Citation
Thoracic Muscle Respiratory Load Compensation in Conscious Animals and Human Respiratory Perception after Spinal Cord Injury

Material Information

Title:
Thoracic Muscle Respiratory Load Compensation in Conscious Animals and Human Respiratory Perception after Spinal Cord Injury
Creator:
Jaiswal, Poonam B
Place of Publication:
[Gainesville, Fla.]
Florida
Publisher:
University of Florida
Publication Date:
Language:
english
Physical Description:
1 online resource (184 p.)

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Veterinary Medical Sciences
Veterinary Medicine
Committee Chair:
DAVENPORT,PAUL W
Committee Co-Chair:
REEP,ROGER L
Committee Members:
BOLSER,DONALD CLEMENTZ
HAYWARD,LINDA F
FULLER,DAVID
MARTIN,ANATOLE D,III
Graduation Date:
5/3/2014

Subjects

Subjects / Keywords:
Breathing ( jstor )
Diaphragm ( jstor )
Electromyography ( jstor )
Hypercapnia ( jstor )
Muscles ( jstor )
Physical trauma ( jstor )
Rats ( jstor )
Respiratory mechanics ( jstor )
Respiratory muscles ( jstor )
Spinal cord ( jstor )
Veterinary Medicine -- Dissertations, Academic -- UF
cervical -- conscious -- cord -- external -- injury -- intercostal -- loading -- rat -- respiratory -- spinal
Genre:
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Veterinary Medical Sciences thesis, Ph.D.

Notes

Abstract:
The external intercostal (EI) muscles are primary muscles of inspiration and play a significant role during normal and loaded breathing. Intrinsic transient tracheal occlusion (ITTO) is a conscious animal model used to induce respiratory load compensation, which is a muscle mediated response aimed at restoring ventilatory homeostasis. Respiratory muscle activity is modulated by the conscious state. This doctoral dissertation presents results from three studies aimed to assess the neurophysiology of the EI muscle in a conscious rat model. Accordingly, the first aim of this study was to investigate the neurophysiological activity pattern of the EI muscle in adult conscious rats. Our primary finding was that ITTO elicits a load compensation response in the EI muscles that is characterized by an increase in EMG activity. The control of thoracic motorneurons is via direct input from the contralateral VRG and polysynaptic ipsilateral pathways from the DRG and VRG via cervical and thoracic interneurons. Cervical spinal cord injury (c-SCI) disrupts these descending inputs to the respiratory motorneurons. This in turn affects the normal EI muscle functioning and may alter their respiratory load compensation response. In our second aim, we studied respiratory load compensation in the EI muscles by means of ITTO conditioning in conscious rats with unilateral c-SCI using a cervical hemisection (C2HS) model. The results of this study indicate that although bilateral EI muscle activity is reduced one week after c-SCI, ITTO elicits a load compensation response in these muscles and repeated conditioning may improve EI muscle functioning. The purpose of our third aim was to assess the impact of ITTO conditioning on EI muscle neurophysiology. We exposed ITTO conditioned, chronically injured c-SCI animals to hyperoxic-hypercapnia and analyzed the corresponding EI muscle EMG activity. Repeated acute intermittent hypoxia (AIH) exposure is known to improve the ventilatory status in SCI animals. In our final aim, we evaluated respiratory load compensation and magnitude estimation abilities of inspiratory resistive loads (IRL) in an individual with chronic c-SCI before and ten days after AIH treatment. AIH did not alter the perceptual sensitivity and induced increases in ventilatory load compensation ability in our study subject. ( en )
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.
Thesis:
Thesis (Ph.D.)--University of Florida, 2014.
Local:
Adviser: DAVENPORT,PAUL W.
Local:
Co-adviser: REEP,ROGER L.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2016-05-31
Statement of Responsibility:
by Poonam B Jaiswal.

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Embargo Date:
5/31/2016
Classification:
LD1780 2014 ( lcc )

Downloads

This item has the following downloads:


Full Text
xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID EO6UGTUFE_6BUVP4 INGEST_TIME 2014-10-03T21:51:11Z PACKAGE UFE0046641_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES



PAGE 1

THORACIC MUSCLE RESP IRATORY LOAD COMPENS ATION IN CONSCIOUS ANIMALS AND HUMAN RESPIRATORY PERCEPTI ON AFTER SPINAL CORD INJURY By POONAM B JAISWAL A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFIL LMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2014

PAGE 2

2014 Poonam B Jaiswal

PAGE 3

To Shanu

PAGE 4

4 ACKNOWLEDGEMENTS My graduate school journey has been quite an undertaking that would not be possible wit hout the guidance, encouragement and support of a number of people. Above all I am extremely grateful for the enduring love and support of my husband, Shanu Sharma. Thank you for sharing this journey with me and seeing me through the highs and lows of it s various demands. It is said that success is when opportunity and preparation come together. I would like to thank my mentor, Dr Paul W Davenport, for giving me the opportunities to realize my dream and preparing me to become success ful in my career. Th ank you for providing to me the time and opportunity to pursue research in my area of interest and for your continued support, patience and guidance as I found my way Thank you for believing in me, I am forever grateful to you. Members of my supervisory committee played a key role in my progress and development from a graduate student into an independent scientist. I would like to thank Dr. David D Fuller for his collaborations and continual, genuine interest in my progress. I would like to thank Dr. Dona ld C Bolser for his perspective during my training, which helped me pay attention to the many details involved in research. I would like to thank Dr. Roger L Reep for encouraging me to think beyond the scope of my research, and also, for teaching me how to be a pro active and patient teacher. I would like to thank Dr. Linda Hayward for always raising the bar with her challenging research questions and for being an inspiration as a successful woman of science. I would like to thank Dr. Danny Martin for bring ing my attention to the statistical side of research and helping me refine my experiments.

PAGE 5

5 I was very fortunate to have the opportunity to work with Drs Hsiu Wen Tsai, Barbara K Smith, Vipa Bernhardt, Teresa E Pitts, Kathryn M Pate and Mark Hotchkiss at v arious stages of my doctoral studies and would like to thank them for their perspective and encouragement. I would like to thank, Dr. Milapjit S Sandhu for surgical training and for being a good friend and peer mentor Also a big t hank you to Dr. Nicole J Tester for her collaboration o n human spinal cord injury research. A big thanks to Dr. D and Cherith Davenport for being so warm, welcoming and making me feel at home here in Gainesville, FL. Thank you, also f or the countless happy memories, of summer ba rbecues, Christmas cookies and everything in between I would like to thank all my friends at UF and in India for their support through these years. Special thanks to my dear friends, Dr. Divya Garg and Kalyani Punyala for helping me stay optimistic and fi nd humor in the most unlikely of situations. Last, but definitely not least, I would like to thank my parents Brij and Nutan Jaiswal and sister Pari Jaiswal for always believing in my potential T hanks to my adorable kitties Cookie and Mickey for being the best study companions I could ever ask for.

PAGE 6

6 TABLE OF CONTENTS page ACKNOWLEDGEMENTS ................................ ................................ ............................... 4 LIST OF TABLES ................................ ................................ ................................ .......... 10 LIST OF FIGURES ................................ ................................ ................................ ........ 11 LIST OF ABBREVIATIONS ................................ ................................ ........................... 13 ABSTRACT ................................ ................................ ................................ ................... 14 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 16 Respiratory Muscles of the Chest Wall ................................ ................................ ... 17 Activity of External Intercostal Muscles (EI) ................................ ...................... 18 Innervation of the EI M uscles ................................ ................................ ........... 20 Afferent Modulation of EI Muscle Activity ................................ ......................... 21 Propriospinal Connectivity of Thoracic Spinal Cord ................................ .......... 22 Respiratory Load Compensation ................................ ................................ ............ 23 Respiratory Load Compensation under Anesthesia ................................ ......... 23 Intrinsic Transie nt Tracheal Occlusion: Model of Respiratory Load Compensation in Conscious Animals ................................ ............................ 24 Cervical Spinal Cord Injury (c SCI) ................................ ................................ ......... 25 Incid ence of c SCI ................................ ................................ ............................ 27 Cervical Spinal Hemisection (C2HS) ................................ ................................ 28 Respiratory Chemoreception ................................ ................................ .................. 31 Hypercapnia ................................ ................................ ................................ ..... 31 Acute Intermittent Hypoxia ................................ ................................ ............... 32 Effect of Conscious State on Respiration ................................ ................................ 33 Voluntary Control of Respiratory Activity ................................ .......................... 33 Respiratory Sensation ................................ ................................ ...................... 34 2 NEUROPHYSIOLOGY OF THE EXTERNAL INTERCOSTAL MUSCLE IN CONSCIOUS RATS ................................ ................................ ................................ 39 Materials and Methods ................................ ................................ ............................ 44 Animals ................................ ................................ ................................ ............. 44 Study Design ................................ ................................ ................................ .... 44 Surgical Protocol ................................ ................................ .............................. 44 Experimental Protocol ................................ ................................ ...................... 45 Data Analyses ................................ ................................ ................................ .. 46 Integrated EMG analysis ................................ ................................ ............ 46 Power spectral analysis ................................ ................................ ............. 48

PAGE 7

7 Statistical analysis ................................ ................................ ...................... 48 Results ................................ ................................ ................................ .................... 48 Characteristics of Applied ITTO Stimulus and Effect on Animal Weight ........... 48 Conscious Rats ................................ ................................ ............................. 49 Peak to ................................ ............................... 52 Characteri Response ................................ ................................ ................................ ...... 52 ................................ ............. 53 Variability of EI Muscle EMG Responses to ITTO in Conscious Rats .............. 53 Conditioning ................................ ................................ ................................ .. 54 Discussion ................................ ................................ ................................ .............. 55 Modulation by Conscious and Affective State ................................ .................. 57 Breath Phase and Lung Volume ................................ ................................ ....... 59 .............................. 59 ................................ ......... 60 Effect of Body Weig ................................ ........................ 60 Technical Considerations ................................ ................................ ................. 62 3 RESPIRATORY LOAD COMPENSATION OF EXTERNAL INTERCOSTAL MUSCLES IN CONSCIOUS RATS WITH UNILATERAL c SCI .............................. 74 Materials and Methods ................................ ................................ ............................ 78 Animals ................................ ................................ ................................ ............. 78 Study Design ................................ ................................ ................................ .... 78 Surgical Instrumentation ................................ ................................ ................... 79 Cervical Hemisection Surgery (C2HS) ................................ ............................. 80 Data Collection ................................ ................................ ................................ 81 Experimental Protocol ................................ ................................ ...................... 82 Cervical Spinal Cord Histology ................................ ................................ ......... 82 Data Analyses ................................ ................................ ................................ .. 83 Integrated EMG analysis ................................ ................................ ............ 83 Statistical analysis ................................ ................................ ...................... 84 Results ................................ ................................ ................................ .................... 84 Histological Verification of Complete C2HS ................................ ...................... 84 Animal Weight and Characteristics of Applied ITTO ................................ ......... 84 Rats ................................ ................................ ................................ ............... 85 Unilateral c SCI ................................ ................................ ............................. 86 Respiratory Load Compensation of the EI Muscles in Conscious Rats One Week After c SCI ................................ ................................ .......................... 8 7 Effect of ITTO Conditioning on the Respiratory Load Compensation Re sponses of EI Muscles in Conscious Rats After c SCI ............................. 88 Discussion ................................ ................................ ................................ .............. 89 Effect of Unilateral c SCI on Eupneic EI EMG Activity in Cons cious Rats ........ 89

PAGE 8

8 SCI ............. 90 Effect of ITTO Conditioning on EI EMG Responses to ITTO in Conscious Rats with c SCI ................................ ................................ ............................. 92 Influences on Observed EI EMG Responses ................................ ................... 92 4 IMPACT OF INTRINSIC TRANSIENT TRACHEAL OCCLU SION CONDITIONING ON THE ACTIVITY OF EXTERNAL INTERCOSTAL MUSCLE DURING EUPNEA AND HYPERCAPNIA IN CONSCIOUS RATS WITH UNILATERAL c SCI ................................ ................................ .............................. 108 Materials and Methods ................................ ................................ .......................... 110 Animals ................................ ................................ ................................ ........... 110 Study Design ................................ ................................ ................................ .. 110 Hypercapnia Challenge ................................ ................................ .................. 110 Data Analyses ................................ ................................ ................................ 111 Statistical Analyses ................................ ................................ ........................ 111 Results ................................ ................................ ................................ .................. 112 Body Weight and CO 2 Levels ................................ ................................ ......... 112 112 Effect of IT TO Conditioning on Changes in Amplitude and Peak to Peak ............................. 114 During Hyperca pnia ................................ ................................ .................... 114 Discussion ................................ ................................ ................................ ............ 115 5 EFFECT OF ACUTE INTERMITTENT HYPOXIA TREATMENT ON VENTILATORY LOAD COMPENSATION AND MAGNITUDE ESTIMATION OF INSPIRATORY RESISTIVE LOADS IN AN INDIVIDUAL WITH CHRONIC INCOMPLETE c SCI CASE STUDY ................................ ................................ .. 126 Materials and methods ................................ ................................ .......................... 128 Case His tory ................................ ................................ ................................ ... 128 Pulmonary and Respiratory Muscle Function ................................ ................. 128 Acute Intermittent Hypoxia Treatment (AIH) ................................ ................... 129 Inspiratory Resistive Load (IRL) ................................ ................................ ..... 130 Magnitude Estimation Procedure ................................ ................................ ... 130 Data Analysis ................................ ................................ ................................ 131 Results ................................ ................................ ................................ .................. 131 Pulmonary and Respiratory Muscle Function ................................ ................. 131 Respiratory Lo ad Compensation ................................ ................................ .... 132 Magnitude Estimation ................................ ................................ ..................... 133 Discussion ................................ ................................ ................................ ............ 133 6 SUMMA RIES AND CONCLUSIONS ................................ ................................ .... 145 Study #1 Summary ................................ ................................ ............................... 145 Study #2 Summary ................................ ................................ ............................... 146

PAGE 9

9 Study #3 Summary ................................ ................................ ............................... 147 Study #4 Summary ................................ ................................ ............................... 148 Discussion ................................ ................................ ................................ ............ 149 EI Muscle Respiratory Neurophysiology in Intact Conscious Rats ................. 149 EI Muscle Respiratory Load Compensation in Conscious Rats After Unilateral c SCI ................................ ................................ ........................... 151 Respiratory Load Compensation and Magnitude Estimation in an Individual with Chronic Incomplete c SCI ................................ ................................ .... 156 Mechanism of Action of ITTO Mediated EI Muscle Responses after c SCI .... 157 Significance ................................ ................................ ................................ .......... 159 Future Directions ................................ ................................ ................................ .. 161 LIST OF REFERENCES ................................ ................................ ............................. 165 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 184

PAGE 10

10 LIST OF TABLES Table page 2 1 Body weight of the animal groups.. ................................ ................................ ..... 66 2 2 Pressure and duration of ITTO presentations.. ................................ ................... 66 3 1 Body weights of animals across study time periods.. ................................ ......... 94 3 2 ITTO Duration. ................................ ................................ ................................ .... 95 3 3 Occlusion pressure during ITTO. ................................ ................................ ........ 95 3 4 Phases used for normal ization of Pre Injury percentage change in EI EMG data.. ................................ ................................ ................................ .................. 95 3 5 Data normalization for percentage change in eupneic EI EMG amplitude.. ...... 98 3 6 Data normalization for ITTO m ediated percentage change in EI EMG amplitude on Days 1 and 10 .. ................................ ................................ ........... 102 5 1 Summary of results for pulmonary function test.. ................................ .............. 138 5 2 Summary of results for respiratory muscle pressure generating capacity test.. 138

PAGE 11

11 LIST OF FIGURES Figure page 1 1 Schematic representation of the r espiratory neural control.. .............................. 37 1 2 Innervation of thoracic spinal cord.. ................................ ................................ .... 38 2 1 Schematic representation of the surgical preparati on.. ................................ ....... 65 2 2 Occlusion pressure during phases of ITTO trial.. ................................ ................ 65 2 3 for each phase of the ITTO trial.. ................................ ................................ ...................... 67 2 4 ..... 68 2 5 esponse pattern to ITTO.. ................................ ................................ ... 69 2 6 Peak to ........................... 70 2 7 itude on Days 1, 3, 5, 7 and 10 normalized to ITTO day 1 Before values.. ................................ ................................ ............. 71 2 8 Power spectral density analysis of eupneic EI muscle EMG.. ............................ 72 2 9 Frequency characteristics of power spectral data of EI muscle EMG during eupneic breathing.. ................................ ................................ ............................. 73 3 1 Pre ................................ ............................. 96 3 2 Effect of unilateral c SCI on the eupneic EMG activity of bilateral EI muscles, in conscious rats on ITTO Day 1.. ................................ ................................ ...... 97 3 3 Effect of unilateral c SCI on eupneic pe to ................................ ....................... 99 3 4 Representative ITTO responses in a conscious rat one week after unilateral c SCI/Sham surgery.. ................................ ................................ ....................... 101 3 5 Respiratory load compensation of EI muscles in conscious rats with c SCI on ITTO Day 1.. ................................ ................................ ................................ ..... 103 3 6 Respiratory load compensation of EI mus cles in conscious rats with c SCI on ITTO Day 10.. ................................ ................................ ................................ ... 105 3 7 Representative histological section of a complete hemisection (C2HS).. ......... 107 4 1 Schematic of the experimental set up for hyperoxic hypercapnia challenge.. .. 119

PAGE 12

12 4 2 ................................ ... 120 4 3 EI motor effort responses during Baseline and Hypercapnia.. .......................... 121 4 4 Effect of ITTO conditioning on the peak to Baseline and Hypercapnia.. ................................ ................................ .............. 122 4 5 conscious rats with c SCI.. ................................ ................................ ............... 123 4 6 Effect of ITTO conditi oning on breath times for the hypercapneic response of conscious rats with c SCI.. ................................ ................................ ............... 124 4 7 Representative EI EMG data traces from all treatment groups.. ...................... 125 5 1 Experimental set up for ME of IRL. ................................ ................................ ... 137 5 2 Ventilatory load compensation after AIH treatment: Pressure.. ........................ 139 5 3 Ventilatory load compensation after AIH treatment: Airflow.. ............................ 141 5 4 Magnitude estimation of IRL after AIH treatment.. ................................ ............ 143 6 1 Mechanism of action of ITTO mediated EI muscle responses in conscious rats after unilateral c SCI ................................ ................................ .................. 163

PAGE 13

13 LIST OF ABBREVIATIONS AIH Acute intermittent hypoxia C2HS Cervical hemisection at the C2 level c SCI Cervi cal spinal cord injury DRG Dorsal respiratory group EI External intercostal EMG Electromyography IRL Inspiratory resistive loads ITTO Intrinsic transient tracheal occlusions VRG Ventral respiratory group

PAGE 14

14 Abstract of Dissertation Presented to the G raduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy THORACIC MUSCLE RESP IRATORY LOAD COMPENS ATION IN CONSCIOUS ANIMALS AND HUMAN RESPIRATORY PERCEPTI ON AFTER SPINAL CORD INJURY By Poonam B Jaiswal May 2014 Chair: Paul W Davenport Major: Veterinary Medical Science s The external intercostal (EI) muscles are primary muscles of inspiration and play a significant role during normal and loaded breathing. Intrinsic tra nsient tracheal occlusion (ITTO) is a conscious animal model used to induce respiratory load compensation which is a muscle mediated response aimed at restoring ventilatory homeostasis Respiratory muscle activity is modulated by the conscious state. This doctoral dissertation presents results from thr ee studies aimed to assess the neurophysiology of the EI muscle in a conscious rat model. Accordingly, the first aim of this study was to investigate the neurophysiological activity pattern of the E I muscl e in adult conscious rats Our primary finding was that ITTO elicits a load compensation response in the EI muscles that is characterized by an increase in EMG activity. The control of thoracic motorneurons is via direct input from the cont ralateral VRG and polysynaptic ipsilateral pathways from t he DRG and VRG via cervical and thoracic interneurons. Cervical spinal cord injury (c SCI) disrupts these descending inputs to the respiratory motorneuron s This in turn affects t he normal EI muscle functioning and may alter their respiratory load compensation response In our second

PAGE 15

15 aim we studied respirat ory load compensation in the EI muscl es by means of ITTO conditioning in conscious rats with unilateral c SCI using a cervical hemisection (C2HS) model The results of this study indicate that although bilateral EI muscle activity is reduced one week after c SCI, ITTO elicits a load compensation response in these muscles and repeated conditioning may improve EI muscle functioning. The purpose of ou r third aim was t o assess the impact of ITTO conditioning on EI muscle neurophysiology W e exposed ITTO conditioned, chronically injured c SCI animals to hyperoxic hypercapnia and analyz e d the corresponding EI muscle EMG activity. Repeated a cute intermitt ent hypoxia (AIH) exposure is known to improve the ventilatory status in SCI animals In our final aim, w e evaluated respiratory load compensation and magnitude estimation abilities of inspiratory resistive loads (IRL) in an individual with chronic c SCI b efore and ten days after AIH treatment. AIH did not alter the perceptual sensitivity and induced increases in v entilatory load compensation ability in our study subject

PAGE 16

16 CHAPTER 1 INTRODUCTION Respiration can be divided into two phases, inspiration and ex piration. Primary inspiratory muscles act mec hanically to inflate the lung. Primary expiratory muscles have a deflation action on the lung. These muscles are activated in a rhythmic pattern to maintain the cycle of respiration and receive descending respir atory drive from bulbospinal (Porter, 1895) and corticospinal (Rikard Bell et al., 1985a) pathways and are unde r behavioral control (Orem and Netick, 1986) Control of the respiratory system is summarized schematically in Fig ure 1 1 These descending inputs t erminate at various levels in the spinal cord that innervate the inspiratory diaphragm (phrenic, C3 C6) and external intercostal muscles (thoracic, T1 T12) as well as expiratory internal intercostal (thoracic, T1 T12) and abdominal (thoraco lumbar, T 6 L 3 ) muscles (Lane, 2011) Disease or trau ma to the central nervous system can directly impact respiratory muscle functioning. Spinal cord injury (SCI) disrupts these descending spinal motor pathways and causes an impaired ventilatory function (Sandhu et al., 2009) The resulting respiratory deficits depend on the level of the S CI Injury to the cervical spinal cord (c SCI) is the most common type of SCI reported (Winslow and Rozovsky, 2003) with impairment in diaphragm (Golder et al., 2001a, Zimmer et al., 2007) intercostal (Dougherty et al., 2012a) and abdominal (Baydur et al., 2001) muscle activitie s The loss of diaphragm function after c SCI is compensated by chest wall and abdominal muscles (Katagiri et al., 1994) These compensatory respiratory mechanisms are fundamental adaptations to c SCI. Humans with SCI are usually studied in the conscious state (Axen and Bergofsky, 1977, Sinderby et al., 1992, Ben Dov et al., 2009, Aslan et al., 2013) However, animal models of SCI are usually studied under

PAGE 17

17 anesthesia (Golder et al., 2001b, Full er et al., 2008, Dougherty et al., 2012a) This dissertation addresses the thoracic muscle respiratory compensation in normal and c SCI injured conscious rats Respiratory Muscles of the Chest Wall The chest wall is comprised of different muscles includi ng the internal, exter nal and parasternal intercostal pectoralis, and triangularis sterni muscles that act in a coordinated manner with the diaphragm during eup neic and loaded breathing to produce ventilation The inspiratory intercostal muscle s along wit h the diaphragm and hypoglossal muscles receive an integrated rhythmic drive from the brainstem respiratory central pattern generator (Rice et al., 2011) The pr esence of a common respiratory motor input of supraspinal origin to the bilateral diaphragm muscle in healthy awake humans (Bruce an d Goldman, 1983) as well as to the thoracic intercostal motorneurons at different levels in the spinal cord of anesthetized cats (Kirkwood et al., 1982a, Kirkwood et al., 1982b) has been established This coordina tion is required for eupneic activity (Feldman, 1986) integration of voluntary behaviors (Dobbins and Feldman, 1994) and is important for generation of airway protective behaviors (Lasserson et al., 2006) T he diaphragm acts on the chest wall such tha t its caudal displacement has an inspiratory effect on the lung (De Troyer, 2012) The internal intercostal muscles are primarily expirato ry and the external intercostal muscles are primarily inspiratory in their action during normal eupneic breathing in supine humans (Taylor, 1960) The parasternal intercostal muscle s shorten during spontaneous and tracheal obstructed breathing and are inspiratory agonists in their action (De Troyer et al., 1988) as are the external inte rcostal muscle s (De Troyer and Farkas, 1990) The triangularis sterni

PAGE 18

18 muscle is recruited during expulsive maneuvers and also during phonation, cough and laughing (De Troyer et al., 1987) Coordinated activity of the parasternal intercostal and triangularis sterni muscles is responsible for inspiratory elevation of the rib cage in supine dogs (Ninane and De Troyer, 1988) However, in supine conscious human subjects, the triangularis stern i muscle is recruited during forced expiratory maneuvers al ong with the abdominal muscles and aids in deflation of the rib cage (De Troyer et al., 1987) Therefore coordinated activities of all individual, chest wall respiratory muscles with the diaphragm are important for normal respiratory movement s. The inspiratory external intercostal muscles in particular contribute substantially to tidal volume generation (Pagliardini et al., 2012) Activity of External Intercostal Muscles (EI) The EI muscles are primarily inspiratory in their mechanical action (Taylor, 1960) and play a n important role during normal and load compensatory breathing (Lane, 2011) Their differential activation is required for efficient respiration (Saboisky et al., 2007) The EI muscles have a rostral to caudal and a dorsal to ventral gradient of activity (De Troyer et al., 2005) and the organization of this gradient is reported to be at the spinal cord level (Hudson et al., 2011) This gradient is used for 1) elevation and outward m ovement of the rib cage (De Troyer et al., 1983) and 2) maintenance of a stable chest wall during normal breathing (Feldman, 1986) This gradient in activity and varied responses to load of the intercostal muscles can be attributed to the three types of synchronization observed at the thoracic spinal cord level (Kirkwo od et al., 1982a, Kirkwood et al., 1982b) Two of these three types, short term synchronization and high frequency oscillation, occur due to the synchronization of descending respiratory drive (Kirkwood et al., 1982 a) whereas the third type, broad peak synchronization, is said to

PAGE 19

19 occur due to modulation via interneuronal activity (Kirkwood et al., 1982b) H owever, segmental deafferentation of the thoracic spinal cord does not i nfluence EI muscle activity gradient at that le vel, one level above or below the denervation (Bonaert et al., 2012) in anesthetized rabbits During norma l breathing, the EI muscles have an inspiratory effect and internal intercostal muscle s have an expiratory effect (De Troyer et al., 2005) However, this activ ity is highly coupled to lung volume. A t low lung volumes, both external and internal intercostals act mechanic ally to elevate the rib cage. A t high lung volumes both these muscles have an expiratory mechanical advantage (De Troyer et al., 1985) Therefore, the EI muscles have both inspiratory and ex piratory function s that are related to the ventilatory status. d e Almeida and Kirkwood have recorded both inspiratory and expiratory excitatory potentials from individual thoracic motorneurons (de Almeida et al., 2010) A single breath airway occlusion in anesthetized dogs has been shown to increase EI and levator costae muscle EMG activity (De Troyer, 1991) A study of the relationship between respiratory muscle strength and effective cough production shows that inspiratory muscle streng th is important in SCI patients, which suggests that EI muscles may be important in generation of an effective cough (Park et al., 2010) The intercos tal muscle s are actively involved in trunk rotation movements (Whitelaw et al., 1992) and their activity is depende nt on pos ture during sleep (Dick et al., 1984) It is important to note that in their study of intercostal muscle activity in sleeping cats, Dick et a l demonstrate d an absence of a significant correlation between postural and phasic respiratory activity. Thus a change in posture will result in an overall change in both

PAGE 20

20 these activities (Dick et al., 1984) Unlike anesthetized animals, t he activity of the intercostal muscles is influenced by sleep wake status is highly variable and changes variably during different sleep states and transiti ons between states (Dick et al., 1982) This variability in activity in unanesthetized animals may be due to modulation by muscle spindle tendon organs and joint receptor afferent feedback Innervation of the EI M uscles The EI muscles are innervated by thoracic motorneurons located in the ve ntrolateral region of the ventral horn of the cat spinal cord (Rikard Bell et al., 1985b) While the cat has been used for most early studies on intercostal neuromuscular function, the rat has increasingly become the model of choice to study neurophysiology and functioning of the respiratory system in normal, disease or trauma conditions (Bianchi et al., 1995) T horacic spinal motorneurons in cats and rats similarly receive bilateral bulbospinal projections from the dorsal (DRG) and ventral (VRG) respiratory groups (La rnicol et al., 1982, Feldman et al., 1985, Rikard Bell et al., 1985b) and also corticospinal projections (Rikard Bell et al., 1985a) Cro ss correlation studies have shown that the connections between the thoracic intercostal and upper cervical inspiratory neurons (Tian and Duffin, 1996a) and inspiratory bulbospinal neurons of the ventral respiratory group (VRG) (Tian and Duffin, 1996b) are via interneurons. Also reported, is an absence of direct monosynaptic input from the VRG (Tian and Duffin, 1996b, a) and from the Btzinger complex (Kanj han et al., 1995) to the thoracic spinal cord motorneurons in rats. Propriospinal interneurons play a major role in inter segmental control of thoracic cord motorneurons (Kirkwood et al., 1988) In their earlier experiments, Merrill and Lipski demonstrated that monosynaptic connections between the medullary respiratory neurons of nucleus retro ambigualis (NRA) and thoracic

PAGE 21

21 mot orneuron pools were extremely rare (Merrill and Lipski, 1987) However, segmental input from interneurons played an important role in respiratory output (Bellingham and Lipski, 1990) The presence of the DRG has been confirmed in the rat (de Castro et al., 1994) and its connections with VRG neurons via axon collaterals have been reported (Lipski et al., 1994) However, very few direct connections between the DRG and thoracic spinal cord motorneuron s have been reported in the rat in contrast to the cat (Duffin and Lipski, 1987) Recently, de Almeida et al. have described the a n atomy of the caudal intercostal muscle segment (de Almeida et al., 2010) and reported expira tory bulbospinal medullary projections to the EI motorneurons in the thoracic spinal cord of rats (de Almeida and Kirkwood, 2010, 2013) Innervation of the thoracic spinal cord with emphasis on data obtained from r ats is outlined in Fig ure 1 2. Afferent Modulation of EI Muscle Activity EI m uscle activity is modulated via afferent feedback from muscle spindle fibers and tendon organs, which are abundant in the EI muscles. This afferent feedback mechanism acts segmen tally on spinal motorneuron s and supraspinally on medullary respiratory neurons (Bolser and Remmers, 1989) Afferents from intercostal mechanoreceptors in hibit inspiratory phrenic activity (Remmers, 1970) and t endon organs are responsible for the inspiratory inhibition reflex (Bolser et al., 1987) Afferent information from the intercostal and abdominal muscle tendon organs inhibits caudal VRG expiratory n euron activity and may be responsible for the decreased descending expiratory drive to the abdominal muscles (Hernandez et al., 1989) M uscle spindles are rhythmically active during nor mal and artificial breathing (Critchlow and von Euler, 1963, Leduc and De Troyer, 2003) and their afferents terminate in the 3a region of the contralateral somatosensory cortex (Davenport et al., 1993) Joint receptors act to

PAGE 22

22 increase inspiratory activity on caudal displacement of the rib, irrespective of muscle length (De Troyer, 1997) This feedback control from the intercostal muscles is also important for their coordinated activity with the diaphragm and other respirat ory muscles. Thoracic dorsal root r hizotomies eliminate intercostal proprioceptor and mechanoreceptor afferent input s thereby changing the pattern of respiratory activity in anesthetized vagotomized cats (Shannon, 1977) Propriospinal Connectivity of Thor acic Spinal Cord A strong inhibitory phrenic intercostal reflex is responsible for decreased EI muscle activity in response to increasing phrenic nerve stimulation (Brichant and De Troyer, 1997, De Troyer, 1998) O n the other hand, stimulation of intercostal nerve s results in an increase in phrenic activity (Decima et al., 1969, Leduc and de Troyer, 2002) Activity from this excitatory intercostal phrenic reflex is influence d variably by administration of morphine (Millhorn et al., 1985) A polysynaptic intercostal intercostal reflex, that is under inhibitory descending control, has also been reported and i ts action is to excite and facilitate mutual intercostal muscle activity (Downman and Hussain, 1958) Thoracic spinal interneurons are extremely important in normal functioning of chest wall and abdominal muscles and may participate in these reflex pathways These interneurons are extensively distribut ed throughout the spinal gray matter (Conta Steencken and Stelzner, 2010) and many interconnect with the motorneuron pool (Kirkwo od et al., 1988) Antidromic stimulation (Kirkwood et al., 1988) and single cell retrograde labeling (Saywell et al., 2011) studies have shown the presence of contralateral interneuronal projections between segments of the thoracic cord. These projections may extend at least three to four segments caudal to the c ell soma

PAGE 23

23 (Kirkwood et al., 1988) implying their role in inter segmental control. This afferent activity along with afferen t feedback from the diaphragm and abdominal muscles is responsible for the respiratory load compensation response (Davenport et al., 1991, Holt et al., 2002, Davenport et al., 2010) As a part of th is dissertation neurophysiological characteristics of the EI muscles in conscious animals have been investigated Respiratory Load C ompensation Respiratory Load Compensation under Anesthesia Various loading paradigms have been used to study inspiratory (Zechman and Davenport, 1978, Martin and De Troyer, 1982) and expiratory (Koehler and Bishop, 1979) load compensation. The primary and accessory muscles of respiration are the key mediators of the load compensation response (Lopata et al., 1983, Katagiri et al., 1994) Modifications in the activities of diaphragm, intercostal, abdominal and/ or upper airway muscles are needed for load compen sation. Respiratory load compensation has been demonstrated in the cat anterolateral abdominal muscles (Bolser and Davenport, 2000) dog EI muscles (De Troyer, 1991) and rat upper airway muscles (Bailey et al., 2001) In anesthetized cats, measurement of integrated respiratory muscle activity is a good indicator of the central respiratory activity (Siafakas et al., 1981) an d is influenced by temperature, pH (Trippenbach and Milic Emili, 1977) an d thoracic volume (Eldridge and Vaughn, 1977) The timing and intensity of the muscle response to a respiratory load is determined by a variety of feedback mechanisms. Many investigators have demonstrated modulation by vagal afferen ts (Clark and von Euler, 1972, Phillipson, 1974, Zechman et al., 1976) muscle afferents (Lumsden, 1923b, D'Angelo et al., 1976,

PAGE 24

24 Shannon et al., 1985) and chemoreceptors (Bailey et al., 2001, Golder et al., 2003, Fuller et al., 2006) The contributions of vagal and thoracic dorsal root afferents to the intercostal muscle load compensation reflex in anesthetized cats have been systema tically documented (Shannon and Zechman, 1972, Shannon, 1977) Elimination of vagal feedback results in an inability to recruit new motor units required to elicit the load compensation reflex and r hizotomies elimin at e thoracic dorsal root afferent feedback thereby causing a delay in firing of intercostal motor units when a load is applied (Shannon and Zechman, 1972) Both vagal a nd thoracic afferent feedbacks are required for an increase in activity of the diaphragm and intercostal muscles. (Shannon and Zechman, 1972) However, these studies utilized anesthetized animal preparations C onscious animal load compensation is less well understood Load compensation in conscious animals adds behavioral load compens ation to reflex diaphragmatic responses reported in anesthetized a nimals. Also, unanesthetized load compensation requires more than just a reflex diaphragmatic response; hence it is important to understand the activity and functional characteristics of tho racic muscles during load compensation in conscious animals. This dissertation investigates the respiratory load compensation responses of the EI muscles in conscious rats. Intrinsic Transient Tracheal Occlusion: Model of Respiratory Load Compensation in C onscious Animals Measurement of occlusion pressure in conscious hu man s is dependent on the neuronal discharge and the ability of respiratory muscles to shorten effectively and thus may give an indication of the respiratory center output (Whitelaw et al., 1975, Altose et al., 1976) Our lab has developed a model of intrinsic transient tracheal occlusions ( IT TO) to investigate respiratory load compensation responses in conscious rats (Pate,

PAGE 25

25 2010, Pate and Davenport, 2012a) In this conscious animal model of ITTO a n occlusion increases the central respiratory drive and the o utput is increased activation of primary respiratory muscles. Responses to IT TO can be measured using chr onically implanted EMG electrodes in the muscles of interest. Although, new techniques to evaluate recovery of muscle function are being investigated (Dow et al., 2009) electromy ography (EMG) remains a classic, well studied approach to evaluate improvements in neuromuscular function in normal and injured animals and human subjects Meaningful comparisons of EMG data collected via longitudinal investigations using chronic electrode placements are possible by utilizing analyses methods that reduce intra and inter animal variability (Mantilla et al., 2011) Exposure to IT TO causes significant increases in diaphragmatic EMG activity (Pate and Davenport, 2012b) and an associated increase in basal neural activation in the nucleus tract us solitarius (nTS) and rostral ventral respiratory group (rVRG) (Pate and Davenp ort, 2012a) Significant increases in diaphragm muscle fiber diameter after IT TO conditioning ha ve also been reported (Smith et al., 2012) Repeated expo sure to tracheal occlusions has been shown to produce anxiety (Pate and Davenport, 2012a) and to up regulate activity of glycinergic neurons in the nucleus tractus solitarius (Tsai and Davenport, 2014) and genes associat ed with anxiety and depression in the medial thalamus (Bernhardt et al., 2011a, Bernhardt et al., 2011b) To better understand the neural control of the EI muscles in conscious animals the ITTO model has been utilized in this study to elicit respiratory load compensation C ervical S pinal C ord I njury ( c SCI ) Respiratory rhythm generated in the ponto medullary respiratory centers is transmitted from the brainstem to the respiratory motorneuron s in the spinal cord

PAGE 26

26 (Lumsden, 1923a) This rhythm is then transmitted to the respective m uscles of respiration (diaphragm, intercostals, abdominals and upper airway) via spinal cord motorneuron s. Trauma to the cervical spinal cord above t he level of the phrenic (C3 C6) nucleus interrupts the descending bulbospinal motor drive to the respirator y spinal motorneuron s (Porter, 1895) and results in respiratory muscle dysfunction (Laghi and Tobin, 2003) Further, denervation of the thoracic musculature results in paradoxical collaps e of the chest wall during inspiratory contraction of the diaphragm. This may lead to difference s in distribution of alveolar ventilation and change the ventilation perfusion dynamics (Shannon and Zechman, 1972, Sha nnon, 1977) T he intercostal muscles have a rostral to caudal and a dorsal to ventral gradient of activity. This activity gradient and the varied response s to applied load s in the intercostal muscles can be attributed to the three types of synchronization observed at the thoracic spinal cord level (Kirkwood et al., 1982a, Kirkwood et al., 1982b) Two of these three types, short term synchronization and high frequency oscillation, occur due to the synchronization o f descending respiratory drive whereas the third type, broad peak synchronization, is said to occur due to modulation via interneuronal activity. A n acute lesion to the spinal cord promotes the occurrence of broad peak synchronized responses thereby implyi ng modulation of intercostal muscle response s via increase in inhibitory inte rneuronal activity post injury (Kirkwood et al., 1982a, Kirkwood et al., 1982b) In cats with chronic thoracic spinal cord lesions, a ret urn of this synchronized activity, which was stronger than in the normal condition, was observed as early as two to four days after injury (Kirkwoo d et al., 1984) Following loss of neural input due to SCI, respiratory muscles exhibit changes in their structural and functional characteristics (Rowley et al., 2005)

PAGE 27

27 and corresponding changes in expression of serotonin and glutamate receptors in the phrenic motorneuron pool have been reported (Mantilla et al., 2012) Incidence of c S CI About 12000 new cases of spinal cord injuries (SCI) are reported every year in the United States (NSCISC, 2013) Injury to any level of the spinal cord can cause respiratory muscle dysfunction, with injury at higher spinal levels producing deficits of greatest severity. More than half of the reported SCI cases are at the cervical spinal cord (c SCI) level (Winslow and Rozovsky, 2003) and pulmonary complications remain the major cause of death i n this patient population (DeVivo et al., 1999, Zimmer et al., 2007) The life expectancy after SCI decreases with increasing age and injury severity (NSCISC, 2013) ; and these outcomes have not improved over the years (Strauss et al., 2006, van den Berg et al., 2010) The most common complicat ions in SCI patients are pneumonia and lung atelectasis (Jackson and Groomes, 1994) Pneumonia can arise from the inability of patients to clear their airways due to impaired cough function In SCI patients, weakness of the chest wal l and abdominal muscles may negatively affect the ability to produce cough (Bolser et al., 2009) Rehabilitation m ethods to clear the airway include postural drainage (Park et al., 2010) mechanical in/exsuffulation devices (CoughAssist, JH Emerson, Cambridge MA), spinal nerve microstimulators (Lin et al., 2008) electrical stimulation of the abdominal wall (Butler et al., 2011) chest percussions, bronchoscopy and suction catheters (Brown et al., 2006) However, these methods are less e ffective than normal cough and are often invasive in nature. Also, long term studies of efficacy and safety of these devices have not been conducted.

PAGE 28

28 In animal studies, a decrease in augmented breath volume has been reported in chronically injured c SCI r ats (Golder et al., 2005a) Augmented breaths are important in restoring lung airway patency thereby preventing atelectasis (Reynolds, 1962, Vlemincx et al., 2013) Atelectasis is defined as the collapse of a part of a lung or the entire lung. This may arise due to an increase in lung elastic recoil or reduced passive chest wall recoil. In SCI patients both of these conditions exist; the former due to de nervation of the diaphragm and the latter most likely due to denervation and inadequate intercostal and abdominal muscle function. A reduction in respiratory muscle activity due to weakness of chest wall muscles has been reported in patients with SCI (Estenne et al., 1983) Expiratory muscle weakness exacerbates symptoms of respiratory dysfunction. SCI patients use binders for abdominal support, which in turn h elps in rib cage expansion during inspiration. With abdominal muscle support the abdominal compliance decreases and pressure increases, both of which are required for efficient inspiration. Cervical Spinal H emisection (C2HS) Due to their similarity to pr imates in respiratory and locomotor control pathways, rodents are the animal model of choice to study respiratory recovery and rehabilitation following experimentally induced c SCI (Kastner and Gauthier, 2008) Various animal models of c SCI including spinal cord hemisect ion, transection and contusion have been used to investigate respiratory recovery after c SCI (Zimmer et al., 2007, Lane et al., 2008) Cervical hemise ction (C2HS) is a reliable, reproducible and widely accepted model among investigators studying respiration (Goshgarian, 1979, Golder et al., 2001a, Golder et al., 2001b, Zimmer et al., 2007, Fuller et al., 2008) Although not a clinically relevant phenomenon, c SCI provides an opportunity to investigate basic mechanisms involved in respiratory recovery after c SCI Researchers have mostly

PAGE 29

29 focused on the diaphragm function as a measure of respiratory recovery in c S CI animals (Fuller et al., 2006, Alilain and Silver, 2009, Dow et al., 2009) The diaphragm needs a stable chest wall as well as abdominal muscle tone to function efficiently. Some studies conducted in humans with SCI have reported changes in tidal volume and frequency to added mechanical loads that are similar to load compensation responses in uninjured humans (Axen, 1982) EMG data from tetraplegics has shown th at intercostal muscle activity is present acutely after injury and becomes more marked months after injury (Silver and Lehr, 1981) However, characteristics of the EMG response in patients with SCI are different when compared to uninjured subjects. Individuals with low c SCI have a faster rat e of rise in their diaphragmatic EMG activity (Kelling et al., 1985) A variety of different interventions to rehabilitate respiratory muscle motor function after SCI have been reported (Alilain and Silver, 2009) Nerve and muscle pacing techniques have been used to overcome muscle we akness and improve respiratory function. Intercostal nerve pacing in anesthetized dogs (DiMarco et al., 1 989) and humans (DiMarco et al., 1994) and combined intercostal and phrenic nerve pacing (DiMarco et al., 2005) produce some improvements in respiratory function. Experimental spinal cord sti mulation at the thoracic level has been shown to produce physiologically similar inspiratory pattern in the diaphragm (Gandevia and Kirkwood, 2011, Dimarco and Kowalski, 2013a, b) However, long term complications of these methods include increased risk of respiratory infections, non specific stimulation hyperesthesia and autonomic d ysreflexia with high stimulus intensities (Brown et al., 2006) In animal studies, training of respiratory pathways by using hypoxia (Doperalski

PAGE 30

30 and Fuller, 2006) and hypercapnia (Fuller et al., 2003) have demonstrated increa ses in phrenic activity post C2HS. Respiratory plasticity seen after SCI may be due to neuroplasticity at the spinal cord level (Johnson and Creighton, 2005) Neuroplasticity in the respiratory system can also be induced by a variety of different chemical and mechanical perturbations to the system or spontaneously after injury to the ne rvous system (Mitchell and Johnson, 2003) Endogenous neuroplasticity leads to slight recovery in ipsilateral diaphragm function, attributed to the spontaneous crossed phrenic phenomenon (sCPP) (Goshgarian, 2003, Fuller et al., 2008, Zimmer et al., 2008, Lane et al., 2009, Sandhu et al., 2009) H owever, compensation from other respiratory muscles, like the intercostals, makes the contribution of sCPP to respiratory recovery effectively neg ligible (Dougherty et al., 2012b) Dougherty et al have proposed a crossed intercostal circuit that enables spontaneous recovery of the intercostal muscle activity ipsilater al to C2HS injury (Dougherty et al., 2012a) Spontaneous improvement in diaphragm function together with improvements in shoulder and upper arm muscles have also been reported in humans with c SCI (Axen et al., 1985) Affective and conscious states play a critical role in determining the type of respiratory load compensation response generated. The resulting respiratory perception further modulates respiratory pattern in individuals with SCI. Current literature on the influence of chest wall denervation on the perception of respiratory effort is conflicting. In patients with low c SCI denervation of chest wall afferents does not se em to influence the perception of breathlessness during arm exercise and hypercapnia (Oku et al., 1997) In anesthetized cats, partial de afferentation of thoracic dorsal roots does not

PAGE 31

31 alter the a ctivity of individual thoracic motorneuron s (Kirkwood et al., 1982b) however complete denervation by thoracic dorsal root rhizotomy alters the intercostal muscle elect romyographic response to applied respiratory loads (Shannon and Zechman, 1972) Other studies report that, thoracic afferent s are involved in the perception of respiratory force production in normal human subjects exposed to various elastic and resistive loads (Axen and Haas, 1982) but these are not req uired for the initiation of load compensation in SCI humans (Axen, 1982) However, these afferents are responsible for driving a consistent phrenic motor output in response to added respiratory loads to maintain homeostatic tidal volume generation (Axen and Bergofsky, 1977) Also, rib cage afferents play an important role in the perceived intensity of respiratory sensation during sub maximal ventilatory loading. Disruptio n of this afferent input a fter SCI results in a blunted sensation to resistive and elastic loads (Gottfried et al., 1984) Therefore, it is hypothesized that the disruption of afferent feedback from the chest wall and abdomen due to c SCI will greatly influence the type and p attern of respiratory muscle recruitment in conscious animals. Respiratory Chemoreception Hypercapnia The respiratory system functions to maintain a highly regulated homeostasis of oxygen and carbon dioxide levels in the blood and cerebral spinal fluid (Clancy and McVicar, 1996) During a hypercapnia challenge, this balance is disrupted by an elevated arterial P CO2 and decreased pH Peripheral (Schlaefke et al., 1979) and central (Saint John, 1975) chemoreceptors are activated and stimulate increases in respiratory drive to increase minute ventilation by eliciting increased respiratory muscle activity (Y asuma et al., 1993) Increases in minute ventilation increase the rate of CO 2

PAGE 32

32 exhalation to restore O 2 and CO 2 homeostasis. The retrotrapezoid nucleus and serotonergic raphe nucleus are involved in regulation of the central chemoreceptor response to expos ure to high levels of CO 2 (Guyenet et al., 2012) Exposure to hypercapnia during development (Bavis et al., 2006) and in adulthood (Schaefer et al., 1963) has been shown to induce tra ns ient respiratory plasticity. However, p revious exposure to stressful stimuli, like immobilization, decreases the ventilatory response to hypercapnia in conscious rats (Kinkead et al., 2001a) Hypercapnia has long been used as a chemical challenge to evaluate respiratory motor responses (Arita and Bishop, 1983b, a, Oliven et al., 198 5) In patients with low c SCI where the intercostal muscles are affected but diaphragm function is preserved the response to hypercapnia exposure is dependent on the posture of the individual (Ben Dov et al., 2009) Since hypercapnia can be used to challenge the respiratory system and elicit increased central neural respiratory drive related muscle responses, hypercapneic challenge is an important tool to investigate the hypothesized benefits of ITTO conditioning. If ITTO conditioned animals with c SCI have improved function of EI muscles then it is hypothesiz ed that EI muscle EMG will increase bilaterally during hypercapneic challenge. Acute Intermittent Hypoxia Acute intermittent hypoxia (AIH) has been shown to induce respiratory plasticity and improve ventilation in animal models of SCI (Wilkerson et al., 2008, Wilkerson and Mitchell, 2009, Lovett Barr et al., 2012) by means of respiratory long term facilitation (LTF) (Vinit et al., 2009, Dale Nagle et al., 2010) Repetitive exposure to AIH while sustaining an elevated level of carbon dioxide (CO 2 ) has been shown to induce

PAGE 33

33 ventilatory LTF in healthy conscious subjects (Harris et al., 2006, Mateika and Sandhu, 2011) and subjects with ch ronic SCI (Tester et al., 2014) Thus, A IH along with elevated level of CO 2 may improve the ventilat ory status in individuals with incomplete SCI. Hence, it is hypothesized that AIH with increased CO 2 treatment in SCI patients will increase respiratory load compensation. Effect of Conscious State on Respiration Voluntary Control of Respiratory Activity R espiration is an automatic process however voluntary modulation of breathing (Hlastala an d Berger, 2001) and integration of behavioral responses (Orem and Netick, 1986) regulate respiratory pattern in conscious states Studies instructi ng human subjects to perform respiratory tasks like breath hold (Hanly et al., 1989) airflow targeting (Alexander Miller and Davenport, 2010) Valsalva and Mueller maneuvers ( Hanly et al., 1989) all require voluntary control of breathing pattern. V oluntary control of breathing enables the performance of these and many other daily tasks Consciousness is required for modulation of respiratory activity by voluntary control. The refore, it is not surprising that c ortical structures are involved in the processing of neural information generated in response to internal and external applied respiratory stimuli. Apart from producing the required motor response, t hese brain areas proce ss information from the respiratory system and also generate an affective response to the applied stimulus including respiratory sensation s. Thus the conscious state significantly influences the sensory motor responses of an individual to internal and ext ernal respiratory stimuli.

PAGE 34

34 Respiratory Sensation Respiratory sensation occurs when a subject consciously detects an internal or external respiratory stimulus that is equal to or l arger than a threshold stimulus required to generate a cognitive response (von Leupoldt et al., 2013) Two main subcortical and cortical processes mediate respiratory sensations 1) discriminative processing, which is the ability to detect and respond to a stimulus based on its quantitative aspects and 2) affective processing, which are the qualitative attributes threat and reward feelings associated with the applied stimulus (Davenport and Vovk, 2009) Detection of inspiratory resistive loads has bee n demonstrated in trained dogs (Davenport et al., 1991) healthy human subjects (Zechman and Davenport, 1978, Huang et al., 2009) and children with life threatening asthma (Julius et al., 2002) Quantification of respiratory sensa tions is often measured using magn itude estimation of the applied stimulus. Magnitude estimation is a scaling technique where a number assigned to the perceived load is proportional to the load intensity. The reported magnitude estimation can then be used to calculate the respiratory perceptual n is a power fun ction of the load magnitude n is the power exponent (sensitivity) and K is a constant (Stevens, 1957) Respiratory percept ual sensitivity is unique to a stimulus modality (Zechman and Davenport, 1978, Davenport and Vovk, 2009, Huang et al., 2009) Respiratory sensation and magnitude estimation of respiratory resistive loads is related to the respiratory load compensation in response to the applied load (Knafelc and Daven port, 1999) Mechanisms underlying the perception of respiratory sensations are though t to be modulated by a combination of changes in diaphragmatic pressure (Zechman and Davenport, 1978) mouth pressure (W hitelaw et al., 1975) feedback

PAGE 35

35 from afferent modalities including phrenic (Davenport et al., 1985, Davenport et al., 2010) and intercostal (Davenport et al., 1993) mechanoreceptors. Respiratory sensations are influenced by cortical and sub cortical pathways (Davenport and Vovk, 2009) neural attention resource availability (Van den Bergh et al., 1998, von Leupoldt et al., 2010, von Leupoldt et al., 2011) affective state and resp iratory muscle strength (Kellerman et al., 2000, Huang et al., 2009) Repeated exposure to respiratory sti muli may result in respiratory learning. This respiratory learning is influenced by the memory of acquired psychosomatic symptoms but not by the physiological responses elicited by the respiratory stimulus (Van d en Bergh et al., 1998) In healthy subjects, signals associated with neural processing of respiratory stimuli decrease in parallel with the r eported r espiratory sensations on repeated presentation s of a single inspiratory occlusions (von Leupoldt et al., 2011) Due to t his process of habituation repeated respiratory sensory information seems r edundant and of lesser importa nce thereby decreasing perceived respiratory sensation inten s ity (Revelette and Wiley, 1987) However in cases where repeated respiratory stimuli make breathing progressi vely difficult, habituation does not occur and respiratory sensations and cognitive awareness are consequently heightened (von Leupoldt et al., 2010) In individuals with respiratory defic its due to traumatic insults to the spinal cord accurate identification of respiratory sensations is important not only in the generation of appropriate respiratory load compensation response s but also to signal airway and lung defensive behaviors Previou s studies have reported a significant ly reduced respiratory perceptual sensitivity in low c SCI individuals (Gottfried et al., 1984) In addition loss of

PAGE 36

36 breathlessness sensation in response to induced apnea has been reported in an individual with h igh cervical transection (Roncoroni, 1972) To our knowledge, s tudies investigating the changes in respiratory sensations and respiratory load compensation of individua ls with high cervical spinal cord injury are lacking. Thus, the studies in this dissertation are designed to systematically investigate thoracic respiratory muscle (EI) responses after c SCI and the compensation to respiratory challenges of increased mecha nical load (ITTO) and hypercapnia. In summary, t he external intercostal ( EI ) muscles are primary muscles of inspiration and play a significant role during normal and loaded breathing. Intrinsic transient tracheal occlusion (ITTO) is a conscious animal mode l used to induce respiratory load compensation, which is a muscle mediated response for restoring ventilatory homeostasis. Respiratory muscle activity is modulated by the conscious state. The control of thoracic motorneurons is via direct input from the co ntralateral VRG, polysynaptic ipsilateral pathways from the DRG and VRG via cervical and thoracic interneurons. Cervical spinal cord injury (c SCI) disrupts these descending inputs to the respiratory motorneuron s. This in turn affects the normal EI muscle functioning and may alter their respiratory load compensation response. This doctoral dissertation presents results from three studies aimed to assess the neurophysiology of the EI m uscle s in a conscious rat model. In addition respiratory load compensati on and magnitude estimation (ME) responses to inspiratory resistive loads (IRL) in an individual with chronic incomplete c SCI before and ten days after AIH treatment are also investigated

PAGE 37

37 Figure 1 1. Schematic representation of the respiratory neura l control Respiratory rhythm generated in the brainstem is transmitted to the motorneuron s of the phrenic and thoracic nuclei of the spinal cord, via bulbospinal and propriospinal pathways. These pontomedullary respiratory centers are direct ly control led by higher brain centers via cortico spinal tracts. Activity of the respiratory muscles results in thoracic pressure changes and lung movements generating ventilation. Afferent modulation of these muscles is via multiple afferent modalities: vagal, muscle sp indles, ten don organs and joint receptors Respiratory related a fferent s modulate respiratory neural drive at the brainstem and spinal cord level s. Afferent vagal modulation from the lung projects to the brainstem to modulate central neural output that dri ves respiratory muscle activity. Respiratory chemoreception of CO 2 O 2 and pH is via peripheral and central chemoreceptors that terminate in the brainstem and modulate the respiratory neural output to the respiratory muscles (Feldman, 1986, Bianchi et al., 1995)

PAGE 38

38 Figure 1 2 Innervation of thoracic spinal cord. ( A) In rats, descending drive to thoracic spinal cord is mainly via the contralateral VRG Projections from the ipsilateral VRG contralateral DRG and VR G terminate o n upper cervical (C1 C2), phrenic (C3 C6) and lower cervical (C7 C8) spinal cord motorneuron s. Propriospinal interneurons at these levels modulate thoracic motorneurons, generating an inter segmental control of intercostal and abdominal muscle activity. There are p rojections between DRG, VRG and the Btzinger complex ( B) Thoracic interneurons are extensively distributed in the cat spinal cord gray matter and interconnect with the motorneuron pool. Interneuron projections are found at least th r ee segmental levels below the cell soma. This system also allows for integrated, complex inter segmental control of respiratory muscle motorneurons (Kirkwood et al., 1988, de Castro et al., 1994, Lipski et al., 1994 Tian and Duffin, 1996b, a, de Almeida and Kirkwood, 2013)

PAGE 39

39 CHAPTER 2 NEUROPHYSIOLOGY OF THE EXTERNAL INTERCOSTAL MUSCLE IN CONSCIOUS RATS The thoracic intercostal muscle s are primary respiratory muscles (Lane, 2011) with the external intercostal muscles (EI) primar il y inspiratory in actio n during eupnea (Taylor, 1960, De Troyer et al., 2005) The EI muscles have a rostral caudal and dor sal ventral gradient of activation (Feldman, 1986, De Troyer et al., 2005 ) and segmental reflexes do not contribute to this gradient (Bonaert et al., 2012) The inspiratory activity of EI muscles is inhibited by increasing lung volume (De Troyer et al., 2005) and this inhibition is somewhat reversed by increasing levels of arterial CO 2 (Bailey et al., 2001) The EI muscles also play a prominent role in trunk movements (Whitelaw et al., 1992) and t heir activity is dependent on posture (Dick et al., 1982, Dick et al., 1984, Whitelaw et al., 1992, Hudson et al., 2011) The EI muscles act together with other muscles of the chest wall to produce respiratory pres sure changes for breathing (Cappello and de Troyer, 2000) However, these observations are based on studies in anesthetized, reduced preparations in cats (Sears, 1964) a nd dogs (De Troyer et al., 1985, De Troyer and Ninane, 1986) sleeping cats (Dick et al., 1982, Dick et al., 1984) or conscious (W hitelaw et al., 1992, Hudson et al., 2011) and sleeping (Henke et al., 1992) healthy humans The function o f intercos tal muscles in conscious animals remains poorly understood. The rat has increasingly become the model of choice to study t he functioning of the respiratory system with disease or trauma (Bianchi et al., 1995) Recently, de Almeida et al. have described the anatomy of the rat caudal intercostal muscle segment (de Almeida et al., 2010) and reported that expiratory bulbospinal medullary neurons project to the EI motorneurons in the thoracic spinal cord (de Almeida and Kirkwood,

PAGE 40

40 2010, 2013) Results from previous studies suggest that in rats medullary p r ojections from the ipsilateral VRG contralateral DRG and cont ralateral VRG terminate o n upper cervical (C1 C2), phrenic (C3 C6) and lower cer vical (C7 C8) spinal cord motor neurons (Tian and Duffin, 1996b, a) I nterneurons at these levels can modulate thoracic motorneurons, e stablishing a major role in inter segmental control of intercostal and abdominal muscle activity (Kirkwood et al., 1982a, Kirkwood et al., 1982b, Tian and Duffin, 1996b, Saywell et al., 2011, de Almeida and Kirkwood 2013) Cross correlation studies have shown that the se interneurons have connections between the upper cervical spinal inspiratory neurons (Tian and Duffin, 1996a) medullary inspiratory bulbospinal neurons of the VRG (Tian and Duffin, 1996b) and the thoracic spinal intercostal motorneur ons Thus t he descending drive to thoracic spinal cord is a n integrated complex comb ination of direct and indirect inputs from the VRG (Lipski et al., 1994, Kanjhan et al., 1995) DRG (de Castro et al., 1994) cervical spinal cord (Lipski et al., 1993, Tian and Duffin, 1996b, a) and cortical mo tor tracts (Rikard Bell et al., 1985a, b, Saji and Miura, 1990) Intercostal muscles are mod ulated by afferent feedback from their muscle spindles (Critchlow and von Euler, 1963, Remmers, 1970, Holt et al., 2002) tendon organs (Bolser et al., 1987, Bolser and Remmers, 1989) and joint receptors (De Troyer, 1997) These afferents act segmentally at the spinal cord level and on medullary inspiratory neurons to inhibit inspiratory activity (Bolser et al., 1987, Bolser and Remmers, 1989) and at the level of the cortex (Davenport et al., 1993) may modulate intercostal muscle activity Reflex connections between thoracic spinal segments (Downman and Hu ssain, 1958) involving phrenic and intercostal motorneuron al systems

PAGE 41

41 (Brichant and De Troyer, 1997, Leduc and de Troyer, 2002) have also been identified. Thus intercostal muscle activity is modulated via respirato ry muscle afferent feedback on a breath by breath basis. Ventilation is the rhythmic and coordinated activity of primary and accessory respiratory muscles to generate minute ventilation When internal or external mechanical stimuli act to perturb the syst em, respiratory load compensation response s act to restore ventilatory homeostasis (Clark and von Euler, 1972, Zechman et al., 1976) Respiratory load compensation is a motor response mediated by the primary and ac cessory muscles of respiration (Lopata et al., 1983) and involve s increases in activity of primary respiratory muscles (Romaniuk et al., 1992, Pate and Davenport, 2012b) and/ or recruitmen t of accessory respiratory muscles (De Troyer and Farkas, 1989) The majority of animal load compen sation studies have been performed in anesthetized animals, making the translation to conscious load compensation behavior (such as occurs in humans) difficult. Intrinsic transient tracheal occlusion (ITTO) in our lab is an established model to study respi ratory load compensation in conscious rats (Jaiswal et al., 2010, Jaiswal et al., 2011, Pate and Davenport, 2012a) The ITTO model allows transient reversible and repeatable tracheal occlusion that elicits activa tion of the respiratory load compensation response s in conscious animals. ITTO is applied by inflating a vascular cuff implanted around the extra thoracic trachea with sufficient pressure to cl ose of the lumen of the trachea Deflating the cuff restores th e trachea l lumen to its original state with no evidence of residual damage Du e to the short duration, transient nature of an ITTO sti mulus minimal fluctuations in arterial blood gasses occur (Lewis et al., 1980) Respiratory load compensation responses have been

PAGE 42

42 reported in conscious rats for the diaphragm (Pate, 2010) but not for the EI muscles The purpose of the present study is to determine the ITTO load compensation responses of EI muscles in conscious rat s Consciousness significantly influences respiratory activity and is fundamental for voluntary control of breathing (Hlastala and Berger, 2001) and the integration of behavior al respiratory responses (Orem and Netick, 1986) Several investigators have used s urgical implantation of electromyography ( EMG ) electrodes under aseptic conditions for purposes of longitudinal data collection in sleep ing (Dick et al., 1982, Dick et al., 1984, BuSha and Stella, 2002, Fraigne and Orem, 2011) and awake (Orem and Netick, 1986, Pate, 2010, Uga et al., 2010, Mantilla et al., 2011) animals This method allows for c hronic assessments of EMG activity and analys es of respiratory muscle neurophysiology over time and across different behaviors in conscious animals (Mantilla et al., 2011) The EMG signal allows for the assessment of muscle properties and behavior (Lindstrom and Magnusson, 1977) EMG signals can be contaminated by noise due to interference from signals of adjacent and overlying muscles, heart rate, ambient electrical artifacts and movement resulting in mechanical motion artifacts (Sinderby et al., 1995, ATS. and ERS., 2002, Reaz et al., 2006) Approp riate filtering of the raw EMG signal help s eliminate these influences and integrated EMG analysis can provide insight on the relative amount of muscle activity generated in response to the experimental stimulus (Re az et al., 2006, Mantilla et al., 2010) I ntegrated EMG analysis alone does not provide information on motor unit recruitment and frequency coding of individual motor units that shape the EMG signal (Seven et al., 2013) Power spectral density (PSD) analysis of an EMG signal can provide insight on these p arameters. PSD

PAGE 43

43 has been utilized to investigate motor unit recruitment (Seven et al., 2013) shifts in frequency content of an EMG signal (Mannion and Dolan, 1994, Spahija et al., 2005) and evaluation of muscle fatigue (Lind strom et al., 1970, Beck et al., 1997) PSD is influenced by the conduction velocity of the nerve fibers innervating the muscle under study (Lindstrom et al., 1970, 1971) and thus may also provide insight on healt h and impact of conditioning by applic ation of a repeated stimulus on the central nervous system. PSD is calculated by utilizing fast Fourier transformation (Lindstrom and Magnusson, 1977) of the raw EMG and results are displayed as power (volt 2 ) vs. frequency (Hz) cont ent of the EMG signal. Median and centroid frequency values of the EI muscles have not been previously reported. Assessment of PSD in conscious rats is essential for unanesthetized comparisons between animals in normal and injured states (Chen et al., 1996) The multiple sources of descending input as well as muscle afferent feedback, phrenic intercostal, intercostal intercostal and abdominal intercostal connectivity all c ontribute to the complex neural control of the intercostal motorneuron system Using integrated EMG and PSD analyses provides insight on the neurophysiology of EI muscles in conscious rats. We hypothesized that conscious rats exposed to ITTO will recruit t he EI muscles with an increased EMG activation. We also hypothesized that repeated ITTO for ten days would potentiate the baseline EMG activity of this muscle in normal conscious rats. Our results demonstrate t hat conscious rats exposed to ITTO respond by recruiting the EI muscle with an increased EMG activation. This response to occlusion was consistent over ten days of ITTO conditioning with little or no ITTO conditioning effect on the baseline EMG activity of EI muscles. In addition f ifty percent

PAGE 44

44 of con scious rats up on presentation with an ITTO. The remaining half i nitially decreased subsequently increased as ITTO was sustained over a few breaths Materials and Methods Animals The Institutional A nimal Care and Use Committee at the University of Florida, Gainesville, reviewed and approved all procedures. A total of 24 adult male Sprague Dawley rats (300 450g) were studied (Harlan Laboratories, Indianapolis, IN). The animals were housed in a 12 hour light/ 12 hour dark cycle with free access to standard rat pellets and water. Study Design This study took place over a period of 16 days. The animals were surgically instrumented at day 0 and allowed one week of recovery from the surgical procedure (desc ribed below). Starting at day 7 (ITTO day 1), animals underwent 20 minute trials of ITTO every day for ten days, ending at day 16 (ITTO day 10). Animals in the no ITTO group underwent the same procedure but did not receive any ITTO presentations. Surgical Protocol Animals (Figure 2 1) were initially anesthetized using isoflurane anesthesia (3 5% in O2) in a whole body chamber. Depth of anesthesia was assessed by an absence of withdrawal to noxious toe web pinch and an absence of corneal blink reflex. Subcut aneous injections of Carprofen (5mg/kg body weight) and Buprenorphine (0.03 mg/kg body weight) were administered pre operatively for management of pain and discomfort. Incision sites were shaved and sterilized with betadine antiseptic solution. Animals wer e placed on a heating pad to maintain body temperature. Anesthesia was

PAGE 45

45 maintained by isoflurane gas delivered through a nose cone. A half inch incision was made on the dorsal skin surface between the scapulae. The animal was then placed in supine a positio n. A one inch midline incision was made on the ventral surface of the neck. After isolating the extra thoracic trachea a saline filled inflatable cuff (Vivo Metric, Fine Science Tools) was placed around the trachea and the ends of the cuff sutured together The actuator tube of the cuff was externalized by routing the tube subcutaneously to the incision between the scapulae Small cutaneous incisions were made bilaterally on the chest wall at level T5 T7. The EI muscles were visualized by blunt dissecti on An area between the parasternal and anterior axillary lines was exposed and bipolar wire electrodes were sutured through the exposed EI muscles (Omnetics Connector Corporation, Minneapolis, MN). The ends of the EMG wires were also routed subcutaneously to the dorsal incision between the scapulae. The incision in the dorsal scapular surface was closed with sutures with the externalized actuator tube and connector head accessible for use during experiments The incisions on the ventral surfaces were sut ured. Rats were administered warm normal saline (0.01 0.02 ml/g body weight), Penicillin (0.1 ml/kg PenG 30,000 units/ ml) and gradually weaned off the isoflurane anesthesia. Animals were allowed to recover in a separate cage over heating pad and returned to the animal care facility once they regained full mobility. Postoperative analgesia was administered once every 24 hours for three days using Carprofen (5mg/kg body weight) and Buprenorphine (0.03 mg/kg body weight). Animals recovered for one week before the experimental protocols were initiated. Experimental Protocol Instrumented animals were randomly divided into two groups: ITTO group (n=16) and no exposure to ITTO control group (n=8). Conscious animals were placed in a

PAGE 46

46 whole body restrainer for the en tire experimental trial duration. The externalized EMG connector was connected to the recording apparatus. The EMG signal s were amplified (P511 series, Grass Instruments Quincy, MA ) and band pass filtered (30 1000 Hz). Analog output s were digitized at 5 k Hz ( Model 1401, Cambridge Electronics Design ), computer processed (Spike2, Cambridge Electronics Design) and stored for subsequent analyses. For the ITTO conditioning group, rats were placed in the restrainer and left undisturbed for 2 minutes of baseline recording. After 2 minutes the ITTO rats received 3 5 seconds of tracheal occlusion (ITTO) via cuff inflation using saline pressure followed by 10 1 5 seconds of unoccluded breathing The 3 5 second ITTO was repeated for a total of 20 minutes. Cuff pressure was recorded with a differential pressure transducer connected to the actuator tube and syring e The cuff pressure was calibrated and monitored to ensure compression and occlusion of the trachea. A total of ~35 40 ITTOs were presented over the 20 minutes After ITTO trials were completed the animals were allowed to recover for 2 minutes post ITTO activity recordings. Control animals were placed in the restrainer, EMG activity recorded and no ITTOs presented throughout the 24 minute trial du ration. At the end of the trial, the animals were removed from the restrain er and returned to their cage. The r estrainer was cleaned wit h alcohol wipes between each trial and between each animal This procedure was repeated every day f or a total of ten days Animals were euthanized after ten days of experimental trials by overdosing with isoflurane gas anesthesia (5% in O 2 ) Data Analyses Integrated EMG a nalysis Raw EI EMG was FIR digital high pass filter ed (300 Hz; Spike2, Cambridge Electronic Design) to minimize the ef fect of heart rate and movement artifacts. DC

PAGE 47

47 remove, rectification and smoothing (50ms) functions were applied to the filtered data. 1, 3, 5, 7 and 10 were analyzed trace was divided into: Before 2 minutes baseline activity prior to ITTO exposure; Onset initial breath response during each ITTO presentation; Phasic all remaining breaths during each ITTO presentation; 2s after 2s time period immediately aft er offset of each ITTO presentation; Between time period in between each ITTO presentations excluding the 2s after period After 2 minutes after completion of ITTO exposure. T hese phases are illustrated in Figure 2 2 Peak amplitude and peak to peak frequency values w ere obtained from Before and A fter and a minimum of 5 10 randomly selected ITTO presentations for Days 1, 3, 5, 7, and 10 for each animal. Data measures greater than () 3 standard deviations were considered as outliers an d eliminated from further analysis (Osborne and Overbay, 2004) Peak ampli tude were normalized to B efore values on : a) Day 1, and b) within each day (1, 3, 5, 7, and 10). Based on the O nset response on D ay 1 animals were divided into two groups, for post hoc analysis : a) High responders those that showed an incre amplitude (n=7), and b) Low responders those that showed a decrease in percentage amplitude and group data for peak to peak frequency were analyzed for statistical significance (SigmaPlot 12.5, Systat Software, In c).

PAGE 48

48 Power spectral a nalysis The Before and A fter period s of the r aw EI EMG trace (defined above) were used in a subset of animals for PSD analysis using fast Fourier transformation (FFT) in Spik m and c were calculated. m, was defined as the frequency at which sum of PSD above is greater than or equal to sum of PSD below (Seven et al., 2013) c was defined as the frequency at which area under curve was half of the total area under the curve (Seven et al., 201 3) m c values were calculated for ITTO group (n=8) and con trol group (n=5) for data from D ays 1 and 10. Statistical a nalysis amplitude and peak to peak frequency were used for statistical analyses. O ne way RMANOVA was used fo r within group analysis across D ays 1, 3, 5, 7, and 10. One way ANOVA was used to statistically compare data between ITTO and control groups. Multiple pair wise comparison procedure using Student Newman Keuls method was used to id entify group differences. PSD Day 1 and Day m c were also analyzed using one way RMANOVA for within group and one way ANOVA for between group analyses. Data were considered significant for All data are presented as Mean standard error of mean (unless noted otherwise). Results Characteristics of A pplied ITTO St imulus and E ffect on A nimal W eight Results presented here are from n=14 for the ITTO group and n=8 for the control group (Ta ble 2 1 and 2 2). Tw o animals from ITTO group with mean amplitude values

PAGE 49

49 greater than ( ) 3 standard deviations were outliers (Osborne and Overbay, 2004) and eliminated from analyses Pre surgical weight (Table 2 1) of the animals was not significantly different between the ITTO (348.4 13 .0 g) and control (381.4 21 .0 g) group s. Both groups showed significant de creases in body weight on ITTO D ay 1: ITTO ( 316 .0 13. 7g, ) and control (355. 4 19. 6 g, ) when compared to pre surgical weight. Animals gained weight over the post surgical days and showed continuous increases in body weight: ITTO D ay3, ITTO (325.8 18 .0 g, ) and control (359.8 17.5 g, ); ITTO D ay 5, ITTO (324.5 15.3g, ) and co ntrol (366.6 16. 4 g, ); ITTO D ay 7, ITTO (333. 3 17 .0 g, ) and control (377. 4 1 4 .0 g, 0. 0 5 ); ITTO Day 10 ITTO (358 .0 11. 4 g ) and control (384. 3 1 3 .0 g). By ITTO D ay 10 all rats had regained their body weight and were not significantly di fferent between groups (Table 2 1) The mean ITTO durations (Table 2 2) were not significantly different across days : ITTO Day 1 (4.7 0.3 s), ITTO Day 3 (4. 4 0.2 s), ITTO Day 5 (4.8 0.2s), ITTO Day 7 (4.8 0. 2 s), ITTO D ay 10 (4.9 0. 2 s ) The mean oc clusion pressures during applied ITTO were not significantly different across days (Table 2 2). ITTO M ediated Respiratory Load C ompensation in of C onscious R ats The responses to ITTO of experimental and control animals on D ay s 1 3, 5, 7 and 10 are presented in Figure 2 3 Before d ata normalized to Da y 1 of ITTO trial presentation resulted in p ercentage changes in Before on Day 3 ( 10.94 17 26 %) Day 5 ( 40.18 26.19 %) Day 7 (39.25 29.87%) and Day 10 (31.05 19.89 %) The

PAGE 50

50 baseline activity of the EI muscles was increased before IT TO presentation began on trial D ay s 5, 7, and 10 compared to Day 1 Onset and Phasic were the two phases corresponding to responses observed during the application of a n ITTO ITTO animals showed a s ignificant load compensation response to applied ITTO on all days. Percentage cha nges in O nset response on the time points were: Day 1 (5.64 12.92 %) Day 3 ( 37.78 54.28 %) Day 5 (58.57 28.11%) Day 7 ( 11.07 2 4 40 %) and Day 10 (1 8. 16 17.66 %) Percentage changes in P hasic response were : Day 1 ( 3.20 10. 59 %) Day 3 ( 21.46 18.85 %) Day 5 ( 17.0 0 2 5.15 %) Day 7 (18.17 21.08%) and Day 10 (13.11 1 4 84 %) The Onset and Phasic response s on all Days were significantly greater than the After response on the same day : Day 1 ( 36.13 9.49 %), Day 3 ( 49.65 14. 00 %), Day 5 ( 31.78 10.48%), Day 7 ( 28 .00 13.3 0 %) and Day 10 ( 35.68 8.59 %) Percentage changes in 2s after response were : Day 1 ( 4 5 6 10.10 %) Day 3 ( 3 9.69 8.4 4%) Day 5 (1 2. 5 9 2 0.64 %) Day 7 (19.38 19.53%) and Day 10 (3.09 1 3 47 %) The 2 s a fter response on each day was significantly greater than the After response on the same day : Day 1 ( 36.13 9.49 %), Day 3 ( 49.65 14. 00 %), Day 5 ( 31.78 10.48%), Day 7 ( 28 .00 13.3 0 %) and Day 10 ( 35.68 8.59 %) The percentage changes in Between response were ; Day 1 ( 2 3.24 8 .48 %) Day 3 ( 45.12 9.2 1%) Day 5 ( 8 0 0 17.83 %) Day 7 (17.85 23.22%) and Day 10 ( 14.02 12.63 %) During this phase, percentage change in EMG amplitude was significantly greater than that during After on a D ays 1, 7 and 10 ; Day 1 ( 36.13 9.49 %), Day 3 ( 49.65 14. 00 %), Day 5 ( 31.78 10.48%), Day 7 ( 28 .00 13.3 0 %) and Day 10 ( 35.68 8.59 %)

PAGE 51

51 Percentage changes in the Aft er response were : Day 1 ( 3 6.13 9 4 9 %) Day 3 ( 49.65 14.00 %) Day 5 ( 31.77 10.48%) Day 7 ( 28.00 13.3 0 %) and Day 10 ( 35.68 8.59 %) Responses during After ; Day 3 ( 49.65 14. 00 %), Day 5 ( 31.78 10.48%), Day 7 ( 28 .00 13.3 0 %) and Day 10 ( 3 5.68 8 .59 %) were significantly smaller than thos e during Before : Day 3 ( 10.94 17.26%), Day 5 (40.18 26.19 %), Day 7 ( 39.25 29.87 %) and Day 10 (31.05 19.89 %) on all Days ( Figure 2 4 presents the data normalized to Before values on the same day, for example, EMG amplitude for Onset response from Day 3 was normalized to EMG am plitude Before values on Day 3 Percentage change s on Day 1 during Onset ( 5.64 1 2.92 %) Phasic ( 3.2 0 10. 59 %) 2s after ( 4.56 10.10 %) and Between ( 2 3 24 8 .49 %) were significantly greater ( ) than After ( 36.13 9.49 %) Percentage changes on Day 3 during Onset ( 14.51 20.18 %) 2s after ( 17 .04 1 4.67%) and Between ( 31. 84 6. 8 3%) were significantly greater ( ) than After ( 42.33 7.46 %). Percentage changes on Day 5 during Onset ( 10.92 19.38 %) Phasic ( 14.9 0 10.51 %) and 2s after ( 1 7 .15 1 1 .7 3 %) were significantly greater ( ) than After ( 5 0 55 4.95 %) Percentage changes on Day 7 during Onset ( 2.58 17.83 %) Phasic ( 11.53 11.17%) 2s after ( 8.01 12.61%) and Between ( 12.67 13.68%) were significantly greater ( than After ( 41.59 6.56%) P ercentage changes on Day 10 during Onset ( 3.25 9.17 %) Phasic ( 5.94 10.64%) 2s after ( 14.99 9.05%) and Between ( 30.85 6.94%) were significantly greater ( ) than After ( 3 9.86 9.09%) The After response s for the control group on : Day 1 ( 21.38 10.30%) Day 3 ( 29. 34 11 09 %) Day 5 ( 44.28 1 0.23 %) Day 7 ( 33.46 20.45%),

PAGE 52

52 Day 10 ( 20.64 1 4.52 %) and for the ITTO group were not significantly different between groups and across days. Peak to P eak F requency The peak to peak EMG frequency during eupneic Before phase of the trial was not significantly different across days and between groups ( Figure 2 6 ) During an ITTO trial, changes in peak to peak EMG frequency were not significant ly different on D ays 1, 5 and 7. On Day 3, 2s after (16 8 .0 18.3 bursts/min) showed a significantly higher ( frequency than After (131.2 16.4 bursts/min ) in the ITTO group. O n Day 10, peak to peak EMG frequency was significantly decreased ( ) during Phasic (119 .0 6. 9 bursts /min) 2s after (136.3 8.9 bursts/min), Betw een (122. 6 5.7 bursts/min ) and After (150.6 17.9 bursts/min ) when compared to Before (163. 6 7. 9 bursts/min). Peak to peak frequency in the control animal group remained stable over Days 1, 3, 5 and 7. There was a significant decrease ( ) during After (144.9 13. 6 bursts/min) compared to Before (182.1 13.5 bursts/min) on Day 10 in the control group. Characteristics of ITTO M ediated uscle Load Compensation R esponse The time required to elicit the Onset response when an ITTO was present ed is termed the latency to O nset response. The number of breaths comprising the Phasic response during each ITTO presentation was measured The la tency to O nset response ( Figure 2 5 B) on Day 1 ( 0. 5 0.03s) Day 3 (0.4 0.04s) Day 5 (0.6 0.04s) Day 7 (0.5 0.05s) and Day 10 (0.5 0.04 s) and number of phasic breaths ( Figure 2 5 A) on Day 1 (6.5 0. 5 ) Day 3 (7. 5 0.5) Day 5 (7.1 0.2) Day 7 (7.5 0. 4 ) and Day 10 (6.4 0.4) w ere not significantly different Therefore, repeated presentatio ns of ITTO did not alter the pattern of the EI muscle load compensation response.

PAGE 53

53 Effect of ITTO C onditioning on E upneic Figure 2 3 presents the percentage changes in EMG across days with data normalized to Before values from Da y 1. The Before value on Day 3 ( 10.94 17.26%) is a decreased percentage change and on Day 5 (40.18 26.2%), Day 7 (39.25 29.87%) and Day 10 (31.05 19.89%) are all increased percentage change s EMG This indicates that EMG was increased (Days 5, 7 and 10 ) as compared to the Before trial phase on Day 1. The After values on Day 1 ( 36.13 9.49%), Day 3 ( 49.65 14.01%), Day 5 ( 31.77 10.48%), Day 7 ( 28.00 13 .3 0%) and Day 10 ( 35.68 8.59 %) we re all decreased percentage change s. Thus the After EMG decreased on D ays 1, 3, 5, 7, and 10 relative to the Before trial phase on Day 1. Normalized Before value on each d ay was significantly greater than After on the same day ( ). Control animals were not exposed to ITTO and therefore, data from correspo nding Before and After are used for analyses. Percentage changes increased for control animals Before response s on Day 3 (117.96 130.29 %), Day 5 (1.65 9.06%), Day 7 ( 11.18 19.14%) and Day 10 (136.24 100.55%). T he percentage changes in the After I EMG responses decreased on Day 1 ( 21.38 10.3 0 %), Day 5 ( 42.04 11. 5 0 %), Day 7 ( 41.6 0 18.28%) but increased on Day 3 (32.47 73.01%) and Day 10 (56.64 103.12%). P ercentage changes in the After EMG amplitude response were not significantly d ifferent compared to Before, across the study days in control animals Variability of EI Muscle EMG R esponses to ITTO in C onscious R ats To assess the variability i n responses to ITTO observed, post hoc analyses were performed ( Figure 2 7 ) Animals were s eparated into High responders ( Figure 2 7A) and Low responders ( Figure 2 7B) based on their Onset responses on Day 1. Seven of

PAGE 54

54 the animals were H igh responders and the remaining six were Low responders on ITTO Day 1. This segregation of animals into High and Low responder groups remained at ~50% throughout the ten days of ITTO trials. In the High responder group on ITTO Day 1, the Onset response (42.85 8.38%) w as significantly greater ( than the After ( 23.01 14.6 0 %) response. T he Onset respons e on ITTO Day 1 for the High responder group (42.85 8.38%) was significantly greater ( ) than the Low responder group ( 37.77 8.36 %) No other significant differences were found between these groups and across time points in this study Power Sp ectral D ensity of efore and A fter Ten days of ITTO C onditioning The power spectral density analyses ( PSD ) of EI muscles were applied to eight ITTO and five Control conscious ra ts The variability in the power of t he signal across its frequency dis tribution is shown in Figure 2 8 This figure illustrates the variability in the activity of EI muscles during eupneic breathing in conscious rats. The m ( Figure 2 9 ) were : ITTO D ay 1 Before (311. 7 35. 2 Hz) and After (319. 5 46.2 Hz); C ontrol Day 1 Bef ore (329.4 54.6Hz) and After (372. 8 55.8Hz); ITTO D ay 10, Before (331.7 3 1.0 Hz) and After (379.3 36.2 Hz); C ontrol Day 10 Before ( 297.8 51.9Hz) and After ( 2 78 3 5 2 5 Hz) The c (Figure 2 9) were: IT TO D ay 1 Before ( 321.8 34.3 Hz) and After ( 3 30.0 45.5 Hz), C ontrol Day 1 Before (3 42. 5 5 4.0 Hz) and After (3 82. 7 54.9 Hz); ITTO Day 10, Before ( 339.9 32.8Hz) and After (374 2 37. 2 Hz); C ontrol Day 10 Before ( 302. 8 47.9 Hz) and After ( 295.6 5 1 6 Hz). There were no significant difference s in m and c between groups and across days

PAGE 55

55 Discussio n The result s from this study demonstrate that conscious rats c onsistently recruit the EI muscles in their load compensation response to ITTO Increased EI activity has been previously demonstrated as a part of the respiratory load compensation response in rabbits (D'Angelo et al., 2010) dogs (De Troyer et al., 1988, De Troyer and Farkas, 1989, 1990, De Troyer, 1991, Brich ant and De Troyer, 1997) and humans (Whitelaw et al., 1975, Altose et al., 1976, De Troyer and Estenne, 1984, Whitelaw et al., 1992) The present study also demonstrated the EI motor pattern of the load compensati on responses in conscious rats. Consistent with our hypothesis, the EI muscles of conscious rats were repeatedly recruited in response to an increased respiratory drive during an applied ITTO It is likely that the EI muscle recruitment acts to increase ve ntilator pump forces by stabilizing the thoracic wall thereby in creasing the mechanical function of diaphragmatic contraction. The net effect of the conscious load compensation is to incr ease respiratory muscle output to attempt to maintain minute ventilat ion despite increased respiratory mechanical load. In the current study, presentation of an ITTO resulted in recruitment and increased EMG activation of the EI muscles on D ays 1, 3, 5, 7, and 10. The percentage change in was consistently and repeatedly increased during an ITTO presentation, as compared to the baseline activity. Thus, repeated presentations of ITTO, both in a single trial and over ten days recruited the EI muscles with an increased amplitude activity with no evidence of habituation The pattern of the responses to an in dividual ITTO latency to Onset response and the peak to EMG frequency during Phasic activity did not change across trial days. This suggests that repeated e xposure to ITTO did not cause neuromuscular potentiation (increased

PAGE 56

56 activity over days) or habituation (decreased activity over days). However, Smith et al using a similar ITTO conditioning paradigm reported over a 30% increase in intercostal muscle cross sectional area (Smith et al., In press) This suggests that ten days of ITTO conditioning may result in increased respiratory muscle contraction force as a result of hypertrophy of the intercostal muscles. Thus, the neuromuscular drive to the EI muscles in creased to compensate for the increased respiratory load but the neural drive for mechanical load compensation response did not need to change by Day 10 due to ITTO EI muscle hypertrophy. The activities of the EI muscles are dependent on their segmental l evel and lung volume The EI muscles of the upper rib cage are reported to be inspiratory in action (Dimarco et al., 1990) at volumes encompassing the vital capacity range. On the other hand, EI muscles of the caudal rib cage are reported to have an expiratory action as lung volume increases (De Troyer et al., 1985) In the present study, the EI mus cle s between thoracic segment s five and seven a transition between cranial and caudal rib cage, were studied. The neurophysiological characteristics of the se EI muscles in conscious rats during eupneic and load compensat ion breathing are likely different fro m anesthetized rats and intermediate between cranial and caudal EI muscles In addition, n o attempts were made to differentiate between inspiratory and expiratory function of the EI muscle activity. Future studies with corresponding diaphragm (Mantilla et al., 2011) and abdominal (Abe et a l., 1996) muscle activity correlated with respiratory volume changes (Walker et al., 1997) are required to determine the breath phase dependent functional c haracteristics of EI muscle load compensation in conscious rats.

PAGE 57

57 The response to ITTO was characterized by an increase in amplitude with no change in the peak to peak frequency across days. However, after ten days of ITTO conditioning, the ITTO animals showed a significant reduction in peak to peak frequency during all phases of the trial when compare d to Day 10 Before values. This was also the case for the control animals in our study. Both the ITTO and control animals were restrained at all times during the trial Restraint is a form of immobilization, which is a stress stimulus and influence s the co ntrol of respiratory pattern (Kinkead et al., 2001b) Therefore, one possible explanation for this res ult could be that after ten days restraint the acute restraint effects at the beginning of trial that caused the animals to breathe with a higher frequency habituated by Day 10 As the trial progressed however, this frequency increase was not maintained a nd no further significant differences were noted. The primary function s of the EI muscle s are to maintain mechanical stability of the chest wall and to aid in the upward and outward movement of the rib cage in concert with contraction of the diaphragm (Feldman, 1986, De Troyer et al., 2005) In the present study, the EI muscles were always recruited with each ITTO presentation. This suggests that in conscious animals the EI muscles are activated to maintain chest w all stability as part of the resp iratory load compensation response However the magnitude of EI muscle activation was variable indicating that activity in response to ITTO may be dependent hase of breath during which ITTO was applied and modulation via afferent feedback. Modulation by C onscious and A ffective S tate Consciousness significantly influences respiratory activity by incorporating voluntary control (Hlastala and Berger, 2001) and integration of different behaviors

PAGE 58

58 (Orem and Netick, 1986) with breathing. Thus, conscious state influences the sensory motor responses to internal and external respiratory stimuli. Sniffing, vocalization and whole body movements affecte d the results from this study with conscious rats In addition, ITTO is an aversive stimulus eliciting escape behavior that may account for the variable responses of EI muscle activity. O bservations during the trial s elicited these behaviors and the corre sponding EI data could not be used for respiratory pattern analyses Hence activity of EI muscles conscious animals is behaviorally unique and dynamically modulated when compared to anesthetized rats From previous studies in our lab, it is known that ITT O conditioning increase s stress, anxiety and associated neural changes in conscious rat s (Pate and Davenport, 2012a) Afferent processing of respiratory stimuli is modality specific (Zechman and Davenport, 1978) and c hanges in affectiv e state m odulate sensory gating and cortical processing of conscious respiratory sensations (Chan et al., 2012) O ur lab has shown that ITTO conditioning in conscious rats causes anxiety and associated changes in gene expression in the thalamus (Bernhardt et al., 2011b) These in turn may influence respiratory act ivity pattern of the EI and other primary and accessory respiratory muscles. The possibility of learned responses to IT TO, however, is also likely but m asked by the current study use of restrained versus freely moving rats The ITTO is a n unexpected, inesc apable stimulus In freely moving rats, ITTO has been suggested to elicit learned helplessness (Pate, 2010) For stimuli such as ITTO, that result in progressive difficulty to breathe, habituation does not occur and respiratory awareness is heightened (von Leupoldt et al., 2011) T he random application of an ITTO and the effect of repeated ITTO on conscious state may have contributed to the variability in the

PAGE 59

59 data in conscious respiratory load compensation responses and may have differentiated the rats into High and Low responders. Breath P hase and L ung V olume Each ITTO presented was of a consistent duration and the cuff occlusion pressure was also maintained rela tively constant within each IT TO trial and across days. No significant differences were observed in these parameters However, we did not selectively apply the ITTO to a breath phase The respiratory rhythm is cyclic in nature (von Euler, 1977) and application of a load during inspiratory or expiratory phases modulates the immediate and subsequent breaths differently (Clark and von Euler, 1972, Zechman et al., 1976) This modulation is dependent on activity of vagal afferents, which in turn is depende nt on lung volume during respiratory loading (Zechman et al., 1976, Davenport et al., 1984) Future studies with corresponding breath phase differentiation will aid in the understanding of modulation of EI muscle activity by lung volume during respiratory loading in conscious rats. Effect of A fferent M odulation on ITTO responses Afferent feedback from intercostal muscle spindles (Critchlow and von Euler, 1963, Remmers, 1970, Holt et al., 2002) tendon organs (Bolser et al., 1 987, Bolser and Remmers, 1989) and joint receptors (De Troyer, 1997) modulate s EI muscle activity. These afferents act segmentally ( Remmers, 1970, Kirkwood, 1988 ) and at the level of the cortex (Davenport et al., 1993) t o drive appropriate EI muscle activity to maintain ventilatory homeostasis. Modulation from reflex pathways between intercostal segments (Downman and Hussain, 1958) and those involving phrenic and intercostal systems (Brichant and De Troyer, 1997, Leduc and de Tr oyer, 2002) further ensure that EI muscle activity is modulated on a breath by breath basis. The results from the present

PAGE 60

60 study do not allow the evaluation of influences from eac h of these afferent mechanisms on these conscious animals. Postural I nfluence s EI muscles have both respiratory (Hudson et al., 2011) and postural (Whitelaw et al., 1992) function s Dick et al demonstrated in cats sleeping in a curled, semi prone posture that EI muscle activity on the upward side was always gre at er than the opp osite downward sid e (Dick et al., 1984) T hese authors also report ed that the activity of the inspiratory intercostal muscle s variably change d from awake to non REM sleep states in supine restrained cats (Dick et al., 1982) This is contrary to reports in healthy adolescent human subjects where intercostal muscle activity increased during non REM sleep in supine position (Tabachnik et al., 1981) .Therefore, although EI muscle activity is modulated by changes in posture, species differences in the pattern and type of modulation also exist s In the present study, the animals were restrained and in the prone position. The effect of this posture on b aseline activity is likely present but could not be specifically differentiated from respiratory activity. Further, the EI muscle activity recorded in the present study was in conscious (non sleeping) rats, which is also different from many previous consci ous animal and human studies. Thus, due to the need for restraint and the conscious state of the animal, influences from postural component of EI muscle activity are likely to contribute to the present outcome measures Effect of Body W tivity A change in body weight and associated changes in muscle fiber mass would (Mantilla et al., 2011) The weight of the animals in our study was significantly decreased one week after

PAGE 61

61 surgical instrumentation proc edure. However, both groups of animals consistently gained weight over the study time period and no significant differences in body weight were observed between groups and the body weights returned to pre surgical levels by the end of our study. Therefore, although the potential effect of changed body weight be due to loss of body weight. However, there may have been an increase in intercostal muscle fiber cross sectional area and related EI motor unit output in these animal s, similar to the hypertrophy effects reported by Smith et al. (Smith et al., In press). A combination of the conscious state, afferent feedback modulation, restraint, posture and EI muscle hypertrophy potentially influe nced the results of the present stu dy. We propose that these factors modulate the brainstem load compensation response not EMG activity in conscious rats To understand the distribution of the variability in the observed responses we divided the ITTO animals into High and Low responders based on their Onset responses to an ITTO presentation. Approximately, 50% percent of conscious rats were H igh responders with an increase in immediately upon presentation with an ITTO Approximately 50% of conscious rats were Low responders with MG at the Onset of the ITTO but subsequently increased as ITTO was sustained This pattern was observed on the Day 1 trial and persisted throughout the ten days of ITTO. The between animal, within species/ strain difference in Onset response is consistent with anxiety measures (Gomes Vde et al., 2013) with high and low anxiety rats within a strain and is usually only observed in conscious studies. The subgroups of High and L ow responders may potentially result from this segregation in

PAGE 62

62 anxiety responses and further increase the inheren t variability of activity (Dick et al., 1982) and load compensation responses in the conscious state Technical C onsiderations Activity from underlying internal intercostal muscles may contribute to the EI EMG recordings, especially during ITTO. We used fine wire electrodes and surgical technique s to minimize contact with internal intercostal muscles, but electrical activity from the internal intercostal muscles can be conducted to the electrodes. P lacement of wire electrodes in the EI muscle was confirmed post mortem and results presented in this stu dy are from successful EI muscle implants only. In our initial experiments, many animals damaged externalized ele ctrode wires by chewing on them. We subsequently used a solid connector (Omnetics Connector Corporation, Minneapolis, MN) which decreased the loss of electrodes due to damage caused by animal. This enabled succe ssful chronic longitudinal studies using the same EMG electrodes implanted in the same location with no replacement of the electrodes required It is also important to consider movement o f electrodes over time as a possible change in recording site (Mantilla et al., 2011) By suturing the electrode wires in place we minimized the possibility of differences in recordings across days due to electrode displacement. Also, normalized EMG data analyses allow for statistica l comparisons between animals an d within the same animal over days We normalized the data to two different tim e points: ITTO Day 1 (Figure 2 3) and to each day (Figure 2 4) to decrease between and within animal variability Post mortem macroscopic analysi s of EI muscle surrounding electrode placement site confirmed the stability of electrode implant sites and an absence of fibrosis

PAGE 63

63 An importa nt result from this study is the median and centroid frequency values from the PSD analyses of the EI muscle EMG ac t ivity during conscious eupneic breathing. Currently, t here are no published reports for these parameters for EI muscles in conscious rats. PSD has been utilized to investigate motor unit recruitment (Seven et al., 2013) shifts in frequency content of an EMG signal (Mannion and Dolan, 1994, Spahija et al., 2005) evaluation of muscle fatigue (Lindstrom et al., 1970, Beck et al., 1997) and the conduction velocity of nerve fibers innervating the muscle influence s the PSD (Lindstrom et al., 1970, 1971) The results of this study show no effect of ITTO on the PSD m and c within a trial and across days. This suggests the EI motor recruitment remains the same during unloaded breathing an d does not show plasticity with ITTO conditioning In summary, we have determined the respiratory load compensation responses of the EI muscles in response to ITTO in conscious rat s. Activity of the EI muscles is not only dependent on descending drive (Feldman, 1986, De Troyer et al., 2005) but is also influenced by lung volume (De Troyer et al., 1985) breath phase (Zechman et al., 1976) afferent modulation (Shannon, 1977) and conscious state (Dick et al., 1982, Orem and Netick, 1986) The present investigation focused on the intercostal spaces loc ated between thoracic levels five and seven These EI muscles are intermediate between the cranial and c audal thoracic levels and have different load compensation responses in the anesthetized state The conscious rats had a respiratory load compensation response characterized by increased EI muscle activity on presentation of ITTO. There was significant variability in the load compensation response that is likely due to conscious state and responder subgroups within the same strain of rats

PAGE 64

64 used in this study. Further studies are required to understand the load compensation response of the EI muscles at all the different thoracic levels and their integrated, functional relationship with other respiratory muscles that are critical for sustaining ventilation during increased respiratory mechanical load The results from the present study demonstrate the importance of EI muscles in eupneic breathing as well as during respiratory load compensation in conscious rats.

PAGE 65

65 Figur e 2 1 Schematic representation of the surgical preparation EMG electrodes were implanted in the EI muscles at the T5 T7 level of the rib cage, between the parasternal and anterior axillary lines. The tracheal occluder was secured carefully ar ound the ext ra thoracic trachea. Figure 2 2 Occlusion pressure during phases of ITTO trial The trace is the internal tracheal cuff pressure. A minimum of 200 mmHg cuff pressure is required to fully occlude the tracheal lumen. The phases of ITTO trial are defin ed on the pressure trace.

PAGE 66

66 Table 2 1 Body weight of the animal groups Body weight ( grams ) for both ITTO and control groups, on study Days 1, 3, 5, 7 and 10 are mean standard error The indica tes p<0.05 compared to pre p for body weights compared to ITTO D ay 10. Time point Pre surgery ITTO Day 1 ITTO Day 3 ITTO Day 5 ITTO Day 7 ITTO Day 10 ITTO 348.4 13.0 316.013.7 333.3 17 .0 358.0 11.4 Control 381.4 21.0 355.4 19.6 377.4 14.0 384.3 13.0 Table 2 2. Pressure and duration of ITTO presentations ITTO tracheal cuff pressure (mmHg) and ITTO duration (seconds) for the trial days are mean standard error. There were n o significant differences across days. ITTO Day 1 ITTO Day 3 ITTO Day 5 ITTO Day 7 ITTO Day 10 Pressure 954.2 11.6 949.5 15.8 950.9 12.4 958.2 12.2 943.4 29.5 Duration 4.7 0.3 4.4 0.2 4. 8 0.2 4.8 0. 2 4.9 0.2

PAGE 67

67 Fig ure 2 3 Percent c hange EMG amplitude normalized to Day1 Before for each phase of the ITTO trial. The compared to A fter values of the same day. No significant differences were found in the control group within day s

PAGE 68

68 Fig ure 2 4 Percent c h EMG amplitude normalized to the same day Before Each phase of the ITTO trial for the ITTO group and only the After phase for the control g roup on D ays 1, 3, 5, 7 and 10, were normalized to same day B efore significance w hen compared to A fter of the same day. No significant differences were found in the control group across all day s

PAGE 69

69 Figure 2 5 EI EMG r esponse pattern to ITTO Panel A is the number of Phasic breaths and Panel B is the latency to Onset of EI EMG respon se with application of an ITTO The responses w ere measured on D ays 1, 3, 5, 7, and 10. There were no significant differences b efore, during and after ten days of ITTO conditioning

PAGE 70

70 Figure 2 6 Peak to peak frequency on D ays 1, 3, 5, 7, and 10. In ITTO animals on Day 3, peak to peak frequency during 2s after was significantly greater than After. In IT TO animals on Day 10, peak to peak frequency during Phasic, 2s after, Between and After significantly decreased when compared to Before, in the experimental group. For control animals on Day 10, peak to peak frequency After was

PAGE 71

71 Fig ure 2 7 ormalized to ITTO day 1 Before values. Panel A, High responders: animals had during Onset on ITTO Day 1 and Panel B, Low responders: animals had itude (%) during Onset on ITTO D ay 1. The indi cates p 0.05 for O ns et compared to After on ITTO D ay 1, and # indicates p 0.001 for O nset response in H igh vs. Low responders on ITTO D ay 1.

PAGE 72

72 Figure 2 8 Power spectral density analysis of eupneic EI muscle EMG. ITTO and control group data on (A) Da y 1 and (B) Day 10.

PAGE 73

73 Figure 2 9 Frequency characteristics of power spectral data of EI muscle EMG during eupneic breathing. Panel A, m and Panel B centroid frequency c There were no significant differences in m and c a cross days and between time points

PAGE 74

74 CHAPTER 3 RESPIRATORY LOAD COMPENSATION OF E XTERNAL I NTERCOSTAL MUSCLES IN CONSCIOUS RATS WITH UNILATERAL c SCI Injury to the cervical spinal cord (c SCI) produces r espiratory neuro plasticity at the spinal cord level (Johnson and Creighton, 2005) which may be spontaneous (Goshgarian, 2003, Zimmer et al., 2007) or induced by a variety of different chemical and mechani cal perturbations to the system (Mitchell and Johnson, 2003) Spontaneous improvement in diaphragm function together with improvem ents in shoulder and upper arm muscles have been reported in humans with c SCI (Axen et al., 1985) E ndogenous neuroplasticity leads to slight recovery in ipsilateral diaphragm function in the rat, and has been attributed to the spontaneous crossed phrenic phenomenon (sCPP) (Goshgarian, 2003, Fuller et al., 2008, Zimmer et al., 2008, Lane et al., 2009, Sandhu et al., 2009) However, compensation from other respiratory muscles, such as the intercostal muscle s, makes the contribution of sCPP to respiratory recovery effectively negligible (Dougherty et al., 2012b) Dougherty et al have proposed a crossed intercostal circuit that enables spontaneous recovery of the intercostal muscle activity ipsilateral to c SCI (Dougherty et al., 2012a) Investigators studying the anatomy (de Almeida et al., 2010) and neurophysiology (de Almeida et al., 2010, de Almeida and Kirkwood, 2010, 2013) of the intercostal motor system in the rat and intercostal muscle afferent control in other species (Remmers, 1970, Shannon, 1977, Kirkwood et al ., 1982b, Kirkwood et al., 1984) have demonstrated the basis for a crossed intercostal neuronal circuit. The descending respiratory drive to the rat thoracic spin al cord is complex with many levels of neural control. Anatomical connectivity of the rat ros tral ventral respiratory group (VRG) is similar to that of the cat showing dense reciprocal connections with the Klliker

PAGE 75

75 Fuse nucleus, medial and lateral parabrachial nucleus and prominent projections from the nucleus tractus solitarius (NTS) (Ellenberger and Feldman, 1990) The presence of the dorsal respiratory group (DRG) has been confi rmed in the rat (de Castro et al., 1994) and its connections between VRG neurons via axon collaterals have been reported (Lipski et al., 1994) However, in contrast to the cat, very few direct connections between the DRG and t he t horacic spinal cord hav e been reported (Duffin and Lipski, 1987) Labeled projections to the thoracic spinal cord from the VRG (Lipski et al., 1994) upper cervical inspiratory neurons (Lipski et al., 1993) and phrenic motorneuron s (Lipski et al., 1994) have been previously identified However, there is an absence of direct monosynaptic connections from the VRG (Tian and Duffin, 1996b) upper cervical inspiratory neurons (Tian and Duffin, 1996a) and Btzinger complex (Kanjhan et al., 1995) to the thoracic spinal cord motorneurons based on cross correlation s tudies in rats. Results from several studies in rats suggest that projections from the ipsilateral VRG contralateral DRG and contralateral VRG terminate in upper cervical (C1 C2), phrenic (C3 C6) and lower cervical (C7 C8) spinal cord motorneuron s (Tian and Duffin, 1996b, a) I nterneurons at these levels then modulate thoracic motorneurons, playing a major role in inter segmental control of intercostal and abdominal muscle activity (Kirkwood et al., 1982a, Kirkwood et al., 1982b, Tian and Duffin, 1996b, Saywell et al., 2011, de Almeida and Kirkwood, 2013) Cross correlation studies have shown the connections between the upper cervical inspiratory neurons (Tian and Duffin, 1996a) inspiratory bulbospinal neurons of the VRG (Tian and Duffin, 1996b) and the intercostal motorneurons are via interneurons. Therefore these interneuronal connections between

PAGE 76

76 the phrenic and thoracic motorneuron pool s relay both descending medullary dri ve and spinal reflex pathways. Interneuron connections are thought to cross above the first thoracic segment and play a n import ant role in the activation and modulation of diaphragm and thoracic muscle activity (Dimarco and Kowalski, 2013a, b, Kowalski et al., 2013) Thus, the descending respiratory drive to the thoracic spinal cord is a n integrated complex combination of direct and indirect inputs from the VRG (Lipski et al., 1994, Kanjhan et al., 1995, de Almeida and Kirkwood, 2013) DRG (de Castro et al., 1994) cervical spinal cord (Lipski et al., 1993, Tian and Duffin, 1996b, a) and cortical motor tracts (Rikard Bell et al., 1985a, b, Saji and Miura, 1990) This interconnectivity is the basis for coordinated and rhythmic drive from the central respira tory pattern generator to the intercostal muscles along with the diaphragm and hypoglossal muscles in rats (Rice et al., 2011) and is functionally similar to act ivation of bilateral diaphragm muscle in healthy awake humans (Bruce and Goldman, 1983) and between the thoracic intercostal motorn eurons at different spinal levels of anesthetized cats (Kirkwood et al., 1982a, Kirkwood et al., 1982b) Injury to the cervical spinal cord will impair intercostal muscle functioning by disrupting the se rhythmic a nd synchronized inputs (Doug herty et al., 2012a) However, results from anesthetized preparation s are similar to respiratory activity in the sleep state (Pagliardini et al., 2012) where intercostal muscle contribution to breathing is conside rably reduced (Stradling et al., 1985) It is well know n that c onscious and affective states not only influence respiratory motor activity but also are required for the postural function and integration of voluntary behaviors associated with respiratory activity (Orem and Netic k, 1986, Hlastala and Berger, 2001) EI muscle activity has

PAGE 77

77 been studied in slee ping cats (Dick et al., 1982, Dick et al., 1984, Fraigne and Orem, 2011) but conscious cat and rat studies are lacking especially af ter c SCI The external intercostal (EI) muscles are primary inspiratory muscles (Taylor, 1960) active during eupnea (Hudson et al., 2011 Jaiswal et al., 2012) respond to respiratory load s (Jaiswal and Davenport, 2012) and also have postural functions (De Troyer et al., 2005) EI muscles display a gradient of activity (Feldman, 1986) that is thought to be organized at the spinal cord level (Hudson et al., 2011) These muscles are modulated by a fferent feedback from their muscle spindles (Critchlow and von Euler, 1963, Remmers, 1970, Holt et al., 2002) tendon organs (Bolser et al., 1987, Bolser and Remmers, 1989) and joint receptors (De Troyer, 1997) A fferent feedback activitie s act segmentally at the spinal cord level and on medullary respiratory neurons to modulate respi ratory central neural activity (Bolser et al., 1987, Bolser and Remmers, 1989) A t the level of the cortex (Davenport et al., 1993) these intercostal muscle afferents may modulate behavioral control of intercostal muscle activity. Reflex pathways between intercostal thoracic spinal segments (Downman and Hussain, 1958) and phrenic and intercostal systems (Brichant and De Troyer, 1997, Leduc and de Troyer, 2002) have also been identified. Thus intercostal muscle activity is modulated via afferent feedback on a breath by breath basis. This modulation may be especially imp ortant after c SCI, where respiratory muscle function is greatly compromised (Fuller et al., 2008, Dougherty et al., 2012a) to maintain breath by breath respiratory homeostasis by shaping the respiratory pattern r equired for co ordination of intercostal, diaphragm and abdominal muscle activities

PAGE 78

78 In the present study, w e have utilized cervical hemisection (C2H S) as an animal model of unilateral c SCI (Golder et al., 2003, F uller et al., 2008, Sandhu et al., 2009, Dougherty et al., 2012a) and respiratory load compensation elicited by intrinsic transi ent tracheal occlusion (ITTO) to determine th e role of EI muscles in respiratory motor control of conscious rats (Jaiswal and Davenport, 2012, Jaiswal et al., 2012, Jaiswal et al., 2013) In the present study we hypothesized that unilateral c SCI will decrease bilateral EI EMG amplitude activity during eupneic breathing and ITTO respirato ry load compensation activities in conscious rats. Additionally, we investigated the effect of ten days of ITTO conditioning on EI muscles pattern in conscious rats with unilateral c SCI. The ITTO was hypothesized to promote neuroplasticity in the EI muscl es after ten days of ITTO conditioning and increase the respiratory load compensation response. C2HS animals not exposed to ITTO were used to control for spontaneous neuroplasticity of the intercostal mo tor system in the conscious rats Materials and Meth ods Animals The Institutional Animal Care and Use Committee at the University of Florida, Gainesville, FL, reviewed and approved all procedures. A total of 21 adult male Sprague Dawley rats ( 300 350g) were used (Harlan Laboratories, Indianapolis, IN). The animals were housed in a 12 hour light/ 12 hour dark cycle with free access to standard rat pellets and water. Study Design This study was conducted over a total period of 23 days. The animals were surgically instrumented with EMG wire electrodes and trac heal occluder under aseptic conditions (as described below) on Day 7. They were allowed one week of recovery

PAGE 79

79 from this surgery. On Day 1, animals were randomized into ITTO (n=10) and no ITTO groups (n=11) and pre injury group data were collected. On Day 0, animals were randomized into C2HS (n=13) and Sham (n=8). Animals in the C2HS group received a complete left C2 hemisection and Sham animals were subjected to a laminectomy at the C2 level as described below. Animals were further randomized into four gr oups: C2HS+ITTO (n=8), C2HS+no ITTO (n=5), Sham+ITTO (n=4) and Sham+no ITTO (n=4). Animals were allowed one week of recovery from spinal cord injury surgery. Starting at day 7 (ITTO day 1), C2HS+ITTO and Sham+ITTO animals underwent 20 minute trials of ITTO every day for ten days, ending at day 16 (ITTO day 10). C2HS+no ITTO and Sham+no ITTO animals underwent the same procedure but did not receive any ITTO presentations Surgical I nstrumentation The surgical instrumentation method for EMG and tracheal cuff implantation are described in C hapter 2. Briefly, s urgical instrumentation consisted of implantation of tracheal occluder cuff around the extra thoracic trachea and bilateral EI muscle EMG wire electrodes implanted at level T5 T7 (Figure 2 1). Animals (n=2 1) were anesthetized using isoflurane anesthesia (3 5% in O2) in a whole body chamber. Subcutaneous injections of Carprofen (5mg/kg body weight) and Buprenorphine (0.03 mg/kg body weight) were administered pre operatively for management of initial pain and discomfort. Anesthesia was maintained by isoflurane gas through a nose cone. A one inch midline incision was made on the ventral surface of the neck. After isolating the extra thoracic trachea a saline filled inflatable cuff (Vivo Metric, Fine Science Too ls) was inserted and the ends were sutured together, around the trachea. Small incisions were

PAGE 80

80 made bilaterally on the chest wall at level T5 T7. The EI muscles were visualized by blunt dissection of the superficial pectoral and scalene muscles. An area bet ween the parasternal and anterior axillary lines was exposed and bipolar wire electrodes were sutured through the exposed EI muscles (Omnetics Connector Corporation, Minneapolis, MN). The actuator tube of the cuff and i mplanted wires were routed subcutaneo usly to the dorsal surface between the scapulae. A ll incisions were sutured and r ats were administered warm normal saline (0.01 0.02 ml/g body weight), Penicillin (0.1 ml/kg PenG 30,000 units/ ml) and gradually weaned off the isoflurane anesthesia. Postope rative analgesia was administered every 24 hours for three days using Carprofen (5mg/kg body weight) and Buprenorphine (0.03 mg/kg body weight). Animals were allowed to recover for one week before undergoing C2HS/ Sham surgery, as described below. Cervical Hemisection Surgery (C2HS) One week after the tracheal cuff and EMG electrode instrumentation procedure, animals were randomized into C2HS (n=13) or Sham (n=8) group. C2HS surgical procedures are consistent with prior reports from Fuller et al ( Fuller et al., 2008, Sandhu et al., 2009 Dougherty et al., 2012a) Anim als were anesthetized using Ketamine ( 120 mg/kg intra peritoneal, FortDodge Animal Health, IA, USA ) and Xylazine ( 10 mg/kg sub cutaneous ) and prepared for surgery. A satisfactory anesthetic level was verified by absence o f corneal blink and withdrawal to noxious toe pinch O phthalmic ointment was applied to prevent corneal drying. The incision sites, head & neck, were shaved and sterilized using alterna ting alcohol and betadine wipes (3x). The animal was placed on a heating pad and covered with sterile drape and body temperature was maintained at 37.5 o C A longitudinal incision measuring approximately 1.5 inches was made in the

PAGE 81

81 dorsal neck midline with a scalpel blade starting just rostral to the base of the skull. The overlying muscles were separated by blunt dissection. A left laminectomy was performed at the C2 vertebral level, removing the entire rostral caudal extent of the vertebra. The dura was cut (~2mm) on left dorsal side using a tapered No. 11 scalpel blade. The spinal lesion was initiated using a micro scalpel to cut into the spinal cord roughly mincing the tissue. Gentle aspiration was used to make a hemisection cavity (~1mm). For animals unde rgoing Sham surgery no lesions of the spinal cord were made after cutting the dura. The area above the cut dura was covered with dura film. The separated overlying muscle s were sutured using absorbable suture (PDS 4 0) and the skin closed using wound clips Animals were given subcutaneous injections of Lactated Ringers (5 ml) to prevent dehydration and yohimbine (1.2 mg/kg) to reverse the action of Xylazine. Also, Carprofen (5mg/kg) and Buprenorphine (0.03 mg/kg) were administered to provide analgesic suppo rt both post operatively and as post surgical care for a total of 3 days Post surgical care included administration of the analgesic support, Pen icillin (PenG 30,000 units/ml; 0.1 ml/kg dose), lactated Ringers solution (5 ml/day, subcutaneously) and oral Nutri cal supplements (1 3 ml, Webster Veterinary, MA, USA), as needed, until animals returned to normal eating and drinking behavior Animals recovered for one week before any experimental protocols were initiated. Data Collection Data were collected as p er experimental protocol on Day 1, i.e. on e day prior to undergoing C2HS/ Sham surgery. Animals were randomized into ITTO (n=10) and no ITTO (n=11) groups for this pre injury data point. On day 7, one week after recovery from C2HS/ Sham surgery, animals we re randomized into C2HS+ITTO (n=8), C2HS+no ITTO (n=5), Sham+ITTO (n=4) and

PAGE 82

82 Sham+no ITTO (n=4). Experimental protocol (described below) was followed every day from Day 7 (ITTO Day 1) to Day 16 (ITTO Day 10). Experimental Protocol Instrumented animals were randomly divided into four groups Conscious animals were placed in a whole body restrainer. The externalized EMG connector was connected to the recording apparatus. The signal was amplified (P511 series, Grass Instruments) and band pass filtered (30 1000 Hz). Analog outputs were digitized at 5 kH z (Model 1401, Cambridge Electronics Design ), computer processed (Spike2, Cambridge Electronics Design) and stored for subsequent analyses. For the ITTO conditioning group, rats were placed in the restrainer and le ft undisturbed for 2 minutes of baseline recording. After two minutes the rats received 3 5 seconds of ITTO via cuff inflation followed by 10 15 seconds of unoccluded breaths, ITTOs were repeated for a total of 20 minutes. The cuff pressure was calibrated to ensure complete compression of the implanted occluder cuff and therefore, complete occlus ion of the trachea. A total of about 35 40 ITTOs were presented over 20 minutes. T he animals were subsequently allowed to recover for another 2 minutes to obtain p ost ITTO recordings. Control animal s were placed in the restrainer; EMG activity recorded and no ITTOs were presented throughout the 24 minute trial duration. At the end of the trials animals were removed from the restrain er and returned to their cage. The r estrainer was cleaned with alcohol wipes between animals and trials This procedure was repeated every day for a total of ten days. Cervical Spinal Cord Histology After ten days of ITTO tri als, the animals were subjected to 8 % CO 2 challenge (data presen ted in Chapter 4). Animals were euthanized by overdosing with isoflurane

PAGE 83

83 gas anesthesia (5% in O 2 ) and intra cardiac perfusion using 4% paraformaldehyde to fix brain and spinal cord tissue, which was then stored at 4 o C. The fixed spinal cord tissue was u sed for histological v erification of the C2HS Tissue sections were mounted on glass slides (Fisher Scientific, Pittsburgh, PA, USA), stained with Cresyl violet and evaluated by light microscopy. The complete absence of white and gray matter in the ipsilat eral C2 spinal cord indicated an anatomically complete C2HS (Golder et al., 2001b, Fuller et al., 2006) Data Analyses Integrated EMG a nalysis Data from p re injury ITTO Day 1 and Day 10 time points were analyzed Two animals were euthanized as they reac hed pre set end point weight loss criteria on ITTO Day 1. Therefore, for ITTO Day 10 data were unavailable for analyses from one animal each in C2HS+ITTO and C2HS+no ITTO groups Raw EI EMG was FIR digital high pass filter (300 Hz; Spike2, Cam bridge Electronic Design) to minimize influence of heart rate and movement artifacts. DC remove, rectification and smoothing (50ms) functions ce was divided into: Before 2 minutes of baseline activity prior to ITTO exposure; IT TO response during entire duration of each ITTO presentation; Recovery time period immediately after each and between two consecutive ITTO presentation s ; After 2 minutes of baseline activity after completion of ITTO exposure s From each animal peak amplitude and peak to peak frequency were obtained for B efore and A fter as well as a minimum of 5 10 randomly

PAGE 84

84 selected ITTO presentations Peak amplitude d ata were normalized to Before amplitu de was calculated. Statistical a nalysis Peak to pea min) and normalized percentage (SigmaPlot 12.5, Systat Software, In c). Ipsilateral and contralateral sides were analyzed separately to determine effect of ITTO conditioning after c SCI in conscious rats. One way RMANOVA was used for within group analysis for pre injury, ITTO D ays 1 and 10. One way ANOVA was used to statistically compare ITTO and control groups on a given day Multiple pair wise comparison procedure using Student Newman Keuls method was used to identify group differences. Data were data are presented a s m ean standard error of mean Results Histological Verification of C omplete C2HS Transverse sections (40 m) of fixed spinal cord tissue stained with Cresyl violet an d evaluated by light microsc opy demonstrated complete absence of white and gray m atter on the left side indicating a complete C2HS ( Figure 3 7). Completeness of C2HS was confirmed for all animals and they were included in data reported below Animal W eight and Characteristics of A ppl ied ITTO Body weights of all grou ps in this study are listed in T able 3 1. On the surgical i nstrumentation time point Sham+ no ITTO group had significantly lower body weight compared to other all treatment groups. All animal s regardless of experimental grou p lost significant weight post surgic al instrumentation at the C2HS/ Sham surgery time point, Sham+no ITTO was the group with lowest mean body weight compared to other

PAGE 85

85 treatment groups. All treatment group s lost additional weight after C2HS/S ham surgery S ham+no ITTO group showed a slight increase in mean body weight (Table 3 1) Animals in all treatment groups gained weight between ITTO Days 1 and 10. The ITTO duration is listed in T able 3 2. Occlusion cuff pressure of the applied ITTOs is listed in T able 3 3. No significan t differences were found in thes e parameters within and between groups at pre injury, ITTO Day 1 and Day 10 time points. Pre injury C haracteristics of the EI EMG R esponse s to ITTO in Conscious R ats Numbers of animals used and phases us ed for data normalization are described in Table 3 4. Responses to ITTO at the pre injury time point are presented in F igure 3 1. observe d during ITTO (left 54.141 7.13%; right 52.3831.79 %) and on left side d uring Recovery (left 8.4713.42 %; right 0.2312.09 %) compare d to After (left 29.5511.03%, 15.0323.98 %) in the ITTO group. Peak to group decreased significantly during A fter (10 0 6 4.9 bursts/ min) compared to Be fore (15 3.315. 1 bursts/ min ) Peak to frequency in the ITTO group decreased significantly during ITTO (117.47.4 bursts/min) compared to Before (15 3.3 15.1 The After response in the ITTO group (100.6 4.9 bursts/min ) was significantly lower than the After respo nse in the no ITTO group (149. 8 12.6 bursts/min No statistical differences were found in the control group perce ntage change in peak to peak frequency within group

PAGE 86

86 E upneic EI EMG M uscle Activity in Conscious Rats After U nilateral c SCI Numbers of animals used for analyses and data normalization to pre injury time point are descr ibed in Table 3 5 To determine the effect of unilateral c to peak frequenc 3 ) and Sham (n=8) an imals on ITTO Day 1 were pooled and normalized to pre injury Before values On ITTO Day 10, C2HS+ITTO (n= 7), C2HS+no ITTO (n=4 ), Sham+ITTO (n=4) and Sham+no ITTO (n=4) were used for comparisons Data from two animals was not available as they were euthanized when they reached study end point criteria In the C2HS group, percentage change in values during eupnea decreased bilaterally on ITTO Day 1 ipsilateral side ( 51.5411.06 %, the contralateral side ( 39.4 1 14.06 %) when compa red to pre injury (ipsilateral 17.657.96 % and contralateral 12.3513.52 %), within C2HS group ( Figure s 3 2 and 3 3). Percentage ased in C2HS+ITTO ( 53.5614.97 %, ared to Sham+ITTO ( 140.1767.24 %) on ITTO Day 10. No other statistical differences were found. Ipsilateral peak to different across study time points, within either C2HS or Sham groups. Contralateral peak to I EMG frequency during eupnea was not statistically different across study time points, within either C2HS or Sham groups. Contralateral peak to EMG frequency during eupnea on ITTO Day 10 in the C2HS+ITTO ( 183.7 1 6.0 bursts/ min, icantly greater than Sham+ITTO (120. 718.7 bursts/ min) group.

PAGE 87

87 contribution to eupnea is significantly decreased in conscious rats one week after unilateral c SCI. Respiratory Load Compensation of the EI M uscles in Conscious Rats One W eek A fter c SCI Table 3 6 describes the numbers of animals in each group and the phases of ITTO trial used for data normalization. Figure 3 5 presents the responses of conscious rats to ITTO one week a fter unilateral c SCI. Although we observed a bilateral decrease SCI, these muscles were recruited in response to applied ITTO with increased activation. However, n o significant differences were observed in ITTO responses bet ween and within groups on the ipsilateral side Ipsilateral peak to uring ITTO (117.87. 9 bursts/ ) and After (123. 59 .0 bursts/ significantly decreased compared to Before (172. 3 15.2 bursts/ min) within group. Ipsilateral peak to 8.0 1 1.6 bursts/ min) in the C2HS+ITTO group was significantly higher than in the Sham ITTO group (102. 6 11.6 bursts/ On the contralateral side, ITTO res ponse ( 85.52 51.52 %) in the C2HS+ITTO group was significantly greater than Recovery ( 3.9 7 1 6 .9 8 %, 2 9 87 1 4 4 0 %, ( 3.9 7 16.98 % ) in the C2HS+ITTO group was sig nificantly greater than After ( 29.8714 .4 0 % within group.

PAGE 88

88 Contralateral p eak to ITTO, Recovery (143. 2 7.4 2 4 bursts/ min) and After (13 3.06.9 bursts/ min) were significantly decreased compared to Before (191.215.7 bursts/min, Effect of ITTO Conditioning on the Respiratory Load Compensation Responses of EI M uscles in Conscious R ats A fter c SCI Table 3 6 describes the numbers of animals in each group and the phases of ITTO trial used for data normalization. Effect of ITTO condi tioning on peak amplitude and peak to peak frequency are presented in F igure 3 6. Ipsilateral ITTO response ( 18.79 16.9 7 %) in the C2HS+ITTO group was signifi cantly greater than Recover y ( 7.2810.76 %, 13.1210.93 %, 1) within group. ITTO response ( 32.386.84 %) in the Sham+ITTO group was significantly greater than Recovery ( 50.074.6 0 %, and After ( 55.449.47 %, response in the C2HS+ITTO ( 13.1210.93 %) C2HS+no ITTO ( 29.7813.37 %) and Sham+ITTO ( 55.45 9.47 %) groups was significantly decreased compared to Sham+no ITTO (50.3220.8 3 group. Ipsilateral Peak to during ITTO (105.59.3 bursts/ min) was significantly dec reased compared to Before ( 14 7.0 23.4 bursts/ min, Contralateral ITTO response (40.4229.29 %) in the C2HS+ITTO group was sig nificantly greater than After ( 22.1013 .49 %, statistical differences were foun d. Contralateral p eak to e C2HS+ITTO group d uring ITTO (116.213.4 bursts/ Recovery (15 4.0 13. 6 bursts/ and After (138. 1 10 6 bursts/ min,

PAGE 89

89 Before (183. 7 16.0 bursts/ min) within group. Also, in the C2HS+ITTO group during ITTO (116.213.4 bursts/ min) contralateral peak to were signific antly decreased than Recovery (15 4.013.6 bursts/ min, Discussion Effect of Unilateral c S CI on Eupneic EI EMG Activity in Conscious Rats The results of this study demonstrate EI muscles and their contribution to eupnea is significantly decreased one week after unilateral c SCI. The EI muscles are inn ervated by the thoracic spinal cord which receives a n integrated complex combination of direct and indirect inputs from the VRG (Lipski et al., 1994, Kanjhan et al., 1995, de Almeida and Kirkwood, 2013) DRG (de Castro et al., 1994) cervical spinal cor d (Lipski et al., 1993, Tian and Duffin, 1996b, a) and cortical motor tracts (Rikard Bell et al., 1985a, b, Saji and Miura, 1990) Injury to the spinal cord above the thora cic level, such as in c SCI disrupts this descending drive. Previous studies have reported a loss of intercostal muscle eupneic activity and its recovery to non injured levels two week s after c SCI in anesthetized rats (Dougherty et al., 2012a) Our results demonstrate the activity of EI muscles during eupnea and respiratory load compensation one week after c SCI in conscious rats. Therefore, these results indicate that EI muscles are active during eupnea as early as one week after c SCI in the conscious state (Pagliardini et al., 2012) No significant differences were found in peak to points analyzed. However, C2HS+ITTO group had a significantly higher contralateral peak to

PAGE 90

90 EI EMG Responses to ITTO in Conscious Rats with Unilateral c SCI One week after unilateral c SCI the bilateral EI muscles are recruited in response to an ITTO with significant increases in peak amplitude activity on the contralateral side. The descending respirat ory drive to the thoracic spinal cord is from various cortical structures (Rikard Bell et al., 1985a) medullary respiratory neurons (Tian and Duffin, 1996b) and from upper cervical inspiratory neurons (Tian and Duffin, 1996a) C2HS disrupts this descending drive and results in a significant decrease of ipsilateral EI EMG amplitude both during eupnea and respiratory loading, as observed in the present study. The exact path of the direct descending inputs from cortex and medullary respiratory group s, including the leve l at which they cross is not known. However, from our results it can be speculated that these descending inputs cross below the C2 level and may be the drive responsible for presence of ipsilateral EI muscle activity observed after c SCI. Application of I TTO may mediate increases in the level of this descending drive thereby causing bilateral increase s in EI EMG activity, which was significantly greater on the contralateral side one week after c SCI. Breath by breath modulation of EI muscle activity is via EI muscle afferent s and co ordination between phrenic (Decima et al., 1969) intercostal (Kirkwood et al., 1984) and abdominal (Saywell et al., 2011) motorneuron pools. Feedback from the muscle spindles (Shannon and Zechman, 1972, Shannon, 1977) tendon organs (Bolser et al., 1987) and joint receptors (De Troyer, 1997) contribute to control of EI muscle EMG activity Afferents from the intercostal muscles also terminate in the sensory motor cortex (Davenport et al., 1993) and may mediate increases in descending respiratory drive to the intercostal muscles Th e activation of at least one, and more likely a

PAGE 91

91 combination of these afferent populations likely contributes to eliciting EI muscle respiratory load compensation in the c SCI injured rats. A major proportion of the respiratory drive to the thoracic spinal cord is via descending projections from ipsilateral VRG contralateral DRG and contralateral VRG that terminate in the upper cervical (C1 C2), phrenic (C3 C6) and lower cervical (C7 C8) spinal cord motorneurons (Ti an and Duffin, 1996b, a) This descending resp iratory drive responsible for activity of EI muscles is modulated by segmental interneuronal connections between the phrenic and intercostal motorneuron pools (Kirkwood et al., 1982a, Kirkwood et al., 1982b, Tian and Duffin, 1996a, Saywell et al., 2011, de Almeida and Kirkwood, 2013) C2HS interrupts this ipsilateral descending input to the phrenic and lower cervical motorneuron s and in turn decreases the proportion of respiratory drive to the ipsilateral intercostal motorneuron s These interneuronal interconnections are thought to travel in the lateral funiculi just above the first thoracic spinal segment and are responsible for EMG activation of diaphragm muscle in C1 transected anesthetized animals in response to high frequency stimulation of the thoracic spinal cord (Dimarco and Kowalski, 2013a, b, Kowalski et al., 2013) In the present study, ITTO in conscious rats resulted i n bilaterally increased EI EMG amplitude activity significant on the contralateral side one week after c SCI Application of ITTO repeatedly such as during ITTO conditioning in conscious rats may d rive mutual activation of intercostal and diaphragm muscles via this phrenic int ercostal interconnectivity and may therefore induce neuroplastic changes in thoracic spinal cord i n c SCI rats

PAGE 92

92 Effect of ITTO Conditioning on EI EMG Responses to ITTO in Conscious Rats with c SCI Ten days of ITTO conditioning resulted in similar pattern of activation between ipsilateral and contralateral side of EI m uscles in the C2HS+ITTO group. On the other hand, t he ITTO Day 10 peak in the C2HS+no ITTO group represents the influence of endogenous plasticity in the thoracic spinal cord and natural recovery of EI muscles a t approximately two weeks after c SCI. In this group, ipsilateral remained significantly decreased and some improvements were observed in the contralateral T herefore, ITTO conditioning may induce neuroplastic changes in thoracic spinal cord of c SCI injured rats by activation of load related sensory motor pathways (Davenport et al., 1985, Davenport et al., 1993, Davenport et al., 2010) and via existing anatomical interconnectivity between phrenic, lower cervical spinal cord and intercostal motorneurons (Tian and Duffin, 1996b, a, Dimarco and Kowalski, 2013a, b) Peak to peak frequency was significantly decreased compared to baseline values within a group ITTO conditioning significantly decreases the peak to peak frequency in normal conscious rats (Chapter 2) which is similar to or results in c SCI animals see in this study Therefore, we suggest t hat after c SCI, conscious rats respond to ITTO and ITTO conditioning primarily characterized by modulation of the amplitude of activity during ITTO mechanical loading Influence s on Observed EI EMG Responses A ge matched animals were found to lo se a significant amount of weight due to multiple surgical procedures and nature of the study. ITTO in conscious rats increases their stress and anxiety states (Bernhardt et al., 2011b, Pate and Davenport, 2012a)

PAGE 93

93 that may have contributed to weight loss in ITTO conditioned animal groups. However, meaningful comparisons can be made between the two C2HS and two Sham groups separately, on ITTO Days 1 and 10 because they were not si gnificantly different in size at these time points (Mantilla et al., 2011) However, the body weight of animals in the C2HS groups was lower than those in the Sham groups which may be due to atrophy of muscle mass and may have affected their EMG responses. In addition, genetic variab ility among rats used in this study (Chapter 2) may have influenced responses and outcomes between treatment groups (Golder et al., 2005b) Therefore, our results should be interpreted with caution. In summary, our results indicate that c SCI alters the activity of the EI muscles. A bilateral decrease, with significant ipsilateral decreases i n peak found during eupnea one week after c SCI. Additionally, we have investigated the respiratory load compensation of EI muscles after c SCI and the effect of ten days of ITTO conditioning on these responses. We conclude that ITTO is an effective animal model to elicit and investigate respiratory load compensation in EI muscles of conscious animals with unilateral c SCI

PAGE 94

94 Table 3 1 Body weights of animal s across study time period s Body weight (grams), for all treatment groups; C2H S+ ITTO Sham+ITTO ( ) and Sham+ no ITTO ( ) study time points ; surgical instrumentation (a), C2HS/ Sham surgery (b), ITTO Day 1 (c) and ITTO day 10 (d). Symbols denote significance ; within animal group a 1 /b 1 /c 1 and a 2 / b 2 / c 2 0.01 between animal groups and Surgical Instrumentation (a) C2HS/ Sham Surgery (b) ITTO Day 1 (c) ITTO Day 10 (d) C2HS+ITTO 336.5 4.8 321 .04. 6 a 1 266. 8 6.2 a 2 310.18.1 a 2 c 2 C2HS+no ITTO ( ) 345.610. 8 300 .0 8. 7 a 1 253 .0 11. 2 a 2 b 2 310.88. 7 a 1 c 2 Sham+ITTO ( ) 334.5 6.9 315 .0 7.6 a 2 310. 3 6.0 a 2 359.3 7.4 a 2 b 2 c 2 Sham+no ITTO ( ) 309.3 6.6 278.511.4 a 2 294. 3 9. 2 338.51 2.0 a 2 b 2 c 2

PAGE 95

95 Table 3 2 ITTO Duration Mean duration of the tracheal cuff in flation on ITTO Days 1 & 10 Pre Injury ITTO Day 1 ITTO Day 10 C2HS + ITTO (sec) 5.60. 1 5.5 0. 2 5. 2 0. 1 Sham + ITTO (sec) 5. 5 0. 3 5.6 0.1 5.2 0.1 Table 3 3 Occlusion pressure during ITTO. Mean occlusion pressure durin g tracheal cuff inflat ion on ITTO Days 1 & 10 Pre Injury ITTO Day 1 ITTO Day 10 C2HS + ITTO (mmHg) 93 6.0 0.8 934.0 1. 7 934. 1 0.6 Sham + ITTO (mmHg) 935.3 0.6 933.4 1.3 934.41. 2 T able 3 4 Phases used for normalization of Pre Injury percentage change in EI EMG data Numbers of animals in each group are listed in parenthesis. ITTO Group (n=10) no ITTO G roup (n=11) Left Side Right Side Left Side Right Side ITTO L ITTO/L Before R ITTO/R Before N/A N/A Recovery L Recovery/L Before R Recovery/R Before N/ A N/A After L After/L Before R After/R Before L After/L Before R After/R Before

PAGE 96

96 Figure 3 1. Pre s to ITTO. Pre injury (A) percentage change in to Significant increase s in bilateral percentage change in observed during ITTO and on left side during Recovery compared to After (B) Peak to After phase compared to Before (***p 0.001) and Recovery (# as well as during ITTO compared to Before (*p in the ITTO group. After response in the ITTO group was significantly lower than After response in the no ITTO group ( p 0.01).

PAGE 97

97 Figure 3 2 Effect of unilateral c SCI on the e upne ic EMG activity of bilateral EI muscles in conscious rats on ITTO Day 1 From top to bottom, occlusion pressure (Occl P) integrated ( ) and raw L EI EMG.

PAGE 98

98 Table 3 5 Data normalization for percentage change in eupneic EI EMG ampl itud e. Phases used for normalization are described below; data were normalized to Pre Injury Before values within side and within group. Numbers of animals in each group are listed in parenthesis. Pre Injury ITTO Day 1 ITTO group Day 10 no ITTO group Day 10 C2HS Pre Injury After/Pre Injury Before (n=1 3 ) ITTO Day 1 Before/ Pre Injury Before (n=1 3 ) ITTO Day 10 Before/ Pre Injury Before (n= 7 ) ITTO Day 10 Before/ Pre Injury Before (n= 4 ) Sham Pre Injury After/Pre Injury Before (n=8) ITTO Day 1 Before/ Pre Inj ury Before (n=8) ITTO Day 10 Before/ Pre Injury Before (n=4) ITTO Day 10 Before/ Pre Injury Before (n=4)

PAGE 99

99 Figure 3 3 Effect of unilateral c SCI on eupneic peak peak to peak frequency in conscious rats. EMG activity on the (A) ipsilateral, (B) contralateral sides for pre injury ITTO Days 1 and 10 (A) eupnea on ITTO Day 1 compared to Pre injury with in the C2HS group was observed. O n ITTO Day 10, eupneic activity in C2HS+ITTO group No other significant differences were observed in (A) and (B). (C) Ipsilateral peak to y different across study time points within either C2HS or Sham groups (D) Contralateral peak to across study time points within either C2HS or Sham groups. Contralateral peak to peak

PAGE 100

100

PAGE 101

101 Figure 3 4 Representative ITTO responses in a conscious rat one week after unilateral c SCI / Sham surgery ITTO responses in (A) Sham+ITTO and (B) C2HS+ITTO groups on ITTO Day 1. F or each panel f rom top to bottom occlusion pressure (Occl P) abdominal mot ion trace (Mvmt) and raw L EI EMG from (A) Sham and ( B ) C2HS injured conscious rats.

PAGE 102

102 T able 3 6 Data normalization for ITTO mediated percentage change in EI EMG amplitude on Days 1 and 10 Phases used for normalization are described below; data were normalized to Before values within group, within side and within Day (1 and 10) Numbers of animals in each group on ITTO Day s 1 and 10 are listed in parenthesis. ITTO Recovery After C2HS+ITTO ( Day 1 n=8 Day 10 n=7 ) ITTO/Before Recovery/Before After/Before C2HS+no ITTO ( Day 1 n=5 Day 10 n=4 ) ITTO/Before Recovery/Before After/Before Sham+ ITTO ( Day s 1 and 10 n=4) ITTO/Before Recovery/Before After/Before Sham+no ITTO ( Day s 1 and 10 n=4) ITTO/Before Recovery/Before After/Before

PAGE 103

103 Figure 3 5 Respiratory load compensation of EI muscles in conscious rats with c SCI on ITTO D ay 1 Percentage c (A) ipsilateral, (B) contralateral sides. Peak to the (C) ipsilateral and (D) contralateral sides in conscious rats with c SCI.on ITTO Day 1. (A) No significant differences were o bserved in ITTO responses between and within groups. (B) ITTO response in the C2HS+ITTO group was Recovery response in the C2HS+ITTO group was significantly greater than After to significantly decreased compared to Before within group. Peak to peak in the C2HS+ITTO group to ITTO, Recovery and After were significantly decreased compared to Before

PAGE 104

104

PAGE 105

105 Fi gure 3 6 Respiratory load compensation of EI muscles in con scious rats with c SCI on ITTO D ay 10 on the (A) ipsilateral, (B) contralateral sides. Peak to peak frequency on the (C) ipsilateral a nd (D) contralateral sides in conscious rats with c SCI after ten days of ITTO conditioning. (A) ITTO response in the C2HS+ITTO group was significantly greater than Recovery (*p 0.05) and After (**p 0.01) within group. ITTO response in the Sham+ITTO group was significantly greater than Recovery and After (*p 0.05) within group. After response in the C2HS+ITTO, C2HS+no ITTO and Sham+ITTO groups was significantly decreased C2HS+ITTO group wa No other statistical differences were found. (C) Peak to EMG frequency in the C2HS+ITTO group during ITTO was significantly (D) Peak to peak decreased compared to Before within group. Peak to EMG frequencies in the C 2HS+ITTO group during ITTO were significantly

PAGE 106

106

PAGE 107

107 Figure 3 7 Representative histological section of a complete hemisection (C2HS). Transverse section (40m cryostat) of the cervical spinal cord at the C2 level stained with cresyl violet A complete hemisection was defined as an absence of any spinal cord tissue on the left injured side the contralateral side remained intact; CC, central canal; DH, dorsal horn; VH, ventral horn; LF, lateral funiculus; VF, ventral funic ulus.

PAGE 108

108 CHAPTER 4 IMPACT OF INTRINSIC TRANSIENT TRACHEAL OCCLUSION CONDITIONING ON THE ACTIVITY OF EXTERNAL INTERCOSTAL MUSCLE DURING EUPNEA AND HYPERCAPNIA IN CONSCIOUS RATS WITH UNILATERAL c SCI R epeated r espiratory load ing has been used as a respiratory strength training paradigm in humans and is mechanistically similar to locomotor strength training Respiratory muscle strengthening can be either expiratory or inspiratory phase specific and is targeted based on patient symptoms such as, patients with eff ort related dyspnea would benefit from inspiratory muscle strength training. On the other hand, expiratory muscle strength training may improve expiratory muscle function and related behaviors such as cough (Sapienz a and Wheeler, 2006) Investigators have successfully used repeated inspiratory muscle strength training to wean mechanically ventilated medically complex patients (Martin et al., 2002, Smith et al., 2014) I mprov ements in cough were reported in sarcopenia patients after repeated expiratory muscle strength training (Kim et al., 2009) and variable effects were observed in a preliminary study with cervical/ thoracic spinal cord injured individuals (Fitsimones et al., 2004) Intrinsic transient tracheal occlusion (ITTO) is a conscious animal model for respiratory muscle strength training. Repeated ITTO condition ing of conscious rats has been shown to induce muscle hypertrophy in the diaphragm (Smith et al., 2012) and parasternal intercostal muscles (Smith et al., in press ) Ten days of repeated ITTO conditioning has also been shown to improve diaphragm function (Pate, 2010) in conscious rats. S tudies as a part of this dissertation provide evidence that repeated ITTO presentations elicit respirat ory load compensation in the external intercostal (EI) mus cles of conscious normal rats (Chapter 2) and rats with unilateral c SCI (Chapter 3) However, it is important to note that, due to the high respiratory frequency of

PAGE 109

109 conscious rats, ITTO in conscio us rats can not be targeted to a specific respiratory phase. Accordingly the purpose of this study was to determine the effects of ten days of ITTO conditioning on the EI muscle functioning during a hypercapnia challenge Hypercapnia has been used as a ch emical challenge to evaluate functional respiratory responses (Arita and Bishop, 1983b, a, Oliven et al., 1985, Gautier et al., 1986, Pokorski et al., 2005) A hypercapneic challenge disrupts the homeostasis of oxy gen (O 2 ) and carbon dioxide (CO 2 ) levels that the respira tory system works to maintain during normal breathing (Clancy and McVicar, 1996) Peripheral (Schlaefke et al., 1979) and central (Saint John, 1975) chemoreceptors are activated by increased CO 2 which in turn stimulate increases in respiratory drive thereby eliciting respiratory muscle responses ( Yasuma et al., 1993) Increases in minute ventilatio n should increase V CO2 thereby eliminating e xcess CO 2 levels re establishing O 2 and CO 2 homeostasis. However, previous exposure to stressful stimuli, like immobilization, decreases the ventilatory respo nse to hypercapnia in conscious rats (Kinkead et al., 2001a) The intercostal motor system is highly plastic (Dougherty et al., 2012a) and the lon g term facilitation of its respiratory activity after carotid sinus nerve stimulation has also been demonstrated (Fregosi and Mitchell, 1994) Thus by exposing conscious rats with unilateral c SCI to a hypercapneic challenge we hypothesize that the ITTO conditioned group will exhibit greater neuroplastic changes in the bilateral EI muscle functionin g compared to the non conditioned group s We analyzed peak to peak EMG frequency, peak amplitude activity (Terada and Mitchell, 2011) and motor effort (Fraigne and Orem, 2011) of the EI muscles to determine the effect of ten

PAGE 110

110 days of ITTO conditioning on the hyper capnia induced increased respiratory motor drive Materials and Methods Animals Data pre sented here are from animals also used in Chapter 3. A total of 18 male Sprague Dawley rats (300 350g) were used as follows, C2HS+ITTO (n=7), C2HS+no ITTO (n=4), Sham+ITTO (n=3) and Sham+no ITTO (n=4) Data from one animal in the Sham+ITTO group could not be recorded due to loss of electrodes. Study Design ITTO conditioned conscious rats were subjected to a 5 minute hypercapneic (7% CO 2 ) challenge, four hours after completion of their final ITTO trial. Hypercapnia Challenge The experimental set up for the hyperoxic hypercapneic challenge is presented in Figure 4 1. C onscious rats were placed in a plexi glass chamber and allowed to breathe room air. The rats were allowed to accommodate to the chamber (typically 5 7 minutes) before the recording was initiated After collecting 5 minutes of baseline eupnea a mixture of 8 % CO 2 and balance O 2 was slowly bled in to the chamber. The outflow from the chamber was sampled, allowing continuous monitoring of the level of CO 2. (Datex 223 CO2 monitor, Puritan Bennett Cor poration). The measured c h amber CO 2 equilibrium time was about 25 30 seconds from when the g as mixture was first introduced. The CO 2 increase was a smooth transition rather than a sudden step change. After the concentration of CO 2 in the chamber stabilized the animals breathed this hyperoxic hypercapneic gas mixture for 5 minutes. After 5 minutes, the CO 2 and O 2 gas valves were turned off room air was bled into the chamber and t he lid of the

PAGE 111

111 chamber was opened to allow rapid dissipation of the hyperoxic h ypercapneic gases. Animals continued to breathe room air for an additional 5 minutes, after which the recording was terminated. The total trial time was about 20 minutes per animal Data Analyses Raw EI EMG was FIR digital high pass filter ed (300 Hz; Spike 2, Cambridge Electronic Design) to minimize influence of heart rate and movement artifacts. DC remove, rectification and smoothing (50ms) functions were applied to the filtered data. EI EMG d ata from one minute of eupneic activity (referred to as Baseline) after ITTO conditioning trial on Day 10 (After phase, refer to Chapter 3) and one minute of EI muscle activity during stable hyperoxic hypercapneic challenge (referred to as Hypercapnia) wer e analyzed Values for peak am plitude, peak to peak frequency, time to peak (Ti ), time between peak of one and onset of next EMG burst (Te) and total burst time (Ttot =Ti+Te ) were obtained from this analysis. The values for duty cycle (Ti/ Ttot) and EI effort ( Peak ampl itude/ Ti) were calculated. Values for peak amplitude, peak to peak frequency and EI effort were averaged for each animal. Changes in peak amplitude, peak to peak frequency and EI effort were calculated by normalizing to the corresponding Baseline values (%Baseline) for each animal. These were then averaged to obtain group data. Statistical Analyses Statistical comparisons were made between treatment groups (C2HS+ITTO, C2HS+no ITTO, Sham+ITTO and Sham+no ITTO) using one way A NOVA with Student Neuman (SigmaPlot version 12.5 Systat Software Inc.). Changes from Baseline in respiratory variables (peak amplitude, peak to

PAGE 112

112 peak frequency and EI motor effort) were compared between treatment groups for ipsilateral and contralateral sides separately by one way ANOVA and Student Neuman Ti, Te, Ttot and dut y cy c l e values during Baseline and Hypercapnia were compared between treatment groups using one way ANOVA and within group using one way RMANOVA with Student Neuman for multiple comparisons. Differences indicated as statistically significant w ere p 0.05. All values are expressed as means standard error of mean Results Body Weight and CO 2 L evels Both the C2HS groups were significantly lower in body weight as compared to Sham +ITTO group, no other differences were found ; C2HS+ITTO 310.1 8.1 g 0.001 C2HS+ no ITTO 310. 8 9.7 g Sham+ ITTO 352. 3 7.4 g and Sham+no ITTO 338 5 1 2.0 g. There were no significant differences between treatment groups in % CO 2 that the animals received: C2HS+ITTO 8.4 0.1 %, C2HS+ no ITTO 8.60. 2 %, Sham+ ITTO 8. 6 0. 3 % and Sham+no ITTO 8. 6 0.2 %. Effect of ITTO C onditioning o n the R esponse s D uring Hypercapnia Representative data traces from all treatment groups during Baseline and Hypercapnia are presented in Figure 4 7 EI EMG amplitude expressed as aribtra ry units (a.u.) for both i psilateral and c ontralateral sides are presented in Figure 4 2 No significant differences in EI EMG were found within and between groups on the ipsilateral side during Baseline or Hypercapnia. Contralateral EI EMG was significa ntly higher during; Baseline in the C2HS+ITTO ( 0.11 0.01 a.u.) group compared to

PAGE 113

113 Bas eline values of C2HS+no ITTO ( 0.030.0 2 a.u. 01) and Sham+noITTO (0.040.02 a.u., in the C2HS+IT TO (0.1 2 0.0 3 a.u.) group compared to Hype rcapnia values of Sham+ITTO ( 0.0 4 0.0 2 Thus, c ontralateral EI EMG wa s significantly greater in the IT TO conditioned rats wi th c SCI compared to all other groups. EI motor effort was calculated using peak EI EMG amplitude and time to peak (Ti) and is presented for both ipsilateral and ocntralateral sides in Figure 4 3 No significant differences in EI motor effort were found w ithin and between groups on the ipsilateral side during Baseline or Hypercapnia. Contralateral EI motor effort was significantly higher during; Baseline in the C2HS+ITTO group (0.48 0.06 mV/sec.) compared to Baseline values of C2HS+no ITTO (0.180.07 mV/s 5 ) Sham+ITTO (0.110.08 mV/sec ) C ontrala teral EI motor effort was higher during Hypercapnia in the C2HS+ITTO group (0. 49 0. 11 mV/sec ) compared to Hyperc apnia values of Sham+noITTO group (0.0 8 Peak to Peak to peak frequency ( Figure 4 4 ) for the C2HS+ITTO group ( 90.2 11. 6 bursts/min ) during Hypercapnia was significantly lower tha n, within group Baseline (119 .0 7 .2 bursts/min, during Hypercapnia ( 143.412.3 bursts/min, no ITTO group during Hypercapnia ( 154.7 4.9 bursts/min, Peak to peak frequency in Sham+ no ITTO group ( 154.75.0 bur s ts/min ) during Hypercapnia was significan tly greater than during Baseline within group ( 108 .72.8

PAGE 114

114 bursts/min, fferences were found with Sham+ ITTO group ( Baseline 100 .8 5.8 bursts/min and Hypercapnia 127.019. 1 bursts/min ). Effect of ITTO C onditioning o n C hanges in Am plitude and Peak to Peak F requency esponses D uring Hypercapnia Ipsilateral and contralateral changes in ( Figure 4 5 A and B), EI effort ( Figure 4 5 C and D) were not significantly different during Hypercapnia between the treatment groups. P eak to peak frequency ( Figure 4 5 E) in the C2HS+ITTO ( 25.28 6.66%) was significantly decreased compared to C2HS+noITTO (10.1421.32 %, %, 3 7.17%, Effect of ITTO C onditioning on the Breath Time EI EMG R esponse s D uring Hypercapnia Group data for Ti, Te, Ttot and Duty cycle are presented in Figure 4 6. Ti was significantly decreased in the Sham+no ITTO group during Hypercapnia ( 0.150.01 sec, c omp ared to Baseline (0.220.02 sec) Ti d ecreased during Hypercapnia for groups; C2HS+noITTO ( 0.180.01 sec ) as compared to Baseline (0.220.0 3 sec, p=0.16) but statistical significance were not reached, Sham+ITTO ( 0.19 0.02 sec ) decreased compared to Baselin e (0.270.0 2 sec, p=0.06), but statistical significance were not reached. Te was significantly decreased in the Sham+no ITTO group during Hypercapnia ( 0. 250.01 sec, (0.410.05 sec) Te in the C2HS+ITTO group was significantly greater during Hypercapnia (0.49 0.07 sec, when compared to Baseline (0.310.02 sec) Te in the C2HS+ITTO (0.49 0.07 sec) group was significantly greater t han C2HS+noITTO ( 0.260.03 sec,

PAGE 115

115 Sham+no ITTO ( 0.250.01 during Hypercapnia Ttot was significantly greater during Hypercapnia in the C2HS+ITTO group (0.750.1 0 sec) when compared to C2HS+noITTO ( 0.440.04 sec, ITTO ( 0.39 0.01 sec, Ttot was significantly less in the Sham+no ITTO group (0.39 0.01 sec) compared to within group Baseline ( 0.630.03 sec, The duty cycle of the E I muscle i n the C2HS+ITTO group was significantly decreased during Hyperc apnia (0.340.02 a.u.) compared to Baseline ( 0.440.03 a.u., F or the other groups duty cycle was not significantly different, C2HS+noITTO ( Baseline 0.470.01 a.u. and Hypercapnia 0.410.02 a.u., p =0.09), Sham+IT TO (Baseline 0.440.01 a.u. and Hypercapn ia 0.380.05 a.u., p=0.4), Sham+no ITTO (Baseline 0.370.04 a.u. and Hypercapnia 0.38 0. 02 a.u., p=0.72) between Baseline and Hypercapnia conditions Discussion The result s of this study demonstrate that ITTO conditioned conscious rats with unilateral c S CI have frequency modulation of their EI muscle responses without significant changes in the amplitude responses during a hyperoxic hypercapnia challenge. Responses to a hyperoxic hypercapnia challenge in this study predo minantly reflect the stimulation fr om hypercapnia mediated central chemoreceptor activity in the conscious state, which is counteracted by hyperoxia mediated decrease in peripheral chemoreceptor activity (Gautier et al., 1986) The presence of background hyperoxia in a hypercapneic cha llenge significantly blunts frequency responses in urethane anesthetized mice and this is attributed to suppression of carotid body functioning by O 2 and is not influenced by the CO 2 content of the inhaled gas mixture (Pokorski et al., 2005) Studies in conscious cats report that ventilatory responses to hyperoxic

PAGE 116

116 hypercapnia are inconsistent, with occasional decreases in respiratory freque ncy and increases in tidal volume, but not significantly different than their responses to normoxic hypercapnia (Gautier et al., 1986) The Sham+ no ITTO group in our study reflects the intact conscious rats response to a hyperoxic hypercapnia challen ge. No significant differences were found in the raw EI EMG amplitude ( Figure 4 2) in this group. However, Ti, Te and Ttot were significantly decreased and consequently the peak to peak frequency of EI EMG was significantly increased in this group ( Figure s 4 4 and 4 6 ). On the other hand, ITTO conditioning in the C2HS animals did not change Ti and Ttot but significantly increased Te, decreased the duty cycle and decreased the peak to peak frequency ( Figure s 4 4 & 4 6) These changes in peak to peak frequency were signifi cantly different between C2HS+ ITTO and other treatment groups ( Figure 4 5 ). Responses from the C2HS+ no ITTO group reflect the endogenous plasticity that occu rs in the intercostal motor system (Dougherty et al., 2012a) Therefore, left to recover without any intervention, the EI muscles of conscious rats with unilateral c SCI respond to Hypercap nia similar to rats in the Sham+ITTO and Sham+ no ITTO groups. Dougherty et al found that inter costal muscle EMG recovers to non injured values two weeks after c SCI (Dougherty et al., 2012a) Our results are similar to the above finding and are evident from the activity of the EI muscles in the C2HS+ no ITTO group. Repeated ITTO in conscious rats is known to induce stress an d anxiety responses (Bernhardt et al., 2011a, Bernhardt et al., 2011b) Previous exposure to stress stimuli is known to blunt the hypercapneic response in conscious rats (Kinkead et al., 2001a) Differences in body weight may also influence the EI EMG values observed

PAGE 117

117 in this study. Animals with higher body weight have more muscle m ass and therefore capable of higher EMG responses (Mantilla et al., 2011) Also genetic variability among rats (Chapter 2) may have influenced responses and outcomes between treatment groups (Golder et al., 2005b) Therefore, our results should be interpreted with caution. Although a bilateral decrease in amplitude was observed in animals with unilateral c SCI, the c amplitude was always slightly greater than ipsilateral response to ITTO (see Chapter 3). Values of expressed as arbitrary units as seen in Figure 4 2 show similar effects on eupneic and hypercapneic EI EMG and supports this previous finding. Therefore, we suggest that ITTO conditioning induces neuroplastic changes in the thoracic spinal cord of conscious rats with unilateral c SCI. However these changes are predominantly directed toward improvements in contralateral EI EMG function. Intercostal motor activity is modulated via interneuronal connections between the phrenic and thoracic and within different levels of thoracic motorneur on pools (Kirkwood et al., 1988, de Almeida and Kirkwood, 2013) This anatomical connectivity has been recently attributed with the physiological activation of diaphragm activity during high frequency stimulation o f the thoracic spinal cord in C1 transected anesthetized rats (Kowalski et al ., 2013) Therefore, it is likely that increases in contralateral EI EMG activity may drive respiratory recovery of ipsilateral diaphragm and/ or bilateral intercostal muscles at other thoracic levels, through the existing phrenic intercostal and intercost al intercostal pathways Furthermore, changes in activity of one muscle group such as EI may not be a reliable predictor of the effects of ITTO conditioning on the functioning of the respiratory system

PAGE 118

118 as a whole. Further studies are required to ascertain whether ITTO conditioning in rats with c SCI is clinically beneficial. In conclusion, we have demonstrated that, ten days on ITTO conditioning alters eupneic and hypercapneic responses of EI EMG in conscious rats with unilateral c SCI. The observed changes in suggest that the improvements in EI muscle function are predominantly directed towards the contralateral side. These rats respond primarily by decreasing the peak to peak frequency during Hypercapnia as compared to Baseline and other tr eatment groups during Hypercapnia.

PAGE 119

119 Figure 4 1 Schematic of the e xperimental set up for hyperoxic hypercapnia challenge Animals were exposed to 8 % CO 2 balance O 2 challenge for 5 minutes and EI muscle EMG was recorded. Eupneic EI muscle activities wer e also recorded before and after exposure to hypercapneic challenge. Total trial time was about 20 minutes per animal.

PAGE 120

120 Figure 4 2 responses during Baseline and Hypercapnia. amplitude expressed as arbitrary units (a.u.) for (A) Ipsilateral and (B) Contralateral EI No significant differences were found within and between groups on the ipsilateral side either during Bas eline or Hypercapnia. Contralateral was significantly greater during Baseline in the C2HS+ITTO group compared to Baseline values 1) and was significantly higher during Hy percapnia in the C2HS+ITTO group compared to

PAGE 121

121 Figure 4 3 EI motor effort responses during Baseline and Hypercapnia. EI motor effort was calculated using peak I EMG amplitude and time to peak (Ti) values and is expressed as mV/ sec for (A) Ipsilateral and (B) Contralateral EI. No significant differences were found within and between groups on the ipsilateral side either during Baseline or Hypercapnia. Contralater al EI motor effort was significantly greater during Baseline in the C2HS+ITTO group compared to Baseline values of C2HS+noITTO ( 5 ) Sham+ITTO ( ^p 0.05) and EI motor effort was significantly higher during Hypercapn ia in the C2HS+ITTO group compared to Hypercapnia values of Sham +noITTO ( ).

PAGE 122

122 Figure 4 4 Effect of ITTO conditioning on the peak to peak frequency during Baseline and Hypercapnia Peak to peak frequenc ies during Baseline were not significantly different. Peak to peak frequency for the C2HS+ITTO ( ) group during Hypercapnia was significantly lower within group Baseline (* 0.001), C2HS+no ITTO group ( ) during Hypercapnia no ITTO ( ) group during Hyperca pnia (# # Peak to peak frequency in Sham+ no ITTO group during Hypercapnia was significantly greater than during Baseline, within group No di fferences were noted in Sham+ ITTO group ( )

PAGE 123

123 Figure 4 5 Effect of ITTO conditioni ng on the conscious rats with c SCI. Panels A and B show change in amplitude (%Baseline) (B) Contralateral. Panels C and D present the change in EI motor effort (%Baseline) during Hypercap nia (C) Ipsilateral, (D) Contralateral. No significant differences were found Panel E presents the change in peak to peak frequency. C2HS+ITTO group was significantly lower in peak to peak frequency response Sham+no ITTO (# #

PAGE 124

124 Figure 4 6 Effect of ITTO conditioning on breath time s f or the hypercapneic response of conscious rats with c SCI. (A) Ti, (B) Te (C) Ttot and (D) Duty cycle arbitrary ivity during Baseline and Hypercapnia Ti and Te in the Sham+no ITTO group were significantly decreased during H ypercapnia compared to Baseline Te in the C2HS+ITTO group was significantly greater during Hypercapnia when compared to Baseline (**p Sham+no ITTO significantly greater during Hypercapnia in the C2HS+ITTO group compared Sham+no ITTO significantly decreased in the Sham+no ITTO group as c ompared to Baseline T he C2HS+ITTO group had significantly reduced duty cycle during 0.05 ).

PAGE 125

125 Figure 4 7 Representative EI EMG data traces from all treatment groups For each panel, the top trace is EI EM G, the second trace is R EI EMG (a.u.) the third trace is EI EMG and the fourth trace is L EI EMG (a.u.) for (A) Baseline and (B) Hypercapnia.

PAGE 126

126 CHAPTER 5 EFFECT OF ACUTE INTERMITTENT HYPOXIA TREATMENT ON VENTILATORY LOAD COMPENSATION AND MAGNITUDE ES TIMATION OF INSPIRATORY RESIST IVE LOADS IN AN INDIVIDUAL WITH CHRONI C IN C OMPLETE c SCI CASE STUDY An estimated 12000 new cases of spinal cord injury (SCI) arise every year in the United States (NSCISC, 2013) and respiratory dysfunction is the leading cause of mortality in this patient population (Winslow and Rozovsky, 2003, Brown et al ., 2006) A disruption o f the neural efferent out put to respiratory muscles due to SCI results in a breathe and compensate to increased respiratory loads (Axen, 1982, Kelling et al., 1985) Also, depending on level and severity of injury, SCI interrupts the ascending afferent input from thoracic and accessory respiratory muscles. This afferent feedback plays a key role in the modulation of breathing (von Euler, 1973) Therefore, rehabilitation aft er SCI necessitates restoration of the respiratory motor function as well as ensuring that the respiratory perceptual sensitivity of the injured individual does not hinder their capabilit y to respond to mechanical and/ or chemical challenges to breathing Magnitude estimation (ME) is a technique used to cognitively scale the size of a given stimulus by assigning a proportional number to the perceived magnitude The : K n The estimated magnitude is a power function of the load magnitude n is the power exponent and K is a constant (Stevens, 1957) The slope of the log log plot of the reported magnitude estimation and mouth pressure generated in respon se to an inspiratory resistive load short term exposure to a respiratory load of high magnitude has been shown to reduce the magnitude estimation of IRL in healthy huma n subjects, and may act by reducing

PAGE 127

127 the sense of effort needed to breathe against the applied load (Revelette and Wiley, 1987) Manning et al have shown that healthy human s exposed to high and low levels of CO 2 (Manning et al., 1994) and changes in inspiratory flow rate (Manning et al., 1995) do not show any changes in the magnitude estimation of tidal volume However, in quadriplegics presented with IRL the magnitude estimation of respiratory force is diminished (Gottfried et al., 1984) but that of inspired volume is unchanged (DiMarco et al., 1982) Acute intermittent hypoxia (AIH) has been shown to induce respiratory plasticity and improve ventilation in animal models of SCI (Wilkerson et al., 2008, Wilkerson and Mitchell, 2009, Terada and Mitchell, 2011, Lovett Barr et al., 2012) by means of respiratory long term facilitation (LTF) (Vinit et al., 2009, Dale Nagle et al., 2010) R epetitive exposure to AIH while sustaining an el evated level of carbon dioxide ( CO 2 ) w as shown to induce ventilatory LTF in healthy conscious subjects (Harris et al., 2006, Mateika and Sandhu, 2011) and subjects w ith chronic spinal cord injury (Tester et al., 2014) Thus, AIH along with elevated level of CO 2 may improve the ventilatory status in individuals with incomplete SCI. We hypothesized that ten days of AIH with elevated CO 2 would improve ventilatory load compensation and in crease the respiratory perceptual sensitivity to IRL, in an individual with chronic incomplete c SCI. The present study was performed on an individual with chronic, incomplete, cervi cal SCI. The procedure for ME of IRL has been previously used to demonstrate improvements after inspiratory muscle strength training (Kellerman et al., 2000) assess gender differences (Alexander Miller and Davenport, 2010), investigate the impact of emotional state (Kellerman et al., 2000, Alexander Miller and Davenport, 2010, Tsai et

PAGE 128

128 al., 2013) on the respiratory per ceptual sensitivity in normal subjects and in children with life threatening asthma (Kifle et al., 1997, Julius et al., 2002) Materials and methods All institutional and governmental regulations concerning the e thical use of human volunteers in research using University of Florida and Brain Rehabilitation Research Center, Veterans Affairs Medical Center (VAMC), Gainesville, FL were followed during this study. The University of Florida Institutional Review Board r eviewed and approved the study. Case History The study participant was a 55 year old female with an AIS impairment score of D (Kirshblum et al., 2002) with a C4 chronic (4 years & 8 months), incomplete SCI. The injury was classified as Brown Sequard (Maynard et al., 1997) and was without tracheostomy or assisted ventilation. Upper motorneuron signs were present. A study physician and a therapist confirmed the absence of heart and lung complications after obtaining medical authorization from the subject. The subject participated in all of the study testing (listed below) at four time points; when nave to any testing (Baseline), after tw o days of elevated CO 2 exposure (Post Sham), after exposure to AIH and elevated CO 2 (AIH treatment) on days one (AIH Day 1) and ten (AIH Day 10). Lung and respiratory muscle function testing was carried out after the IRL procedure. N o prior history of smok ing was reported. Pulmonary and Respiratory Muscle Function A digital spirometer (Futuremed, Granada Hills, CA) and a respiratory pressure meter (Micro Direct, Inc, Lewiston, ME) were used to assess pulmonary and respiratory muscle function. The subject w as given directions for task performance. The digital

PAGE 129

129 spirometery inspiratory and expiratory pressure m easurement s were conducted three times each. Average and standard deviation values for the outcomes were calculated. Acute Intermittent Hypoxia Treatme nt (AIH) Protocols for sham and AIH were delivered via a non rebreathing system and a facemask during wakefulness. The subject maintained a supine position throughout trial durations. Partial pressures of carbon dioxide (P ET CO 2 ) and oxygen (P ET O 2 ) were sa mpled from a port in the facemask and adjusted in real time (models 17515 and 17518, respectively, VacuMed, Ventura, CA). Heart rate was continuously monitored using an electrocardiogram (model 17032, VacuMed, Ventura, CA) and O 2 saturation was continuousl y monitored via pulse oximetry (Biox 3740, Ohmeda, Boulder, CO). The subject was blinded to 2 sham sessions for 2 days before the 10 days of treatment with AIH. During each of these sessions the subject breathed room air for 10 min to establish eupneic ba seline values. Supplemental CO 2 was added to the inspirate and P ET CO 2 was elevated 3.8 0.09 to 4.3 0.19 mmHg above baseline and maintained for the remainder of the treatment session. After the 10 min period with elevated CO 2 levels, eight 2 min episode s of 8% O 2 / balance N 2 were administered while maintaining P ET O 2 2 On days when subject underwent sham sessions, room air was used instead of 8%O 2 / balance N 2 Every hypoxic/sham episode was terminated abruptly by ad ministering one breath of 100% O 2 to rapidly normalize P ET O 2 This was followed by a 2 min recovery period where the subject breathed room air. After the eighth hypoxic/ sham session the subject was monitored for an additional 30 min end recovery period. Th e subject tolerated all the treatment and sham sessions with n o signs of distress.

PAGE 130

130 Inspiratory Resistive Load (IRL) The experimental set up is illustrated in Fig ure 5 1. The IRL manifold was placed at the end of the experimental set up and was attached to the inspiratory side of the non rebreathing valve via a reinforced flexible plastic tube (~30cm long). The manifold was placed away from the view of the participant so she was not aware what load was presented. Four different grades of IRL (5, 15, 30 and 50cmH2O/L/sec) and a no load were applied five times each, in a randomized block design. The subject and the investigator recording the subject reported magnitude estimation were blinded to the sequence of IRL being applied. Magnitude Estimation Procedure The subject reclined comfortably on a flat bench, with her back supported. With nose clips on, she was asked to breathe through a mouthpiece, which was attached to the non rebreathing valve. Four different grades of IRL (5, 15, 30 and 50cmH2O/L/sec) and a no load were applied five times each (seven times each during baseline), in a randomized block design. During baseline the first two blocks of IRL were presented to familiarize the subject to the respiratory load sensation testing device and protocol. Th e data from these presentations were not used for analysis. A light cued the subject that her next inspiratory effort may (randomized graded IR L) or may not (no load) have an IRL. When the light cue was turned off, the subject was asked to rate her perceiv ed magnitude of breathing effort for the light cued breath A modified Borg scale (Borg, 1982) ranging from 0 (nothing) to 1 0 (very, very severe) was used to record the magnitude estimation ( ME ) rating for the IRL. One of the investigators recorded these ME rating aloud to ensure that it was communicated

PAGE 131

131 properly. The subject was unaware of the magnitude of IRL presented and not allowed to see any of the previous ratings. Each IRL was presented for a single inspiration only. Mouth pressure (P) was measured fr om a port in the center of the non rebreathing valve and airflow (AF) was measured with a pneumotachograph (Vernier Software & Technology) attached to the inspiratory port of the non rebreathing valve The data were recorded with a portable signal processo r and transferred to a computer and stored for subsequent data analysis. Data Analysis The data collection software (Vernier Software & Technology) generated numerical P (mbar), AF (L/sec) and time (sec) data corresponding with the recorded respiratory p attern The experimenter marked each single presentations of IRL, during the recording session P mbar were multiplied by a correction factor (1.019744) and converted to P cmH 2 O values, used for all further P analyses. P (cmH2O) and AF (L/sec) values and l og transformed P and ME values were used for further statistical analysis. Mean slopes for P vs. Resistance, AF vs. Resistance and Log ME vs. Log P were calculated. All data were analyzed by RMANOVA and Holm Sidak test for multiple comparisons (SigmaPlot 1 2.5). Data are provided as mean standard error of mean (unless noted otherwise) and significance was set for p 0.05. Results Pulmonary a nd Respiratory Muscle F unction Table 5 1 summarizes subject data from pulmonary function tests and Table 5 2 summariz es subject data from maximal inspiratory (MIP) and expiratory (MEP) pressure tests. Values for percent predicted forced vital capacity (FVC) increas ed significantly ( able 5 1) on Post Sham (74.66 3.05 %), AIH Days 1 (76.33 0.5 8 %) and 10

PAGE 132

132 (80.00 2.00 %) when compared to baseline (67 .33 4.04 %). Forced expiratory volume in one second (FEV1) also increased significantly ( Table 5 1) on Post Sham (63.66 2. 5 1%), AIH Days 1 (60.00 2.00%) and 10 (67.00 2.65 %) when compared to baseline (46 .33 12.66 %). These results indicate an improvement in pulmonary function after treatment with AIH and elevated CO 2 The MIP and MEP were not significantly different from baseline values (Table 5 2). This treatment paradigm did not alter respiratory muscl e pressure generating capacity in the study subject. Respiratory Load C ompensation The results for P and AF in response to IRL indicate increased respiratory load compensation in the study subject after ten days of AIH with elevated CO 2 treatment. At the lower load levels (0 15 cmH2O/L/sec) the P results were not significant ly different on AIH Day 1 (p=0.06 at 0cmH2O/L/sec) and Pos t Sham (p=0.19 at 5cmH2O/L/sec) compared to B aseline values. The subject generated a significantly higher P at the 30 cmH2O/L/se c load on AIH Day 1 ( 8.28 0.36 cmH2O, Figure 5 2A) compared to B aseline ( 5.61 0.36 cmH2O) At the highest load of 50cmH2O/L/sec the P values were not significantly different when compared to B aseline, AIH Day 1(p=0.15) and AIH Day 10 (p=0.11). The mean slopes for the pressure vs. re sistance plot demonstrated a gradual decrease. The mean slope for AIH Day 10 ( 0.17 0.02 a.u.) was significantly lower ( Figure 5 2B) when compared to mean slope at B aseline ( 0.12 0.02 a.u.) Post Sham (p=0.075) and AIH Day 1 (p=0.09) were not statistically significant. The subject produced a significantly larger Figure 5 3A) AF in response to a 15 cmH2O/L/sec IRL on Post Sham (0.280.01 L/sec) and AIH Day 1 ( 0.310.03 L/sec) ; a 30cmH2O/L/sec I RL on Post Sham (0.280.02 L/sec) and AIH Da y 1

PAGE 133

133 (0.310.02 L/sec) and a 50cmH2O/L/sec IRL on Post Sham (0.250.003 L/sec) AIH Day 1 (0.250.02 L/sec) and AIH Day 10 ( 0.270.02 L/sec ). The results for AF were not significan tly different at, 0 and 5cmH2O/L/sec IRL on any of the time points studied and at 15cmH2O/L/sec on AIH Day 10 (p=0.06). The mean slope for the AF vs. resistance was significantly greater on AIH Day 10 ( 0.00040.0015, 0.05, Fig ure 5 3B) compared to B aseline ( 0.0030.0012) No significan t difference was found for mean slope on Post Sham (p=0.06) and AIH Day 1 (p=0.65) compared to Baseline Magnitude E stimation The ME for each IRL were log transformed (Log ME) and plot ted against log transformed values of P generated against each load (Log P). The Log ME at 50cmH2O/L/sec load on AIH Day 1 ( 0.640.02 a.u. Figure 5 4A ) was significantly lower than B aseline (0.71 0.3 a.u.) The mean slopes of Log ME vs. Log P ( Fig ure 5 4B) were not significantly different between all conditions in this study. Discussion The key result from this study is an improvement in ventilatory load compensation and pulmonary function after ten days of treatment with AIH and elevated levels o f CO 2 in an individual with chronic, incomplete cervical SCI. However, this paradigm did not demonstrate an AIH effect on the ME of IRL and therefore the perceptual sensitivity to IRL, in the study subject. E xposure to AIH with elevated levels of CO 2 has been shown to induce ventilatory LTF in animal models of SCI (Vinit et al., 2009, Dale Nagle et al., 2010) healthy awake humans (Harris et al., 2006, Mateika and Sandhu, 20 11) and humans with chronic SCI (Tester et al., 2014) Based on our results, a decrease in mean slo pe for P vs. resistance plot and a corresponding increase in mean slope for AF vs.

PAGE 134

134 resistance, on AIH Day 10 demonstrate that the subject in our study improved her ability to ventilatory compensation for an increased respiratory load Also, pulmonary funct ion tests showed a significant and consistent improvement in %FVC and %FEV1 after AIH treatment. During baseline testing the first two blocks of IRL were presented to familiarize the subject to the testing device and protocol. The data from these presentat ions were not used for analysis in this report. However, one cannot rule out the possibility that the reported improvements in P and AF may be due to the combined influence of AIH treatment and a learning effect of repeated exposure to the IRL protocol. No improvements in respiratory muscle pressure generating capacity were observed. Values for MIP and MEP were not significantly different on any of the time points measured in our study. This suggests that the increased pulmonary function, AF and P are not due to increased respiratory pump function and may be due to increased central respiratory drive. Posture significantly affects the ventilatory response to added loads in both normal and SCI individuals (Loveridge et al., 1992) Supine posture has been shown to have a mechanical advantage over upright sitting in tetraplegics (Ben Dov et al., 2009) The subject in our study was supine during all AIH treatment trials and reclined comfortably with her back supported and legs str aight on a flat bench. It is likely that the P and AF responses to lower intensities of IRL (0, 5cmH2O/L/sec) were influenced by posture. However, consistent increases in AF at the higher intensities of IRL (30, 50cmH2O/L/sec) with the same posture suggest the increased ventilatory load compensation response was not the result of the respiratory mechanical effects due to posture.

PAGE 135

135 The baseline perceptual sensitivity of our study subject was slightly lower but comparable to that of healthy conscious adults, previously reported by our lab (Tsai et al., 2013) Previous studies with quadriplegics have reported a much greater reduction in the value of the respiratory loa d sensitivity exponent (Gottfried et al., 1984) The perceptual sensitivity is dependent on the respi ratory drive, breathing pattern and respiratory muscle fatigue (Gandevia et al., 1981 ) In our case, spinal injury severity also plays a key role in determining the perceptual sensitivity to IRL. The subject in our study has a chronic and less severe form cervical SCI that may have partly preserved the afferent and efferent pathways from the thoracic muscle receptors that modulate respiratory effort responses (von Euler, 1973, Gottfried et al., 1984) The presentation of a single breath IRL does not cause any respiratory muscle fatigue (von Leupoldt et al., 2011) In addition, d iaphragm muscle fatigue does not influence the respiratory effort perceptual sensitivity o f normal subjects (Bradley et al., 1986) Thus, the perceptual response of this SCI patient is likely the result of spared neur omuscular function and not respiratory muscle fatigue. In patients with sleep apnea, the ventilatory response to AIH and the resulting LTF is depende nt on time of day when the subject was exposed to AIH treatment (Gerst et al., 2011) However, repeated exposure to AIH with elevated CO 2 levels has been shown to augment the hypoxic ventilatory response, irrespective of time of day (Gerst et al., 2011) To our knowledge there is no study determining the optimal tim e of day for conducting the ME o f IRL protocol. However, w e contro lled for this effect and maintained a consistent time of the day when the patient underwent respiratory testing and AIH.

PAGE 136

136 In summary, we conclude that chronic exposure to AIH with elevated CO 2 levels improved respiratory load compensation without altering r espiratory muscle function in a n individual with chronic incomplete c SCI The ventilatory load compensation response to added IRL increased but the perceptual sensitivity to IRL was not affected after AIH treatment. Further studies are required to determi ne the applicability of AIH treatment as a rehabilitative intervention for SCI individuals. Study Limitations A learning effect, due to repeated exposure to the IRL protocol, may have significantly influenced the results of our study. However, we also se e improvements in % FVC and % FEV1 values, which suggest that the study paradigm may have a beneficial effect on the subject's pulmonary function and associated lung mechanics Also, the subject reported no changes in perceptual sensitivity. This would sugge st that our study paradigm might affect the discriminative but not affective processing of IRL (Davenport and Vovk, 2009) Thus, even if the subject learned to differentiate between loads (vs Baseline) and generate greater negative pressure in the ventilatory load compensation response, t he affective response to each load remained the same over the treatment time. This is consistent with findings from a previous study demonstrating that respiratory learning is not influenced by the elicited physiological responses (Va n den Bergh et al., 1998) Improvements in pulmonary function tests and ventilatory load compensation response suggest that this paradigm may be a potentially beneficial rehabilitation intervention for individuals with chronic incomplete c SCI However, f urther studies are warranted to determine the clinical applicability of AIH treatment.

PAGE 137

137 Fig ure 5 1 Experimental set up for ME of IRL

PAGE 138

138 Table 5 1 Summary of r esults for pulmonary function test. Values for percent predicted forced vital capacity (FVC) a nd forced expiratory volume in 1 second Pulmonary Function Test Baseline Post Sham AIH Day 1 AIH Day 10 Forced Vital capacity (FVC%) 67 .33 4.04 74.66 3.05 ** 76 .33 0.58 ** 80 00 2 .0 0 *** Forced Expiratory Volume (FEV%) 46 33 12. 6 6 63 66 2. 5 1 60.00 2.00 67.00 2.65 Table 5 2 Summary of r esults for respiratory muscle pressure generating capacity test. No significant difference s were observed after AIH treatment in maximal inspiratory (MIP) and expiratory (MEP) pressur e values. Respiratory Muscle Pressure Generating Capacity Baseline Post Sham AIH Day 1 AIH Day 10 Maximal Inspiratory Pressure (MIP, cmH 2 O) 56 .00 3.61 57 .33 1.53 57 .00 6.56 59 .33 3.21 Maximal Expiratory Pressure (MEP, cmH 2 O) 63 .00 8.88 6 5.66 3.05 73 .00 3.46 75 .00 4 .00

PAGE 139

139 Figure 5 2. Ventilatory load compensation after AIH treat ment: Pressure (A) Pressure (cmH2O) vs. Resistance (cmH2O/L/sec) for Baseline, Post Sham, AIH Day 1 and AIH Day 10. The subject generated significantly greater negat ive IRL; (B) Mean slope of Pressure vs. Resistance. Mean slope of this plot on AIH Day 10 was significantly less than Baseline

PAGE 140

140

PAGE 141

141 Figure 5 3. Ventilatory load compensati on af ter AIH treatment: Airflow (A) Airflow (L/sec) vs. Resistance (cmH2O/L/sec) for Baseline, Post Sham, AIH Day 1 and AIH Day 10. The subject generated significantly greater airflows when 05), Mean slope of Airflow vs. Resistance. Mean slope of this plot on AIH Day 10 was significantly greater than B

PAGE 142

142

PAGE 143

143 Figure 5 4 Magnitude estimation of IRL after AIH treatment. (A) Log log plot for magnitude estimation (ME) vs. pressure. Log ME was significantly greater than Baseline on AIH Day 1 w hen subject was presented with the 50cmH2O/L/sec log plot of ME vs. pressure. No significant differences were found in the perceptual sensitivity to IRL in this subject, after AIH treatment.

PAGE 144

144

PAGE 145

145 CHAPTER 6 SUMMARIES AND CONCLUSIONS Study #1 Summary The purpos e of this study (Chapter 2) was to determine the load compensation response of the external intercostal (EI) muscles in normal conscious rats. We used the model of intrinsic transient tracheal occlusions (ITTO) to elicit respiratory load compensation in th e EI muscles in chronically instrumented conscious rats. It was hypothesized that conscious rats exposed to ITTO would recruit the EI muscles with an increased EMG activation. The results from this study demonstrate that conscious rats consistently recruit their EI muscles, with an increased EMG amplitude activation, in their respiratory load compensation response to ITTO This response to ITTO was maintained over the ten days of ITTO conditioning with no potentiation or habituation Previous reports from o ur lab have demonstrated respirato ry muscle hypertrophy in intercostal (Smith et al., In press) and diaphragm (Smith et al., 2012) muscles as a result of repeated respiratory load compensation via ITTO Accordingly, w e hypothesized that ten days of ITTO conditioning would potentiate the baseline EMG activity of EI mus cle s in normal conscious rats. However, contrary to our hypothesis, no potentiation of baseline EI EMG activity was found after ten days of ITTO conditioning The activity of EI muscles is inherently variable due to the modulatory effects of conscious and affective states posture EI muscle afferents and vagal feedback exerted on a breath by breath basis. In this study, 50% of conscious rats respo nded with an (Onset) on presentation with an ITTO and these were termed as High responders The remaining animals ( Onset) but subsequently increased (Phasic) during sustained applicatio n of an

PAGE 146

146 ITTO and were termed as Low responders This segregation of animal groups in to High and Low responders observed on the first day of ITTO trial s persisted throughout our study time period of ten days. Additionally, this is the first study to desc ribe the frequency content of the EI EMG data in conscious rats. Power spectral density analyses of eupneic EI EMG data revealed its median and centroid frequency distribution, which did not change after ten days of ITTO conditioning. Study #2 Summary Cerv ical hemisection (C2HS) is an animal model of unilateral c SCI (Moreno et al., 1992) C2HS disrupts the descending respiratory drive to the EI muscles ipsilateral to injury thereby impairing normal EI muscle activi ty. Our results (Chapter 3) indicate that one week after c present but significantly decreased only on the side ipsilateral to injury, in conscious rats. activity is also decreased on the side contralateral to injury but is not si gnificantly different than the pre injury values. Also, the purpose of this study (Chapter 3) was to determine the respiratory load compensation to ITTO of the EI muscles in conscious rats with cervical spinal cord injury (c SCI). We hypothesized that ten days ITTO conditioning would result in increase s in bilateral EI EMG amplitude activi ty in conscious rats with c SCI. ITTO effectively elicited respiratory load compensation responses after c SCI and this response was amplitude activity during applied I TTO as compared to that during After phase of trial. ITTO r esponses were robust on the contralater al side, in the C2HS+ITTO group, on ITTO Day 1, one week after unilateral c SCI. However, by Day 10, ITTO responses

PAGE 147

147 showed a balanced co activation of both ip silateral and contralateral sides in the C2HS+ITTO group. Ten days of ITTO conditioning elicited respiratory load compensation responses similar to those observed during ITTO D ay 1. EI EMG amplitude activity was significantly greater during ITTO compared to Recovery and After phases in the C2HS+ITTO and Sham+ITTO groups. We conclude that, ITTO is an effective conscious animal stimulus to elicit respiratory load compensation of the EI muscle s in rats with unilateral c SCI Repeated ITTO may have beneficial effects on EI muscle functioning in conscious rats with unilateral c SCI. Study #3 Summary The final animal study (Chapter 4) was conducted to determine the impact of ITTO conditioning on the hyperoxic hypercapnia (Hypercapnia) response in c SCI injured c onscious rats. We hypothesized that unilateral c SCI injured conscious rats with ITTO conditioning would demonstrate a greater bilateral EI EMG amplitude activity during H ypercapnia than those without ITTO conditioning However, the results of this study demonstrate that the ipsilateral EI EMG values for C2HS+ITTO group during Hypercapnia were not significantly different compared to Baseline. Also, changes in EI EMG amplitude were not significantly different between the treatment groups. We also found th at, contralateral EI EMG wa s significantly greater in the C2HS+ITTO group compared to all the other treatment groups. Our results show that c onscious rats with unilateral c SCI after ten day s of ITTO conditioning exhibit frequency modulation of their EI m uscle responses without significant changes in the amplitude responses during a H ypercapnia challenge. These rats respond primarily by decreasing the peak to peak frequency during

PAGE 148

148 Hypercapnia as compared to Baseline and other treatment groups durin g Hypercapnia. On the other hand, S ham animals respond by increasing their peak to peak frequency. We suggest that ten days of ITTO conditioning drives neurop lastic changes in the EI muscle directed predominantly towards the contralateral side T h e functional benefits of this finding are not entirely clear at this time. S tudy #4 Summary Our primary goals f or study #4 (Chapter 5) w ere to test the hypotheses that ten days of AIH with elevated CO 2 would increase ventilatory load compensation ability and increase the respiratory perceptual sensitivity to inspiratory resistive loads (IRL), in a human subject with chronic incomplete c SCI Changes in mouth pressure (P) and airflow (AF) generated against the applied IRLs indicated respiratory load compen sation response and those in the forced vital capacity (%FVC) and forced expiratory volume (%FEV) reflected the status of the pulmonary function. The study subject showed a progressive improvement in respiratory load compensation and pulmonary fu nction after ten days of treatment with AIH and elevated levels of CO 2 Post AIH treatment FVC increased by 19.4% and FEV by 44.61%. To evaluate changes in the perceptual sensitivity a modified Borg scale was utilized to rate the difficulty to breathe agai nst applied IRLs. Our results indicate that this AIH paradigm did not have a significant effect on the magnitude estimation ( ME ) of IRL in the study subject Thus we conclude that c hronic exposure to AIH with elevated CO 2 levels improved pulmonary functi on without altering respiratory muscle pressure generating capacity in the study subject Also, the ventilatory load compensation response to added IRL improved but the perceptual sensitivity to IRL was not affected by AIH

PAGE 149

149 treatment. Consistent improvement s in pulmonary function tests and ventilatory load compensation response suggest that this paradigm may be a potentially beneficial rehabilitation intervention for individuals with chronic incomplete spinal cord injury. Further studies are needed to determ ine the clinical applicability of AIH treatment as a rehabilitative intervention for individuals with chronic c SCI Discussion EI Muscle Respiratory Neurophysiology in Intact Conscious Rats The EI muscles are primary inspiratory muscles (Lane, 2011) The present study provides new informati on on the eupneic activity and ITTO mediated respiratory load compensation responses of EI muscles in conscious rats. Consistent with our hypothesis, the EI muscles of conscious rats were repeatedly recruited in response to an increased respiratory drive e licited during ITTO (F igures 2 3, 2 4) P resentation of an ITTO resulted in recruitment and increased EMG activation of the EI muscles on D ays 1, 3, 5, 7, and 10. The percentage change in the activity of the EI muscle was consistently and repeatedly increa sed during an ITTO presentation, as compared to the basel ine activity. Thus, repeated presentations of ITTO, both in a single trial and over activity. The pattern of responses to an indi vidual ITTO including latency to Onset resp onse and the frequency of Phasic EI muscle activity during an ITTO did not change across trial days ( Figure 2 5) ITTO conditioning had no effect on EI muscle peak to peak frequency on D ays 1, 3, 5 and 7 in most phases of the trial On ITTO day 10 however, peak to peak frequency decreased during ITTO trial both in the ex perimental and control groups (F igure 2 6).

PAGE 150

150 The primary functions of the EI muscle s are to maintain mechanical stability of the chest wall and to aid in the upward and outward movem ent of the rib cage in concert with diaphragmatic contraction (Feldman, 1986, De Troyer et al., 2005) In our study, the EI muscles were always recruited on ITTO presentation. This suggests that in conscious animal s EI muscles are activated to maintain chest wall stability as part of the respiratory load compensation response However, the magnitude of EI muscle dependent on the ani during which ITTO was applied and influenced by modulation via EI muscle afferent s and vagal feedback. To better understand the distribution of the variability in the observed responses we divi ded the animals into H igh and L ow responding groups based on their Onset resp onses to an ITTO presentation (F igure 2 7). 50% percent of conscious rats were termed as H igh responders. The remaining animals L ow responders, decreased as ITTO was sustained for multiple breaths This pattern observed on the first day of trial persisted throughout our study time period of t en days. These High and L ow responding groups of animals further demonstrate in conscious animals (Dick et al., 1982) Repeated exposure to ITTO, a stressful respiratory stimu lus, has been shown to generate anxiety in conscious rats (Pate and Davenport, 2012a) The between animal, within species/ strain difference in Onset re sponse is consistent with anxiety measures (Gomes Vde et al., 2013) of high and low anxiety rats within a strain

PAGE 151

151 usually only observed in conscious studies. The subgroups of High and L ow responders may potentially result from this segregation in anxiety responses. W e also analyzed th e power spectral density (PSD) of the EI EMG during eupneic breathing for ITTO days 1 and 10. No significant differences were found in the median m ) and centroid c ) frequencies after ITTO conditioning in the EI EMG during eupnea ( Figure 2 9). This sugg ests that the EI muscle motor recruitment does not show plasticity with ITTO conditioning and thus, remains unchanged during unloaded breathing. The variability in the power of the signal across its f requency distribution is shown in Figure 2 8 This figur e reflects the variability seen in the activity of intercostal muscles during eupneic breathing in conscious rats. Currently, there are no published reports describing similar data in conscious rats. PSD has been utilized to investigate motor unit recruitm ent (Seven et al., 2013) shifts in frequency conte nt of an EMG signal (Mannion and Dolan, 1994, Spahija et al., 2005) and evaluation of muscle fatigue (Lindstrom et al., 1970, Beck et al., 1997) The conduction velocity of the nerve fibers inner vating the muscle influence PSD (Lindstrom et al., 1970, 1971) and future comparisons in diseased or injured animals can provide valuable insight on the health of the central nervous system, and resulting effects on the EI muscle PSD. EI Muscle Respiratory Load Compensation in Conscious Rats After Unilateral c SCI The EI muscles are innervated by the thoracic spinal cord which receives a n integrated complex combination of direct and indirect inputs from the VRG (Lipski et al., 1994, Kanjhan et al., 1995, de Almeida and Kirkwood, 2013) DRG (de Castro et al., 1994) cervical spinal cord (Lipski et al., 1993, Tian and Duffin, 1996b, a) and cortical mo tor tracts (Rikard Bell et al., 1985a, b, Saji and Miura, 1990) Injury to the spinal cord

PAGE 152

152 above the thoracic level, such as in c SCI disrupts this descending drive. The results of this study demonstrate that MG amplitude activity of bilateral EI muscles and their contribution to eupnea is significantly decreased o ne week after unilateral c SCI. Previous studies have reported a loss of intercostal muscle eupneic activity and its recovery to non injured levels t wo week s after c SCI in anesthetized rats (Dougherty et al., 2012a) Our results demonstrate that activity in EI muscles during eupnea and respiratory load compensation is present one week after c SCI in conscious rats earlier that that observed in anesthetized animals (Dougherty et al., 2012a) In the prese nt study, application of ITTO in conscious rats resulted in bilaterally increased EI EMG amplitude activity, significant on the contralateral side one week after c SCI. Additionally, t en days of ITTO conditioning resulted in a similar pattern of activation between ipsilateral and contralateral side of EI muscles in the C2HS+ITTO group On the other hand, the ITTO Day 10 peak in the C2HS+no ITTO group represents the influence of endogenous plasticity in the thoracic spinal cord and natural recovery of EI muscles at approximately two weeks after c SCI. In this group, ip silateral peak remained significantly decreased and some improvements were observed in the contralateral peak T herefore, ITTO conditioning may induce neuroplastic changes in the thoracic spinal cord of c SCI injured rat s by activation of load related sensory motor pathways (Davenport et al., 1985, Davenport et al., 1993, Davenport et al., 2010) and via existing anatomical interconnectivity between phrenic, lower cervical spinal c ord and intercostal motorneurons (Tian and Duffin, 1996b, a, Dimarco and Kowalski, 2013a, b) Peak to peak frequency was significantly decreased compared to baseline values,

PAGE 153

153 within a group. ITTO conditioning significantly decreases the peak to peak frequency in normal conscious rats (Chapter 2) which is similar to o u r results in c SCI animals. T herefore, we suggest that after c SCI, conscious rats respon d to ITTO and ITTO conditioning primarily by modulation of the amplitude activity during ITTO mechanical loading The contribution of EI muscles to respiratory activity is greater in the c onscious state (Pagliardini et al., 2012) and the results of this study support with this finding Application of ITTO repeatedly such as during ITTO conditioning in conscious rats may drive mutual activation of intercostal and diaphragm muscles via phrenic intercostal interconnectivity and may therefore induce neuroplastic c hanges in thoracic spinal cord i n c SCI rats To test this hypothesis, we determined the effect of ITTO conditioning on the EI muscle activit y during a H ypercapnia challenge. ITTO conditioned conscious rats with unilateral c SCI exhibit frequency modulation of their EI muscle responses without significant changes in the amplitude responses during a H ypercapnia challenge. In the present study, t hese responses predominantly reflect the stimulating component of central chemoreceptor activity in the conscious state, which is counteracted by hyperoxia effects on peripheral chemoreceptors (Gautier et al., 1986) The presence of background hyperox ia in a hypercapneic challenge significantly blunts frequency responses in urethane anesthetized mice and is attributed to suppression of carotid body functioning by O 2 and is not influenced by the CO 2 content of the inhaled gas mixture (Pokorski et al., 2005) Investigations in conscious cats report that ventilatory responses to H ypercapnia are inconsistent, with occasional decreases in r espiratory frequency and increases in tidal volume, but not significantly different than

PAGE 154

1 54 their responses to normoxic hypercapnia (Gautier et al., 1986) The Sham+ no ITTO group in our study reflects the intact conscious rat response to a H ypercapnia ch allenge. No significant differences were found in the EI EMG amplitude ( Figure 4 2) in this group. However, Ti, Te and Ttot were significantly decreased and consequently the peak to peak frequency was significantly increased in this group ( Figure s 4 4 and 4 6 ). On the other hand, ITTO conditioning in the C2HS animals did not change Ti and Ttot but significantly increased Te, decreased the duty cycle and decreased the peak to peak fr equency ( Figure s 4 4 and 4 6 ). These changes in frequency were signi ficantly different between C2HS+ ITTO and othe r treatment groups ( Figure 4 5 ). Responses from the C2HS+ no ITTO group reflect the endogenous plasticity that occurs in the intercostal motor system after unilateral c SCI (Dougherty et al., 2012a) Therefore, left to recover without any intervention, the EI muscles of conscious rat s with unilateral c SCI respond to Hypercapnia similar to r ats in the Sham+ITTO and Sham+ no ITTO groups. Dougherty et al found that intercostal muscle EMG recovers to non injured values two weeks after c SCI (Dougherty et al., 2012a) Our results are similar to the above finding and are evident from the activity of the EI muscles in the C2HS+ no ITTO group. Although a bilateral decrease in peak EI EMG amplitude was observed in animals with unilateral c SCI, the contralateral peak slightly greater than ip silateral peak ea and in response to ITTO ( Chapter 3). Values of e xpressed as arbitrary units as seen in Figure 4 2 and calculated EI motor effort in Figure 4 3 illustrate similar effects on eupneic and hypercapneic EI EMG and le nd support to this previous finding. Therefore, we suggest

PAGE 155

155 that ITTO conditioning induces neuroplastic changes in the thoracic spinal cord of conscious rats with unilateral c SCI. However these changes are predominantly directed toward improvements in con tralateral EI EMG function. Intercostal motor activity is heavily modulated via interneuronal connections between the phrenic and thoracic spinal cord levels and within different levels of the thoracic motorneuron pools (Kirkwood et al., 1988, de Almeida and Kirkwood, 2013) This anatomical connectivity has been attributed with the physiological activation of diaphragm activity during high frequency stimulation of the thoracic spinal cord in C1 transected anestheti zed rats (Kowalski et al., 2013) Therefore, it is likely that increases in contralateral peak amplitude activity observed in this study may drive respiratory recovery of ipsilateral diaphragm and/ or bilateral intercostal m uscles at other thoracic levels through the existing phrenic intercostal and intercostal intercostal circuitry. Furtherm ore, changes in activity of one muscle group such as EI may not be a reliable predictor of the effects of ITTO conditioning on the functioning of the respiratory motor system as a whole. Therefore, ITTO conditioned conscious rats with unilateral c SCI resp ond to Hypercapnia primarily by decreasing the peak to peak frequency compared to Baseline within group and across groups during Hypercapnia. In conclusion, we have demonstrated that, ten days on ITTO conditioning alters eupneic and hypercapneic responses in conscious rats with unilateral c SCI. F uture studies to investigate the underlying mechanisms of ITTO conditioning and its influence of other respiratory muscles are necessary

PAGE 156

156 R espiratory Load Compensation and Magnitude Estimation in an Individual with Chronic Incomplete c SCI Exposure to a cu te intermittent hypoxia (AIH) with elevated levels of CO 2 has been shown to induce ventilatory LTF in both animal models (Vinit et al., 2009, Dale Nagle et al., 2010) and awake humans (Harris et al., 2006, Mateika and Sandhu, 2011, Tester et al., 2014) Based on our results, a decrease in mean slope for P vs. resistance plot (Figure 5 2) and a corresponding increase in mean slope for AF vs. resistance (Figure 5 3) on AIH Da y 10 demonstrates that the subject in our study improved her ability to load compensate. Also, pulmonary function tests showed a significant and consistent improvement in %FVC (19.4%) and %FEV1 (44.61%) after AIH treatment (Table 5 1) However, no improvem ents in maximal inspiratory (MIP) and expiratory (MEP) pressures were noted (Table 5 2) Values for MIP and MEP were not significantly different on any of the time points measured in our study. T he possibility that the reported improvements in P and AF may be due to the combined influence of AIH treatment and a learning effect of repeated exposure to the IRL protocol cannot be ruled out However, we also see improvements in FVC and FEV1 values, which suggest that the study paradigm may have a beneficial eff ect on the sub ject's ventilatory functioning. Th e subject reported no changes in perceptual sensitivity (Figure 5 4) The baseline perceptual sensitivity of our study subject was slightly lower but comparable to that of healthy conscious adults from previo us reports from our lab (Tsai et al., 2013) Previous reports on quadriplegics have reported a much greater reduction in the value of the power exponent (Got tfried et al., 1984) The perceptua l sensitivity is greatly depende nt on the respi ratory drive, breathing pattern and respiratory muscle fatigue (Gandevia et al., 1981) In the study subject injury severity also plays a key role in

PAGE 157

157 determining the perceptual sensitivity to IRL. The subject in our study has a chronic and less severe form of SCI that may have partly preserved the afferent and efferent pathways from the thoracic muscle receptor s that are required to modulate respiratory responses (von Euler, 1973, Gottfried et al., 1984) This would indicate that our study paradigm might affect the discriminative but not affective processing of IRL (Davenport and Vovk, 2009) Thus, even if the subject learned to differentiate between loads better than Baseline and generate higher airflows and greater negative pressure in response to the loads the perceptual response to each load remained the same over the treatment time. This is consistent with findings from a previous study demonstrating that respiratory learning is not i nfluenced by the physiological responses it elicits (Van den Bergh et al., 1998) In summary, we conclude that chronic exposure to AIH with elevated CO 2 levels improved pulmonary function without altering respi ratory muscle pressure generating capacity in the study subject Also ventilatory load compensation in response to added IRL improved and the perceptual sensitivity to IRL wa s not affected by ten days of AIH treatment. Consistent improvements in pulmonary function tests and ventilatory load compensation response s suggest that this paradigm may be a potentially beneficial rehabilitation intervention for individuals with chronic incomplete c SCI However, further studies are needed to determine the clinical applicability of AIH treatment in individuals with chronic SCI Mechanism of Action of ITTO Mediated EI Muscle Responses after c SCI The research presented in this dissertation determined the physiological activity of EI muscles in normal conscious animals and the mechanism of action of ITTO mediated EI responses in conscious rats with out (Chapter 2) and with unilateral c SCI

PAGE 158

158 (Chapter 3 and Figure 6 1 ) Respiratory rhythm generated in the brainstem is transmitted to the motorneuron s of the phrenic and thora cic spinal cord, via bulbospinal and propriospinal pathways. These centers are also under direct control from higher brain centers via the corticospinal tracts. Propriospinal connections from the phrenic motorneuron pool modulate thoracic motorneuron activ ity. The phrenic and thoracic motorneuron pools innervate the diaphragm and EI muscles, respectively. Activity of the EI muscles provides mechanical stability to the chest wall and aids in chest wall movement s Thus the activity of EI and diaphragm muscle s together results in lung movement and thereby ventilation. Application of an ITTO temporarily restricts this lung movement thereby driving increases in the central respiratory drive. Afferent modulat ion of the EI muscles is via muscle spindles, tendon or gans and joint recepto rs, which act at spinal cord, brainstem and cortical levels. Afferent vagal feedback from the lung also modulate s respiratory muscle activity. Respiratory chemoreception is by careful regulation of CO 2 O 2 and pH via peripheral and ce ntral chemoreceptors that terminate in the brainstem and cortex, and drive a djustments to the respiratory neural output. The collective integrated input of these afferent mechanisms at the segmental, brainstem and cortical levels is res ponsible for increas e s in central respiratory drive on application of ITTO which in turn, increase s respiratory muscle output as evidenced by increases in EI EMG amplitude activity. Injury to the cervical spinal cord at the C2 level (C2HS) disrupts the descending input to the phrenic and thoracic motorneuron pools. Therefore unilateral c SCI results in a reduction of input from the central respiratory drive to the phrenic and thoracic

PAGE 159

159 respiratory motorneuron pools. T his results in a decrease of EI muscle activity during baseline and respiratory load compensation. However, the level at which these corticospinal, bulbospinal and propriospinal descending inputs cross the midline is un known. From our results, it can be speculated that the level of this crossing is below the C2 spinal cord level. This explains the presence of bilateral EI EMG activity, significantly reduced on the ipsilateral side and also the rec ruitment of bilateral EI EMG with significant increases on the contralateral side, one week after unilateral c SCI. Application of ITTO repeatedly such as during ITTO conditioning in conscious rats may strengthen the descending pathways carrying central respiratory drive activity and facilitate mutual activation of intercostal and diaphragm muscles via the phrenic intercostal interconnectivity thereby inducing neuroplastic changes in thoracic spinal cord i n conscious rats after c SCI Significance The EI muscles are primary inspiratory muscles (Lane, 2011) and most of what is known about their activity is from studies in anesthetized animal studies (DiMarco et al., 1989, Romaniuk et al., 1992, De Troyer et al., 2005, Dimarco and Kowalski, 2013a, b) Anesthesia abolishes the cortical motor and affective modulation of respiratory activity (Kir kwood et al., 1982b) and depresses EI muscle respiratory activity (Pagliardini et al., 2012) Rats have long been preferred as animal models to study functioning of the respiratory system after disease or trauma (Bianchi et al., 1995, Kastner and Gauthier, 2008) and little is know n about EI respiratory muscle function in conscious rats. The research presented in this dissertation provides new information on the eupneic acti vity and respiratory load compensation responses of the EI muscles in conscious rats (Figure 6 1) By utilizing intrinsic transient tracheal occlusions (ITTO) to elicit respiratory

PAGE 160

160 load compensation the studies in this dissertation have demonstrated EI mus cle responses in normal r ats in the conscious state (Chapter 2) Injury to any level of the spinal cord can cause respiratory muscle dysfunction, with damage to higher levels producing deficits of greatest severity. About fifty percent of reported SCI cas es are at the cervical spinal cord level (Winslow and Rozovsky, 2003) and pulmonary complications remain the major cause of death in this patient population (DeVivo et al., 1999, Zimmer et al., 2007) The life expectancy after SCI decreases with increasing age and injury severity (NSCISC, 2013) ; and these outcomes have not improved over the years (Strauss et al., 200 6, van den Berg et al., 2010) The most common complications in SCI patients are pneumonia and lung atelectasis (Jackson and Groomes, 1994) Pneumonia can arise from the inability of patients to clear their airways with cough. Atele ctasis is defined as the collapse of a part or entire lung. This may arise due to an increase in lung elastic recoil or reduced passive chest wall recoil. In SCI patients both of these conditions exist the former due to denervation of the diaphragm and the latter most likely due to denervation and inadequate intercostal and abdominal muscle function. In animal studies, a decrease in augmented breath volume has been reported in chronically injured SCI rats (Golder et al., 2005a) This dissertation describes the effect s of unilateral c SCI (Figure 6 1) on the EI EMG responses during eupnea and ITTO mediated respiratory load compensation of conscious rats (Chapter 3) Also, we have determined the im pact of ten days of ITTO conditioning on EI muscle neurophysiological function in conscious rats with unilateral c SCI (Chapter 4).

PAGE 161

161 A disruption o f the efferent input to respiratory muscles due to SCI resu lts in reduced motor function which in turn alters compensate (Axen, 1982, Kelling et al., 1985) Also, depending on level and severity of injury, SCI interrupts the ascending afferent input from respiratory and accessory re spiratory muscles. This afferent feedback plays a key role in the modulation of breathing (von Euler, 1973) Respiratory sensations and respiratory system functioning are highly influenced by each other (Davenport and Vovk, 2009, von Leupoldt et al., 2010) Therefore, rehabil itation after SCI necessitates restoration of the respiratory motor function as well as ensuring that the respiratory perceptual sensitivity of the injured individual does not hinder their capability to respond to mechanical and/ or chemical challenges. Ou r research has shed light on the respiratory load compensation abilities and perceptual sensitivity to graded inspiratory resistive loads in an individual with chronic incomplete c SCI. Improvements in these responses were observed after ten days of AIH tr eatment Thus experiments performed as a part of this dissertation have provided information on the functioning of EI muscles in normal and unilateral c SCI conscious rats (Figure 6 1) T he applicability of ITTO as a respiratory muscle strengthening para digm after unilateral c SCI is discussed. Further research is needed before clinical applicability of this model can be established. Finally, we have describe d the importance of evaluating respiratory perceptual responses in individuals with c SCI and the influence of AIH treatment is discussed (Chapter 5) Future Directions The progression of studies to determine the interactions between the respiratory pump muscles (diaphragm and intercostals) with the abdominal (external oblique and

PAGE 162

162 rectus abdominus) mus cles is essential Cross correlation analyses of their EMG activities during eupnea and respiratory loading may provide valuable information on their activation patterns and help detail compensatory mechanisms in conscious rats. Additionally, between anima l variability arising from the influence of high and low anxiety groups observed in conscious animals and its impact on the variability of EI muscle responses needs to be further investigated. To establish the clinical applicability of ITTO for use after unilateral c SCI, further studies that determine respiratory volume changes corresponding to respiratory muscle EMG activities are required. Als o, the impact s of ITTO conditioning on diaphragm and abdominal muscle neurophysiology are need ed for further app lication of ITTO in conscious rats with c SCI.

PAGE 163

163 Figure 6 1. M echanism of action of ITTO mediated EI muscle responses in conscious rats after unilateral c SCI

PAGE 164

164

PAGE 165

165 LIST OF REFERENCES Abe T, Kusuhara N, Yoshimura N, Tomita T, Easton PA (Differential respiratory activity of four abdominal muscles in humans. J Appl Physiol 80:1379 1389.1996). Alexander Miller S, Davenport PW (Perception of multiple breath inspiratory resistive loads in males and females. Biol Psychol 84:147 149.2010). Ali lain WJ, Silver J (Shedding light on restoring respiratory function after spinal cord injury. Front Mol Neurosci 2:18.2009). Altose MD, Kelsen SG, Stanley NN, Levinson RS, Cherniack NS, Fishman AP (Effects of hypercapnia on mouth pressure during airway occ lusion in conscious man. J Appl Physiol 40:338 344.1976). Arita H, Bishop B (Firing profile of diaphragm single motor unit during hypercapnia and airway occlusion. J Appl Physiol 55:1203 1210.1983a). Arita H, Bishop B (Responses of cat's internal intercost al motor units to hypercapnia and lung inflation. J Appl Physiol 54:375 386.1983b). Aslan SC, Chopra MK, McKay WB, Folz RJ, Ovechkin AV (Evaluation of respiratory muscle activation using respiratory motor control assessment (RMCA) in individuals with chron ic spinal cord injury. J Vis Exp.2013). ATS., ERS. (ATS/ERS Statement on respiratory muscle testing. Am J Respir Crit Care Med 166:518 624.2002). Axen K (Ventilatory responses to mechanical loads in cervical cord injured humans. J Appl Physiol 52:748 756.1 982). Axen K, Bergofsky EH (Thoracic reflexes stabilizing loaded ventilation in normal and cord injured man. J Appl Physiol 43:339 346.1977). Axen K, Haas SS (Effect of thoracic deafferentation on load compensating mechanisms in humans. J Appl Physiol 52:7 57 767.1982). Axen K, Pineda H, Shunfenthal I, Haas F (Diaphragmatic function following cervical cord injury: neurally mediated improvement. Arch Phys Med Rehabil 66:219 222.1985). Bailey EF, Jones CL, Reeder JC, Fuller DD, Fregosi RF (Effect of pulmonary stretch receptor feedback and CO(2) on upper airway and respiratory pump muscle activity in the rat. J Physiol 532:525 534.2001). Bavis RW, Johnson RA, Ording KM, Otis JP, Mitchell GS (Respiratory plasticity after perinatal hypercapnia in rats. Respir Phys iol Neurobiol 153:78 91.2006).

PAGE 166

166 Baydur A, Adkins RH, Milic Emili J (Lung mechanics in individuals with spinal cord injury: effects of injury level and posture. J Appl Physiol 90:405 411.2001). Beck J, Sinderby C, Lindstrom L, Grassino A (Diaphragm interfere nce pattern EMG and compound muscle action potentials: effects of chest wall configuration. J Appl Physiol 82:520 530.1997). Bellingham MC, Lipski J (Respiratory interneurons in the C5 segment of the spinal cord of the cat. Brain Res 533:141 146.1990). Ben Dov I, Zlobinski R, Segel MJ, Gaides M, Shulimzon T, Zeilig G (Ventilatory response to hypercapnia in C(5 8) chronic tetraplegia: the effect of posture. Arch Phys Med Rehabil 90:1414 1417.2009). Bernhardt V, Garcia Reyero N, Vovk A, Denslow N, Davenport P W (Tracheal occlusion modulates the gene expression profile of the medial thalamus in anesthetized rats. J Appl Physiol (1985) 111:117 124.2011a). Bernhardt V, Hotchkiss MT, Garcia Reyero N, Escalon BL, Denslow N, Davenport PW (Tracheal occlusion condition ing in conscious rats modulates gene expression profile of medial thalamus. Front Physiol 2:24.2011b). Bianchi AL, Denavit Saubie M, Champagnat J (Central control of breathing in mammals: neuronal circuitry, membrane properties, and neurotransmitters. Phys iol Rev 75:1 45.1995). Bolser DC, Davenport PW (Volume timing relationships during cough and resistive loading in the cat. J Appl Physiol 89:785 790.2000). Bolser DC, Jefferson SC, Rose MJ, Tester NJ, Reier PJ, Fuller DD, Davenport PW, Howland DR (Recovery of airway protective behaviors after spinal cord injury. Respir Physiol Neurobiol 169:150 156.2009). Bolser DC, Lindsey BG, Shannon R (Medullary inspiratory activity: influence of intercostal tendon organs and muscle spindle endings. J Appl Physiol 62:104 6 1056.1987). Bolser DC, Remmers JE (Synaptic effects of intercostal tendon organs on membrane potentials of medullary respiratory neurons. J Neurophysiol 61:918 926.1989). Bonaert A, Voisin V, Caron N, Legrand A (Do segmental reflexes play a role in the d istribution of external intercostal EMG activity in the rabbit? Respir Physiol Neurobiol 183:1 9.2012). Borg GA (Psychophysical bases of perceived exertion. Med Sci Sports Exerc 14:377 381.1982).

PAGE 167

167 Bradley TD, Chartrand DA, Fitting JW, Killian KJ, Grassino A (The relation of inspiratory effort sensation to fatiguing patterns of the diaphragm. Am Rev Respir Dis 134:1119 1124.1986). Brichant JF, De Troyer A (On the intercostal muscle compensation for diaphragmatic paralysis in the dog. J Physiol 500 ( Pt 1):245 253.1997). Brown R, DiMarco AF, Hoit JD, Garshick E (Respiratory dysfunction and management in spinal cord injury. Respir Care 51:853 868;discussion 869 870.2006). Bruce EN, Goldman MD (High frequency oscillations in human respiratory electromyograms duri ng voluntary breathing. Brain Res 269:259 265.1983). BuSha BF, Stella MH (State and chemical drive modulate respiratory variability. J Appl Physiol (1985) 93:685 696.2002). Butler JE, Lim J, Gorman RB, Boswell Ruys C, Saboisky JP, Lee BB, Gandevia SC (Post erolateral surface electrical stimulation of abdominal expiratory muscles to enhance cough in spinal cord injury. Neurorehabil Neural Repair 25:158 167.2011). Cappello M, de Troyer A (Interaction between left and right intercostal muscles in airway pressur e generation. J Appl Physiol (1985) 88:817 820.2000). Chan PY, von Leupoldt A, Bradley MM, Lang PJ, Davenport PW (The effect of anxiety on respiratory sensory gating measured by respiratory related evoked potentials. Biol Psychol 91:185 189.2012). Chen R, Collins SJ, Remtulla H, Parkes A, Bolton CF (Needle EMG of the human diaphragm: power spectral analysis in normal subjects. Muscle Nerve 19:324 330.1996). Clancy J, McVicar A (The respiratory system and homeostasis. Br J Theatre Nurs 6:16 20, 22.1996). Cla rk FJ, von Euler C (On the regulation of depth and rate of breathing. J Physiol 222:267 295.1972). Conta Steencken AC, Stelzner DJ (Loss of propriospinal neurons after spinal contusion injury as assessed by retrograde labeling. Neuroscience 170:971 980.201 0). Critchlow V, von Euler C (Intercostal Muscle Spindle Activity and Its Gamma Motor Control. J Physiol 168:820 847.1963). D'Angelo E, Miserocchi G, Agostoni E (Effect of rib cage or abdomen compression at iso lung volume on breathing pattern. Respir Phys iol 28:161 177.1976).

PAGE 168

168 D'Angelo E, Monaco A, Pecchiari M (Motor control of the diaphragm in anesthetized rabbits. Respir Physiol Neurobiol 170:141 149.2010). Dale Nagle EA, Hoffman MS, MacFarlane PM, Satriotomo I, Lovett Barr MR, Vinit S, Mitchell GS (Spina l plasticity following intermittent hypoxia: implications for spinal injury. Ann N Y Acad Sci 1198:252 259.2010). Davenport PW, Dalziel DJ, Webb B, Bellah JR, Vierck CJ, Jr. (Inspiratory resistive load detection in conscious dogs. J Appl Physiol 70:1284 12 89.1991). Davenport PW, Freed AN, Rex KA (The effect of sulfur dioxide on the response of rabbits to expiratory loads. Respir Physiol 56:359 368.1984). Davenport PW, Reep RL, Thompson FJ (Phrenic nerve afferent activation of neurons in the cat SI cerebral cortex. J Physiol 588:873 886.2010). Davenport PW, Shannon R, Mercak A, Reep RL, Lindsey BG (Cerebral cortical evoked potentials elicited by cat intercostal muscle mechanoreceptors. J Appl Physiol 74:799 804.1993). Davenport PW, Thompson FJ, Reep RL, Freed AN (Projection of phrenic nerve afferents to the cat sensorimotor cortex. Brain Res 328:150 153.1985). Davenport PW, Vovk A (Cortical and subcortical central neural pathways in respiratory sensations. Respir Physiol Neurobiol 167:72 86.2009). de Almeida A T, Al Izki S, Denton ME, Kirkwood PA (Patterns of expiratory and inspiratory activation for thoracic motoneurones in the anaesthetized and the decerebrate rat. J Physiol 588:2707 2729.2010). de Almeida AT, Kirkwood PA (Multiple phases of excitation and inh ibition in central respiratory drive potentials of thoracic motoneurones in the rat. J Physiol 588:2731 2744.2010). de Almeida AT, Kirkwood PA (Specificity in monosynaptic and disynaptic bulbospinal connections to thoracic motoneurones in the rat. J Physio l 591:4043 4063.2013). de Castro D, Lipski J, Kanjhan R (Electrophysiological study of dorsal respiratory neurons in the medulla oblongata of the rat. Brain Res 639:49 56.1994). De Troyer A (Differential control of the inspiratory intercostal muscles durin g airway occlusion in the dog. J Physiol 439:73 88.1991). De Troyer A (Role of joint receptors in modulation of inspiratory intercostal activity by rib motion in dogs. J Physiol 503 ( Pt 2):445 453.1997). De Troyer A (Respiratory effect of the lower rib di splacement produced by the diaphragm. J Appl Physiol (1985) 112:529 534.2012).

PAGE 169

169 De Troyer A, Estenne M (Coordination between rib cage muscles and diaphragm during quiet breathing in humans. J Appl Physiol 57:899 906.1984). De Troyer A, Farkas GA (Inspirator y function of the levator costae and external intercostal muscles in the dog. J Appl Physiol 67:2614 2621.1989). De Troyer A, Farkas GA (Linkage between parasternals and external intercostals during resting breathing. J Appl Physiol (1985) 69:509 516.1990) De Troyer A, Farkas GA, Ninane V (Mechanics of the parasternal intercostals during occluded breaths in dogs. J Appl Physiol 64:1546 1553.1988). De Troyer A, Kelly S, Macklem PT, Zin WA (Mechanics of intercostal space and actions of external and internal intercostal muscles. J Clin Invest 75:850 857.1985). De Troyer A, Kelly S, Zin WA (Mechanical action of the intercostal muscles on the ribs. Science 220:87 88.1983). De Troyer A, Kirkwood PA, Wilson TA (Respiratory action of the intercostal muscles. Physio l Rev 85:717 756.2005). De Troyer A, Ninane V (Respiratory function of intercostal muscles in supine dog: an electromyographic study. J Appl Physiol 60:1692 1699.1986). De Troyer A, Ninane V, Gilmartin JJ, Lemerre C, Estenne M (Triangularis sterni muscle u se in supine humans. J Appl Physiol 62:919 925.1987). De Troyer AD (The canine phrenic to intercostal reflex. J Physiol 508 ( Pt 3):919 927.1998). Decima EE, von Euler C, Thoden U (Intercostal to phrenic reflexes in the spinal cat. Acta Physiol Scand 75:56 8 579.1969). DeVivo MJ, Krause JS, Lammertse DP (Recent trends in mortality and causes of death among persons with spinal cord injury. Arch Phys Med Rehabil 80:1411 1419.1999). Dick TE, Parmeggiani PL, Orem J (Intercostal muscle activity during sleep in th e cat: an augmentation of expiratory activity. Respir Physiol 50:255 265.1982). Dick TE, Parmeggiani PL, Orem JM (Intercostal muscle activity of the cat in the curled, semiprone sleeping posture. Respir Physiol 56:385 394.1984). DiMarco AF, Budzinska K, Su pinski GS (Artificial ventilation by means of electrical activation of the intercostal/accessory muscles alone in anesthetized dogs. Am Rev Respir Dis 139:961 967.1989).

PAGE 170

170 Dimarco AF, Kowalski KE (Activation of inspiratory muscles via spinal cord stimulation Respir Physiol Neurobiol 189:438 449.2013a). Dimarco AF, Kowalski KE (Spinal pathways mediating phrenic activation during high frequency spinal cord stimulation. Respir Physiol Neurobiol 186:1 6.2013b). Dimarco AF, Romaniuk JR, Supinski GS (Mechanical ac tion of the interosseous intercostal muscles as a function of lung volume. Am Rev Respir Dis 142:1041 1046.1990). DiMarco AF, Supinski GS, Petro JA, Takaoka Y (Evaluation of intercostal pacing to provide artificial ventilation in quadriplegics. Am J Respir Crit Care Med 150:934 940.1994). DiMarco AF, Takaoka Y, Kowalski KE (Combined intercostal and diaphragm pacing to provide artificial ventilation in patients with tetraplegia. Arch Phys Med Rehabil 86:1200 1207.2005). DiMarco AF, Wolfson DA, Gottfried SB, Altose MD (Sensation of inspired volume in normal subjects and quadriplegic patients. J Appl Physiol 53:1481 1486.1982). Dobbins EG, Feldman JL (Brainstem network controlling descending drive to phrenic motoneurons in rat. J Comp Neurol 347:64 86.1994). Do peralski NJ, Fuller DD (Long term facilitation of ipsilateral but not contralateral phrenic output after cervical spinal cord hemisection. Exp Neurol 200:74 81.2006). Dougherty BJ, Lee KZ, Gonzalez Rothi EJ, Lane MA, Reier PJ, Fuller DD (Recovery of inspir atory intercostal muscle activity following high cervical hemisection. Respir Physiol Neurobiol 183:186 192.2012a). Dougherty BJ, Lee KZ, Lane MA, Reier PJ, Fuller DD (Contribution of the spontaneous crossed phrenic phenomenon to inspiratory tidal volume i n spontaneously breathing rats. J Appl Physiol 112:96 105.2012b). Dow DE, Zhan WZ, Sieck GC, Mantilla CB (Correlation of respiratory activity of contralateral diaphragm muscles for evaluation of recovery following hemiparesis. Conf Proc IEEE Eng Med Biol S oc 2009:404 407.2009). Downman CB, Hussain A (Spinal tracts and supraspinal centres influencing visceromotor and allied reflexes in cats. J Physiol 141:489 499.1958). Duffin J, Lipski J (Monosynaptic excitation of thoracic motoneurones by inspiratory neuro nes of the nucleus tractus solitarius in the cat. J Physiol 390:415 431.1987).

PAGE 171

171 Eldridge FL, Vaughn KZ (Relationship of thoracic volume and airway occlusion pressure: muscular effects. J Appl Physiol Respir Environ Exerc Physiol 43:312 321.1977). Ellenberge r HH, Feldman JL (Brainstem connections of the rostral ventral respiratory group of the rat. Brain Res 513:35 42.1990). Estenne M, Heilporn A, Delhez L, Yernault JC, De Troyer A (Chest wall stiffness in patients with chronic respiratory muscle weakness. Am Rev Respir Dis 128:1002 1007.1983). Feldman JL (1986) Neurophysiology of breathing in mammals. In: Handbook of Physiology: The Nervous System, vol. 4 (Vernon B Mountcastle, F. E. B., Stephen R Geiger, ed), pp 463 524: American Physiological Society. Feldm an JL, Loewy AD, Speck DF (Projections from the ventral respiratory group to phrenic and intercostal motoneurons in cat: an autoradiographic study. J Neurosci 5:1993 2000.1985). Fitsimones L, Sapienza CM, Daveport PW (2004) The effect of expiratory muscle strength training on low cervical and thoracic SCI patients. No URL available. Fraigne JJ, Orem JM (Phasic motor activity of respiratory and non respiratory muscles in REM sleep. Sleep 34:425 434.2011). Fregosi RF, Mitchell GS (Long term facilitation of in spiratory intercostal nerve activity following carotid sinus nerve stimulation in cats. J Physiol 477 ( Pt 3):469 479.1994). Fuller DD, Doperalski NJ, Dougherty BJ, Sandhu MS, Bolser DC, Reier PJ (Modest spontaneous recovery of ventilation following chroni c high cervical hemisection in rats. Exp Neurol 211:97 106.2008). Fuller DD, Golder FJ, Olson EB, Jr., Mitchell GS (Recovery of phrenic activity and ventilation after cervical spinal hemisection in rats. J Appl Physiol 100:800 806.2006). Fuller DD, Johnson SM, Olson EB, Jr., Mitchell GS (Synaptic pathways to phrenic motoneurons are enhanced by chronic intermittent hypoxia after cervical spinal cord injury. J Neurosci 23:2993 3000.2003). Gandevia SC, Killian KJ, Campbell EJ (The effect of respiratory muscle fatigue on respiratory sensations. Clin Sci (Lond) 60:463 466.1981). Gandevia SC, Kirkwood PA (Spinal breathing: stimulation and surprises. J Physiol 589:2661 2662.2011).

PAGE 172

172 Gautier H, Bonora M, Gaudy JH (Ventilatory response of the conscious or anesthetized cat to oxygen breathing. Respir Physiol 65:181 196.1986). Gerst DG, 3rd, Yokhana SS, Carney LM, Lee DS, Badr MS, Qureshi T, Anthouard MN, Mateika JH (The hypoxic ventilatory response and ventilatory long term facilitation are altered by time of day and rep eated daily exposure to intermittent hypoxia. J Appl Physiol 110:15 28.2011). Golder FJ, Davenport PW, Johnson RD, Reier PJ, Bolser DC (Augmented breath phase volume and timing relationships in the anesthetized rat. Neurosci Lett 373:89 93.2005a). Golder F J, Fuller DD, Davenport PW, Johnson RD, Reier PJ, Bolser DC (Respiratory motor recovery after unilateral spinal cord injury: eliminating crossed phrenic activity decreases tidal volume and increases contralateral respiratory motor output. J Neurosci 23:249 4 2501.2003). Golder FJ, Reier PJ, Bolser DC (Altered respiratory motor drive after spinal cord injury: supraspinal and bilateral effects of a unilateral lesion. J Neurosci 21:8680 8689.2001a). Golder FJ, Reier PJ, Davenport PW, Bolser DC (Cervical spinal cord injury alters the pattern of breathing in anesthetized rats. J Appl Physiol 91:2451 2458.2001b). Golder FJ, Zabka AG, Bavis RW, Baker Herman T, Fuller DD, Mitchell GS (Differences in time dependent hypoxic phrenic responses among inbred rat strains. J Appl Physiol (1985) 98:838 844.2005b). Gomes Vde C, Hassan W, Maisonnette S, Johnson LR, Ramos A, Landeira Fernandez J (Behavioral evaluation of eight rat lines selected for high and low anxiety related responses. Behav Brain Res 257:39 48.2013). Goshgari an HG (Developmental plasticity in the respiratory pathway of the adult rat. Exp Neurol 66:547 555.1979). Goshgarian HG (The crossed phrenic phenomenon: a model for plasticity in the respiratory pathways following spinal cord injury. J Appl Physiol 94:795 810.2003). Gottfried SB, Leech I, DiMarco AF, Zaccardelli W, Altose MD (Sensation of respiratory force following low cervical spinal cord transection. J Appl Physiol 57:989 994.1984). Guyenet PG, Abbott SB, Stornetta RL (The respiratory chemoreception conu ndrum: Light at the end of the tunnel? Brain Res.2012).

PAGE 173

173 Hanly PJ, George CF, Millar TW, Kryger MH (Heart rate response to breath hold, valsalva and Mueller maneuvers in obstructive sleep apnea. Chest 95:735 739.1989). Harris DP, Balasubramaniam A, Badr MS, Mateika JH (Long term facilitation of ventilation and genioglossus muscle activity is evident in the presence of elevated levels of carbon dioxide in awake humans. Am J Physiol Regul Integr Comp Physiol 291:R1111 1119.2006). Henke KG, Badr MS, Skatrud JB, Dempsey JA (Load compensation and respiratory muscle function during sleep. J Appl Physiol (1985) 72:1221 1234.1992). Hernandez YM, Lindsey BG, Shannon R (Intercostal and abdominal muscle afferent influence on caudal medullary expiratory neurons that driv e abdominal muscles. Exp Brain Res 78:219 222.1989). Hlastala MP, Berger AJ (2001) Physiology of Respiration. Seattle: Oxford University Press. Holt GA, Johnson RD, Davenport PW (The transduction properties of intercostal muscle mechanoreceptors. BMC Physi ol 2:16.2002). Huang CH, Martin AD, Davenport PW (Effects of inspiratory strength training on the detection of inspiratory loads. Appl Psychophysiol Biofeedback 34:17 26.2009). Hudson AL, Gandevia SC, Butler JE (Common rostrocaudal gradient of output from human intercostal motoneurones during voluntary and automatic breathing. Respir Physiol Neurobiol 175:20 28.2011). Jackson AB, Groomes TE (Incidence of respiratory complications following spinal cord injury. Arch Phys Med Rehabil 75:270 275.1994). Jaiswal PB, Davenport PW (Respiratory Load Compensation Responses of External Intercostal Muscle in Conscious Rats. http://www .fasebj.org/cgi/content/meeting_abstract/26/1_MeetingAbstracts/1147. 10?sid=7eceb2f4 0d4e 4d23 8717 e31d84fd2b4c Last accessed 02/04/2014.2012). Jaiswal PB, Pate KM, Deoghare H, Scheuer D, Davenport PW (2010) Cardiovascular responses to acute and chronic tracheal occlusions in conscious rats. URL http://www.a bstractsonline.com/Plan/ViewAbstract.aspx?sKey=c1700654 f795 4b1a bf99 598c795575f9&cKey=2bc73081 4d40 44ae ac22 c46ed56ecee4&mKey=%7bE5D5C83F CE2D 4D71 9DD6 FC7231E090FB%7d Last accessed 02/04/2014.

PAGE 174

174 Jaiswal PB, Sandhu MS, Fuller DD, Davenport PW (2012 ) Effect of Cervical Spinal Hemisection on Intercostal and Abdominal Motor Patterns during Eupneic Breathing in Conscious Rats. http://www.isarp.org/mediapool/86/867512/dat a/ISARP_2012_Program.pdf Last accessed 02/04/2014. Jaiswal PB, Sandhu MS, Fuller DD, Davenport PW (2013) Increased Bilateral External Intercostal EMG Activity during Tracheal Occlusions (TO) in Conscious Rats with Unilateral Cervical Spinal Cord Injury ( c SCI). URL http://www.fasebj.org/cgi/content/meeting_abstract/27/1_MeetingAbstracts/930.1 0?sid=10b1a749 2b8e 41f9 89d8 a4384a6aafb0 Last accessed 02/04/2014. Jaiswal PB, Tsai HW, Davenport PW (2011) Expiratory Muscle Response to Tracheal Occlusions in Conscious Rats. URL http://www.fasebj.org/cgi/content/meeting_abstract/25/1_MeetingAbstracts/1111. 17?sid=7eceb2f4 0d4e 4d23 8717 e31d84fd2b4c Last accessed 02/04/2014. In: FASEB J. Johnson SM, Creighton RJ (Spinal cord injury induced chang es in breathing are not due to supraspinal plasticity in turtles (Pseudemys scripta). Am J Physiol Regul Integr Comp Physiol 289:R1550 1561.2005). Julius SM, Davenport KL, Davenport PW (Perception of intrinsic and extrinsic respiratory loads in children wi th life threatening asthma. Pediatr Pulmonol 34:425 433.2002). Kanjhan R, Lipski J, Kruszewska B, Rong W (A comparative study of pre sympathetic and Botzinger neurons in the rostral ventrolateral medulla (RVLM) of the rat. Brain Res 699:19 32.1995). Kastne r A, Gauthier P (Are rodents an appropriate pre clinical model for treating spinal cord injury? Examples from the respiratory system. Exp Neurol 213:249 256.2008). Katagiri M, Young RN, Platt RS, Kieser TM, Easton PA (Respiratory muscle compensation for un ilateral or bilateral hemidiaphragm paralysis in awake canines. J Appl Physiol 77:1972 1982.1994). Kellerman BA, Martin AD, Davenport PW (Inspiratory strengthening effect on resistive load detection and magnitude estimation. Med Sci Sports Exerc 32:1859 18 67.2000). Kelling JS, DiMarco AF, Gottfried SB, Altose MD (Respiratory responses to ventilatory loading following low cervical spinal cord injury. J Appl Physiol 59:1752 1756.1985).

PAGE 175

175 Kifle Y, Seng V, Davenport PW (Magnitude estimation of inspiratory resisti ve loads in children with life threatening asthma. Am J Respir Crit Care Med 156:1530 1535.1997). Kim J, Davenport P, Sapienza C (Effect of expiratory muscle strength training on elderly cough function. Arch Gerontol Geriatr 48:361 366.2009). Kinkead R, Du penloup L, Valois N, Gulemetova R (Stress induced attenuation of the hypercapnic ventilatory response in awake rats. J Appl Physiol 90:1729 1735.2001a). Kinkead R, Dupenloup L, Valois N, Gulemetova R (Stress induced attenuation of the hypercapnic ventilato ry response in awake rats. J Appl Physiol (1985) 90:1729 1735.2001b). Kirkwood PA, Munson JB, Sears TA, Westgaard RH (Respiratory interneurones in the thoracic spinal cord of the cat. J Physiol 395:161 192.1988). Kirkwood PA, Sears TA, Stagg D, Westgaard R H (The spatial distribution of synchronization of intercostal motoneurones in the cat. J Physiol 327:137 155.1982a). Kirkwood PA, Sears TA, Tuck DL, Westgaard RH (Variations in the time course of the synchronization of intercostal motoneurones in the cat. J Physiol 327:105 135.1982b). Kirkwood PA, Sears TA, Westgaard RH (Restoration of function in external intercostal motoneurones of the cat following partial central deafferentation. J Physiol 350:225 251.1984). Kirshblum SC, Memmo P, Kim N, Campagnolo D, M illis S (Comparison of the revised 2000 American Spinal Injury Association classification standards with the 1996 guidelines. Am J Phys Med Rehabil 81:502 505.2002). Knafelc M, Davenport PW (Relationship between magnitude estimation of resistive loads, ins piratory pressures, and the RREP P(1) peak. J Appl Physiol 87:516 522.1999). Koehler RC, Bishop B (Expiratory duration and abdominal muscle responses to elastic and resistive loading. J Appl Physiol 46:730 737.1979). Kowalski KE, Hsieh YH, Dick TE, DiMarco AF (Diaphragm activation via high frequency spinal cord stimulation in a rodent model of spinal cord injury. Exp Neurol 247:689 693.2013). Laghi F, Tobin MJ (Disorders of the respiratory muscles. Am J Respir Crit Care Med 168:10 48.2003).

PAGE 176

176 Lane MA (Spinal respiratory motoneurons and interneurons. Respir Physiol Neurobiol.2011). Lane MA, Fuller DD, White TE, Reier PJ (Respiratory neuroplasticity and cervical spinal cord injury: translational perspectives. Trends Neurosci 31:538 547.2008). Lane MA, Lee KZ, Fu ller DD, Reier PJ (Spinal circuitry and respiratory recovery following spinal cord injury. Respir Physiol Neurobiol 169:123 132.2009). Larnicol N, Rose D, Marlot D, Duron B (Spinal localization of the intercostal motoneurones innervating the upper thoracic spaces. Neurosci Lett 31:13 18.1982). Lasserson D, Mills K, Arunachalam R, Polkey M, Moxham J, Kalra L (Differences in motor activation of voluntary and reflex cough in humans. Thorax 61:699 705.2006). Leduc D, de Troyer A (Effect of chest wall vibration on the canine diaphragm during breathing. Eur Respir J 19:429 433.2002). Leduc D, De Troyer A (Mechanical effect of muscle spindles in the canine external intercostal muscles. J Physiol 548:297 305.2003). Lewis G, Ponte J, Purves MJ (Fluctuations of Pa, CO 2 with the same period as respiration in the cat. J Physiol 298:1 11.1980). Lin VW, Deng X, Lee YS, Hsiao IN (Stimulation of the expiratory muscles using microstimulators. IEEE Trans Neural Syst Rehabil Eng 16:416 420.2008). Lindstrom L, Magnusson R, Peter sen I (Muscular fatigue and action potential conduction velocity changes studied with frequency analysis of EMG signals. Electromyography 10:341 356.1970). Lindstrom L, Magnusson R, Petersen I (The "dip phenomenon" in power spectra of EMG signals. Electroe ncephalogr Clin Neurophysiol 30:259 260.1971). Lindstrom LH, Magnusson RI (Interpretation of Myoelectric Power Spectra: A Model and Its Applications. Proceedings of the IEEE 65:653 662.1977). Lipski J, Duffin J, Kruszewska B, Zhang X (Upper cervical inspir atory neurons in the rat: an electrophysiological and morphological study. Exp Brain Res 95:477 487.1993). Lipski J, Zhang X, Kruszewska B, Kanjhan R (Morphological study of long axonal projections of ventral medullary inspiratory neurons in the rat. Brain Res 640:171 184.1994).

PAGE 177

177 Lopata M, Onal E, Ginzburg AS (Respiratory muscle function during CO2 rebreathing with inspiratory flow resistive loading. J Appl Physiol 54:475 482.1983). Loveridge B, Sanii R, Dubo HI (Breathing pattern adjustments during the firs t year following cervical spinal cord injury. Paraplegia 30:479 488.1992). Lovett Barr MR, Satriotomo I, Muir GD, Wilkerson JE, Hoffman MS, Vinit S, Mitchell GS (Repetitive intermittent hypoxia induces respiratory and somatic motor recovery after chronic c ervical spinal injury. J Neurosci 32:3591 3600.2012). Lumsden T (Observations on the respiratory centres. J Physiol 57:354 367.1923a). Lumsden T (The regulation of respiration: Part I. J Physiol 58:81 91.1923b). Manning HL, Molinary EJ, Leiter JC (Effect o f inspiratory flow rate on respiratory sensation and pattern of breathing. Am J Respir Crit Care Med 151:751 757.1995). Manning HL, Slogic S, Leiter JC (Tidal volume perception in normal subjects: the effect of altered arterial PCO2. Respir Physiol 96:99 1 10.1994). Mannion AF, Dolan P (Electromyographic median frequency changes during isometric contraction of the back extensors to fatigue. Spine (Phila Pa 1976) 19:1223 1229.1994). Mantilla CB, Bailey JP, Zhan WZ, Sieck GC (Phrenic motoneuron expression of s erotonergic and glutamatergic receptors following upper cervical spinal cord injury. Exp Neurol 234:191 199.2012). Mantilla CB, Seven YB, Hurtado Palomino JN, Zhan WZ, Sieck GC (Chronic assessment of diaphragm muscle EMG activity across motor behaviors. Re spir Physiol Neurobiol 177:176 182.2011). Mantilla CB, Seven YB, Zhan WZ, Sieck GC (Diaphragm motor unit recruitment in rats. Respir Physiol Neurobiol 173:101 106.2010). Martin AD, Davenport PD, Franceschi AC, Harman E (Use of inspiratory muscle strength t raining to facilitate ventilator weaning: a series of 10 consecutive patients. Chest 122:192 196.2002). Martin JG, De Troyer A (The behaviour of the abdominal muscles during inspiratory mechanical loading. Respir Physiol 50:63 73.1982). Mateika JH, Sandhu KS (Experimental protocols and preparations to study respiratory long term facilitation. Respir Physiol Neurobiol 176:1 11.2011).

PAGE 178

178 Maynard FM, Jr., Bracken MB, Creasey G, Ditunno JF, Jr., Donovan WH, Ducker TB, Garber SL, Marino RJ, Stover SL, Tator CH, Wa ters RL, Wilberger JE, Young W (International Standards for Neurological and Functional Classification of Spinal Cord Injury. American Spinal Injury Association. Spinal Cord 35:266 274.1997). Merrill EG, Lipski J (Inputs to intercostal motoneurons from ven trolateral medullary respiratory neurons in the cat. J Neurophysiol 57:1837 1853.1987). Millhorn DE, Eldridge FL, Kiley JP, Waldrop TG (Excitatory and inhibitory effects of morphine on the intercostal to phrenic respiratory reflex. Respir Physiol 62:79 84. 1985). Mitchell GS, Johnson SM (Neuroplasticity in respiratory motor control. J Appl Physiol 94:358 374.2003). Moreno DE, Yu XJ, Goshgarian HG (Identification of the axon pathways which mediate functional recovery of a paralyzed hemidiaphragm following spi nal cord hemisection in the adult rat. Exp Neurol 116:219 228.1992). Ninane V, De Troyer A (Mechanics of parasternals and triangularis sterni in upright vs. supine dogs. J Appl Physiol 65:452 459.1988). NSCISC (2013) Spinal Cord Injury Facts and Figures at a Glance, SCIMS/ NDIRR. Oku Y, Kurusu M, Hara Y, Sugita M, Muro S, Chin K, Mishima M, Ohi M, Kuno K (Ventilatory responses and subjective sensations during arm exercise and hypercapnia in patients with lower cervical and upper thoracic spinal cord injurie s. Intern Med 36:776 780.1997). Oliven A, Deal EC, Jr., Kelsen SG, Cherniack NS (Effects of hypercapnia on inspiratory and expiratory muscle activity during expiration. J Appl Physiol 59:1560 1565.1985). Orem J, Netick A (Behavioral control of breathing in the cat. Brain Res 366:238 253.1986). Osborne JW, Overbay A (The power of outliers (and why researchers should always check for them). Practical Assessment, Research & Evaluation 9:9.2004). Pagliardini S, Greer JJ, Funk GD, Dickson CT (State dependent mod ulation of breathing in urethane anesthetized rats. J Neurosci 32:11259 11270.2012). Park JH, Kang SW, Lee SC, Choi WA, Kim DH (How respiratory muscle strength correlates with cough capacity in patients with respiratory muscle weakness. Yonsei Med J 51:392 397.2010).

PAGE 179

179 Pate KM (2010) Respiratory load compenstaion responses in concious animals. "doctoral dissertation" retrieved from University of Florida library catalog. In: Physiological Sciences, vol. PhD, p 144 Gainesville: University of Florida. Pate KM, D avenport PW (Tracheal Occlusion Conditioning Causes Stress, Anxiety and Neural State Changes in Conscious Rats. Exp Physiol.2012a). Pate KM, Davenport PW (Tracheal occlusions evoke respiratory load compensation and neural activation in anesthetized rats. J Appl Physiol 112:435 442.2012b). Phillipson EA (Vagal control of breathing pattern independent of lung inflation in conscious dogs. J Appl Physiol 37:183 189.1974). Pokorski M, Izumizaki M, Homma I (Transient O2 dependent effects of CO2 on ventilation in the anesthetized mouse. J Physiol Pharmacol 56:447 454.2005). Porter WT (The Path of the Respiratory Impulse from the Bulb to the Phrenic Nuclei. J Physiol 17:455 485.1895). Reaz MB, Hussain MS, Mohd Yasin F (Techniques of EMG signal analysis: detection, p rocessing, classification and applications (Correction). Biol Proced Online 8:163.2006). Remmers JE (Inhibition of inspiratory activity by intercostal muscle afferents. Respir Physiol 10:358 383.1970). Revelette WR, Wiley RL (Plasticity of the mechanism su bserving inspiratory load perception. J Appl Physiol 62:1901 1906.1987). Reynolds LB, Jr. (Characteristics of an inspiration augmenting reflex in anesthetized cats. J Appl Physiol 17:683 688.1962). Rice A, Fuglevand AJ, Laine CM, Fregosi RF (Synchronizatio n of presynaptic input to motor units of tongue, inspiratory intercostal, and diaphragm muscles. J Neurophysiol 105:2330 2336.2011). Rikard Bell GC, Bystrzycka EK, Nail BS (Cells of origin of corticospinal projections to phrenic and thoracic respiratory mo toneurones in the cat as shown by retrograde transport of HRP. Brain Res Bull 14:39 47.1985a). Rikard Bell GC, Bystrzycka EK, Nail BS (The identification of brainstem neurones projecting to thoracic respiratory motoneurones in the cat as demonstrated by re trograde transport of HRP. Brain Res Bull 14:25 37.1985b). Romaniuk JR, Supinski G, DiMarco AF (Relationship between parasternal and external intercostal muscle length and load compensatory responses in dogs. J Physiol 449:441 455.1992).

PAGE 180

180 Roncoroni AJ (Lack of breathlessness during apnea in a patient with high spinal cord transection. Chest 62:514 515.1972). Rowley KL, Mantilla CB, Sieck GC (Respiratory muscle plasticity. Respir Physiol Neurobiol 147:235 251.2005). Saboisky JP, Gorman RB, De Troyer A, Gandev ia SC, Butler JE (Differential activation among five human inspiratory motoneuron pools during tidal breathing. J Appl Physiol 102:772 780.2007). Saint John WM (Differing responses to hypercapnia and hypoxia following pneumotaxic center ablation. Respir Ph ysiol 23:1 9.1975). Saji M, Miura M (Thoracic expiratory motor neurons of the rat: localization and sites of origin of their premotor neurons. Brain Res 507:247 253.1990). Sandhu MS, Dougherty BJ, Lane MA, Bolser DC, Kirkwood PA, Reier PJ, Fuller DD (Respi ratory recovery following high cervical hemisection. Respir Physiol Neurobiol 169:94 101.2009). Sapienza CM, Wheeler K (Respiratory muscle strength training: functional outcomes versus plasticity. Semin Speech Lang 27:236 244.2006). Saywell SA, Ford TW, Me ehan CF, Todd AJ, Kirkwood PA (Electrophysiological and morphological characterization of propriospinal interneurons in the thoracic spinal cord. J Neurophysiol 105:806 826.2011). Schaefer KE, Hastings BJ, Carey CR, Nichols G, Jr. (Respiratory Acclimatizat ion to Carbon Dioxide. J Appl Physiol 18:1071 1078.1963). Schlaefke ME, See WR, Herker See A, Loeschcke HH (Respiratory response to hypoxia and hypercapnia after elimination of central chemosensitivity. Pflugers Arch 381:241 248.1979). Sears TA (The Slow P otentials of Thoracic Respiratory Motoneurones and Their Relation to Breathing. J Physiol 175:404 424.1964). Seven YB, Mantilla CB, Zhan WZ, Sieck GC (Non stationarity and power spectral shifts in EMG activity reflect motor unit recruitment in rat diaphrag m muscle. Respir Physiol Neurobiol 185:400 409.2013). Shannon R (Effects of thoracic dorsal rhizotomies on the respiratory pattern in anesthetized cats. J Appl Physiol Respir Environ Exerc Physiol 43:20 26.1977). Shannon R, Shear WT, Mercak AR, Bolser DC, Lindsey BG (Non vagal reflex effects on medullary inspiratory neurons during inspiratory loading. Respir Physiol 60:193 204.1985).

PAGE 181

181 Shannon R, Zechman FW (The reflex and mechanical response of the inspiratory muscles to an increased airflow resistance. Resp ir Physiol 16:51 69.1972). Siafakas NM, Chang HK, Bonora M, Gautier H, Milic Emili J, Duron B (Time course of phrenic activity and respiratory pressures during airway occlusion in cats. J Appl Physiol Respir Environ Exerc Physiol 51:99 108.1981). Silver JR Lehr RP (Electromyographic investigation of the diaphragm and intercostal muscles in tetraplegics. J Neurol Neurosurg Psychiatry 44:837 841.1981). Sinderby C, Ingvarsson P, Sullivan L, Wickstrom I, Lindstrom L (The role of the diaphragm in trunk extensio n in tetraplegia. Paraplegia 30:389 395.1992). Sinderby C, Lindstrom L, Grassino AE (Automatic assessment of electromyogram quality. J Appl Physiol 79:1803 1815.1995). Smith BK, Gabrielli A, Davenport PW, Martin AD (Effect of training on inspiratory load c ompensation in weaned and unweaned mechanically ventilated ICU patients. Respir Care 59:22 31.2014). Smith BK, Martin AD, Vandenborne K, Darragh BD, Davenport PW (Chronic intrinsic transient tracheal occlusion elicits diaphragmatic muscle fiber remodeling in conscious rodents. PLoS One 7:e49264.2012). Spahija J, Beck J, Lindstrom L, Begin P, de Marchie M, Sinderby C (Effect of increased diaphragm activation on diaphragm power spectrum center frequency. Respir Physiol Neurobiol 146:67 76.2005). Stevens SS (O n the psychophysical law. Psychol Rev 64:153 181.1957). Stradling JR, Chadwick GA, Frew AJ (Changes in ventilation and its components in normal subjects during sleep. Thorax 40:364 370.1985). Strauss DJ, Devivo MJ, Paculdo DR, Shavelle RM (Trends in life e xpectancy after spinal cord injury. Arch Phys Med Rehabil 87:1079 1085.2006). Tabachnik E, Muller NL, Bryan AC, Levison H (Changes in ventilation and chest wall mechanics during sleep in normal adolescents. J Appl Physiol Respir Environ Exerc Physiol 51:55 7 564.1981). Taylor A (The contribution of the intercostal muscles to the effort of respiration in man. J Physiol 151:390 402.1960). Terada J, Mitchell GS (Diaphragm long term facilitation following acute intermittent hypoxia during wakefulness and sleep. J Appl Physiol (1985) 110:1299 1310.2011).

PAGE 182

182 Tester NJ, Fuller DD, Fromm JS, Spiess MR, Behrman AL, Mateika JH (Long term facilitation of ventilation in humans with chronic spinal cord injury. Am J Respir Crit Care Med 189:57 65.2014). Tian GF, Duffin J (Con nections from upper cervical inspiratory neurons to phrenic and intercostal motoneurons studied with cross correlation in the decerebrate rat. Exp Brain Res 110:196 204.1996a). Tian GF, Duffin J (Spinal connections of ventral group bulbospinal inspiratory neurons studied with cross correlation in the decerebrate rat. Exp Brain Res 111:178 186.1996b). Trippenbach T, Milic Emili J (Temperature and CO2 effect on phrenic activity and tracheal occlusion pressure. J Appl Physiol Respir Environ Exerc Physiol 43:44 9 454.1977). Tsai HW, Chan PY, von Leupoldt A, Davenport PW (The impact of emotion on the perception of graded magnitudes of respiratory resistive loads. Biol Psychol 93:220 224.2013). Tsai HW, Davenport PW (Tracheal Occlusion Evoked Respiratory Load Compe nsation and Inhibitory Neurotransmitter Expression in Rats. J Appl Physiol (1985).2014). Uga M, Niwa M, Ochiai N, Sasaki S (Activity patterns of the diaphragm during voluntary movements in awake cats. J Physiol Sci 60:173 180.2010). van den Berg ME, Castel lote JM, de Pedro Cuesta J, Mahillo Fernandez I (Survival after spinal cord injury: a systematic review. J Neurotrauma 27:1517 1528.2010). Van den Bergh O, Stegen K, Van de Woestijne KP (Memory effects on symptom reporting in a respiratory learning paradig m. Health Psychol 17:241 248.1998). Vinit S, Lovett Barr MR, Mitchell GS (Intermittent hypoxia induces functional recovery following cervical spinal injury. Respir Physiol Neurobiol 169:210 217.2009). Vlemincx E, Abelson JL, Lehrer PM, Davenport PW, Van Di est I, Van den Bergh O (Respiratory variability and sighing: a psychophysiological reset model. Biol Psychol 93:24 32.2013). von Euler C (The role of proprioceptive afferents in the control of respiratory muscles. Acta Neurobiol Exp (Wars) 33:329 341.1973) von Euler C (The functional organization of the respiratory phase switching mechanisms. Fed Proc 36:2375 2380.1977). von Leupoldt A, Bradley MM, Lang PJ, Davenport PW (Neural processing of respiratory sensations when breathing becomes more difficult and unpleasant. Front Physiol 1:144.2010).

PAGE 183

183 von Leupoldt A, Chan PY, Esser RW, Davenport PW (Emotions and neural processing of respiratory sensations investigated with respiratory related evoked potentials. Psychosom Med 75:244 252.2013). von Leupoldt A, Vovk A Bradley MM, Lang PJ, Davenport PW (Habituation in neural processing and subjective perception of respiratory sensations. Psychophysiology 48:808 812.2011). Walker JK, Lawson BL, Jennings DB (Breath timing, volume and drive to breathe in conscious rats: c omparative aspects. Respir Physiol 107:241 250.1997). Whitelaw WA, Derenne JP, Milic Emili J (Occlusion pressure as a measure of respiratory center output in conscious man. Respir Physiol 23:181 199.1975). Whitelaw WA, Ford GT, Rimmer KP, De Troyer A (Inte rcostal muscles are used during rotation of the thorax in humans. J Appl Physiol 72:1940 1944.1992). Wilkerson JE, Mitchell GS (Daily intermittent hypoxia augments spinal BDNF levels, ERK phosphorylation and respiratory long term facilitation. Exp Neurol 2 17:116 123.2009). Wilkerson JE, Satriotomo I, Baker Herman TL, Watters JJ, Mitchell GS (Okadaic acid sensitive protein phosphatases constrain phrenic long term facilitation after sustained hypoxia. J Neurosci 28:2949 2958.2008). Winslow C, Rozovsky J (Effe ct of spinal cord injury on the respiratory system. Am J Phys Med Rehabil 82:803 814.2003). Yasuma F, Kimoff RJ, Kozar LF, England SJ, Bradley TD, Phillipson EA (Abdominal muscle activation by respiratory stimuli in conscious dogs. J Appl Physiol 74:16 23. 1993). Zechman FW, Davenport PW (Temporal differences in the detection of resistive and elastic loads to breathing. Respir Physiol 34:267 277.1978). Zechman FW, Frazier DT, Lally DA (Respiratory volume time relationships during resistive loading in the cat J Appl Physiol 40:177 183.1976). Zimmer MB, Nantwi K, Goshgarian HG (Effect of spinal cord injury on the respiratory system: basic research and current clinical treatment options. J Spinal Cord Med 30:319 330.2007). Zimmer MB, Nantwi K, Goshgarian HG (Ef fect of spinal cord injury on the neural regulation of respiratory function. Exp Neurol 209:399 406.2008).

PAGE 184

184 BIOGRAPHICAL SKETCH Poonam B Jaiswal was born and raised in Mumbai, India. S he was accepted into Texas A&M University, College Station, Texas and was supported by the Regents Fell biotechnology & graduate school scholarship. She graduated with a master in biotechnology and a diploma in business in fall 2007. Poonam was accepted into the veterinary medical sciences doctoral program at U niversity of Florida in s pring 2009, She was s pring 2010, pursuing research focused on respiratory neurophysiology and spinal cord injury. Poonam received her Doctor of Philosophy in Veterinary Medical sciences in spring 2014. I n addition to support from her mentor Dr Davenport, Poonam has received grants and support from the college of veterinary medicine, graduate student council and the department of physiological sciences for her work. Poonam has also received awards for excellence in basic science research and as an outstanding international student during her time at the University of Florida.