFE and the connected PCV pipe were temporally disconnected from the whole apparatus. The air blower and the MFE were connected t o the entry port and exit port of the LFE, respectively. All of the connecting points were sealed with electrical tape to prevent air leakage. To obtain calibration data, an air blower (under control of a variable transformer) was used to generate airflow through the LFE at different flow rates. Airflow rate data from MFE direct readings and P data generated by a particular airflow were recorded. The calibration process was repeated at the end of the experiment each day. Temperature of airflow in the syste m was measured by a fast response thermocouple probe (SRTC TT T 4036, Omega) installed proximally to the LFE. The thermocouple probe was connected to the meter (DP 41B, Omega) with a direct digital readout for temperature value and a capability for genera ting a digital signal output. The temperature digital signal output was transferred to the signal interface prior to being connected to the analog digital converter. Pressure differential and temperature data were recorded with data acquisition software. A irflow direction control system Airway pressure and airflow direction in the system were controlled by manually actuated PVC ball valves. As mentioned earlier in the system for artificial inspiration, the

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90 inspiratory pressure was continuously monitored dur ing airway inflation, and its pressure was controlled by a 2 NPT manually actuated PVC ball valve. An inline 2 NPT manually actuated PVC ball valve installed between the airflow measurement system and the negative pressure reservoir was used for separat ing the negative pressure reservoir from the airflow measurement component of the system. This valve was also used to control the rapid partial FE maneuver. To induce expiration, the valve was quickly opened to expose a fully inflated airway to the negative pressure reservoir. Difference in pressure between these two compartments generated airflow through the airflow measurement component. Data Acquisition system Pressure and temperature data were acquired and recorded by Windaq Pro+ software (Windaq). The software was kindly provided by Professor James H. Jones, Department of Surgical and Radiological Science, University of California, Davis, for research cooperation on equine exercise physiology. Briefly, signals from the pressure transducers were conditioned with high -gain carrier demodulators (CD 19A, Validyne) installed in a 10-channel -module case (MC1 10, Validyne) prior to being relayed to a signal interface (DP250, Windaq). Analog signals of temperature were relayed directly to the signal interface. All analog signals gathered at the signal interface were transferred to the analog to digital converter (DP702 USB, Windaq). Data were acquired and recorded by the Windaq Pro+ software at a frequency of 1000 Hz with a laptop computer. Diagram of system set up for RP -FE is shown in Figure 3 -2. Animal Preparation for rapid partial forced expiration.maneuver. The horses were chemically restrained with intravenous administration of detomidine hydrochloride (Domosedan, Pfizer) at a dosage of 2030 g/kg. Followi ng administration, horses were left undisturbed for 10 minutes. Body temperature was obtained with a rectal mercury thermometer. The oral cavity was then thoroughly rinsed with a water hose to remove saliva and

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91 food materials. A mouth gag made from an 8 x 1 1/2 NPT PVC pipe (Figure 3 3) was inserted between the lower and upper incisors. One -third of the cuffed end of a 2.6 cm internal diameter endotracheal (ET) tube was lubricated with sterile water -soluble lubricant (KY Jelly, Johnson & Johnson). The ET tube was passed through the mouth with the animals head held in a straight and fully extended position. Once the ET tube was in the trachea, it was advanced to the midcervical trachea and the cuff of the ET tube was inflated with 80100 ml of air. A fiberoptic endoscope, which was preinstalled with a homemade three -way connecter, was inserted into the ET tube until the tip of the endoscope was proximal to the tracheal bifurcation. The homemade three -way connecter was then connected to the ET tube and to the manifold of the artificial inspiration system. All of the connections were sealed with electrical tape to prevent leakage of air. Once the ET tube was connected to the RP -FE apparatus, the airway and lung were artificially inflated with the air blower under the control of the variable transformer. The airway was gradually inflated to a pressure of 25 30 cm H2O, and the variable transformer was turned off. The lung and airway deflated under the force of the lung and chest wall elastic recoil properties u ntil the airway pressure dropped to near zero when the airway pressure relief valve was opened. Artificial inspiration was maintained at a rate of 25 30 times/minute. Induction of Rapid Partial Forced Expiration Prior to initiation of RP FE, the negative p ressure in the reservoir was adjusted to a desired level, and the airway was concurrently inflated to its TLC, at an airway pressure of 30 cm H2O. At this airway pressure, the pressure relief valve and the valve connecting the artificial inspiration manifo ld with the blower were closed and the valve located downstream of the airflow measuring system was quickly opened to expose the inflated airway to negative pressure. The difference in pressure between the vacuum reservoir and the airway emptied the air fr om the

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92 airway, which mimicked voluntary forced expiration. Once the airway was emptied, the FE controlling valve was closed and the valve connecting the artificial inspiration manifold and the blower was opened, and artificial inspiration was resumed. Rapi d partial forced expiration maneuver was conducted with a negative pressure at 25, 50, 75, 100, 150, 200, and 250 Torr. After the last maneuver, the RP FE system was disconnected from the ET tube, the cuff of the ET tube was deflated, and the ET tube removed from the horse. Calculation of the Pulmonary Function Test Parameters Reset the calibrated pressure differential of LFE with pressure/temperature corrected flow rate Airflow rate data from the NIST traceable MFE and P data generated from the airflow, w ere used to confirm a linear relationship between the flow rate and differential pressure. Maximum airflow rate and its pressure differential from MFE/LFE calibration were used to calculate pressure and temperature -corrected airflow rates. The original air flow rate from MFE (at the standard condition: 21 C, 760 Torr, and dry) was corrected with barometric pressure (BP) and body temperature (BT) to reflect airflow rate of the expired air from the horse during RP FE maneuver. Barometric pressure data were obt ained from Gainesville Regional Airport weather station, available online, National Weather Service (http://weather.noaa.gov/weather/current/KGNV.html ). The BP and BT corrected airflow ra te was used to reset the high calibration of the P of the LFE at the same MFE data point, and a P of LFE at no flow was reset to zero. After resetting the high and the low calibrations of LFEs pressure differential with a BT/BP -corrected flow rate, the LFE data represent the airflow rate generated by the RP -FE maneuver. These data were used for computation of PFTPs.

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93 Calculation of expiratory volume from airflow data Volume of airflow generated by the RP FE was obtained by integration of the area under the curve for the relationship between airflow rate and time. Volume integration was computed with the Advanced CODAS add -on module for the Windaq Pro+ data acquisition software. The derivative of the integrated volume calculated by the Advance CODAS was u sed to determine the beginning of the RP FE. Determination of pulmonary function test parameters After computing the integrated volume and calculating its derivative, the PFTPs were computed or obtained either with Windaq waveform browser software or EXC EL. Pulmonary function test parameters of interest include forced vital capacity (FVC), peak expiratory flow (PEF), FEV0.5, FEV0.75, FEV1, FEV1.5, FEV2, FEV2.5, FEV3, forced vital capacity (FVC), and FEV1/FVC ratio. Airflow rates at 25, 50, and 75 % of FVC (MEF25%, 50%, and 75%) were also computed. Determination of suitable negative pressure for the rapid partial forced expiration maneuver During the FE maneuver, negative pressures at 25, 50, 75, 100, 150, 200, and 250 Torr were tested for their capacity to empty air from the airway. The impact of negative pressures on lower airway mucosa was determined by endoscopic examination during the RP -FE maneuver. Statistical Analysis Histamine concentrations causing PC35 delta flow were categorized from 1 to 4 accor ding to the PC35 delta flow interpretation protocol (Table 3 1). Agreement between the 1st and 2nd HB tests of the degree of airway hyper -sensitivity (severe, moderate, mild, and none) was investigated using the Friedman test and Spearman correlation. HB r esults were then categorized into two categories (horses with hyper -sensitive airways and horses without), and agreement between the

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94 1st and 2nd HB tests for identifying airway hyper -sensitivity was investigated using the Friedman test and Spearman correlation. Broncho alveolar lavage fluid cytology data from the 1st and 2nd HB were compared using Wilcoxon Signed Rank test, and the correlation between the two tests was investigated using Spearman correlation. Linear relationships between volumetric flow rate (Q) and differential pressure ( P) of the LFE pressure transducer obtained before RP -FE and after RP -FE were evaluated using regression analysis. Pulmonary function test parameters derived from RP FE at 25, 50, 75, 100, 125, 150, 200, 250 Torr were prese nted in mean SD. Reproducibility of PFTPs was investigated using Wilcoxon Signed Rank test. Analysis of PFTPs reproducibility was conducted on PFTPs data derived from RP -FE at 150, 200, and 250 Torr. Data of horses for which the 1st HB test result agreed with the 2nd HB test (hyper sensitive or non hyper -sensitive) were selected for further analysis. After categorizing the horses into hyper -sensitive and nonhyper -sensitive groups, BALf cytology and PFTPs were recorded as meanSD Differences in BALf cytol ogy of horses with sensitive and horses with non -sensitive airways were compared using Mann-Whitney U test. Pulmonary function test parameters of horses with sensitive airways were compared to those with non -sensitive airways using MannWhitney U test. P v alue Results Subject Twenty -four horses used in this study included six mares and 18 geldings. Mean SD and range of age and body weight were 6.81.7 years (range = 2.5 9) and 52941 kg (range = 412599). When the horses w ere tested for HB and RP -FE, they were clinically normal.

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95 Histamine Bronchoprovocation and Respiratory Inductance Plethysmography and Pneumotachography (Open PlethTM) The test of airway hyper -sensitivity by the Open PlethTM system is a non -invasive techni que for measuring the response of the airway to histamine. The technique also is called histamine bronchoprovocation (HB). Most of the horses tolerated the test procedures well. Only one horse in this study developed extreme anxiety and restlessness after a facemask was put on, and an additional administration of xylazine hydrochloride (100 mg) was required. The test procedure took 35 50 minutes from the first injection of chemical restraint. Xylaxine hydrochloride administration at a dosage of 0.75 mg/kg produced a short period of moderate to strong sedation, which lasted less than 20 minutes. Most of the horses became restless when the sedative effect subsided. Therefore, to immobilize the horses throughout the test, two or three doses of xylazine hydrochl oride were administered. During HB, most of the horses developed clinical signs of dyspnea and respiratory noise originating from the upper airways. There was also an increase in nasal secretion in some horses after HB. Histamine concentrations causing a 3 5% decrease in airway dynamic compliance (PC35 delta flow) from the 1st and the 2nd HB test are presented in Table 3 2, and descriptive statistics are presented in Table 3 3. In the 1st HB test, 18 horses were classified as having airway hyper -sensitivity (interpretation = 1 3), and 6 horses were classified as having normal airways (interpretation = 4). In the 2nd test 11 horses were classified as having airway hyper -sensitivity, and 13 horses were classified as having normal airways. Fifteen horses produc ed the same results in the 1st and the 2nd HB test, and 9 horses did not. Among these 15 horses, 10 possessed hyper sensitivity airways, and 5 possessed normal airways. Airway hyper -sensitivity (severe, moderate, mild, and normal) in the 1st and the 2nd H B tests did not differ significantly (p -value = 0.11), and results between the tests were not

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96 significantly correlated (R = 0.383, pvalue = 0.07). When only the presence of airway hyper sensitivity was considered (horses with airway hyper -sensitivity and horses without), the results of the tests differed significantly (p -value = 0.02) and they were not significantly correlated (R = 0.338, p -value = 0.11) (Table 3 4). Broncho -alveolar Lavage Mean SD percentage of BALf recovery was 57 16 and ranged from 22 t o 81. Measurement of recovered BALf did not include the foamy part of the BALf. Physical appearances of all samples were colorless and slightly turbid. There were no visible flakes of mucus in the BALf. Mean SD percentage of epithelial lining fluid in BALf determined by urea concentration was 1.52 0.73, and ranged from 0.3 to 3.6. Cytology results compared between the 1st HB and the 2nd HB tests were not significantly different. Mean SD BALf cytology results of the 1st HB test and the 2nd HB test and their statistical comparison are presented in Table 3 5. To determine the relationship between the airway hyper -sensitivity and BALf cytology, data from subjects with the same results in the 1st and 2nd HB tests were used, and the degree of hyper -sensitivity was not considered. After excluding subjects with different results in the 1st and 2nd HB tests, 10 horses had hyper -sensitive airway and 5 horses had normal airways. BALf cytology data from horses with hyper -sensitive airways were not significantly different from those of horses with normal airways. However, the percentages of BALf neutrophils in horses with hyper -sensitive airways and horses with normal airways approached significance different in both tests (Table 3 6). Flow -volume loops derived from averag e flow data and volume data of horses with normal airways and horses with hyper -sensitive airway were not different (Figure 3 4).

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97 Rapid Partial Forced Expiration (RP-FE) Maneuver The linear relationship between volumetric flow rate (Q) and differential pre ssure ( P) of the LFE pressure transducer was evaluated using regression analysis. The relationship of the airflow rate data and their P was linear (Figure 3 5). The simple linear regression models of the relationship between airflow rates measured by MFE and their P, before and after RP -FE, were Y = ( 0.056055) + (0.091098)X and Y = ( 0.043768) + (0.090272)X, respectively, with X = MFE airflow and Y = P. When X = 10 to 150, differences in Y between these models were not greater than 0.8%. The maximum nu mber of 150 was tested because RP FE was not expected to produce peak airflow greater than 150 liters/second. Negative pressure at 200 Torr emptied air from the airways without causing visible damage to the lower airways. Lower negative pressures incomplet ely emptied the airways. Integrated volumes from airflow data were positively correlated with the amount of negative pressure. Negative pressure at 250 Torr produced the highest FVC and caused visible airway mucosal damage, which was characterized by sub -m ucosal petechial and echimotic hemorrhage. There was no evidence of visible intra luminal hemorrhage in any horse after the RP -FE. After being chemically restrained with an intravenous administration of 20 -30 g/kg detomidine hydrochloride, endotracheal (E T) tube intubation was successful in all but one horse. This one horse was 2.5 years old, was the youngest of the subjects and was excluded from RP FE study. Endotracheal tube intubation induced coughs in some horses. The cough subsided once the ET tube w as in place. Artificial inspiration caused expansion of the thoracoabdominal wall, which in turn collapsed when the air blower was turned off. There were no major signs of discomfort during artificial inspiration or RP -FE. Negative pressure at 100, 150, 200, and 250 Torr emptied the airways which could be visualized either by endoscope in the trachea or by

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98 contraction of the thoracoabdominal wall when the airways were exposed to the negative pressures. A negative pressure at 250 Torr caused the greatest c ollapse of the trachea and the greatest contraction of the thoraco abdominal wall, but caused endoscopically visible diffused submucosal petechial and ecchymotic hemorrhage at the distal end of the trachea and in major bronchi. Raw data for RP FE from 4 horses were not used in the PFTPs calculation due to an erroneous setting of the LFE pressure transducer, which caused an error in the recording of pressure differential signals generated by the airflow. Figure 3 6 illustrates the data acquisition window of Windaq data acquisition software. Pulmonary function test parameters derived from lower negative pressures were less than the PFTPs derived from the higher negative pressures; i.e., PFTPs25 < PFTPs50 < PFTPs100 < PFTPs125 < PFTPs150 < PFTP200 < PFTP250 (Ta ble 3 7). Figures 3 7 and 38 demonstrate an example of comparison in expiratory flow rate and flow -volume loop generated by different negative pressure. MeanSD of PFTPs from the 1st and the 2nd RP -FE maneuvers at 150, 200, and 250 Torr are presented in T able 3 8. The PFTPs of the 1st and 2nd maneuvers were not significantly different when RP -FE was performed at 150 or 200 Torr. However, PFTPs derived from the 1st and 2nd maneuvers at 250 Torr were significantly different (Table 3 9). Example of FV loop s form the 1st and the 2nd RP -FE maneuvers at 150, 200, and 250 Torr are presented in Figure 3 9. These results demonstrated that PFTPs derived from RP FE of 150 and 200 Torr were repeatable, while the PFTPs of 250 Torr were less repeatable. The potential correlation between the percentage of BALf neutrophils and the PFTPs was investigated. Data on five horses with the highest percentage of BALf neutrophils were compared with data on five horses with the lowest percentage of BALf neutrophils. Horses used in this comparison were selected if their percentage of BALf neutrophils was consistent in both

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99 the 1st and the 2nd BALf collections. Data on horses with highest and lowest percentages of BALf neutrophils were compared to correlate BALf neutrophil with PFTPs alteration. Percentages of BALf neutrophils of these two groups of horses were significantly different (Table 3 10), in view of the previously mentioned resons. Pulmonary function test parameters derived from these selected populations were not significa ntly different at a p -value of 0.05. Statistics of FEV0.5, FEV0.75, PEF, and MEF25% approached 0.1 (Table 3 11). Despite the lack of statistical significance between these two groups, their FV loops generated from average flow data and volume data did diff er (Figure 3 10). Discussion Histamine is a vasoactive amine substance. It is synthesized from amino acid histidine by decarboxylation reaction. The reaction is catalyzed by the enzyme histidine decarboxylase.194 On ce synthesized, it is mostly stored in granules of mast cells or basophils. Histamine plays important role in the local immune responses and controls normal functions of the gastrointestinal system.195,196 Non-mast cell associated histamine can be found in other tissues, for example, in the central nervous system where it acts as a neurotransmitter.197 An increase in the number of mast cells and histamine concentration in BALf has been demonstrated in human asthma.198 Once released, histamine binds to histamine receptors on the bronchial smooth muscle cells and causes contraction, which leads to b ronchoconstriction. Histamine also activates the histamine receptors (H4) on the surface of the mast cells, which intensifies the release of histamine and regulates the mast cell immune response.199 As a result, the effects of histamine are augmented. Lower airway inflammatory diseases caused by non -infectious agents in horses include IAD, RAO, and SPAOPD. They are characterized by chronic inflammation of the lower airways and are thought to associate with TH1 and TH 2 cytokines dys regulation.86,200 Numbers of

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100 neutrophils in BALf increased in affected horses.171 An increase in the number of mast cells also has been demonstrated in some studies, but the results were inconsistent.171,200,201 Elevation in the number of pulmonary tissue mast cells after being challenged with moldy hay suggested that mast cells may play an important role in RAO pathogenesis and may be involved in pulmonary tissue remodeling in chronic RAO.202 Histamine, the mast cell product, is thought to be involved in airway hyper -sensitivity, which is commonly seen in inflammatory diseases of lower airways in both humans and animals.178,187 I n human medicine, the degree of airway hyper -sensitivity to exogenous histamine has been used extensively in the diagnosis of respiratory diseases such as bronchial asthma and COPD.178 Histamine challenge in veterinary medicine is modified from histamine bronchoprocation (HB) in humans. Histamine bronchoprocation is a noninva sive technique of measuring airway hyper -sensitivity. The test is commonly performed in human patients when either obstructive (disease affecting airways that carry gas into and out of the lung such as asthma and COPD) or restrictive (disease affecting pul monary tissue such as neoplasia and fibrosis) pathologies of the pulmonary tissues are suspected.182 Exposure of the affected airways to histamine causes bronchoconstriction and an increase in airway resi stance. In this study, horses without clinical signs of respiratory problems responded to HB inconsistently. Horses with a high percentage of BALf neutrophils were more likely to have hyper -sensitive airways. However, correlations between percentages of BA Lf neutrophils and airway hyper -sensitivity were not significant. The difference in HB results between tests in this study agreed with those of a previous study, suggesting a high variation in individual response to HB.184 Results from the previous study suggested that the HB response in normal horses and horses with low -grade lung disease (with no clinical signs of respiratory tract di stress) were not

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101 significantly different. However, horses with severe pulmonary disease required a significantly lower histamine concentration to cause 35% reduction in lung dynamic compliance.184 The cause of individual variation in HB response is not known. However, a possible explanation is a nonspecific response of upper airways to histamine, especially when a facemask was used during HB test. A previous report suggested that upper airway resistance in normal horses is approximately 50% of the total pulmonary resistance.203 Abnormalities of either pharyngeal or laryngeal functions, such as displacement of soft palate and laryngeal hemiplegia, increase resistance of airflow in the upper airways.204206 Temporary malfunc tion of these structures can be found in some horses following the administration of chemical restraints. This may be due partly to the effect of generalized muscle relaxation, which resulted in a relaxation of pharyngeal and laryngeal muscle tone.207 Xylazine hydrochloride being used in this study during HB is an 2 adrenoceptor agonist. It produces bradycardia and alters rhythm of cardiac contraction.208 After administration, it initially produced hypertension followed by a prolonged hypotension, decrease in cardiac output, and respiratory depression.208 Xylazine hydrochloride caused the head carriage position to be less than 180 (w hen 180 was defined as a position when both head and heck are parallel to the horizontal plane).209 This resulted in an increase in t he resistance in both the upper and lower airways. However, when the head and neck were re -positioned to 180 the previous increase in airway resistance from low head carriage was restored to values seen prior to the beginning of sedation.210 During HB in this study the head and neck positions were maintained close to the horizontal plane by using a support at the mandible. Histamine not only induces the contraction of the smooth muscles of the airways, but also causes vasodilatation of blood vessels in the airways. Vasodilatation leads to edema of the

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102 respiratory mucosa and increases mucus production in both upper and lower airways.211 These results may contribute to an increase in non -specific resistance of the upper airways that potentially increases total r esistance of the respiratory tracts. A pharmacological effect of histamine on mucus production was observed in this study; that is, the vast majority of the horses had an increase in their nasal secretion during and after exposed to histamine. Histamine ca uses immediate bronchoconstriction in humans, which is different from bronchoconstriction naturally occurring in equine lower airway inflammatory diseases (IAD, RAO, and SPAOPD). After challenge with known environmental inciting agents, bronchoconstriction slowly developed over a few hours, and the resolution of clinical signs was delayed after the agents were removed.171 These results suggested that bronchoconstriction in horses affected by lower airway inflammation may depend on an unknown intermediate substrate, which might be chemical substance release from tissue resident inflammatory cells, vascular endothelium, or inflammatory cells present in bronchioalveolar fluid.171 Among these cells, alveolar macrophages and neutrophils are the most studied. Besides phagocytic activity, alveolar macrophages release pro inflammatory cytokines in response to exte rnal stimuli. Results from an in vitro study demonstrated that activation of alveolar macrophages and monocytes with LPS and fungal antigen increased their TNF production.144 Tumor necrosis factor alpha can activa te macrophages, endothelial cells, and other immune cells to produce more inflammatory cytokines, which amplify the inflammatory reactions. Neutrophils in BALf are recruited from pulmonary circulation in response to TNF .212 The migration of neutrophils across endothelium requires chemotactic cytokine, includin g interleukin8 (IL 8). Interleukin8 can be produced by endothelial cells, cells from bronchoalveolar lavage, and bronchial epithelium.177,200,213 An increase in IL 8 concentration in BALf following an exposure to hay dust that was accompanied

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103 by an increase in the number of neutrophils in BALf, indicated that IL 8 plays an important role in pathogenesis of lower airway inflammatory diseases in horses.214 Results from this study suggested that the HB test can be used to reinforce diagnosis of equine lower airway inflammatory diseases, but interpretation of HB test results obtained from clinically normal horses should be done with great care. Information derived from an HB te st should be used together with results from other diagnostic information, including BALf cytology, clinical signs, history of illness, and response to therapy. Forced expiration maneuver is another test of pulmonary function modified from human medicine. In human medicine, the test is the most common diagnostic procedure performed in respiratory clinics. Derived parameters reflect the biomechanical characteristics of pulmonary parenchyma and air conductive tissues. The test is voluntary and noninvasive in humans. Quality of the test depends on subject cooperation and ability of the respiratory diagnostician to coach the patient to breath correctly.182 Results from the test are repeatable.183 Forced expiration in horses requires additional maneuvers and is more invasive than that in humans. A des ign for a system that is capable of intervening inspiration and expiration is necessary since coaching on breathing in horses is not possible. In this study, the inspiration maneuver was accomplished by inflating the airways with atmospheric air at a posit ive pressure 30 45 cm H2O at the rate of approximately 25 30 breaths/minute. At this pressure and rate, artificial inspiration suppressed the physiological respiratory drive of the test subjects. The suppression might be due partly to hypocapnea and increa sed partial pressure of blood oxygen from hyperventilation. This, in turn, reduced the respiratory drive governed by the autonomic nervous system. Whether artificial ventilation in this study affected the blood gas parameters is unknown and is worth furthe r investigation.

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104 The RP -FE maneuver in this study lasted 7 10 minutes. However, duration of the maneuver did not reflect the total time required for conducting the test since this experiment included the evaluation of RP -FE generated by various negative pr essures. Less time will be needed if a single negative pressure is used for the maneuver in the future. Other problems in this RP -FE set up included control of airway pressure at TLC prior to an induction of RP -FE. Determination of TLC in this study was ba sed on airway pressure at the end of artificial inspiration, which was expected to 30 cm H2O. Airway pressure data suggested that the inspiratory manifold set up in this study was insufficient to produce repeatable airway pressure at TLC. This might due pa rtly to leakage of air through the connection sites in the manifolds. Manually adjusting the airway pressure also contributed to high variation in airway pressure at TLC. Leakage of air in the manifold upstream of the LFE not only affected the ability to c ontrol the airway pressure, but also produced airflow signals on LFE pressure transducer during RP -FE when airways were already emptied. The flow signals caused by air leakage could interfere with PFTPs calculations. Pulmonary function test parameters from this study were different from values reported by a previous study.175 The differences in PFTPs may be caused by differences in manifold designs, in the negative pressures used to empty airways, or in methods of measuring airflow. Regardless of the total length of the manifold used in the two studies, the major factor that contributed to airflow resistance was the diameter of the manifold. The internal diameter (ID) was determined by the selection of an endotracheal tube (2.6 cm, this study) versus nasotracheal (2.2 cm, previous study). Intubation possessed an advantage over the facemask in obtaining PFTPs, since the tube bypassed the upper respiratory structures, which potentially contributed to a non -specific resistance to airflow. Poiseuilles equation demonstrates the

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105 relationship between flow resistance and geometr ic restriction of flow.193 Resistance is inversely related to the fourth power of the radius: R = 8 4 (3 5) When R = Resistance. = Fluid viscosity. L = length of pipe. R = Radius. Despite seemingly a minor difference in the internal diameter between these two designs, resistance caused by 2.2 cm ID is twice that of 2.6 cm ID. Relationship of the radius to the volumetric flow rate also is demonstrated by a variation of Poiseuilles equation; the amount of the volumetric flow rate is positively related to the fourth power of the radius. Q = ( 4)/8 L (3 6) When Q = Volumetric flow rate. P = Differential pressure. Pulmonary function test parameters derived from different negative pressures indicated that PEF and FVC values were positively correlated with the amount of negative pressure used to generate RP -FE. Negative pressure at 2 00 Torr was selected in this study because it emptied the air from airways without causing observable damage to the tracheal mucosa. The PEF and FVC values derived from 200 Torr in this study were greater than the previously reported values, when FE was in duced by 161.8 Torr ( 220 cm H2O). In this RP -FE experiment, airflow data were obtained from a direct measurement of P generated by airflow. The P data were calibrated with NIST-traceable MFE. Additional calibration with a known -volume injection through the LFE should be considered in future experiments. By comparing the integrated volume (computed from MFE calibration) to a known volume injected through LFE, accuracy of measurements can be determined.

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106 Even though subjects used in this study were clinical ly normal, BALf cytology of some horses possessed a high percentage of BALf neutrophils. In inflamed airways, neutrophils are recruited during an active disease episode, and an increase in their numbers in BALf has been used as an indication of inflammation of the lower airways. Previously reported values for percentages of BALf neutrophils in normal horses varied among studies.176,215 However, numbers greater than 5% in the samples have been used to suggest the pres ence of an inflammation within the lower respiratory tract.201,216 Mean SD percentages of BALf neutrophils from the 1st and 2nd BALf collections in this study were 9.0 4 and 7.7 5, respectively. These results agreed with previously reported values in clinically normal horses (6.82.7215 and 11.58.9176). Despite no clinical signs of respiratory problems, a high percentage of BALf neutro phils might indicate an ongoing inflammatory reaction in the pulmonary tissues. Flow volume loop derived from horses with low percentages of BALf neutrophils (< 6%) was larger than that from horses with a high percentage of BALf neutrophils (>9%). These re sults suggested a possible compromise in the pulmonary function when inflammation is present in the lower airways. However, this study could not confidently identify sub-clinical lower airway inflammation in the selected population. Future investigation of PFTPs derived from clinically active cases of lower airway inflammatory diseases will be highly beneficial.

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107 Figure 3 1. Variable transformer.

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108 Figure 3 2. Diagram of device setup to perform rapid partial forced expiration mane uver in horses and component symbols. MC1 10 DP 205 DP 702 DP41 B Vacuum Pump AC power supply DP41 B MC1 10 DP 205 DP 702 Temperature meter Signal conditioner Signal interface Analog to digital converter Rigid polypro pylene tube Thermocouples Connecting cable Electrical power cord Negative pressure reservoir Air blower Variable transformer Analog pressure gauge Laminar flow element Filter Pressure transducer Manually actuated PVC valve Computer Component s ymbols

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109 Figure 3 3. Mouth gag made from an 8 x 1 1/2 NPT PVC pipe. Hand guard made from black rubber curry comb.

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110 0 10 20 30 40 50 60 70 0 20 40 60 80 100 120 140 Flow rate (liters/second) Volume (liter) Normal airway Hyper sensitive airway Figure 3 4. Flow -volume loops derived from average flow data and volume data of horses with normal airw ays and horses with hyper -sensitive airway. 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 0 10 20 30 40 50 60 Direct reading of MFE airflow (liters/second) LFE differential pressure (cm H2O) Before RP-FE After RP-FE Figure 3 5. Relationship of airflow rates measured by NIST -calibrated mass flow element (MFE) and differential pressure generated before and after rapid partial forced expiration (RP -FE).

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111 Figure 3 6. Exa mple of data acquisition window of Windaq Pro+ software. Data from pressure transducer connecting to the negative pressure reservoir, the laminar flow element, and the airway were recorded in channels 1, 2 and 4, respectively. Channel 3 was set for recordi ng the temperature of air in the inspiratory manifold. Channel 5 is the forced expiratory volume calculated from integration of airflow data as a function of forced expiratory time. Channel 6 represents the derivative of the forced expiratory volume.

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112 0 20 40 60 80 100 120 140 160 0 0.5 1 1.5 2 2.5 3 Forced expiratory time (second) Forced expiratory flow (liter/second) 25 50 75 100 125 150 200 250 Fi gure 3 7. Example of forced expiratory flow rates generated by different negative pressures. 0 10 20 30 40 50 60 70 80 0 20 40 60 80 100 120 140 160 Forced expiratory airflow rate (liters/second) Forced expiratory volume (liter) 25 50 75 100 125 150 200 250 Figure 3 8. Example of flow -volume loops generated by different negative pressures.

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113 0 10 20 30 40 50 60 70 80 0 50 100 150 Forced expiratory airflow (liters/second) Forced expiratory volume (liter) 1st 150 2nd 150 1st 200 2nd 200 1st 250 2nd 250 Figure 3 9. Example of flow -volume loops generated by negative pressur es at 150, 200, and 250 Torr. 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00 Airflow (liters/second) Volume (liter) Horses with low % BALf Neu Horses with high % BALf Neu Figure 3 10. Flow -volume loops derived from average flow data and volume data of horses with low percentages of BALf neutrophils and horses with high percentages of BALf neutrophils.

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114 Table 3 1. Interpretation of histamin e concentrations that cause 35% increase in delta flow (modified from operation manual of Open PlethTM system). Histamine concentration that causes PC35 delta flow Interpretation of airway hyper sensitivity/ category 0 2 mg/ml Severe / 1 2 4 mg/ml Mode rate / 2 4 8 mg/ml Mild / 3 8 >32 mg/ml Normal / 4 (Source:. Operation manual of Open PlethTM System. Ambulatory Monitoring, Inc, 2006.186) Table 3 2. Histamine concentration causing PC 35 delta flow and their interpretations from the 1st and the 2nd HB tests. Horse PC 35 delta flow of the 1st test Interpretation of the 1st test* PC 35 delta flow of the 2nd test Int erpretation of the 2nd test* H1 0.88 1 1.62 1 H2# > 32.00 4 15.00 4 H3 0.92 1 7.38 3 H4# 12.87 4 9.00 4 H5 2.17 2 24.55 4 H6 1.05 1 1.85 1 H7 1.33 1 21.05 4 H8 1.00 1 1.50 1 H9 5.23 3 1.20 1 H10 1.25 1 0.18 1 H11 3.41 2 1.97 1 H12 2.35 2 27.02 4 H13# > 32.00 4 21.94 4 H14# > 32.00 4 21.94 4 H15 1.62 1 1.41 1 H16 3.35 2 1.91 1 H17 0.49 1 14.38 4 H18 1.56 1 29.33 4 H19 6.94 3 9.00 4 H20 6.53 3 12.00 4 H21 3.58 2 8.51 4 H22# 13.00 4 > 32.00 4 H23 0.29 1 2.3 4 2 H24 8.14 4 0.44 1 = Interpretation of the PC 35 delta flow results (1= severe airway hyper -sensitivity, 2 = moderate airway hyper -sensitivity, 3 = mild airway hyper -sensitivity, and 4 = normal airway). Method of interpretation is explained in Table 3 1. HB = histamine bronchoprovocation, = horse with airway hyper -sensitivity regardless of severity, and the result of the 1st test agreed with the 2nd test, # = horse with normal airway, and the result of the 1st test agreed with the 2nd test.

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115 Table 3 3. Descriptive statistics of the degree of airway hyper -sensitivity based on results from the 1st and 2nd tests of histamine bronchoprovocation. Degree of airway hyper sensitivity Frequency of the 1 st test Percent Frequency of the 2 nd test Percent 1 10 41.7 9 37.5 2 5 20.8 1 4.2 3 3 12.5 1 4.2 4 6 25.0 13 54.2 Total 24 100.0 24 100.0 = Interpretation of the PC 35 delta flow results (1= severe airway hyper -sensitivity, 2 = moderate airway hyper -sensitivity, 3 = mild airway hyper -sensitivity, an d 4 = normal airway). Method of interpretation is explained in Table 3 1. Table 3 4. P -values from comparisons of the HB results between the 1st and 2nd tests and their correlations. Degree of airway hyper sensitivity (categorized from 1 4) Result based on presence or absence of airways hyper sensitivity Comparison* Correlation coefficient** Comparison* Correlation coefficient** 0.11 0.383 (p value = 0.07) 0.02 0.338 (p value = 0.11) = Friedman test, ** = Spearmans rho. Table 3 5. MeanSD of BALf cytology results of the 1st and 2nd HB test and p value of Wilcoxon Signed Rank test and Correlations between the tests. Statistics TNC (x10 6 cell/ml) Mac (%) Lym (%) Neu (%) Mast (%) MeanSD 1 st test 29.41 9.16 39.96 7.69 49.14 6.92 9.03 4.17 1.82 1 .27 2 nd test 32.12 13.50 39.06 8.89 50.39 7.89 7.79 5.22 2.32 1.52 P value 0.49 0.29 0.33 0.14 0.25 Correlation coefficient# **0.768 *0.479 0.400 *0.473 0.375 TNC = total nucleated cell, Mac = macrophage, Lym = lymphocyte, Neu = neutrophi l, Mast = mast cell, # = Spearmans rho statistic, ** = correlation is significant at the 0.01 level, = correlation is significant at the 0.05 level.

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116 Table 3 6. MeanSD and MannWhitney U statistics of BALf cytology from horses with hyper sensitive an d normal airways. 1 st test 2 nd test BALf cytology parameters Hyper sensitive Normal P value Hyper sensitive Normal P value TNC (x10 6 cell/ml) 28.24 12.31 30.89 5.24 0.95 36.11 16.86 28.52 6.49 0.37 Mac (%) 38.48 8.55 38.45 6.52 0.59 34.78 10.50 44.40 5.89 0.13 Lym (%) 49.35 5.79 51.85 6.75 0.61 52.58 8.10 47.1 5.79 0.27 Neu (%) 10.33 4.71 8.20 1.96 0.19 10.18 6.69 6.15 2.03 0.14 Mast (%) 1.85 1.83 1.45 0.57 1.00 2.15 1.84 1.55 0.44 0.83 BALf = Broncho alveolar lavage fluid, TNC = total nucleated cell, Mac = macrophage, Lym = lymphocyte, Neu = neutrophil, Mast = mast cell.

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117 Table 3 7. MeanSD of PFTPs derived from RP -FE at negative pressures of 25, 50, 75, 100, 125, 150, 200, and 250 Torr (n=19). PFTPs Negative pressure (Torr) 25 50 75 100 125 150* 200* 250* FVE0.5 9.98 0.88 14.12 1.13 18.69 0.92 23.45 0.96 27.68 0.79 34.45 1.18 43.57 1.09 52.44 1.21 FVE0.75 12.71 1.09 17.98 1.49 23.54 1.11 29.36 1.13 34.46 0.96 43.99 1.55 55.44 1.67 66.75 1.95 FEV1.0 14.79 1 .31 20.95 1.80 27.32 1.32 33.80 1.28 39.44 1.39 48.94 3.36 60.49 3.42 71.66 3.31 FEV1.5 17.55 1.71 24.93 2.49 32.15 2.26 38.48 3.09 42.79 2.93 50.70 4.34 62.00 4.01 73.13 3.7 FEV2.0 18.61 2.11 26.53 3.14 33.47 3.06 39.15 3.46 43.18 2. 91 49.49 6.77 62.28 4.01 73.38 3.74 FEV2.5 18.95 2.35 26.84 3.37 33.77 3.18 39.25 3.45 43.32 2.83 51.04 4.38 62.45 4.06 73.44 3.76 FEV3.0 19.01 2.33 26.88 3.43 33.86 3.21 39.25 3.45 43.34 2.82 51.07 4.38 62.47 4.05 73.44 3.77 FVC 19.0 1 2.33 26.88 3.43 33.86 3.21 39.25 3.45 43.34 2.82 51.07 4.38 62.47 4.05 73.44 3.77 FEV0.5/FVC 0.53 0.03 0.53 0.04 0.55 0.04 0.60 0.05 0.64 0.04 0.68 0.04 0.70 0.03 0.72 0.03 FEV0.75/FVC 0.67 0.03 0.67 0.05 0.70 0.05 0.75 0.06 0.80 0 .04 0.86 0.05 0.89 0.04 0.91 0.02 FEV1/FVC 0.78 0.03 0.78 0.05 0.81 0.05 0.87 0.05 0.91 0.04 0.96 0.02 0.97 0.01 0.98 0.01 PEF 32.53 3.87 47.39 4.60 62.62 3.27 78.06 3.00 90.08 2.45 108.15 3.48 133.64 3.44 157.17 3.65 PFTPs = pulmonar y function test parameters, RP FE = rapid partial forced expiration, FEVx = forced expiratory volume at x second, FVC = forced vital capacity, and PEF = peak expiratory flow, = average value of 2 maneuvers.

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118 Table 3 8. Mean SD of PFTPs derived from the 1st and the 2nd RP -FE, when 150, 200, and 250 Torr were used to induced RP -FE. PFTP 150 Torr 200 Torr 250 Torr 1 st 2 nd 1 st 2 nd 1 st 2 nd FVE0.5 34.56 1.10 34.60 1.10 43.60 1.10 43.54 1.10 52.68 1.30 51.89 1.00 FVE0.75 43.97 1.60 44.00 1.60 55.41 1.70 55.47 1.80 67.22 1.80 65.72 2.10 FEV1 48.82 3.44 49.07 3.50 60.46 3.50 60.53 3.50 72.32 3.10 70.25 3.80 FEV1.5 50.48 4.40 50.91 4.70 61.96 4.10 62.03 4.00 73.82 3.40 71.65 4.30 FEV2 50.77 4.40 51.14 4.70 62.25 4.10 62.31 4.00 7 4.06 3.50 71.91 4.30 FEV2.5 50.88 4.40 51.21 4.70 62.35 4.10 62.55 4.10 74.11 3.50 71.97 4.40 FEV3 50.93 4.40 51.21 4.70 62.34 4.10 62.57 4.10 74.12 3.50 71.98 4.30 FVC 50.93 4.40 51.21 4.70 62.37 4.10 62.57 4.10 74.12 3.50 71.98 4.4 0 FEV0.5/FVC 0.68 0.04 0.68 0.05 0.70 0.04 0.70 0.03 0.71 0.03 0.72 0.04 FEV0.75/FVC 0.87 0.04 0.86 0.05 0.89 0.03 0.89 0.04 0.91 0.02 0.91 0.03 FEV1/FVC 0.96 0.02 0.96 0.02 0.97 0.01 0.97 0.01 0.98 0.01 0.98 0.01 PEF 108.00 3.60 108 .51 3.60 133.98 3.50 133.58 3.40 158.00 3.70 155.57 2.90 PFTP = pulmonary function test parameter. RP FE = rapid partial forced expiration. FE = forced expiration. FEV = forced expiratory volume, FVC = fforced vital capacity. PEF = peak expiratory fl ow.

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119 Table 3 9. Wilcoxon Signed Rank test statistics (p -value) of pulmonary function test parameters (PFTPs) from the 1st and the 2nd RP -FE maneuver at 150, 200, and 250 Torr. Vacuum (Torr) FEV 0.5 FEV 0.75 FEV 1.0 FEV 1.5 FEV 2.0 FEV 2.5 FEV 3.0 FVC PEF 150 1.00 0.89 0.94 0.87 1.00 0.90 0.75 0.75 0.19 200 0.54 0.84 0.81 0.81 0.75 0.94 0.98 0.98 0.29 250 0.02 0.01 0.01 0.02 0.02 0.02 0.02 0.02 0.01 P value less than 0.05 indicated that the PFTPs from the 1st and the 2nd RP -FE maneuvers were significa ntly different, RP -FE = rapid partial forced expiration. FEV = forced expiratory volume, FVC = forced vital capacity, and PEF = peak expiratory flow. Table 3 10. MeanSD of percentage of BALf neutrophils of horses with low % Neu and horses with high % Ne u, and MannWhitney U test statistics. Test Horse with high % Neu (n=5) Horse with low % Neu (n=5) P value 1 st 14.052.73 4.801.64 0.01 2 nd 14.956.93 5.850.70 0.01

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120 Table 3 11. MeanSD of PFTPs of horses with low percentage of BALf neutrophils and horses with high percentage of BALf neutrophil and MannWhitney U test statistics. PFTPs Horse with high % Neu (n=5) Horse with low% Neu (n=5) P value FEV0.5 42.804 0.802 43.917 1.345 0.11 FEV0.75 54.302 1.643 56.131 1.725 0.15 FEV1.0 59.325 3 .749 61.625 3.522 0.55 FEV1.5 60.916 4.275 63.138 4.113 0.55 FEV2.0 61.263 4.248 63.390 4.111 0.55 FEV2.5 61.67 4.621 63.458 4.059 0.55 FEV3.0 61.686 4.610 63.476 4.037 0.55 FVC 61.686 4.610 63.476 4.037 0.55 PEF 131.226 1.894 134.828 4. 415 0.15 MEF25% 130.982 1.957 134.576 4.364 0.10 MEF50% 98.636 6.624 99.674 1.836 1.00 MEF75% 54.320 6.176 53.956 2.427 1.00 FEV0.5/FVC 0.697 0.048 0.693 0.024 1.00 FEV0.75/FVC 0.882 0.047 0.884 0.031 0.95 FEV1.0/FVC 0.962 0.015 0.971 0.0 07 0.22 PFTPs = pulmonary function test parameters, BAL = broncho alveolar lavage, FEVx = forced expiratory volume at x second, FVC = forced vital capacity, PEF = peak expiratory flow, MEFx% = forced expiratory flow rate when x% of FVC was exhaled, FEVx/F VC = ratio between forced expiratory volume at x second to the forced vital capacity.

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121 CHAPTER 4 EFFECTS OF ELECTROAC UPUNCTURE ON PULMONA RY FUNCTION AND IMMU NE RESPONSE IN HORSES Introduction and Background The respiratory system plays an important role in equine athletic performance. Unlike the cardiovascular and musculoskeletal system, competence of the lung and respiratory tracts are not changed by exercise training programs. The respiratory system has been postulated to be the major factor limiting th e maximal performance of the horse.217,218 Abnormalities of the respiratory system, which decrease gas transport capacity of the airways, diminish alveolar gas exchange, or both, reduce athletic performance capacity .219 Chronic lower airway inflammatory disease is a naturally occurring respiratory disorder in horses.220 Three forms of this disorder have been described based on the age of affected horse s and seasonal occurrences, including inflammatory airway disease (IAD), recurrent airway obstruction (RAO), and summer pasture associated obstructive pulmonary disease (SPAOPD).221 Inflammatory airway disease is diagnosed in young training racehorses, while the other two d iseases are commonly diagnosed in older horses, and usually associated with poor stable management. Recurrent airway obstruction occurs primarily in horses being kept in stables and in the winter months, and is also called heaves.220 Summer pasture associated obstructive pulmonary disease occurs primarily in horses being kept in pastures, and the clinical signs are more severe in the summer season.222 These diseases cause chronic inflammation of the lower airways. They produce non -specific clinical signs and produce similar broncho alveolar lavage fluid (BALf) cytology, suggesting that they might partly share a common disease mechanism.223 Several etiol ogies have been suggested to cause inflammation of the lower airways, including hay dust and fungal spores in the stable environment.224,225 Laan et al.144 demonstrated an in crease in production of tumor necrosis factor alpha (TNF ) and interleukin 1 beta (IL 1 )

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122 by alveolar macrophages following in vitro challenge with a solution of hyphae and conidia prepared from Aspergillus fumigatus The role of a fungal antigen in pathogenesis of lower airways diseases has been also demonstrated in an in vivo study. After challenging the airways of RAO -susceptible horses with A. fumigatus antigen, the numbers of total nucleated cells and neutrophils in BALf increased.226 The challenge also increased the mRNA expression of TNF IL 1 and IL 8 in alveolar macrophages. These inciting antigens caused immunological responses at both local and systemic levels. An increase in BALf IgA specific to Microsp orum faeni and A. fumigatus in RAO affected horses has been demonstrated.227 There was also a significant increase in serum IgE specific to Aspergillus antigen in RAO affected horses.228 The role of endotoxin in pathogenesis of lower airway inflammatory diseases has also been been shown. Nebulization of bacterial lipopolysaccharide (LPS) solutions into the airways induced neutrophil infiltration in a dose -dependent maner.229 However, LPS nebulization alone did not cause clinical signs of respiratory problems in either normal horses or in RAO affected horses. Fungal antigen such as Aspergillus fumigatus has been suggested to play an impor tant role in equine lower airway inflammatory diseases.144 Further study demonstrated that the increase in the numbers of neutrophils in airways was significantly reduced when the airways were challenged with a LPS -depleted fungal antigen. These results suggested that endotoxins present in the antigens that play an important role in pulmonary inflammation associated with RAO and other lower airway inflammatory diseases in vivo .230 Until recently, ther e was no single diagnostic procedure that could be used to accurately distinguish among these diseases. Currently diagnosis relies on case history, clinical signs, BALf cytology, and response to treatment.170 The ability to definitively diagnose these diseases at their early stage is important. However, early diagnosis is difficult due to a lack of specific clinical

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123 manifestations and, more importantly, the affected horses may not show clinical signs of respiratory problems until the amount of pulmonary tissue affected is greater than the functional reserve. It is believed that most affected horses are undiagnosed because of the sub -clinical nature of the diseases. Several prog ressive diagnostic methods have been developed in attempts to identify early stages of these diseases, including direct measuring of intra -pleural pressure, histamine bronchoprovocation, and forced expiration.172175 Treatments of chronic lower airway inflammatory diseases are complex due to an incomplete understanding of the causes of these diseases and their pathogenesis. Conventional therapy requires long-term medication and an adjustment of the housing environmen t to remove probable inciting causes.86 Significant improvement in clinical signs may be seen in some cases after making changes in the animals environment. Bronchodilator, mucolytic, and anti inflammatory agents alone or in combination together are normally prescribed in order to control the clinical signs of dyspnea and cough.170 Improvement in the clinical signs does not guarantee complete resolution of the disease. Horses with a history of previous lower airway inflammatory disease are more likely to experience a recurrence of clinical signs when inciting factors are re introduced. Failure or delay in delivery of proper treatment at early stages of these diseases may lead to chronic irreversible changes in the histopathological structures of the small airways and pulmonary parenchyma.86,231 An increase in airway resistance and decrease in pulmonary c ompliance and gas exchange capacity are common consequences of these changes. Besides the standard medical management discussed above, alternative treatments such as acupuncture (AC), electroacupuncture (EA), and Chinese herbs have been used as adjunctive therapies.109,232 They are intended to decrease the dosage requirements of bronchodilators and anti inflammatory agents and improve the quality of life of animals suffering from chronic

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124 respiratory diseases.233 In the United States, the integration of AC and EA into conventional veterinary practice is new when compared to their practice in China. They were not widely practiced outside of China until 1974 when the International Veterinary Acupuncture Society was founded.8 In contrast to the use of AC or EA to induce analgesia, modern research data on the use of AC and EA for treating respiratory diseases in horses is limited. Until recently, the benefit of AC and EA in treating equine respiratory problems was inconclusive. Previous research demonstrated an improvement in pulmonary function in RAO affected horses following a single AC treatment, but that was not statistically significant. After treatment, respi ratory rate and pulmonary resistance decreased, while the maximal change in pleural pressure, dynamic compliance, and tidal volume increased. These improvements lasted less than 24 hours.88 In rats with bronchial as thma induced by ovalbulmin, EA at GV 14, BL 13, Fei -shu Ding -chuan, LU 1, CV 17, ST 36, and SP 6 has been shown to significantly reduce lung inflammation relative to a sham treatment.97 Peri -bronchial and peri -vasc ular infiltrations of inflammatory cells were significantly reduced in the EA group. Electroacupuncture treatment also significantly reduced the percentage of polymorphonuclear cells in BALf.97 Application of AC/EA for treating chronic respiratory problems is also supported by the study conducted by the World Health Organization (WHO). The report reviewing data from controlled clinical trials by WHO in 2003 suggested that AC possesses therapeutic benefits for allergi c rhinitis and bronchial asthma.9 More recent scientific investigations also supported this conclusion.87,234,235 Previous research in humans demonstrated an immediate bronchodilating effect after 30 minutes of AC treatment in patients suffering from asthma.87 In this study, patients who received AC treatment had their forced expiratory volume in the first second (FEV1) increased by 11%, while the increase of FEV1 in the sham group was improved less tha n

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125 1%. The benefit of laser -AC combined with pro biotic supplementation as an adjunctive therapy over conventional treatment in children with intermittent asthma has also been reported.234 In this study, acupoints were chosen according to traditional Chinese medicine (TCM) diagnostic methods (questioning, palpation, tongue and pulse diagnosis). Prescription of acupoints for each patient was based on TCM, and a maximum of 16 acupoints were chosen from a preformulated list. Selected acupoints were stimulated with laser light for 20 seconds without skin contact. Study patients were treated with laser -AC once a week for 10 treatments in conjunction with seven weeks of pro -biotic supplementation, and controls given only conventional therapy. At the end of the study, peak flow var iability (PFV) of patients in the treatment group was significantly decreased; PFV is one of the pulmonary function test parameters used to measure hyper sensitivity of bronchi. Moreover, patients in the treatment group had a less severe respiratory tract infections compared to patients in the control group.234 Information on immune modulatory properties of AC/EA was reviewed in Chapter 2. In Chapter 2 the evidence presented suggested that EA at GV 14, LI 4, and LI -10 once a day for three consecutive days suppressed in vitro TNF production from stimulated whole blood. In contrast, the degree of suppression caused by AC was not significantly different compared to the TNF concentration prior to AC. The anti inflammatory effect of EA was thought to be mediated by modulatio n of mononuclear leukocytes mediated innate immune response. This in vitro anti -inflammation action of EA was demonstrated using antigens that have been proported to cause lower airway inflammatory disease, including fungal antigens and bacterial endotoxins. In Chapter 3, a method of performing the rapid partial forced expiration (RP -FE) in horses using a direct measurement of the pressure differential ( P) generated by airflow was demonstrated. One objective of the study was to identify airway obstructions that might be

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126 caused by sub-clinical inflammation of the lower airway in horses. This was done by comparing BALf cytology to the degree of airway hyper -sensitivity [using the histamine bronchoprovocation (HB) test] and the pulmonary function test paramete rs (PFTPs) derived from the RP FE. When the test of airway hyper -sensitivity was repeated within 2 months, horses showed individual variation in the degree of airway hyper -sensitivity. However, the test can be used to identify horses with airway hyper -sens itivity when the degree of hyper -sensitivity is not considered. There was no significant correlation between airway hyper -sensitivity and the percentage of BALf neutrophils, with a p -value greater than 0.1. Flow -volume (FV) loops generated from airflow dat a and volume data from the RP FE maneuver (Chapter 3) in clinically normal horses demonstrated the variability in airflow limitation from horse to horse. Flow -volume loops of some horses possessed a scoop-out characteristic after the peak expiratory flow, and some did not. The scoop-out characteristic is a result of a rapid decrease of airflow due to a narrowing of the small airway. Even though FV loop from horses (n=5) with a high percentage of BALf neutrophils (> 9%) was smaller than that of the horses (n =5) with a low percentage of BALf neutrophils (< 7%), the difference in PTFTs, including forced expiratory volume at 0.5, 0.75 second (FEV0.5, FEV0.75), peak expiratory flow ( PEF), forced expiratory flow rate when 25% of FEV has been expired (MEF25%), and ratio of forced expiratory volume at 1 second to the forced vital capacity (FEV1.0/FVC) between these two groups were not significant at a 95% confidence level. However, the p-value for MEF25% was less than 0.1, and the pvalues for FEV0.5, FEV0.75, and P EF were close to 0.1. Although results from the experiment in Chapter 3 suggested that both HB and RP -FE tests might benefit diagnosis of lower airway inflammatory diseases, they could not confidently be used to identify horses with possible sub -clinical l ower airway inflammation. Some horses

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127 enrolled in the Chapter 3 experiment possessed persistent high percentages of BALf neutrophils. Even though the FV loop derived from average airflow data and volume data of these horses was smaller than that derived fr om data from horses with low percentages of BALf neutrophils, the difference in PFTPs between these two groups was not significant at a 95% confidence level To test the effects of EA on biomechanical properties of lower airways and lung tissues, the RP FE maneuver is more suitable than HB with facemask for the following reasons. Rapid partial forced expiration using endotracheal tube (ET) intubation through the mouth eliminates non -specific airway resistance originating in the upper respiratory tracts. Ther efore, derived PFTPs are thought to truly reflect mechanical properties of lower airways. Intubation with a large ET tube also is superior to that using a smaller tube because the larger tube markedly reduces airflow resistance created by the system manifo ld. Pulmonary function test parameters and FV loop computed from the airflow data and volume data from the RP -FE maneuver are reliable. This factor is critical since the study measured effects of treatment that may cause only subtle changes in pulmonary fu nction. The objective of experiment was to evaluate the effects of EA and sham EA at acupoints regime mimicking acupoints commonly used for managing equine respiratory problems on the PFTPs, anti inflammatory activity and circulating immunoglobulins. Mate rials and Methods Animals Seventeen horses that had been previously used in the study of Chapter 3 were used in this study. Three horses were unable to enroll in this study. Additional three horses were recruited from the herd to yield a total numbers of 20. These 20 horses were regrouped and assigned for individual treatment. Horses were kept outdoors in paddocks in groups of two or four. This pattern was used because of space limitations. Groups of two or four individuals also have been found to be the mo st practical for subject handling. Groups were randomly arranged, and horses were numbered from 1 to 20. Horses with odd numbers were treated with EA, and

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128 horses with even numbers were treated with sham EA. Horses were grouped according to their housing pr ior to assigning numbers to avoid separation anxiety that usually occurs among horses when at least one in their group is taken away. All horses of each group were brought into the experimental room at the same time. At the end of the last treatment, a per iod of 3 weeks wash out was provided prior to the crossover experiment. The horses had no clinical signs of respiratory disease during the past 3 months. Protocol for animal use was approved by the University of Florida Institutional Animal Care and Use Co mmittee (Permit A 130). Electroacupuncture The regime of therapeutic acupoints in this study was formulated according to previous research data and equine clinical acupuncture practice. Acupoints selected in this study mimicked the acupoint regime recommen ded for treating horses with RAO and other chronic lower airway inflammatory problems. Therefore, BL 13, Ding -chuan, Fei -men Fei -pan, Fei shu CV 22, and GV 14 were stimulated.78,97 Thirty two gauge, 2 or 3 inch l ong disposable stainless steel acupuncture needles (Kingli, China) were used. Details about needle size and direction of needle insertion for each acupoint are listed in Table 4 1. Needle insertion was performed by two veterinarians who were certified in e quine acupuncture practice. Information about locations of acupoints was reviewed and the methods of needle insertion were rehearsed by the acupuncturists prior to the beginning of the experiment. Acupoints were bilaterally connected and stimulated with an electrostimulator (Pantheon Research), except GV 14 and CV 22 were connected to one another (Figure 4 1). Acupoints were stimulated with 20 Hz for 10 minutes and then with 200 Hz for 10 minutes. The amplitude of stimulation was adjusted to the point at w hich slight muscle contraction was observed or to a level that the animal could comfortably tolerate. The frequency, duration, and amplitude of EA stimulation were based on the guidelines published by the Council of Acupuncture and Oriental

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129 Medicine Associ ation and previous research.236 238 However, when possible, the amplitude was maximally adjusted to 4 mA (but not greater than this level). Animals were treated once a day for seven consecutive days followed by two treatments per week for another two and a half weeks. Each horse received total 12 treatments. The first seven consecutive days of treatment was designed to investigate acute aggressive effect of EA, while the subsequent less frequent treatment was carried out to investigate effects of chronic treatment. Sham EA was performed using the same procedure except that the needles were taped to acupoints using adhesive tape (duct tape) without skin penetration (Figure 42). The sham EA mimicked animal handling pro cedures during the experiment, including needle placement, electrical wiring, and animal restraint. Data and Sample Collection Blood, plasma, and serum samples were collected one week prior to the beginning of the experiment (Pr), 20 24 hours after the 7t h treatment (Po), and 24 48 hours after the 12th treatment (Ps). Samples collected prior to the beginning of the treatment are referred to as the pretreatment samples. All samples were collected from the left external jugular vein. Twenty -five ml of whol e blood were collected in a 35 ml syringe containing sodium heparin (NDC 0641 247041). The final concentration of sodium heparin was 10 units/ml. This sample was used for in vitro whole blood stimulation. Additional blood samples were collected with parti ally evacuated blood collection tube containing sodium heparin (10 ml), no anti coagulant (10 ml), and EDTA (7 ml) vacutainer tubes (BD Vacutainer). Plasma or serum was harvested from each of these tubes. The samples collected in the EDTA containing tubes were submitted to the clinical pathology diagnostic laboratory of the College of Veterinary Medicine at University of Florida for complete blood counts.

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130 Bronchoalveolar lavage fluid (BALf) samples were collected one week prior to the experiment (Pr) and 2 4 48 hours after the 10th treatment (Po). The percentage of epithelial lining fluid (ELF) in the BALf samples was determined based on the urea dilution method. Details of the method explained in Chapter 3. The percentage of ELF in BALF was used to calculat e the total nucleated cells count relative to 100% ELF (ELF -corrected TNC) and concentrations of immunoglobulins relative to 100% ELF (ELF -corrected BLAf immunoglobulins). The RP -FE test was performed prior to the beginning of the experiment (Pr), 2024 ho urs after the 7th treatment (Po), and 2448 hours after the 12th treatment (Ps). Collection of the Po sample of BALf on the same day when RP -FE was performed was not desirable, since the procedures may affect the PFTPs. Rapid Partial Forced Expiration Mane uver The rapid partial forced expiration (RP -FE) maneuver was performed with the apparatus modified from the system described in Chapter 3. During the previous operation of this apparatus, some technical limitations were encountered. Reproducibility of air way pressure at total lung capacity (TLC). False differential pressure signals in the laminar flow element (LFE) caused by artificial inspiration. Residual signals from the pressure transducer at the end of RP -FE when the airway was empty. Requirement of a dditional personnel to control the vacuum level of the negative pressure reservoir and the initiation of RP FE. During the RP FE maneuver, the TLC of the lung was determined when the airway pressure at the end of the artificial inspiration reached 30 cm H2O. Variability of TLC airway pressure in the previous operation was due partly to a leak in the air direction control system

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131 manifold, lack of an appropriate pressure relief valve, and manual dependence on controlling airflow and initiating the RP FE maneu ver. To improve reproducibility of the airway pressure at TLC, a pressure regulator (EB5NL6, EquiliBar) (Figure 4 3) was installed on the manifold of the artificial inspiration system. The operation of this pressure regulator required an air supply at a se t reference pressure. The set reference pressure was maintained by a set -point regulator (type 41, Belloframe) (Figure 43) connected to an air compressor (Campbell Hausfeld). A manually actuated ball valve controlling the air from the air blower in the ar tificial inspiration system was replaced with a 2.5 NPT solenoid valve (8215A90, AscoRedhat) (Figure 4 4). False pressure differential signals in the laminar flow element (LFE) caused by artificial inspiration were prevented by isolation of the LFE from t he artificial respiration manifold and the negative pressure reservoir by installation of two 2.5 NPT solenoid valves (8215a90, AscoRedhat). One was installed upstream to the LFE, and the other was installed downstream. This eliminated pressure fluctuatio n in the LFE caused by artificial respiration. These two solenoid valves were controlled with a single relay switch for simultaneous operation. Opening these valves initiated the RP FE maneuver. The vacuum level in the negative pressure reservoir was main tained by a vacuum switch (9016 GAW1, Square D) and a 1/4 NPT solenoid valve (8016G, AscoRedhat). Briefly, the vacuum switch was used to inactivate the vacuum pump when the negative pressure of the vacuum reservoir reached 215 Torr, and to actuate the vac uum pump when the vacuum dropped to 130 Torr. The vacuum switch alone could not repeatedly reproduce an accurate negative pressure. Therefore, the negative pressure range (215130 Torr) was preset. Final fine adjustment to lower the vacuum was accomplished by activation of the solenoid valve. This also allowed

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132 the device operator to synchronize inflation of the lung to TLC while adjusting the vacuum of the negative pressure reservoir to a value very close to 200 Torr for the RP FE maneuver. Two hundred Tor r of vacuum was chosen based on the previous experiment, which indicated that this amount of vacuum completely emptied the airway without causing visible damage to the tracheal mucosal. In this experiment, an 865 watts/1500VA battery backup and power surge protector (BR1500, APC) was installed and connected to the module case of the signal conditioner (MC110), temperature meter (DP41 B -A -C24 TC), analog to digital converter (DI720), and a laptop computer. The system setup is illustrated in Figure 4 5. Symbols of the device component are illustrated in Figure 4 6. Airflow calibration of LFE was based on airflow values determined by the mass flow element (MFE). This MFE was routinely calibrated and was National Institute of standards and technology (NIST) tra ceable. Setup of LFE calibration is shown in Figure 4 -7. In this experiment, the accuracy of MFE calibration was tested by comparing integrated airflow signals generated by pulling a known volume of air through the pipe connected downstream of the calibrat ed LFE. A 12.829liter syringe was used to test the integrated airflow signals (Figure 4 8). By comparing the integrated volume derived from the P data to the known volume of the syringe, a correction factor for calibration was computed. Mean SD volume of air from integrated airflow signals generated by pulling 12.829liter of air through the calibrated LFE was 13.63856 0.023401 liters. This result indicated that the volume from integration was greater than the actual volume of the syringe being injected by 6.31%. This means that the volume integration result of 13.639 liters was, in fact, 12.829 liters.

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133 Therefore, the calculated correction factor for MFE/LFE is 1.063105 (13.639/12.829). This correction factor was used in the calculation of calibrated volum es. Arterial Blood Gas Analysis During RP-FE After chemical restraint, a 1.25 inch 21 G arterial catheter (SurflashTM, Terumo) was inserted in the left transverse facial artery on eight horses. Briefly, hairs over the left transverse facial artery were sha ved, and the skin was cleaned and disinfected with a surgical scrub. The catheter, which was pre -flushed with sterile heparinized saline (10 units/ml), was inserted into the transverse facial artery. A 1 -foot extension with a three -ways stop valve, which w as pre -filled with sterile heparinized saline, was connected to the catheter. The catheter hub was secured in place with superglue, and the catheter was flushed with 3 5 ml sterile heparinized saline. An arterial blood sample was collected with a 3 -ml syri nge, which was pre -flushed with heparin. To obtain an arterial blood sample, 5 ml of blood and saline in the extension set was withdrawn and discarded, and 1 ml of arterial blood was withdrawn with the prepared syringe. The syringe with the arterial blood sample was capped and put on ice. The extension cord was flushed with 10 ml sterile heparinized saline. Arterial blood samples were analyzed with a CG8+ cartridge and i STAT blood gas analyzer (Abbott Laboratories) within 10 minutes. Calculation of the Pul monary Function Test Parameters The linear relationship between airflow rate data from a direct reading of NIST traceable MFE and P data from LFE calibrations was tested using regression analysis. Maximum flow rate from NIST traceable MFE and the correspo nding P from the calibration were used for the calculation of pressure and temperature -corrected airflow rates. The original airflow rate from MFE (at the standard condition: 21 C, 760 Torr, and dry) was corrected with barometric pressure (BP) and body t emperature (BT) to reflect the airflow rate of the expired air from the horse

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134 during the RP -FE maneuver. Barometric pressure data were obtained from the weather station of the Department of Physics at the University of Florida, Gainesville. Data were avail able online at http://www.phys.ufl.edu/weather/. Pressure and temperature corrected airflow rate was further adjusted with a correction factor obtained from the syringe calibration. This adjusted rate was used for resetting the high calibration of the P of the LFE at the same MFE data point, and P of LFE at no flow was reset to zero. After resetting the high and low calibrations of LFE P, the LFE data represent the airflow rate generated by the RP -FE maneuver. These airflow data were used for computation of PFTPs as described in Chapter 3. BALf Collection and Preparation The procedure used for BALf collection was performed as described in Chapter 3. Methods used for evaluations of BALf cytology were similar to those explained in Chapter 3. The percentage of the epithelial lining fluid (ELF) recovered in BALf was determined with the urea dilution principle. The method for the urea assay was explained in Chapter 3. The BALf sample from each horse was collected in a sterile glass bottle and kept on ice until processed. Broncho alveolar lavage cells were isolated from BALf by centrifugation at 600 RCF at 4 C for 15 minutes. Supernatant from BALf was aliquoted and stored in 80 C for further analysis. Tumor Necrosis Factor Alpha (TNF ) Production of Whole Bloo d and TNF Assay The anti inflammatory activity of EA was indirectly determined by in vitro TNF production of whole blood after stimulation with selected immunological stimulants. Heparinized -blood stimulation and TNF assay were performed as described in the Chapter 2. Culture extract of A. fumigatus (CE), which was used in Chapter 2, was not used alone in this

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135 study due to low response. Moreover, the results from Chapter 2 showed that whole blood from some horses may not produce TNF after being stim ulated with CE for 6 hours. Immunoglobulin Assay Determination of circulating immunoglobulin isotypes was used as an indicator of humoral immunity. Concentrations of immunoglobulin isotypes (IgA, IgM, IgGa, IgGb, and IgG(T)), in plasma and BALf supernatant were determined with horse immunoglobulin ELISA Quantitation Kits (Bethyl Laboratories, Inc. Texas #E70 116, #E70114, #E70124, #E70127, and #E70 105). Assays were performed according to the protocols provided by the manufacturer as described in Chapte r 2. Cortisol Assay Quantitative analysis of cortisol was performed in serum samples with a commercial cortisol ELISA test kit (Endocrine Technologies). The assay followed the manufacturers protocol. Briefly, 50 l of standards, controls, and samples were added to designated wells in duplicate. One hundred l of the Cortisol Enzyme Conjugate solution was added to each well, except wells that were designated as controls. After incubating the plate at 37C for 1 hour, excess standards, controls, and samples were removed from the plate by inverted snapping. Residual fluid was then removed from the plate by firmly tapping the plate on a clean absorbent paper towel. Each well was washed three times with 300 l of washing solution, and residual washing solution w as removed from the plate by firmly tapping the plate on a clean absorbent paper towel. After washing, 100 l of TMB substrate solution was added to each well, and the plate was incubated at room temperature for 20 minutes. After incubation, the peroxidase reaction of the TMB substrate was terminated by adding 50 l of a stop solution to each well. The stop solution was added in the same sequence as the TMB substrate. The plate was

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136 evaluated with a microtiter plate reader at a wavelength of 450 nm. The conc entrations of cortisol in serum samples were calculated based on a four -parameter logistic curve -fit generated by KC4 software. Statistical Analysis Linear relationships between volumetric flow rate (Q) and differential pressure ( P) of the LFE pressure tr ansducer obtained before RP -FE and after RP -FE were evaluated using regression analysis. Homogeneity of data from pre -EA and pre -sham treatment groups for hematological parameters, BALf cytological parameters, concentrations of plasma immunoglobulins, conc entrations of BALf immunoglobulins, concentrations of TNF from an in vitro whole blood stimulation, PFTPs from RP -FE, and concentrations of serum cortisol were initially tested using Students t test for independent samples. Effects of treatment (EA, sha m), sampling time (Pr, Po, Ps), trial (1st, 2nd), horse (1 20), and sequence (1, 2) on dependent variables were tested using a general linear model. A variable called sequence was created for testing effect of treatment order. Horses receiving EA treatmen t first were assigned as sequence 1. Horses receiving sham treatment first were assigned as sequence 2. The dependent variables included hematological parameters, BALf cytological parameters, concentrations of plasma immunoglobulins, concentrations of ELF -corrected BALf immunoglobulins, concentrations of TNF from an in vitro whole blood stimulation, PFTPs from RP -FE, and concentrations of serum cortisol. Main effects and their interactions were tested using the Type III sum of squares test. Sequence ne sted in horse was treated as a random factor.

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137 Differences among sampling times (Pr, Po, Ps) within dependent variables were compared using the multiple t tests with Bonferroni correction. The Bonferoni correction is used to keep the total chance of erroneously reporting a difference below alpha = 0.05. Differences in BALf cytological parameters and in concentrations of BALf immunoglobulin isotypes between Pr and Po were compared using paired t -test. Statistical analysis was performed using SPSS 17 softwar e for Windows. Data is reported as mean standard error of mean (mean SE). P value 0.05 was used for determining significance. Results Subjects Horses used in this study included 19 geldings and one mare. Means SD of the age and body weight of all horses were 8 1.1 years and 551 37 kg. Means SD of age and body weight of ho rses receiving odd number assignments were 8 0.6 years and 541 41 kg. Means SD of age and body weight of horses receiving even number assignments were 8 1.3 years and 560 32 kg. During the study period, one evennumbered horse (H4) was treated for hoof abs cess, and one odd -numbered horse (H11) was treated for acute abdomen. Multiple dosages of NSAIDs were administered for treatments of these diseases and data derived from these horses were not included in statistical analyses. EA and Sham Treatments Reactio ns of subjects to the EA and the sham treatments were observed. Reactions to sham treatment ranged from no reaction to agitation, restlessness, and trying to remove the needle. Reactions to EA ranged from no reaction to severe agitation. One horse in the o dd numbered group did not accept the EA procedure during the 2nd EA session, and EA was discontinued on this horse. Experimental data from this horse for the EA trial were not included

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138 in statistical analyses. Animal reactions to EA were more prominent tha n those to the sham treatment. A summary of reactions to EA and to sham treatment is presented in Table 4 2. Reactions to needles were greatest at GV 14, Ding -chuan, and BL 13. There was no reaction at CV 22 in either EA or sham treatments. Complete Blood Count and Other Hematological Parameters Mean values of complete blood counts and other hematological parameters of the pre treatment samples of the EA and the sham groups were not significantly different; p -values from independent t -tests white blood cell counts (Wbc) (0.45), percentage of neutrophils (Neu) (0.84), percentage of lymphocytes (Lym) (0.63), percentage of monocytes (Mono) (0.40), percentage of eosinophils (Eos) (0.33), percentage of basophils (Baso) (0.86), red blood cell counts (Rbc) (0.53), hemoglobin (Hb) (0.45), hematocrit (Hct) (0.46), mean corpuscular volume (Mcv) (0.87), mean corpuscular hemoglobin (Mch) (0.76), mean corpuscular hemoglobin concentration (Mchc) (0.96), cellular hemoglobin concentration (Chcm) (0.55), and corpuscular hemog lobin content (Ch) (0.72). P values from the Type III sum of squares test of the main effects and their interactions for the white blood cell parameters are given in Table 4 3. Wbc, mean percentages of Neu, Lym, Mono, Eos, and Baso of all samples were with in normal limits throughout the study (Table 4 4). Pairwise comparisons between Pr, Po, and Ps samples of all the white blood cell indices in the EA group were not significantly different (Table 4 5). In the sham group, even though the Wbc of Po was signi ficantly different from that of the Ps sample (p -value = 0.02), Wbc from both Po and Ps samples were within the normal reference values (Table 4 4). P values from the Type III sum of squares test of the main effects and their interactions for the red blood cell indices are given in Table 4 6. Mean SE of the red blood cell indices are

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139 given in Table 4 7. P -values of pairwise comparisons between Pr, Po, and Ps samples of all the red blood cell indices in the EA and the sham groups are given in Table 4 8. Even though some pairwise comparisons of Rbc, Hct, Hb, Mcv, and Ch in the EA and the sham group were significantly different, mean values of these red blood cell parameters were within the normal reference values. The mean values of Mchc and Chcm were in a hig h normal range or greater than the normal reference values throughout the experiment. Hemoglobin (Hb) values from pre -treatment samples from 6 of 17 horses in the EA group and 7 of 19 horses in the sham group were greater than normal reference values (11.216.2 mg/dL), and ranged from 16.3 18.5 and 16.318.7 mg/dL, respectively. The high Hb values in these horses corresponded to their hematocrit (Hct) values, which were in the upper limit of the normal reference values (30 43%) or greater. Both Hb and Hct o f all horses were within normal limits in samples collected after the 7th and 12th treatments. An increase in Hb in pre treatment samples may result from excitation caused by initial handling. The excitement activates the sympathetic nervous system, which could cause a spleenic contraction and released stored red blood cells. Broncho -alveolar Lavage and BALf Cytology Mean SD percentages of BALf recovered by using a fiberoptic endoscope and percentages of ELF in BALf determined by urea dilution technique are shown in Table 4 9. Mean SD percentage of overall BALf recovered was 75 5.5%, and percentages ranged from 60 to 85%. Measurements of recovered BALf did not include the foamy part of the BALf. The physical appearance of all samples was clear and slightly t urbid. Based on simple observation, there were no visible mucus flakes. Mean values of the BALf cytology indices of the EA and the sham groups were not significantly different; p values from independent t tests the ELF -

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140 corrected TNC (0.39), percentage of m acrophages (Mac) (0.68), Lym (0.67), Neu (0.87), Mast (0.63), and Eos (0.35). P values from the Type III sum of squares test of the main effects and their interactions for the BALf cytological parameters are given in Table 4 10. Mean SE values of the ELF c orrected TNC, Mac, Neu, Mast, and Eos in BALf are given in Table 4 11. Within treatment comparisons of ELF -corrected TNC, Mac, Lym, and Eos of pre treatment and post 10th treatment samples were not significantly different (Table 4 12). The mean percentages of Neu in post 10th treatment samples of the EA group was significantly higher than that of the pre -treatment samples (p -value = 0.04). The mean percentages of Neu in post 10th treatment samples of the sham group also increased, but the increase was not s ignificant (p -value = 0.06). The mean value of Mast in post 10th treatment samples of the EA and sham groups significantly decreased (p -value of the EA group = 0.05, and p-value of the sham group = 0.01). Immunoglobulins Mean concentrations of immunoglobul in isotypes in pre treatment samples of the EA and the sham groups were not significantly different based on independent t tests IgA (0.3), IgM (0.7), IgGa (0.7), IgGb (0.7), and IgG(T) (0.8). P values from the Type III sum of squares test of the main effe cts and their interactions for plasma immunoglobulin isotypes are given in Table 4 13. Mean SE concentrations of plasma immunoglobulin isotypes are given in Table 414. Pairwise comparison between Pr, Po, and Ps samples of all plasma immunoglobulin isotype s are given in Table 4 -15. Even though p -values of the treatment effect for all immunoglobulin isotypes were significant, results of pairwise comparisons between Pr, Po, and Ps samples of all

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141 immunoglobulin isotypes indicated that the significantly differe nt might be due partly to individual subject difference. Mean concentrations of immunoglobulin isotypes in the ELF -corrected BALf of pre treatment samples of the EA and the sham group were not significantly different; p -values IgA (0.5), IgM (0.4), IgGa (0 .7), IgGb (0.9), and IgG(T) (0.9). P values from the Type III sum of squares test of the main effects and their interactions for concentrations of IgA, IgM, IgGa, IgGb, and IgGt in the ELF -corrected BALf are given in Table 4 16. Mean SE concentrations of E LF -corrected BALf immunoglobulins are given in Table 4 17. Withingroup comparisons of ELF -corrected BALf immunoglobulin isotypes of Pr and Po -samples in the EA and the sham groups were not significantly different (Table 4 18). TNF Production of Whole B lood Stimulation After in vitro incubation of whole blood, TNF production with no stimulant added was less than 0.08 x103 pg/ml. There was no detectable TNF by ELISA assay when only PBS was added. Mean concentrations of TNF production from whole bloo d stimulated with LPS, Zym, CA, CA+CE, CA+LPS, CE+LPS, and CA+CE+LPS of the pre -treatment samples of the EA and the sham were not significantly different based on independent t tests; p values LPS (0.3), Zym (0.4), CA (0.9), CA+CE (0.9), CA+LPS (0.6), CE+L PS (0.6), and CA+CE+LPS (0.7). P values from the Type III sum of squares test of the main effects and their interactions for the TNF production from stimulated whole blood are given in Table 419. Mean SE concentrations of TNF production from whole blood when stimulated with each stimulant are given in Table 4 20. EA generally suppressed TNF production when the whole blood was stimulated with all stimulants. The significant of suppressions were greatest between pre treatment and post 7th treatment sam ples, and were greater than those of between

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142 pre treatment and post 12th treatment samples (Table 4 21). Suppression of TNF production from whole blood by EA treatment of post 7th treatment and post 12th treatment samples when the whole blood was stimula ted with LPS, CA, CA+CE, CA+LPS, CE+LPS, and CA+CE+LPS were not significantly different (Table 4 21). In the EA group, TNF production of whole blood stimulated with Zym in the pre treatment samples did not significantly differ from that of post 7th and post 12thtreatment samples (Table 4 21). However, the decrease from the post 7th treatment samples to post 12th treatment samples was significant (p -value individual responses to Zym. The individual variation in response to Zym can be seen from the large standard error of the mean TNF concentration derived from this stimulant (Table 4 20). In the sham treatment group, mean concentrations of TNF production from whole blood samples stimulated with Zym, CA, CA+CE, CA+LPS, CE+LPS, and CA+CE+LPS were not significantly different between sampling times (Table 4 21). W hen LPS was used as a stimulant alone, TNF production from whole blood samples collected after the 12th treatment significantly increased when compared to those of pre treatment and post 7th treatment samples (Table 4 21). Moreover, when CA was used as s timulant alone, TNF production from whole blood samples collected after the 12th sham treatment significantly increased when compared to the post 7th treatment sample, but was not significantly different when compared to the pre treatment samples. Rapid Partial Forced Expiration Maneuver The relationship of the airflow rate to the pressure differential ( P) was linear. Examples of linearity of the airflow rate to P before RP -FE and after RP -FE are shown in Figure 4 9.

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143 After being chemically restrained wi th an intravenous administration of 20 -30 g/kg detomidine hydrochloride, an endotracheal (ET) tube was successfully inserted in each horse. Endotracheal tube intubation induced coughs in some horses. Coughing subsided once the ET tube was in place. Artifi cial inspiration caused expansion of the thoraco abdominal wall, which resolved when the air blower was turned off. There were no major signs of discomfort during artificial inspiration or the RP -FE maneuver. Mean values of PFTPs of the pre treatment sampl es of the EA and the sham groups were not significantly different; p -values from independent t tests FEV0.5 (0.88), FEV0.75 (0.78), FEV1.0 (0.69), FEV1.5 (0.98), FEV2.0 (0.72), FEV2.5 (0.71), FEV3.0 (0.71), FVC (0.71), PEF (0.72), MEF25% (0.79), MEF50% (0. 51), MEF75% (0.48), FEV0.5/FVC (0.75), FEV0.75/FVC (0.54), FEV1.0/FVC (0.44), FEV1.5/FVC (0.29), and FEV2.0/FVC (0.52). P values from the Type III sum of squares test of the main effects and their interactions for PFTPs are given in Table 4 22 and Table 4 23. Mean SE values of PFTPs are given in Table 4 24 and Table 4 25. P -values of pairwise comparisons between Pr, Po, and Ps data of PFTPs in the EA and the sham groups are given in Table 4 26 and Table 4 27. In the EA treatment group, FEV0.75 and FEV1.0 af ter the 7th treatment were significantly greater than those in the pre treatment. When pre treatment PFTPs were compared with those obtained after the 12th treatment, FEV0.75, FEV1.0, FEV1.5, PEF, MEF 25%, MEF 50%, and MFE 75% significantly increased. More over, FEV1.0, FEV1.5, PEF, and MEF 25% obtained after the 12th treatment were significantly greater than those obtained after the 7th treatment (Tables 4 24 and Table 4 25).

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144 In the sham group, when pre -treatment PFTPs were compared with those obtained after the 7th treatment, FEV1.0, PEF, and MEF 25% significantly increased. FEV0.5, FEV0.75, FEV1.0, FEV1.5, FEV2.0, FEV2.5, FEV3.0, FVC, PEF, and MEF 50% obtained after 12th treatment were significantly greater than pre -treatment PFTPs. Moreover, FEV0.75, FEV 1.0, FEV1.5, and PEF obtained after the 12th treatment were significantly greater than values obtained after the 7th treatment (Tables 4 24 and 4 25). Arterial Blood Gas Analysis During RP-FE Results of arterial blood pH, pO2, pCO2, and concentrations of H CO3 (on pre and post RP FE, and 1, 2, and 5 minutes after RP -FE was initiated) were obtained from 8 horses, are presented in Figures 4 10 to 4 13. When artificial inspiration was initiated, the arterial blood pH and oxygen partial pressure (pO2) increased and the concentration of HCO3 and carbon dioxide partial pressure (pCO2) decreased. The results indicated that hyperventilation of the airways suppressed the normal respiratory drive of the horses by increasing blood oxygenation. Low arterial concentrati on of HCO3 and pCO2 suggested hypocapnea. Together with an increase in the arterial blood pH, results from blood gas analysis indicated that artificial ventilation during RP FE induced respiratory alkalosis. Serum Cortisol Pre -treatment serum cortisol conc entrations in the EA and sham groups were not significantly different (p -value = 0.85). P -values from the Type III sum of squares test of the main effects and their interactions for serum cortisol are given in Table 4 28. Mean SE concentrations of serum co rtisol are given in Table 4 29. Pairwise comparisons of serum cortisol from the EA and the sham groups were not significantly different. P -values from pairwise comparisons of Pr Po, Pr -Ps, and Po -Ps of the EA group were 0.71, 1.00, and 0.51. P values

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145 from pairwise comparisons, of Pr -Po, Pr Ps, and Po Ps of the sham group were 0.50, 1.00, and 0.79. Discussion Seventeen of 24 horses that were used in the study detailed in Chapter 3 also were used in this study. Three additional horses were recruited from the research herd to yield a total of 20. None of the horses that had been used in the Chapter 3 experiment possessed significantly different PFTPs from one another, suggesting that these horses have normal pulmonary functions. This may be one of the major fac tors explaining why there were no effects of EA on BALf cytology and PFTPs in this study. To investigate effects of EA on the immune system and pulmonary function, multiple acupoints were used. Twelve acupoints used in this study were chosen from acupoints recommended for treating equine chronic respiratory disorders.78 All acupoints are located on the cranial part of the body or on the lateral thoracic wall. Cutaneous sensations originating from GV14 and Ding -chuan are likely transmitted to the central nervous system via the cutaneous branch of the local spinal nerves.239 Cutaneous sensations from BL 13 and Fei -men are likely transmitted via the dorsal branch of the local thoracic nerve. Cutaneous sensation from Fei -pan is likely transmitted via both dorsal branch of the local thoracic nerve and the intercostobrachial nerve. Cutaneous sensation from Fei -shu is likely transmitted via the intercostal nerve. Cutaneous sensation originating from CV 22 area is likely transmitted via the ventral branch of the 6th cervical nerve and cranial branch of the supraclavicular nerve.169,239 Piercing an acupoint with an acupuncture needle not only generates a nerve signal in the skin, but also activates sensory receptors residing in the subcutaneous tissue and muscles located under the acupoint. When the selected twelve acupoints are stimulated, likely nerve of activation include facial, intercostobrachial, cutaneous branch of the local spinal nerve, and dorsal branches

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146 of local spinal nerve.169,239241 Details of acupoint locations, cutaneous innervations, muscles involved and their innervations are given in Table 4 30. It is obvious that inserting an acupuncture needle through the skin at an acupoint activates both mechanoreceptors and pain receptors in the tissues. Mechanoreceptors in the skin and subcutaneous tissues include Meissners corpuscles, Pacinian corpuscles, and Merkels disks.242 Activation of these receptors generates sensory signals, which are transmitted to the spinal cord via A axon. These receptors have a low threshold of activation. Receptors for pain s ensation, or nociceptors, are free nerve endings that are associated with either C or A axon. They have a high threshold of activation and transmit pain, temperature, and crude touch sensation.242 Electroacupuncture in this study seemed to generate both mechanical and pain sensations. Some horses showed no reaction to EA, while some horses w ere agitated by the procedure. The reactions to EA ranged from mild skin twitching to severe agitation. Chemical restraint with a short acting sedative during needle placement can be used, but this practice was not considered in this experiment. However, w hen additional physical restraint was required, either a lip chain or shoulder twitch was used. Numbers of Wbc and Rbc of all subjects were within normal reference values throughout the study. Results from the differential counts of white blood cells from individual horses were within normal reference values throughout the study. Horses used in this study were handled regularly (3 5 times a week) and were assumed to be accustomed to the research facilities. However, physiological excitation caused by initia l handling for taking pre treatment blood samples was observed. Hemoglobin concentrations of pre treatment samples from 6 of 17 horses in the EA group and 7 of 19 horses in the sham group were greater than normal reference values (11.216.2 g/dL), and rang ed from 16.3 18.5 and 16.318.7, respectively. High values of Hb in

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147 these horses coincided with their high Hct values, which were in the upper limit of the normal reference values (30 43%) or greater. Both Hb and Hct of these horses were within normal lim its after the 7th treatment and after the 12th treatment. It is assumed that the high Hb and Hct values of the pre -treatment samples in this study resulted from excitation caused by initial handling. The excitement activates the sympathetic nervous system, which could cause spleenic contraction and release of stored red blood cells into circulation. Mean Mchc and Chcm of the EA and sham groups were in high normal or greater than the reference values throughout the experiment. High Mchc and Chcm might be cau sed by hemolysis of red blood cells or by the clumping of red blood cells, which led to an under estimate of Rbc numbers. Blood samples in EDTA vacutainer tube for hematology in this study were stored on ice prior to being submitted to the laboratory. In humans with cold agglutinin (an auto -immune disease that caused hemolytic anemia or AIHA), the red blood cells agglutinate and cause high Mchc and Chcm when blood temperature is colder than 37 C. In veterinary medicine, cold agglutinin has been described mo st often in dogs and horses. The condition is idiopathic and may be secondary to chronic infections, other autoimmune disorders, or neoplastic diseases.243,244 Whether this condition present in horses being used in this study was not investigated, but is not likely. Another possibility is rouleaux formation, a physiologic phenomenon in which red blood cells of horses stack together forming a line when the blood sample is still. Rouleaux formation disappears when the blood sample is mixed thoroughly. Percentages of BALf neutrophils in pre treatment samples of the EA and sham groups in the first trial ranged from 0.5 6.5%. This level of percentages in BALf neutrophils has been reported as characteristic of BALf cytolog y of normal horses.215 The results suggested that horses used in this study did not have pulmonary disorders. Percentages of neutrophils were

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148 significantly higher in BALf samples that were collected after the 10th t reatment, which was 8 10 days after the second RP -FE maneuver. Also, percentages of BALf neutrophils in pre -treatment samples from the second trial were greater than those of the first trial. These results together suggested that RP FE induced pulmonary i nflammation, which was carried over to the second trial. Collecting BALf samples and performing the RP -FE maneuver at the same time was avoided during the initial study design. A period of 8 10 days was first thought to be enough for the pulmonary tissues to recover from RP -FE -induced inflammation. However, the results indicated that inflammation in the airways and pulmonary tissues caused by RP FE lasted longer than the period first hypothesized. Despite RP FE induced inflammation in the pulmonary tissues, the PFTPs were not compromised. According to the amount of TNF produced from whole blood stimulation, LPS was more potent than Zym and CA. There was a synergistic effect when CA or CE was individually or both added to LPS for the stimulation. Electroacu puncture, but not the sham treatment, significantly suppressed in vitro TNF production from whole blood when stimulated with LPS, CA+CE, CA+LPS, CE+LPS, and CA+CE+LPS. In the EA treatment group, mean TNF production when whole blood was stimulated with Zym alone decreased over time (Figure 4 14), and it is possible that EA progressively suppressed TNF production. However, the suppression was not shown to be statistically significant due to a large variation in individual response. Tumor necrosis factor alpha is an inflammatory cytokine. It is an important component of the early response in the innate immune system. It is thought to be involved in the pathogenesis of chronic lower airway inflammation in horses.144 Suppression of TNF production from

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149 immune cells after EA treatment, as demonstrated when the whole blood was stimulated with LPS, CA+CE, CA+LPS, CE+LPS, and CA+CE+LPS, may partly play a role in how EA exerts an anti inflammatory action. In RP FE maneuve r, after the ET tube was connected to the FE -system, the RP FE maneuver for each horse took less than five minutes. When the ET tube was connected to the FE system, the artificial inspiration was initiated. Inspiration was accomplished by inflation of the airway to a pressure of 30 cm H2O. Expiration was driven by passive mechanism, which is based on the elastic recoil properties of pulmonary tissues and thoracic walls. Artificial respiration at the rate of 25 30 breaths/minute suppressed the physiological respiratory drive of the tested subjects, and allowed the RP -FE operator to override the normal respiratory rate of the horse. Results from the blood gas analysis suggested that artificial inspiration during RP FE increased blood pH and pO2, while decreasi ng pCO2 and HCO3, which indicated respiratory alkalosis. Pulmonary function test parameters from this study were different from values reported by a previous study.175 The causes of differences in PFTPs were discussed in detail in Chapter 3, and included differences in manifold designs, in the negative pressures used t o empty airways, and in methods of measuring airflow. Mean FVC from both the EA and sham groups in this experiment were greater than the TLC value suggested for equine species (55 liters).192 Possible explanations for high FVC include dec reased density of expired air during RP FE -maneuver. This may have happened when gas in the airways was exposed to the negative pressure. In normal expiration, gas in the lungs and airways is compressed due to the contraction of the thoracic walls by expir atory muscle action. Expiration is a result of an increase in intra -pleural pressure, which also increases airway pressure. When airway pressure is greater than atmospheric pressure, air in the lungs and airways is expired. Artificial expiration in RP -FE

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150 m aneuver is caused by pulling air out from the airways and lungs. At TLC (artificially inflating airways to 30 cm H2O), gas in the airways was compressed. When the compressed gas in the airways and lungs was exposed to the negative pressure, the gas moved a nd expanded at the same time. Another possible explanation for high FVC is that the RP FE maneuver might have completely emptied air from the airways and lungs, including air in the physiological dead spaces. In this study, an individual RP -FE maneuver for each horse was composed of 3 5 artificial forced expirations. Forced vital capacity, PEF and flow -volume loop of the first artificial forced expiration in every maneuver were always greater than those of the subsequent artificial forced expirations (Figur es 4 15 and 4 16). It is possible that some parts of the alveoli and airways were collapsed after the first artificial forced expiration, and were not inflated by the artificial inspiration. Because of the elastic properties of airways and alveoli, which a re located in a collapsible thoracic cavity, it might be possible to completely empty air from the airways and lungs. At the end of the artificial forced expiration, the thoracic and abdominal cavities were severely contracted. This study also investigated the effects of treatments (EA and sham) and RP -FE on serum cortisol concentration. The hormone cortisol is released mainly from the adrenal cortex and has been used as an indicator of stress response in various species, including the horse. Average concen trations of serum cortisol from horses in the EA and sham groups in this experiment were 6.6 and 6.9 g/dL, respectively. These values were less than the reference values ( g/dlL) for normal horses.245 Serum cortisol concentrations among three sampling times (pre after the 7th, and after the 12th treatments) were not significantly different. These results suggested that neither treatments (EA and sham) nor RP FE maneuvers in this study induced change in cortisol.

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151 In summary, the results of this study suggest that EA pro vides anti inflammatory effect as demonstrated by a suppression of TNF production of stimulated whole blood. These effects may help modulate inflammatory response present in several equine diseases, including chronic respiratory diseases. Therefore, we b elieve that EA treatment at GV 14, CV 22, BL 13, Ding chuan, Fei -men Fei -pan, and Fei-shu has merit in the treatment of inflammatory diseases in horses, but requires further study to be fully validated.

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152 Figure 4 1. Electroacupuncture.

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153 Figure 4 2. Sham electroacupuncture.

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154 Figure 4 3. A pressure regulator and a set point regulator installed on the manifold of the artificial inspiration system. Two analog pressure gauges, G1 and G2 were used for monitoring pressure in the manifold of inspirator y system and pressure of the set point regulator. G 1 G2 Pressure regulator

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155 Figure 4 4. Solenoid valve used for controlling the air from the air blower in the artificial inspiration system and for isolating the LFE from the artificial respiration manifold and the negative press ure reservoir.

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156 Figure 4 5. Diagram of the rapid partial forced expiration apparatus. MC1 10 DP 205 DP41 B DP 702 A P C V P

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157 Figure 4 6. Component symbols. DP41 B MC1 10 DP 205 DP 702 Temperature meter Signal conditioner Signal interfa ce Analog digital converter Rigid polypopylene tube Thermocouples Connecting cable Electrical power cord Vacuum switch Manual relay switch Electrical power source V P Negative pressure re servoir Air blower Variable transformer Analog pressure gauge Laminar flow element Filter Pressure transducer Manual actuated PVC valve Computer Solenoid valve Airway pressure relive valve Vacuum Pump

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158 Figure 4 7. Apparatus setup for laminar flow element (LFE) calibr ation using NIST traceable mass flow element (MFE). The LFE, MFE, and air blower were linearly connected and air was sucked out through the MFE. Yellow arrows indicate direction of airflow. LFE MFE

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159 Figure 4 8. The 12.829liter syringe used for testing an accur acy of integrated airflow volume compare with a 60 ml disposable syringe

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160 0 1 2 3 4 5 6 0 10 20 30 40 50 60 Direct reading of MFE airflow (liters/second) LFE differential pressure (cm H2O) Before RP-FE After RP-FE Figure 4 9. Linear relationship of airflow rate to P before RP -FE and after RP -FE. Lines were plotted using calibration data from RP -FE on July 26, 2008. After the initial calib ration, RP -FE was performed in 4 horses. 7.3 7.35 7.4 7.45 7.5 7.55 Pre RP-FE 1 2 5 Post RP-FE Time (minute) Arterial blood pH Figure 4 10. MeanSD arterial blood pH during RP FE maneuver (data were obtained from 8 horses).

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161 25 35 45 55 Pre RP-FE 1 2 5 Post RP-FE Time (minute) Arterial blood pCO2 (mmHg) Figure 4 11. MeanSD arterial blood pCO2 during RP -FE maneuver (data were obtained from 8 horses). 50 70 90 110 130 Pre RP-FE 1 2 5 Post RP-FE Time (minute) Arterial blood pO2 (mmHg) Figure 4 1 2. MeanSD arterial blood pO2 during RP FE maneuver (data were obtained from 8 horses).

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162 22 24 26 28 30 Pre RP-FE 1 2 5 Post RP-FE Time (minute) Arterial blood HCO3 (mmol/l) Figure 4 13. MeanSD arterial blood HCO3 during RP -FE maneuver (data were obtained from 8 horses). 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 Pr Po Ps Sampling time TNF-alpha (pg/ml) EA Sham Figure 4 14. Mean SD TNF production in electroacupuncture ( EA) and sham -EA (sham) groups when whole blood was stimulated with Zymosan. Pr = pre treatment, Po = after the 7th treatment, Ps = after the 12th treatment.

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163 0 20 40 60 80 0 0.5 1 1.5 2 2.5 3 Forced expiratory time (second) Forced expiratory airflow (liters/second) 1st FE 2nd FE 3rd FE 4th FE 5th FE Figure 4 15. Airflow rates from five artificial forced expirations (FE) during rapid partial fo rced expiration maneuvers on one horse in 20 July 2008. 0 10 20 30 40 50 60 70 0 20 40 60 80 Forced expiratory airflow rate (liters/second) Forced expiratory volume (liter) 1st FE 2nd FE 3rd EF 4th FE 5th FE Figure 4 16. Flow -volume loops from five artificial forced expirations (FE) during rapid partial forced expiration maneuvers on one horse in 20 July 2008. Data from same horse as in Figure 5 15.

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164 T able 4 1. Anatomical location of acupoints, their Western medical indication, needle size and method of insertion. Cun = unit of measurement in TCVM. Length of cun is relative to the body size of the animal, and the width of scapula from the cranial to caudal border is 3 cun.246 (Sources: Xie H, Yamagiwa K. Equine Classical Acupoints In: Xie H,Preast V, eds. Xie's veterinary acupuncture. 1st ed. 2007; pages 89127, and Xie H, Trevisanello L. Equine Transpositional Acupoints In: Xie H,Preast V, eds. Xie's veterinary acupuncture. 2007; pages 2787.78,135) Acupoint Location Indication in Western medicine Needle size (inch) Needle insertion method GV 14 C7 T1 dorsal midline cranial to depressio n of wither Fever, cough, heaves, and immune stimulation 2 Perpendicular to skin CV 22 Ventral midline at depression cranial to sternum Cough, heaves, and asthma 2 Perpendicular to skin BL 13 Caudal edge of scapular cartilage (8th intercostals space) 3 cun lateral to dorsal midline Cough, heaves, and asthma 3 Perpendicular to skin Fei men 1/3 distance from top of scapular along cranial border Upper respiratory problem, cough, and asthma 2 Caudoventrally Fei pan 1/3 distance from top of scapular alon g caudal border Lower airway and lung disease 2 Cranioventrally Fei shu 9 th intercostals space on line connecting shoulder and coxofemoral joint Upper airway infection, cough, heaves, asthma, bronchitis, pneumonia, flu, and COPD 2 Ventromedially and para llel to thoracic wall Ding chuan 0.5 cun lateral to GV 14 Cough and asthma 2 Perpendicular to skin

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165 Table 4 2. Numbers of horses categorized by degree of reaction to EA and to sham treatments in the 1st and 2nd trials prior to eliminating the horse that did not accept EA treatment and horses receiving NSAIDs. Study Treatme nt Degree of reaction Total A B C D E 1st trial EA 5 1 1 2 1 10 Sham 7 2 1 0 0 10 2nd trial EA 6 1 0 2 1 10 Sham 8 1 1 0 0 10 A = no reaction, B = mild skin twitching, C = moderate skin twitching, D = intermittent forceful skin twitching, E = co ntinuous forceful skin twitching and restlessness, EA = electroacupuncture, Sham = sham EA. Table 4 3. P -values from the Type III sum of squares test on main effects and their interactions for white blood cell indices. Factor Wbc Neu Lym Mono Eos Baso T reatment 0.91 0.92 0.50 0.10 0.68 0.95 Sampling time 0.19 0.93 0.84 0.58 0.37 0.29 Trial 0.25 0.55 0.74 0.94 0.43 0.84 Sequence 0.13 0.75 0.70 0.87 0.81 0.24 Treatments sampling time 0.16 0.93 0.89 0.29 0.39 0.61 Horse sequence 0.02 0.11 0.64 = interaction between factors, Wbc = white blood cell counts, Neu = percentage of neutrophils, Lym = percentage of lymphocytes, Mono = percentage of monocytes, Eos = percentage of eosinophils, Baso = percentage of basophils.

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166 Table 4 4. MeanSE white blood cell indices, by treatment and sampling time. Normal reference values in parentheses. Tx Sampling time Wbc x103cell/ L (5.5 11.0) Neu% (28.0 82.8) Lym% (19.8 58.9) Mono% (1.4 10.5) Eos% (0 8.7) Baso% (0 2.0) EA Pr 8.77 0.28 61.9 1. 5 29.0 1.3 4.6 0.4 3.9 0.5 0.4 0.1 Po 8.78 0.28 61.9 1.5 28.9 1.3 3.9 0.4 4.6 0.5 0.3 0.1 Ps 8.65 0.28 61.8 1.5 29.0 1.3 4.5 0.4 4.1 0.5 0.3 0.1 Sham Pr 9.07 0.26 61.6 1.4 27.9 1.2 5.1 0.4 4.8 0.5 0.4 0.1 Po 8.20 + 0.26 61.2 1 .4 28.3 1.2 5.3 0.4 4.5 0.5 0.4 0.0 Ps 8.93 0.26 62.3 1.4 28.9 1.2 4.4 0.4 3.8 0.5 0.2 0.0 + = p -value of comparison between Po and Ps treatment, Po = after the 7th treatment, Ps = after the 12th treatment, Tx = treatment, EA = electroacupuncture, Sham = sham EA, Wbc = white blood cell count, Neu = neutrophil, Lym = lymphocyte, Mono = monocyte, Eos = eosinophil, Baso = basophil.

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167 Table 4 5. P -values from pairwise comparisons of white blood cell indices, by treatment and sampli ng time. Tx Pairwise Wbc Neu Lym Mono Eos Baso EA Pr Po 1.00 1.00 1.00 0.51 0.48 1.00 Pr Ps 1.00 1.00 1.00 1.00 0.62 1.00 Po Ps 1.00 1.00 1.00 1.00 0.72 1.00 Sham Pr Po 0.06 1.00 1.00 1.00 1.00 1.00 Pr Ps 1.00 1.00 1.00 0.99 0.71 0.76 Po Ps 0.02 1.00 1.00 0.90 0.65 0.30 Pr = pre treatment, Po = after the 7th treatment, Ps = after the 12th treatment, Tx = treatment, EA = electroacupuncture, Sham = sham EA, Wbc = white blood cell count, Neu = neutrophil, Lym = lymphocyte, Mono = monocyte, Eos = eos inophil, Baso = basophil. Table 4 6. P -values from the Type III sum of squares test on main effects and their interactions for red blood cell indices. Factor Rbc Hb Hct Mcv Mch Mchc Chcm Ch Treatment 0.61 0.68 0.69 0.47 0.74 0.30 0.93 0.68 Sampling tim e Trial 0.02 0.15 0.76 0.03 0.06 Sequence 0.83 0.76 0.82 0.45 0.51 0.71 0.44 0.49 Treatments sampling time 0.33 0.43 0.49 0.13 0.27 0.35 0.07 0.77 Horse sequence 0.01 0.64 = interaction between factors, Rbc = red blood cell count, Hct = hematocrit, Hb = hemoglobin, Mcv = mean corpuscular volume, Mch = mean corpuscular hemoglobin, Mchc = mean corpuscular hemoglobin concentration Chcm = cellular hemoglobin concentration, and Ch = corpuscular hemoglobin content.

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168 Table 4 7. MeanSE red blood cell indices, by treatment and sampling time. Reference values in parentheses. Tx Sampling time Rbc (6.7 10.0x106 cell/ L) Hct (30.043.0%) Hb (11.216.2 g/dL) Mcv (37.550.0 fL) Mch (14.018.7 pg) Mchc (36.438.8 g/dL) Chcm (34.937.6 g/dL) Ch (13.717.9 pg) EA Pr 9.28 0.21 39.6 0.9 #15.3 0.3 *#42.7 0.5 #16.5 0.2 *38.7 0.4 38.6 0.1 *16.5 0.2 Po 8.90 0.21 37.6 0.9 14.7 0.3 42.3 0.5 +16.5 0.2 +39.1 0.4 38.6 0.1 16.4 0.2 Ps 8.71 0.21 36.9 0.9 14.1 0.3 42.3 0.5 16.2 0.2 38.4 0.4 38.7 0.1 16.5 0.2 Sham Pr *#9.49 0.20 *#40.4 0.8 *#15.6 0.3 42.5 0.5 *#16.4 0.2 *38.7 0.4 38.7 0.1 *16.5 0.2 Po 8.69 0.20 36.9 0. 8 14.4 0.3 42.4 0.5 +16.6 0.2 +39.2 0.4 38.3 0.1 +16.4 0.2 Ps 8.93 0.20 37.7 0.8 14.4 0.3 42.3 0.5 16.2 0.2 37.3 0.4 38.9 0.1 16.5 0.2 = p -value of comparison between Pr and Po -value of comparison between Pr and Ps -value of comparison between Po and Ps -treatment, Po = after the 7th treatment, Ps = after the 12th treatment, Tx = treatment, EA = electroacupuncture, Sham = sham EA, Rbc = red blood cell count, Hct = hematocrit, Hb = hemoglobin, Mcv = mean corpuscular volume, Mch = mean corpuscular hemoglobin, Mchc = mean corpuscular hemoglobin concentration, Chcm = cellular hemoglobin concentrati on, and Ch = corpuscular hemoglobin content.

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169 Table 4 8. P -values from pairwise comparisons of the red blood cell indices, by treatment and sampling time. Tx Pairwise Rbc Hct Hb Mcv Mch Mchc Chcm Ch EA Pr Po 0.20 0.08 0.22 1.00 0.03 1.00 Pr Ps 0.12 0.07 0.04 0.02 0.03 0.27 1.00 1.00 Po Ps 1.00 1.00 0.61 1.00 0.60 0.15 Sham Pr Po 1.00 1.00 0.02 Pr Ps 0.03 0.02 0.27 0.13 0.10 1.00 Po Ps 0.54 0.78 1.00 0.18 0.12 0.02 Pr = pre treatment, Po = after the 7th treatment, Ps = after the 12th treatment, Tx = treatment, EA = electroacupuncture, Sham = sham EA, Rbc = red blood cell counts, Hct = hematocrit, Hb = hemoglobin, Mcv = mean cor puscular volume, Mch = mean corpuscular hemoglobin, Mchc = mean corpuscular hemoglobin concentration, Chcm = cellular hemoglobin concentration, and Ch = corpuscular hemoglobin content. Table 4 9. MeanSD and range (in parenthesis) of percentages of recovered BALf and percentages of ELF in BALf samples determined by urea dilution technique. Tx Sampling time Percentage of BALf recovered Percentage of epithelial lining fluid EA Pr 76.35.3 (85.0 65.3) 1.30.4 (2.1 0.6) Po 75.45.0 (83.3 63.3) 1.30.5 (2.2 0.6) Sham Pr 75.66.9 (83.3 60.0) 1.20.5 (2.4 0.6) Po 72.24.1 (80.0 64.0) 1.20.3 (1.9 0.9) Pr = pre treatment, Po = after the 10th treatment, Tx = treatment. EA = electroacupuncture, Sham = sham EA, BALf = broncho alveolar lavage fluid, ELF = epith elial lining fluid.

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170 Table 4 10. P -values from the Type III sum of squares test on main effects and their interactions for broncho alveolar lavage fluid cytological parameters. Factor ELF corrected TNC Mac Lym Neu Mas Eos Treatment 0.59 0.38 0.36 0.59 0. 85 0.26 Sampling time 0.43 0.55 0.23 0.89 Trial 0.28 0.10 0.85 0.67 Sequence 0.04 0.30 0.90 0.75 0.21 0.33 Treatments sampling time 0.45 0.35 0.73 0.78 0.72 0.47 Horse sequence 0.18 0.69 = interaction between factors, ELF = epithelial lining fluid, TNC = total nucleated cell count, Mac = macrophage, lym = lymphocyte, Neu = neutrophil, Mast = mast cell, and Eos = eosinophil. Table 4 11. MeanSE ELF -corrected TNC and differential counts of BALf cells, by treatment and sampling time. Tx Sampling time ELF corrected TNC (x107 cell/ml) Mac (%) Lym (%) Neu (%) Mast (%) Eos (%) EA Pr 2.84 0.27 41.0 7.8 52.3 1.5 *4.5 0.6 *1.8 0.2 0.3 .0 Po 2.81 0.27 51.7 7.8 54.4 1.5 6.1 0.6 1.0 0.2 0 .5 .0 Sham Pr 3.17 0.25 40.7 7.2 51.2 1.3 4.7 0.6 *1.7 0.2 0.8 0.2 Po 2.77 0.25 38.5 7.2 52.3 1.3 6.6 0.6 1.0 0.2 0.6 .0 = p -value of difference between Pr and Po -treatment, Po = after the 10th treatment, Tx = treatment, EA = electroacupuncture, Sham = sham EA, BALf = bronchoalveolar lavage fluid, ELF = epithelial lining fluid, TNC = total nucleated cell count, Mac = macrophage, lym = lymphocyte, Neu = neutrophil, Mast = mast cell, and Eos = eosinophil.

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171 Table 4 12. P -v alues from paired samples t test of BALf cytological parameters by treatment. Tx Pairwise ELF corrected TNC Mac Lym Neu Mast Eos EA Pr Po 0.97 0.47 0.39 0.04 0.05 0.73 Sham Pr Po 0.31 0.27 0.50 0.06 0.01 0.50 Pr = pre treatment, Po = after the 10th trea tment, Tx = treatment, EA = electroacupuncture, Sham = sham EA, BALf = broncho alveolar lavage fluid, ELF = epithelial lining fluid, TNC = total nucleated cell count, Mac = macrophage, lym = lymphocyte, Neu = neutrophil, Mast = mast cell, and Eos = eosinophil. Table 4 13. P -values from the Type III sum of squares test on the main effects and their interactions for plasma concentration of immunoglobulin isotypes. Factor IgA IgM IgGa IgGb IgG(T) Treatment 0.03 Sampling time 0.13 0.76 0.55 Trial 0.06 0.03 0.03 0.98 Sequence 0.37 0.54 0.86 0.82 0.88 Treatments sampling time 0.74 0.97 0.65 0.15 0.66 Sequence horse 0.05 = i nteraction between factors. Table 4 14. MeanSE concentrations of plasma immunoglobulin isotypes (x105 ng/ml), by, treatment and sampling time. Tx Sampling time IgA IgM IgGa IgGb IgG(T) EA Po 12.91 2.46 5.59 0.56 24.24 1.59 48.38 2.76 42.22 6.18 Pr 13.47 2.46 5.62 0.56 25.51 1.59 50.70 2.76 42.69 6.18 Ps 15.80 2.46 5.76 0.56 28.94 1.59 39.62 2.76 43.26 6.18 Sham Po 17.85 2.43 7.99 0.54 28.27 1.52 56.56 2.67 63.62 6.01 Pr 18.69 2.43 8.09 0.54 27.53 1.52 53.41 2.67 60.81 6.01 Ps 19.49 2.43 8.35 0.54 34.09 1.52 49.31 2.67 68.35 6.01 Pr = pre treatment, Po = after the 7th treatment, Ps = after the 12th treatment, Tx = treatment, EA = electroacupuncture, Sham = sham EA.

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172 Table 4 15. P -values from pairwise comparisons of concentration of plasma immunoglobulin isotypes, by treatment and sampling time. Tx Pairwise IgA IgM IgGa IgGb IgG(T) EA Pr Po 0.95 1.00 1.00 1.00 1.00 Pr Ps 0.07 1.00 0.29 0.03 Po Ps 0.34 0.32 Sham Pr Po 1.00 1.00 1.00 1.00 0.12 Pr Ps 1.00 0.06 0.45 0.02 Po Ps 0.19 0.07 0.99 Tx = treatment, EA = electroacupuncture, Sham = sham electroacupuncture, Pr = pre treatment, Po = after the 7th treatment, Ps = after the 12th treatment. Table 4 16. P -values from the Type III sum of squares test on main effects and their interactions for concentration of immunoglobulin isotypes in ELF corrected BALf. Factor IgA IgM IgGa IgGb IgG( T) Treatment 0.84 0.45 0.26 0.71 0.71 Sampling time 0.96 0.94 0.53 0.32 0.25 Trial 0.21 0.44 0.46 0.31 0.12 Sequence 0.36 0.97 0.44 0.96 0.89 Treatments sampling time 0.21 0.83 0.37 0.27 0.42 Horse sequence 0.22 0.43 0.88 0.52 0.31 *= interactio n between factors. Table 4 17. MeanSE concentrations of immunoglobulin isotypes in ELF -corrected BALf (x105 ng/ml), by treatment and sampling time. Tx Sampling time IgA IgM IgGa IgGb IgG(T) EA Po 24.12 2.96 20.32 2.80 9.87 1.09 18.92 2.41 11.57 1. 47 Pr 20.58 2.96 23.61 2.80 8.05 1.09 13.87 2.41 8.82 1.47 Sham Po 20.32 2.80 0.31 0.06 7.71 1.03 14.42 2.28 10.20 1.39 Pr 23.61 2.80 0.31 0.06 7.97 1.03 14.59 2.28 9.68 1.39 Pr = pre treatment, Po = after the 10th treatment, Tx = trea tment, EA = electroacupuncture, Sham = sham EA, BALf = broncho alveolar lavage fluid, ELF = epithelial lining fluid.

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173 Table 4 18. P -values from pairwise comparisons for concentrations of immunoglobulin isotypes in ELF corrected BALf. Tx Sampling time IgA IgM IgGa IgGb IgG(T) EA Pr Po 0.93 0.37 0.86 0.52 0.52 Sham Pr Po 0.67 0.86 0.68 0.72 0.55 Tx = treatment, EA = electroacupuncture, Sham = sham EA, Pr = pre treatment, Po = after the 10th treatment. Table 4 19. P -values from the Type III sum of square s test on main effects and their interactions for TNF production from stimulated whole blood by stimulants. Factor Stimulants None PBS LPS Zym CA CA+ CE CA+ LPS CE+ LPS CA+ CE+ LPS Treatment 0.08 0.04 0.08 Samplin g time 0.18 0.11 0.05 0.02 0.15 0.02 0.02 Trial 0.31 0.05 0.95 0.26 0.12 0.06 0.29 0.14 Sequence 0.34 0.66 0.53 0.82 0.63 0.38 0.20 0.62 Treatments sampling time 0.25 0.03 0.03 0.02 Horse sequence 0.43 0.01 = interaction between factors, None = no stimulant added, PBS = phosphate buffer saline, LPS = lipopolysaccharide, Zym = zymosan, CA = cellular antigen from A. fumigatus CE = culture extract of A. fumigat us

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174 Table 4 20. MeanSE TNF concentrations in whole blood after stimulation, by treatment and sampling time. Concentrations are x103 pg/ml. Tx Sampling time Stimulants None PBS LPS Zym CA CA + CE CA + LPS CE + LPS CA + CE + LPS EA Pr 0.00 0.01 0.0 0 0.00 *5.10 0.29 1.66 0.36 *4.12 0.21 *4.23 0.23 *#5.59 0.27 *#5.32 0.26 *#5.78 0.30 Po 0.04 0.01 0.00 0.00 4.82 0.29 +0.56 0.36 3.38 0.21 3.58 0.23 4.24 0.27 4.24 0.26 4.60 0.30 Ps 0.01 0.01 0.00 0.00 4.94 0.29 0.22 0.36 3.64 0. 21 3.73 0.23 4.69 0.27 4.44 0.26 5.03 0.30 Sham Pr 0.00 0.01 0.00 0.00 #4.90 0.28 1.13 0.35 4.10 0.21 4.22 0.22 5.45 0.26 5.17 0.25 5.69 0.29 Po 0.00 0.01 0.00 0.00 5.11 0.28 1.67 0.35 +4.09 0.21 4.30 0.22 5.36 0.26 5.12 0.25 5.87 0.29 Ps 0.00 0.01 0.00 0.00 5.71 0.28 0.97 0.35 4.65 0.21 4.76 0.22 5.88 0.26 5.79 0.25 6.37 0.29 = p -value of comparison between Pr and Po -value of comparison between Pr and Ps -value of comparison between Po and Ps electroacupuncture, Sham = sham EA, Pr = pre treatment, Po = after the 7th treatment, Ps = after the 12th treatment, None = no stimulant added, PBS = phosphate buffer saline, LPS = lipopolysaccharide, Zym = zymosan, CA = cellular antigen from A. fumigatus CE = culture extract of A. fumigatus

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175 Table 4 21. P -values from pairwise comparisons of TNF concentrations in whole b lood after stimulation, by treatment. Tx Pairwise Stimulants None PBS LPS Zym CA CA + CE CA + LPS CE + LPS CA + CE + LPS EA Pr Po 0.53 0.29 Pr Ps 1.00 0.073 0.12 0.06 0.20 0.03 Po Ps 0.99 1.00 0.79 0.57 0.58 1.00 0.54 Sham Pr Po 0.99 0.10 0.87 1.00 1.00 1.00 1.00 0.21 Pr Ps 0.52 0.05 0.79 0.10 0.29 0.53 0.17 0.0 7 Po Ps 1.00 0.21 0.75 0.04 0.26 0.28 0.11 0.28 Tx = treatment, EA = electroacupuncture, Sham = sham EA, Pr = pre treatment, Po = after the 7th treatment, Ps = after the 12th treatment, None = no stimulant added, PBS = phosphate buffer saline, LPS = lipopolysaccharide, Zym = zymosan, CA = cellular antigen from A. fumigatus CE = culture extract of A. fumigatus

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176 Table 4 22. P -values from the Type III sum of squares test on the main effects and their interactions for FEVx, FVC, and PEF. Factor FEV 0.5 FEV 0.75 FEV 1.0 FEV 1.5 FEV 2.0 FEV 2.5 FEV 3.0 FVC PEF Treatment 0.79 0.79 0.86 0.786 0.79 0.82 0.83 0.83 0.84 Sampling time 0.03 Trial .01 Sequence 0.98 0.76 0.52 0.61 0.77 0.75 0.73 0.73 0.53 Treatments sampling time 0.99 0.96 0.68 0.86 0.95 0.95 0.95 0.95 0.57 Horse sequence 0.89 0.89 0.76 0.68 = interaction between factors, FEVx = forced exp iratory volume at x seconds, FVC = forced vital capacity, PEF = peak expiratory flow. Table 4 23. P -values from the Type III sum of squares test on the main effects and their interaction for MEFx% and FEVx/FVC ratio. Factor MEF 25% MEF 50% MEF 75% FEV0.5 / FVC FEV0.75 /FVC FEV1.0/ FVC FEV1.5/ FVC FEV2.0/ FVC Treatment 0.47 0.87 0.56 0.76 0.71 0.74 0.96 0.48 Sampling time 0.19 0.03 0.35 0.16 0.26 0.96 0.23 Trial 0.45 Sequence 0.71 0.93 0.87 0.78 0.80 0.83 0.94 0.26 Treatments sampling time 0.49 0.37 0.23 0.99 0.93 0.71 0.35 0.28 Horse sequence 0.72 0.2 4 = interaction between factors, MEF% = forced expiratory flow rate after x% of forced vital capacity has been expired, FEVx/FVC = ratio of forced expiratory volume at x seconds/forced vital capacity.

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177 Table 4 24. MeanSE FE Vx, FVC, and PEF obtained by the rapid partial forced expiration maneuver, by treatment and sampling time. Tx Sampling time FEV 0.5 FEV 0.75 FEV 1.0 FEV 1.5 FEV 2.0 FEV 2.5 FEV 3.0 FVC PEF EA Pr 10.34 0.21 *#29.73 0.2 *#45.61 0.23 #62.29 0.42 65.19 0 .66 65.46 0.66 65.76 0.67 65.76 0.67 #80.16 0.35 Po 10.72 0.21 30.49 0.26 +46.59 0.23 +63.15 0.42 65.88 0.66 66.15 0.66 66.45 0.67 66.45 0.67 +81.80 0.35 Ps 10.96 0.21 31.24 0.26 47.66 0.23 64.14 0.42 66.66 0.66 66.94 0.66 67.26 0.6 7 67.26 0.67 83.91 0.35 Sham Pr #10.40 0.19 #29.89 0.24 *#45.86 0.21 #62.26 0.39 #64.76 0.60 #65.00 0.61 #65.29 0.61 #65.29 0.61 *#80.61 0.33 Po 10.81 0.19 +30.53 0.24 +46.56 0.21 +62.93 0.39 65.48 0.60 65.78 0.61 66.12 0.61 66.12 0.6 1 +81.59 0.33 Ps 10.98 0.19 31.19 0.24 47.46 0.21 63.81 0.39 66.56 0.60 66.86 0.61 67.17 0.61 67.17 0.61 83.61 0.33 = p -value of comp arison between Pr and Po -value of comparison between Pr and Ps -value of comparison between Po and Ps electroacupuncture, Sham = sham EA, Pr = pre treatment, Po = after the 7th treatment, Ps = after t he12th treatment, FEVx = forced expiratory volume at x seconds (liters), FVC = forced vital capacity (liters), PEF = peak expiratory flow (liters/second).

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178 Table 4 25. MeanSE MEFx% and FEVx/FVC ratio obtained by the rapid partial forced expiration maneuver, by treatment and sampling time. Tx Sampling time MEF 25% MEF 50% MEF 75% FEV0.5/ FVC FEV0.75 /FVC FEV1.0/ FVC FEV1.5/ FVC FEV2.0/ FVC EA Pr #78.39 1.11 #71.75 0.55 #46.52 0.78 0.15 0.00 0.45 0.01 0.695 0.01 0.94 0.01 0.99 0.00 Po +79.92 1.11 73. 41 0.55 47.47 0.78 0.16 0.00 0.46 0.01 0.70 0.01 0.95 0.01 0.99 0.00 Ps 81.61 1.11 75.50 0.55 49.20 0.78 0.16 0.00 0.46 0.01 0.70 0.01 0.95 0.01 0.99 0.00 Sham Pr *78.73 1.02 #72.57 0.50 47.30 0.71 0.15 0.00 0.45 0.01 0.70 0.01 0.95 0.01 0.99 0.00 Po 80.00 1.02 73.42 0.50 47.57 0.71 0.16 0.00 0.46 0.01 0.70 0.01 0.95 0.01 0.99 0.00 Ps 79.46 1.02 74.81 0.50 47.87 0.71 0.16 0.00 0.46 0.01 0.70 0.01 0.95 0.01 0.99 0.00 = p -value of comparison between Pr and Po -value of comparison between Pr and Ps -value of comparison between Po and Ps electroacupuncture, Sham = sham EA, Pr = pre treatment, Po = after the 7th treatment, Ps = after the 12th treatment, MEFx% = forced expiratory flow rate after x% of forced vital capacity has been expired (liters/second), FEVx/FVC = ratio of forced expiratory volume at x seconds/forced vital capacity.

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179 Table 4 26. P -values from pairwise comparisons of the FEVx, FVC, and PEF o btained by the rapid partial forced expiration maneuver. Tx Pairwise FEV 0.5 FEV 0.75 FEV 0.1 FEV 1.5 FEV 2.0 FEV 2.5 FEV 3.0 FVC PEF EA Pr Po 0.53 0.05 0.03 0.51 1.00 1.00 1.00 1.00 0.19 Pr Ps 0.13 0.14 0.14 0.13 0.13 Po Ps 1.00 0.35 0.05 0.39 0.39 0.39 0.39 Sham Pr Po 0.78 0.32 0.03 0.12 0.46 0.38 0.33 0.33 Pr Ps 0.02 0.02 0.02 0.02 0.02 Po Ps 0.55 0.04 0.02 0 .07 0.07 0.07 0.07 Tx = treatment, EA = electroacupuncture, Sham = sham EA, Pr = pre treatment, Po = after the 7th treatment, Ps = after the12th treatment, FEVx = forced expiratory volume at x seconds (liters), FVC = forced vital capacity (liters), PEF = peak expiratory flow (liters/second). Table 4 27. P -values from pairwise comparisons of the MEFx% and FEVx/FVC ratio obtained by the rapid partial forced expiration maneuver. Tx Pairwise MEF 25% MEF 50% MEF 75% FEV0.5/ FVC FEV0.75 /FVC FEV1.0/ FVC FE V1.5/ FVC FEV2.0/ FVC EA Pr Po 0.27 0.06 0.57 1.00 0.91 0.86 1.00 1.00 Pr Ps 0.02 0.98 0.46 0.39 0.73 1.00 Po Ps 0.19 0.47 1.00 1.00 1.00 1.00 1.00 Sham Pr Po 0.08 0.68 1.00 1.00 1.00 1.00 0.12 Pr Ps 1.00 1.00 0.15 0.14 0.96 1.00 0.19 Po Ps 1.00 0.21 1.00 1.00 1.00 1.00 0.98 1.00 Tx = trea tment, EA = electroacupuncture, Sham = sham EA, Pr = pre treatment, Po = after the 7th treatment, Ps = after the 12th treatment, MEF% = forced expiratory flow rate after x% of forced vital capacity has been expired (liters/second), FEVx/FVC = ratio of forc ed expiratory volume at x seconds/forced vital capacity.

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180 Table 4 28. P -values from the Type III sum of squares test on main effects and their interactions for serum cortisol concentration. Factor (s) P value Treatments 0.93 Sampling time 0.17 Trial 0. 29 Sequence 0.09 Treatments sampling time 0.53 Sequence horse 0.00 = interaction between factors. Table 4 29. Mean SE concentrations of serum cortisol, by treatment and sampling time. Tx Sampling time Concentration of serum cortisol ( g/dl) EA Pr 6.271 0.687 Po 5.408 0.687 Ps 6.847 0.687 Sham Pr 6.992 0.639 Po 5.564 0.639 Ps 6.111 0.639 Tx = treatment, EA = electroacupuncture, Sham = sham EA.Pr = pre treatment, Po = after the 7th treatment, Ps = after the 12th treatment, P -value s of pariwise comparisons; Pr -Po, Pr Ps, and Po Ps in the EA group were 0.71, 1.01, and 0.51, respectively. P values of pairwise comparsons; Pr Po, Pr -Ps, and Po -Ps in the sham group were 0.50, 1.00, and 0.79, respectively.

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181 Table 4 30. Details of cutaneo us and muscle innervations at acupoints used in this study. Acupoint locations are described in Table 4 1. (Source s ; Hackett MS Sack WO, Simmons MA, et al. Forelimb In: Hackett MS,Sack WO, eds. Rooney's guide to the dissection of the horse. 7th ed. 2001;page 112152., Budras K D, Sack WO, Rck S, et al. Selected body systems in tabular form In: Budras K D, Sack WO, Rck S, et al. eds. Anatomy of the horse : an illustrated text. 3rd ed. 2001;page 81102., Fleming P. Transpositional equine acupuncture atlas In: Schoen AM, ed. Veterinary acupuncture : ancient art to modern medicine. 2nd ed. 2001; page 393431., and Blythe LL, Kitche ll RL. Electrophysiologic studies of the thoracic limb of the horse. Am J Vet Res 1982;43;page15111524.169,239241) Acupoint Cutaneous innervations (N = nerve) Muscles/innervations (N = nerve) GV 14 Dorsal branch of 7 th cervical N. and 1st thoracic N. CV 22 Vent ral cutaneous branch of 6 th cervical spinal N and cranial branch of supraclavicular N. Cutaneous coli/Ramus coli of facial N. BL 13 Dorsal branch of thoracic N. Cutaneous trunci/Lateral thoracic and intercostobrachial N. Trapezius thoracic/Dorsal branch o f accessory N. Rhomboideus thoracis/Medioventral branch of local thoracic N. Latissimus dorsi/Thoracodorsal N. Longismus thoracis/Dorsal branch of local spinal N. Fei men Ventral cutaneous branch of C 6 th cervical spinal N. Trapezius cervicis/Dorsal branc h of accessory N. Subclavius/Cranial pectoral N. Longissimus cervicis/Dorsal branch of local spinal N. Fei pan Lateral cutaneous branch of 2 nd and 3 rd thoracic N (component of intercostobrachial N) and lateral cutaneous branch of 4 th thoracic N. Cutaneou s trunci/Lateral thoracic and intercostobrachial N. Triceps (long head)/Radial N. Fei shu Intercostal N. Cutaneous trunci/Lateral thoracic and intercostobrachial N. Intercostal muscle/Intercostal N. (from ventral branch of Thoracic N.). Ding chuan Dorsal branch of local cervical and thoracic spinal N. Trapezius cervicis/Dorsal branch of accessory N. Rhomboideus cervicis/Medioventral branch of cervical N. Splenius/Dorsal branch of local spinal N. and dorsal branch of accessory nerve.

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182 CHAPTER 5 SUMMARY AND CONCLUSI ONS Introduction Cardiopulmonary fitness is an important factor de termining athletic performance of horses. Diseases and abnormalities that alter the normal airflow and gas exchange of the lung may originate from the upper or lower airways.231 Disorders of the upper airway that increase resistance and limit the normal air flow include recurrent laryngeal hemiplegia, pharyngeal lymphoid hyperplasia, dorsal displacement of the soft palate, nasopharyngeal collapse, sub epiglottic cyst, and entrapment of the epiglottis. Abnormalities of the lower airways affect horses at all ag es, of all breeds, and in physiological stages. Severity of the diseases depends on several factors including the age of the animal, immunological status, and the cause of the disease. Important diseases that cause non septic inflammation of the lower airw ays include inflammatory airway disease (IAD), heaves or recurrent airway obstruction (RAO) and summer pasture associated obstructive pulmonary disease (SPAOPD).170 Horses af fected by theses diseases show clinical signs of dry cough, serous nasal discharge, increased expiratory effort, flaring of nostrils, and exercise intolerance.222 Horses that are being kept for competition, sports a nd recreational purposes are unable to participate in their routine training program when affected by these conditions and a decrease in performance capability is seen. Opportunities to develop other pulmonary disease associated complications such as pleur opneumonia, pleuritis, emphysema and fibrosis are increased if appropriate treatment is delayed and may lead to an incomplete recovery. Irreversible changes in the histological structure and severe compromise of the normal respiratory physiological function can lead to the termination of an animals athletic career or, in severe cases, euthanasia.

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183 Until recently, there has been no single diagnostic procedure that can be used to accurately distinguish these diseases and, more importantly, to diagnose these diseases at the earliest stage. Current diagnosis relies on case history, clinical signs, BALf cytology, and response to treatments. Ability to recognize these diseases at an early stage is important. However, it is difficult due to a lack of specific clini cal manifestations. Several diagnostic methods have been developed to identify early disease stages in affected horses, including direct measurement of intra -pleural pressure, histamine broncho-provocation, and forced expiration.172 175 Histamine Bronchoprovocation as a Test for Equine Airways Hyper -sensitivity Histamine bronchoprovocation (HB), also known as histamine challenge, has been used to determine the degree of airway hyper -sensitivity. The hyper -sensitive a irway is though to be a sequel to chronic inflammation of the lung and to be reflective of the severity of airway disease. Using HB result alone as a diagnostic tool for respiratory disease is questionable. Bronchoconstriction in lower airway inflammatory diseases in horses develops over a few hours after exposure to an irritant, and the improvement of clinical signs is delayed following the causes are removed.171 The bronchoconstriction effect of HB is of much shorter duration. Horses without clinical signs of respiratory problems responded to HB inconsis tently as was demonstrated in Chapter 3. Horses with a high percentage of BALf neutrophils were more likely to have hyper -sensitive airways. However, the correlation between percentage of BALf neutrophils and airway hyper -sensitivity was not significant. V ariation in HB test results for individuals, when the test was repeated, agreed with the finding of previous research which suggested a high level of inconsistency in individual response to HB.184,185 Variation in i ndividual responses to HB also has been demonstrated in clinically normal foals.187 Concentrations of histamine causing a decline in lung dynamic compliance by 35% in these foals

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184 ranged from 1 mg/ ml to more than 8 mg/ml. Results of previous research also has suggested that the HB response in normal horses and horses with low grade lung disease (with no clinically evident signs of respiratory tract disease) were not significantly different. However, horses with severe pulmonary disease required a lower histamine concentration to cause a 35% decline in the lung dynamic compliance.184 The cause of variability in individual response to HB is unknown. However, it may be caused by the non-specific response of the upper airway to histamine, especially when a facemask was used to conduct the HB test. Histamine not only induces the contraction of the smooth muscles of the airways, but also causes vasodilatation of blood vessels in the airways. Vasodilatation leads to edema of the respiratory mucosa and increased mucus production in the upper and lower airways.211 These effects may contribute to an increase in a resistance of the upper airways that potentially increases total resistance of the respiratory tracts. A pharmacological effect of histamine on mucus production was observed in the experiment of Chapter 3; that is, the vast majority of the horses had increased nasal secretion during and after exposure to histamine. Using a facemask for delivering histamine is another factor that may affect HB test results. The nebulization rate of histamine solution depends on the pressure an d type of nebulizer being used in the test. With the facemask, the exact amount of histamine reaching the lower airway is unknown.187 The quantity of histamine reaching the lower airways likely de pends on respiratory frequency and depth of breathing. Results in Chapter 3 suggested that the HB test can be used to determine airway hyper sensitivity and may be used to support the diagnosis of equine lower airway inflammatory diseases. However, interpr etation of HB test results obtained from clinically normal horses

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185 requires great care. Results from the HB test should be used in conjunction with other diagnostic information (BALf cytology, clinical signs, history of illness, and response to therapy) for a more reliable diagnosis. Rapid Partial Forced Expiration for Testing Equine Pulmonary Function Rapid partial forced expiration (RP -FE) is another pulmonary function test modified from the forced expiration test in human medicine. The technique is novel in veterinary medicine and not widely practiced. The test requires additional maneuvers and is more invasive than that in humans. A design for a system that is capable of intervening inspiration and expiration is necessary since coaching of breathing in horses to obtain maximal breth excursions is not possible. To perform RP FE test in horses, an airtightsealed airway of the horse was first inflated with atmospheric air at a controlled pressure to total lung capacity (TLC). Then the airway was exposed to a negative pressure reservoir. The difference in pressure between the airway and negative pressure reservoir moved air out of the lung. Negative pressure causes emptying of the air from the lung beyond the effect of elastic recoil properties of the pulmonary tissues and the chest wall. The functional reserve volume of the lung is also emptied, mimicking forced expiration. Pulmonary function test parameters (PFTPs) from RP -FE test results in Chapters 3 and 4 differed from values reported by a previous study.175 The differences in PFTPs may be caused by differences in manif old designs, in the negative pressures used to empty airways, or in the methods of measuring airflow. Regardless of the total length of the manifold used in the two studies, the major factor that contributed to airflow resistance was the diameter of the ma nifold. The internal diameter (ID) was determined by the selection of an endotracheal tube (2.6 cm, study in Chapter 3 and 4) versus a nasotracheal tube (2.2 cm, previous study). Relationship of the radius to the volumetric

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186 flow rate can be demonstrated by Poiseuilles law,193 which states that the amount of the volumetric flow rate is positively related to the fourth power of the radius (Equation 3 6). Results in Chapter 3 demonstra ted that PEF and FVC values were positively correlated with the amount of negative pressure used to generate RP -FE. Negative pressure at 200 Torr was used for RP -FE in Chapter 4 because the studies cited in Chapter 3 suggested that it eventually emptied th e air from airways without causing observable damage to the tracheal mucosa. The PEF and FVC values derived from 200 Torr used for inducing RP -FE in Chapters 3 and 4 were greater than the previously reported values, when FE was induced by a vacuum at 161.8 Torr ( 220 cm H2O).175 Forced expiratory airflow during the RP -FE maneuver in Chapters 3 and 4 was measured directly via the pressure differential generated by the maneuver. This method of measurement differed from that in previous research in which the airflow rate and expiratory volume were indirectly calculated fro m an immediate change in negative pressure in a vacuum reservoir. The P generated by airflow in RP -FE was measured by the MFE -calibrated laminar flow element. The MFE used in the calibration was regularly tested for its accuracy and was NIST (National ins titute of standards and technology) traceable. From Poiseuilles law, in the laminar flow condition the volumetric flow rate is linearly related to the drop in P measured across the flow tube.193 The Equation 36 can be re -written as Equation 3 3. When factors determined by the geometry of the flow restriction are reduced to the K constant, Equation 3 3 can be rewritten as Equation 3 4. This equation shows a linear relationship betw een volumetric flow rate (Q), differential pressure ( P), and fluid viscosity ( ) in a simple form. This simple linear relationship between Q and P was confirmed by pre and post RP -FE calibrations in Chapters 3 and 4.

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187 In Chapter 4, the accuracy of mass f low element (MFE) calibration was tested by comparing integrated airflow signals generated by pulling a known volume of air through the pipe connected downstream of the calibrated laminar flow element (LFE). A 12.829liter syringe was used to test the inte gration of airflow signals. The results indicated that the volume from integration was greater than the actual volume of the syringe being injected by 6.31%. This means that the volume integration result of 13.639 liters was, in fact, 12.829 liters. Theref ore, the calculated correction factor for MFE/LFE was 1.063105 (13.639/12.829). This correction factor was used in the calculation of calibrated volumes in Chapter 4. These results suggested that a carefully calibrated LFE is suitable for measuring airflow during RP FE. Measurement of forced expiratory airflow by LFE in the RP -FE relied on the same principle as that of a pneumotachograph. Commercially available pneumotachographs are superior to an ordinary LFE in that they are electrically heated. This prev ents condensation of moisture from the expired air inside the pneumotachograph. The condensation might have occurred in the LFE of the RP -FE apparatus during the experiments. Laminar flow element used in Chapters 3 and 4 was 4 inches in internal diameter a nd was nearly four times greater than that of the endotracheal tube (1.024 inches), which was a bottleneck of airflow during the RP -FE maneuver. It can be assumed that minor condensation in the LFE would not significantly alter the resistance of the appara tus. The increase in the percentage of neutrophils in the bronchoalveolar lavage fluid (BALf) after RP -FE testing suggested that RP FE induced airway inflammation. Percentages of neutrophils were significantly higher in BALf samples collected after the 10th treatment, which was 8 10 days after the second RP -FE maneuver. Also, percentages of BALf neutrophils in pre treatment samples from the second trial were greater than those in the first trial. These results

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188 together suggested that RP -FE induced pulmona ry inflammation, which was carried over to the second trial. The magnitude of RP -FE induced airway inflammation observed might be due partly to an accumulation of inflammatory reaction when the horses were scheduled for re testing too soon. A period of 81 0 days was first thought to be enough time for the pulmonary tissues to recover from RP FE induced inflammation. However, the increase in the percentages of neutrophils in BALf suggested that the pulmonary tissue had not completely recovered, and that inflammation in the airways and pulmonary tissues caused by RP -FE lasted longer than the period first hypothesized. There were no significantly differences in PFTPs between pre and post RP -FE observed, even in the face of RP -FE induced inflammation in the pul monary tissues. Previous research demonstrated that when airways of rats were ventilated with an end expiratory pressure of 0 cm H2O and with a peak inspiratory pressure of 45 cm H2O for 20 minutes, lung tissues were damaged as indicated by edema of the pu lmonary tissues and an increase in percentages of neutrophils in BALf.247 Concentrations of inflammatory cytokines in BALf, including macrophage inflammatory protein 2, IL 1 heat shock protein70, and matrix metalloproteinase were also increased. Negative p ressure induced pulmonary injury has been documented in humans subjected to general anesthesia.248,249 It is usually caused by attempted ventilations against an acute upper airway obstruction in the perioperative pe riod. Negative intrathoracic pressure develops during such respiratory efforts.250 This results in accumulation of fluid in the pulmonary tissue.251 The mechanism and extent of pulmonar y tissue injury induced by RP FE in horses have never been tested. Artificial inspiration and artificial forced expiration during the RP -FE maneuver caused a rapid swing in intrapleural pressure. A continuous abrupt change in intrapleural pressure has

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189 been observed in horses during strenuous exercise, and has been thought to be one of the factors contributing to exercise induced pulmonary hemorrhage.252 In the future, the RP -FE apparatus may be used in research in th is field. Partial collapse of the small airways and alveoli that might be caused by artificial forced expiration is potentially a problem of the RP -FE maneuver, as demonstrated by the fact that forced vital capacity (FVC), peak expiratory flow (PEF), and f low -volume loop of the first artificial forced expiration were always greater than those of the subsequent artificial forced expirations (Figures 9 and 10 in Chapter 4). Artificial inspiration during RP -FE maneuvers in Chapters 3 and 4 was adequate to over come the physiological drive of normal respiration, but a thorough ventilation of all alveoli could not be ensured, especially after the first artificial forced expiration in each RP FE test. In the future, temporarily disconnecting the RP FE apparatus fro m the endotracheal tube after each artificial forced expiration cycle prior to performing the subsequent RP -FE cycles, may allow the horse to regain physiological control of respiration. Physiologically, inspiration is initiated by expansion of the thoraci c cavity and a decrease in intrapleural pressure, unlike artificial inspiration. Artificial inspiration was initiated by positive pressure generated from inflation of the airways. Allowing the horse to spontaneously breath unassisted may improve ventilatio n of the collapsed small airways and alveoli. Testing equine pulmonary function with RP -FE is an emerging technique for measuring biomechanical properties of the lung. There is no single standard method for obtaining PFTPs, and reference values for horses are lacking. RP -FE maneuver explained in Chapters 3 and 4, and its PFTPs provided more information on the pulmonary function in clinically normal thoroughbred horses. Additional PFTPs from RP FE in horses of different breeds, sex, age, body

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190 weight, and wit h known pulmonary diseases, will be highly beneficial for comparisons of these parameters in horses in general. Acupuncture and Electroacupuncture Efects on Equine Immune Response and Pulmonary Function Acupuncture and electro acupuncture (AC/EA) combined with Chinese herbs have been used as adjunctive therapies for treating equine chronic respiratory disorders.78,232 They are intended to decrease the dosage requirements o f bronchodilators and anti inflammatory agents and improve the quality of life of horses suffering from chronic respiratory diseases.233 Therapeutic results obtained from humans and laboratory animals suggested that AC/EA possess therapeutic benefits for respiratory problems.97,253255 Acupuncture and EA applications in the respiratory system require stimulation of multiple acupoints. The points selected in the treatment strategy are intended to replenish the normal lung function, to reduce cough, to a lleviate clinical signs of heaves and dyspnea, and to improve immune function.78 Selected acupoints can be stimulated with an acupuncture needle, with low voltage electricity, or with an injection of a mild irritant sterile solution such as normal saline or a solution of water -soluble vitamins. Stimulation of acupoints with a laser also has been used.234 Frequently used acupoints and their functions (in traditional Chinese veterinary medicine) are listed in Table 5 1.78,135,256 In respiratory disorders, the affected animals develop clinical signs of respiratory discomfort due to reduced airway ventilation, impaired gas exchange capacity, increased production and accumulation of airway secretions and increased inflammatory reaction in the lower airway.86 Alleviation of one or more of these disease mechanisms may reduce the severity of the clinical signs. Acupuncture treatment in humans sufferi ng from asthma has been shown to improve the pulmonary function and significantly improved the quality of life of patients.87,255

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191 However, a more scientifical based explanation of how AC and EA benefit treatment of respiratory diseases is still needed. By extrapolating from recent scientific research on AC/EA in animal models of disease, it can be proposed that AC/EA benefit the respiratory system by several mechanisms, including: Improved mucociliary action of the a irway epithelium. Reduced airway and pulmonary tissue inflammation. Activation of cholinergic anti -inflammation. Alteration of immune responses. Modulation of autonomic nervous system. Alteration in the peripheral sensory input from inflamed pulmonary tiss ues. Other mechanisms (as yet to be fully defined). Improved Mucociliary Action of the Airway Epithelium An increase in mucus production is a consequence of airway inflammation. Normally, mucus is immediately removed by epithelial mucociliary action. Disr uption of the mucociliary clearance occurs in response to chronic airway inflammation and is thought to be caused by neutrophil -derived elastase.98 The increased production and accumulation of mucus decreases airway diameter and increase total airway resistance. Until recently, no scientific evidence directly demonstrated the effect of AC or EA on mammalian mucociliary clearance. However, Tai et al.99 demonstrated that EA at a cupoints LU 1 and CV 22 significantly increased the rate of tracheal mucociliary transport in quail compared to a control group.99 Moreover, EA at these acupoints significantly reversed the decrease in mucociliary t ransport caused by the administration of human neutrophil -derived elastase. Several mediators have been shown to regulate the rate of mucociliary clearance. These included endogenous nitric oxide (NO) and substance -P.257, 258 However, whether these substances are involved in AC/EA -mediated mucociliary clearance needs further investigation.

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192 Reduced Airway and Pulmonary Tissue Inflammation Analgesic and anti inflammato ry effects of AC and EA are well documented in both somatic and visceral tissues.60,89 Mechanisms associated with these analgesic and anti inflammatory effects have been linked to a release of endogenous opioid subs tances in the central nervous system (CNS) during AC/EA treatment.28 These endogenous substances activate the opioid receptors in the CNS tissues, such as the substantia gelatinosa of the spinal cord and the periaqu eductal grey of the midbrain, and produce analgesia.92, 93 This endogenous opioid dependent anti -nociception has been shown to be modulated via the mu-opioid receptor.93 Immunocytes such as macrophages, monocytes, and polymorphonuclear cells possess opioid receptors on their cell surfaces where mu -opioid receptors predominate.94 Once activated, th e receptor induces an anti -inflammatory response via the down regulation of the transcription factor, nuclear factor kappa-B (NF B).95 The NF B down regulation has been demonstrated to reduce the mRNA expression o f other inflammatory cytokines including tumor necrosis factor alpha (TNF ), IL 1 IL 6, nitric oxide synthase (iNOS), and metalloproteinase.96 Carneiro et al.97 demonstrat ed that EA reduced the inflammatory cell infiltration in the peribronchial tissue and in the pulmonary perivascular spaces in rats with ovalbumin induced bronchial asthma.97 Moreover, the number of total nucleated c ells and the percentages of neutrophilic and eosinophilic leukocytes in bronchoalveolar fluid (BALf) were significantly decreased when compared to control and sham EA groups. In this study, the EA was performed on acupoints GV 14, BL 13, LU 1, CV 17, ST 36, SP 6, and Ding -chuan which mimicked the acupoints used to treat human asthma.

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193 Activation of Cholinergic Anti -inflammation The vagus nerve is a major parasympathetic nerve that contains afferent and efferent nerve fibers for both somatic and visceral tiss ues.79,80 Acethylcholine is a major neurotransmitter in both preganglionic and postganglionic parasympathetic nerve synapses. The cholinergic influence on immunological functions has been demonstrated in laboratory animals.81 Electrical stimulation of the vagus nerve suppressed the in vivo release of TNF and inhibited lipopolysaccharide (LPS) induced endotoxic shock. In this experiment, the authors also demonstrated attenuation in release of IL 1 IL 6, and IL 18, but not IL 10, by acethylcholine in LPS -stimulated human macrophage culture. This cholinergic dependent a nti inflammatory pathway suppresses the non -specific, innate immune response and may explain how AC/EA works in treating other diseases affecting visceral organs. The direct effect of AC/EA on the cholinergic associated anti inflammatory response in viscer al organs has never been demonstrated. However, the effect of EA on the cholinergic anti inflammatory response of somatic tissue has been shown in rats with collagen induced arthritis.60 In this model, EA at ST 36 s howed significant analgesic and anti -inflammatory properties. These analgesic and anti inflammatory activities of EA were suppressed when the muscarinic cholinergic receptor antagonist was co administered with the EA. This suggests that EA at ST 36 may act ivate a local cholinergic anti inflammatory mechanism preventing an inflammatory reaction and thus reducing pain. It is also possible that endogenous opioids and cholinergic pathways work together to create the anti inflammatory action associated with AC/E A. Alteration of Immune Responses When treating chronic respiratory diseases with AC/EA in clinical practice, acupoints that benefit the immune system are routinely stimulated in addition to acupoints that have

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194 specific indications for respiratory problem s. Acupoints that are normally used for this purpose include LI 4, LI 11, ST 36 and GV 14.138 139 259 260 Tian et al.76 studied the anti -inflammatory benefit of EA stimulation at ST 36 in rats with induced colitis.76 He reported a significant decrease in circulating TNF and a down regulation of mRNA coding for TNF ex pression in inflamed colonic tissue. Electroacupuncture treatment in Chapters 2 and 4 generally suppressed TNF production in whole blood cultures. The suppression of TNF production in whole blood cultures when LPS alone was added was more significant ( Chapter 4). The difference in suppression was variable possibly due partly to a different in EA treatment protocol. In Chapter 4, more acupoints were stimulated during the treatment, and the assay for TNF production in whole blood cultures were performed after the 7th and 12th EA treatments (versus after the 3rd EA in Chapter 2). Electroacupuncture in Chapters 2 and 4 involved GV 14 stimulation. This may suggest that GV 14 plays an important role in modulating immune response. Further investigation on thi s acupoint will be beneficial. Electroacupuncture at ST 25 and CV 6 significantly reduced IL 1 IL 6, and TNF secretion by monocytes during experimentally induced colitis in rats and reversed the decrease in apoptosis rate of peripherally circulating ne utrophils.75 Prolonged apoptosis of neutrophils is thought to be a result of the presence of pro-inflammatory triggering substances. Alleviation in the suppression of neutrophil apoptosis may be due to the reduction of inflammation of local tissue and a decrease in the production of inflammatory cytokines.75 Electroacupuncture may possess an activity similar to that of direct electrical stimulation of the vagus nerve, thereby possibly also stimulating the cholinergic anti -inflammatory pathway, inhibiting macrophage activation, and decreasing the production of TNF IL 1 IL 6, and IL -

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195 18.261 A down regulation of TNF released by AC/EA also has been demonstrated in other experimental models.76,262,263 Modulation of Autonomic Nervous Systems Equine airway smooth muscle and lymphoid tissue are innervated by both sympathetic and parasympathetic nervous systems.264 Alterations of the activity in both systems have been demonstrated following AC treatment.265 It has been proposed that the s pinal cord and the 10th cranial nerve are essential to relay AC/EA sensory signals to higher relay centers such as the brain stem and the hypothalamus.266 All the afferent signals converge at these relay centers prior to sendin g a signal to the somatosensory cortex. Bradycardia following AC/EA is commonly seen in both clinical practice and laboratory experiments demonstrating the parasympathomimetic and sympatholytic properties of AC/EA.267,268 Electroacupuncture at ST 36 decreased the excitability of the cardiovascular system manifested by bradycardia, which may be associated with sympathetic inhibition and modification of the central baroreflex arch.269 Modulation of the sympathetic response also has been reported in a study of AC performed at PC 6.270 Chan et al.271 reported that AC can be used to treat post -traumatic sympathetic dystrophy in humans with a 70% improvement in clinical signs.271 In another study, EA at LI 11 and LI 4 produced moderate hypoalgesia in humans concurrent with by a significant increase in muscle sympathetic nerve activity.272 Several studies indicate that AC/EA possess an immediate mild to moderate bronchodilator effect. A clinical study of human asthma found that AC at LU 7, LI 4, PC 6, ST 40, LI 11, and PC 3 for 15 minutes induced bronchodilation m anifested by an increase in forced expiratory volume in the first second (FEV1).87 Impr ovement in pulmonary function parameters (pleural pressure, tidal volume, minute ventilation, peak inspiratory flow and peak expiratory

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196 flow) in RAO affected horses also has been demonstrated after a single AC treatment.88 In this previous study, the authors concluded that the improvements were due to animal handling. Electroacupuncture treatment in Chapter 4 did not cause a change in PFTPs specific to the EA treatment. Results suggested that in clinically normal hor ses, EA has no effect on PFTPs. Horses airways are in the normal state likely maximally dilated. Any bronchodilatory effect is probably mechanically impossible in normal horses. Alteration in the Peripheral Sensory Input From Inflamed Pulmonary Tissues Alt hough the non-myelinated nerve fibers (C fibers) have never been described in the equine airway, they can be identified in several animal species.273,274 Therefore, it can be assumed that this type of nerve fiber is also present in equine species. It is a vagally mediated non -myelinated nerve fiber and functions as a polymodal receptor, activated by tissue damage, edema, or inflammatory mediators.275 Irritations caused by inhaled particles or mediators released by airway resident inflammatory cells activate this nerve fiber. Activation also leads to airway hyper responsiveness, a classical clinical manifestation of lower airway diseases. Attenuation of nociceptive signal conduction by C fibers has been hypothesized as the main analgesic mechanism of AC/EA.29 This is also known as the Gate Control Theory, proposed by Melzack and Wall. In this theory, sensory input from AC conducted by the A myelinated nerve fiber reaches the spinal cord at a faster speed than does the nociceptive signal that travels through the non-myelinated nerve fiber. When the sensory signal reaches the substantia gelatinosa of the spinal c ord, it stimulates the inhibitory interneuron and prevents subsequent conduction of the slow nociceptive signal transmitted by the non-myelinated C fiber and A fiber. Based on this theory, ascending non-nociceptive signals originating from AC/EA

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197 may modul ate the nociceptive input from C fibers from the airway at the level of the spinal cord and alleviate airway hyper responsiveness. Other Mechanisms Positive therapeutic effects following sham AC/EA in human subjects suggest a psychogenic influence and a pl acebo effect.276 Whether a similar placebo effect is present in other animals is questionable. Animals do not expect a positive effect treatment unless the investigator predictably rewards the animal following the treatment. Subjective parameters that require human assessment of an animal, such as appetite and degree of pain, are unavoidably affected by human bias. Also, it should be kept in mind that handling during the experiment potentially plays a major role in producing stress, which is frequently demonst rated by elevation of the endogenous corticosteroid concentrations. In Chapter 4, mean concentrations of serum cortisol in the EA and the sham groups were not significantly changed throughout the study, and the concentrations of serum cortisol of all sampl es were within normal reference values. These results at least suggested that neither treatments (EA and sham) nor RP FE maneuvers in this study induced stress when serum cortisol concentration was used as a stress indicator. Even though the thoracic pain associated with respiratory disease has never been studied in horses, it is thought to be present in horses affected by chronic respiratory diseases.277 Immediate relief in respiratory discomfort following AC/EA could be a result of a non-specific nociceptive inhibition by a diffuse noxious inhibitory control mechanism (DNIC). In DNIC, noxious stimulation, including AC needle insertion, may evoke analgesi a by a non -specific attenuation of the afferent pain sensation.278 Alleviation of respiratory discomfort following AC/EA at acupoints located around the thoracic region, including BL 13, Fei -men Fei -pan, and Ding -c huan, may be the result of this mechanism.

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198 Summary Histamine bronchoprovocation test can be used to reinforce diagnosis of equine lower airway inflammatory diseases, but interpretation of HB test results obtained from clinically normal horses should be don e with great care. Information derived from the HB test should be used together with results from other diagnostic information, including BALf cytology, clinical signs, history of illness, and response to therapy for determining the severity of the equine lower airway diseases. Forced expiration test in horses is an emerging technique of measuring biomechanical properties of the lung. Standard methods for obtaining PFTPs and reference values for horses are still lacking. Rapid partial forced expiration mane uver and its PFTPs provided more information on the pulmonary function in horses. Rapid partial forced expiration possesses great potential for diagnosis of lower airway diseases. Additional data from RP FE in horses of different breeds, sex, age, body we ight, and in horses with known lung disease are likely to be highly informative. Electroacupuncture was more effective than AC in modulation of innate immune response of the horses tested in these studies. This anti inflammatory action was likely governed by modulation of a cellular component of the innate immune system by altering the production of inflammatory cytokine upstream of the inflammatory cascade. Effects of EA on pulmonary biomechanics are still unclear and further study in horses with clinical signs of respiratory disease is needed.

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199 Table 5 1. Acupoints commonly used to treat chronic respiratory diseases of horses. Acupoint Location Attributes and Indications BL 13 At caudal edge of scapular cartilage (8th intercostal space), 3 cun from dors al midline. Lung Back Shu Association point to strengthen lung. BL 23 At 2 nd Lumbar intercostal space (L2 L3), 3 cun from dorsal midline. Kidney Back Shu Association point to strengthen kidney. CV 17 On ventral midline at level of 4 th intercostals spac e (caudal border of elbow). Cough, dyspnea. CV 22 On ventral midline in depression just cranial to manubrium of sternum. Cough, dyspnea. LU 9 On medial side of carpus at junction of radius and first row of carpal bone, at level of accessory carpal bone. Chronic cough, asthma, and heaves. Ding chuan 0.5 cun lateral to midline at level of GV14 (on dorsal midline between C7 T1). Chronic lung problems, cough, asthma, and dyspnea. Fei pan On the caudal edge of the scapula, 1/3 from upper border. Lower airway problems, heaves and cough. Fei men On cranial edge of scapula, 1/3 from upper border. Upper airway disease LI 4 At level of upper one third of cannon bone in a depression between 2nd and 3 rd metacarpal bone. Immune deficiency. LI 11 In depression cran ial to elbow on lateral aspect, in transverse cubital crease cranial to lateral epicondyle of humerus. Immune deficiency ST 36 3 cun distal to ST 35 and 0.5 cum lateral to the cranial aspect of the tibial crest over the cranial tibial muscle. General wea kness. GV 14 Dorsal midline between dorsal spinous processes C7 T1. Cough, heaves, and immune modulation. (Sources: Xie H, Yamagiwa K. Equine Classical Acupoints In: Xie H, Preast V, eds. Xie's veterinary acupuncture. 1st ed. 2007; pages 89 127, Xie H, T revisanello L. Equine Transpositional Acupoints In: Xie H,Preast V, eds. Xie's veterinary acupuncture. 2007; pages 2787, and Xie HS, Preast, V. Appendix D: Acupuncture Point Locations In: Xie HS, Preast, V., ed. Traditional Chinese Veterinary Medicine. 20 02; pages 559581.78,135,256)

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220 BIOGRAPHICAL SKETCH Weerapongse Tangjitjaroen received his degree in Doctor of Veterinary Medici ne from Kasetsart University, Bangkok, Thailand, in 1997 with first class honors. After practicing in equine medicine for two and a half years, he returned to academia by joining the Faculty of Veterinary Medicine, Chiang Mai University, which is located i n the north of Thailand. At Chiang Mai University, he lectured in the fields of equine medicine and surgery as well as supervised veterinary students during their clinical rotation and clinical clerkships. In 2004 he came to the University of Florida to pu rsue graduate study. At the University of Florida, he worked in the Equine Performance Laboratory under the academic supervision of Professor Dr. Patrick T. Colahan. His dissertation focused on investigating the effects of acupuncture and electroacupunctur e on immune responses and pulmonary functions in horses. After completing his Ph.D., he will return to Chiang Mai University.