<%BANNER%>

Spinal Cord Injury and Plasticity in Cervical Motor Systems

Permanent Link: http://ufdc.ufl.edu/UFE0045398/00001

Material Information

Title: Spinal Cord Injury and Plasticity in Cervical Motor Systems
Physical Description: 1 online resource (222 p.)
Language: english
Creator: Gonzalez-Rothi, Elisa
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2013

Subjects

Subjects / Keywords: cervical -- cord -- extremity -- injury -- interneurons -- motor -- plasticity -- respiratory -- serotonin -- spinal -- upper
Rehabilitation Science -- Dissertations, Academic -- UF
Genre: Rehabilitation Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Injury to the cervical spinal cord (cSCI) is often accompanied by impaired upper extremity and respiratory function. Though the potential for spontaneous recovery has been demonstrated, this is often limited. This dissertation presents original research exploring spontaneous plasticity and recovery in cervical motor systems following experimental cSCI and explores potential anatomical substrates. We first characterized upper extremity motor recovery over several months following lateral C2 hemisection. This model has been used extensively to study plasticity in the phrenic system. Early deficits followed by modest functional recovery were observed. Assessment of the neuroanatomical circuitry of the upper limb indicated a change in the distribution of pre-motor interneurons, which we hypothesized were likely candidates for mediating functional recovery. Next we assessed the serotonergic innervation of cervical motor systems. Serotonin, which is correlated with motor function, has been shown to be reduced following SCI. Studies have demonstrated that restoration of serotonin to motoneuron pools improves motor output, however whether serotonin also innervates interneurons has not been investigated. We demonstrated that serotonergic projections innervate phrenic motoneurons and pre-motor interneurons and that 5-HT2A and 5-HT7 receptor subtypes are expressed on these neurons. The contribution of propriospinal interneurons to functional recovery (respiratory and upper extremity) following cervical hemisection was assessed. Early functional deficits were followed by spontaneous recovery and recent evidence (presented in this thesis) suggests this may be mediated by propriospinal interneurons. Focal ablation of propriospinal interneurons in the intermediate gray matter of the cervical cord of chronically injured rats attenuated upper extremity and respiratory functional recovery. In conclusion, the data presented here provide the following novel findings: 1) spontaneous recovery of upper extremity function occurs following cSCI and coincides with recruitment of pre-motor interneurons 2) serotonin innervates cervical motoneuron and pre-motor interneurons of the phrenic circuit, and 3) specific ablation of propriospinal cervical interneurons in the cervical cord further supports their role in mediating functional recovery. Therapeutic strategies that target this intraspinal network of interneurons and promote restoration of serotonergic innervation to motor circuitry may represent a promising avenue for optimizing functional recovery after SCI.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Elisa Gonzalez-Rothi.
Thesis: Thesis (Ph.D.)--University of Florida, 2013.
Local: Adviser: Fuller, David.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2015-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2013
System ID: UFE0045398:00001

Permanent Link: http://ufdc.ufl.edu/UFE0045398/00001

Material Information

Title: Spinal Cord Injury and Plasticity in Cervical Motor Systems
Physical Description: 1 online resource (222 p.)
Language: english
Creator: Gonzalez-Rothi, Elisa
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2013

Subjects

Subjects / Keywords: cervical -- cord -- extremity -- injury -- interneurons -- motor -- plasticity -- respiratory -- serotonin -- spinal -- upper
Rehabilitation Science -- Dissertations, Academic -- UF
Genre: Rehabilitation Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Injury to the cervical spinal cord (cSCI) is often accompanied by impaired upper extremity and respiratory function. Though the potential for spontaneous recovery has been demonstrated, this is often limited. This dissertation presents original research exploring spontaneous plasticity and recovery in cervical motor systems following experimental cSCI and explores potential anatomical substrates. We first characterized upper extremity motor recovery over several months following lateral C2 hemisection. This model has been used extensively to study plasticity in the phrenic system. Early deficits followed by modest functional recovery were observed. Assessment of the neuroanatomical circuitry of the upper limb indicated a change in the distribution of pre-motor interneurons, which we hypothesized were likely candidates for mediating functional recovery. Next we assessed the serotonergic innervation of cervical motor systems. Serotonin, which is correlated with motor function, has been shown to be reduced following SCI. Studies have demonstrated that restoration of serotonin to motoneuron pools improves motor output, however whether serotonin also innervates interneurons has not been investigated. We demonstrated that serotonergic projections innervate phrenic motoneurons and pre-motor interneurons and that 5-HT2A and 5-HT7 receptor subtypes are expressed on these neurons. The contribution of propriospinal interneurons to functional recovery (respiratory and upper extremity) following cervical hemisection was assessed. Early functional deficits were followed by spontaneous recovery and recent evidence (presented in this thesis) suggests this may be mediated by propriospinal interneurons. Focal ablation of propriospinal interneurons in the intermediate gray matter of the cervical cord of chronically injured rats attenuated upper extremity and respiratory functional recovery. In conclusion, the data presented here provide the following novel findings: 1) spontaneous recovery of upper extremity function occurs following cSCI and coincides with recruitment of pre-motor interneurons 2) serotonin innervates cervical motoneuron and pre-motor interneurons of the phrenic circuit, and 3) specific ablation of propriospinal cervical interneurons in the cervical cord further supports their role in mediating functional recovery. Therapeutic strategies that target this intraspinal network of interneurons and promote restoration of serotonergic innervation to motor circuitry may represent a promising avenue for optimizing functional recovery after SCI.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Elisa Gonzalez-Rothi.
Thesis: Thesis (Ph.D.)--University of Florida, 2013.
Local: Adviser: Fuller, David.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2015-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2013
System ID: UFE0045398:00001


This item has the following downloads:


Full Text

PAGE 1

1 SPINAL CORD INJURY AND P LASTICITY IN CERVICAL MOTOR SYSTEMS By ELISA J. GONZALEZ ROTHI A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGR EE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2013

PAGE 2

2 2013 Elisa J. Gonzalez Rothi

PAGE 3

3 To my family

PAGE 4

4 ACKNOWLEDGMENTS The writing of this dissertation has been both an exciting and challenging experience for me. It has been an endeavor that I could not have completed without the help and support of a number of people in my life. My deepest gratitude goes to my advisor, Dr. David Fuller. I am forever grateful to him for his dedicated mentorship and his gen uine excitement for science. I also owe him special thanks for his enthusiasm and support for my research and professional interests I am also incredibly grateful to Drs. Paul Reier and Michael Lane who have been incredible mentors and friends. Their gu idance has been incredibly valuable to me, and their enthusiasm for neuroscience is infectious. I want to express my gratitude to Dr. Krista Vandenborne for her never ending support, as a mentor, program director, and friend. She has been a tremendous role model, and I feel blessed to have had the opportunity to learn from her. I would also like to thank Dr. Andy Judge for his guidance as a committee member and as a professional mentor. I would like to thank all the present and past members of the Fuller, Reier, and Lane labs for all their assistance and guidance over the past few years, as well as for making it a great place to work: Dr. Kun Ze Lee, Dr. Milap Sandhu, Dr. Brendan Dougherty, Dr. Mai Elmallah, Lynne Mercier, Angela Rombola, Celeste Rousseau, Lisbet Fernandez, Natalie Stephens, Shaunn Hussey, Cindy Lopez, Devee Sanchez, Greg Armstrong, Amy Poirier, Nicole Little, Roland Austin Federico, Daniel Ross, Rachel Mattio, Alison Daly, Victoria Spruance and Garret Fitzpatrick An extra thanks to my amaz

PAGE 5

5 have always gone above and beyond, and this dissertation would not have been possible without all their hard work! A special thanks to Lynne Mercier my apparent twin, for her friendship and support, and for all the laugh ter (and occasional tears) we through it with anyone else. I also want to give a special for always encouraging me. She will always hold a special place in my heart, and no matter where my journey ta what would I would like to thank my parents, Ricardo and Leslie, without whom none of this would have been possible. Their tremendous love and support has allowed me to always pursue my dreams. I am so g I also have to thank my beautiful and talented sister, Sara Gonzalez Roth Kronenthal for always being there for me, for being my best friend, and my biggest cheerleader. Also, special thanks to all my wonde rful friends, especially Martina and Ryan Murphy, Corie and Emery Patton, Ginny Little, Chris Gregory Meredith and Stephen Walker, Frank and Nancy Moses, Tiffany Cary and Ashley Maloy, Jill Brooks and all of the Hammond Brooks clan for their friendship an d support. Lastly, I would like to say a big thank you to my husband, Nick, whose support and sacrifices have allowed me to pursue this dream. He is my rock. His patience, strength and never ending love are such a blessing. He has been my best friend and has helped me every step of the way!

PAGE 6

6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ .......... 10 LIST OF FIGURES ................................ ................................ ................................ ........ 11 LIST OF ABBREVIATIONS ................................ ................................ ........................... 15 ABSTRACT ................................ ................................ ................................ ................... 17 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 1 9 The Concept of Plasticity ................................ ................................ ........................ 19 Spinal Cord Injury ................................ ................................ ................................ ... 20 Pathophysiology of Spinal Cord Injury ................................ .............................. 21 Primary injury ................................ ................................ ............................. 21 Secondary injury ................................ ................................ ........................ 21 Axonal Regeneration Following Spinal Cord Injury ................................ .... 24 Gray Versus White Matter Pathology ................................ ......................... 25 Cervical Spinal Cord Injury ................................ ................................ ............... 29 U pper extremity consequences of cervical spinal cord injury ..................... 29 Respiratory consequences of cervical spinal cord injury ............................ 30 Experim ental Models of cervical spinal cord injury ................................ ..... 30 Neural Control of Cervical Motor Function ................................ .............................. 31 Neural Control of Upper Extremity Mo tor Function ................................ ........... 32 Supraspinal control of upper extremity function ................................ ......... 32 Spinal Control of Upper Extremity Function ................................ ............... 34 Neural Control of Respiration ................................ ................................ ........... 36 Brainstem control of respiration ................................ ................................ 37 Cortical c ontrol of respiration ................................ ................................ ..... 40 Spinal Respiratory Control ................................ ................................ ......... 40 Neuromodulatory Control of Respiration ................................ .................... 43 Characterization of Functional Plasticity in Experimental SCI ................................ 45 Assessment of Upper Extremity Function ................................ ......................... 45 Gross motor function ................................ ................................ .................. 45 Fine motor manipulation ................................ ................................ ............ 46 Locomotor function ................................ ................................ .................... 46 Characterization of Ventilatory Function in Experimental SCI .......................... 47 Ventilatory function ................................ ................................ .................... 47 Respiratory electro myography ................................ ................................ ... 48 Phrenic nerve recordings ................................ ................................ ........... 48

PAGE 7

7 Interaction of Muscle and Nerve ................................ ................................ ............. 49 Components of the Motor Unit ................................ ................................ .......... 50 ....................... 50 Skeletal muscle adaptations to spinal cord injury. ................................ ...... 50 ............. 52 2 OUTLINE OF EXPERI MENTS ................................ ................................ ................ 57 Overall Objectives ................................ ................................ ................................ ... 59 Aim One ................................ ................................ ................................ .................. 60 Objective ................................ ................................ ................................ .......... 60 Rationale ................................ ................................ ................................ .......... 60 Experimental Design ................................ ................................ ........................ 60 Aim Two ................................ ................................ ................................ .................. 61 Objective ................................ ................................ ................................ .......... 61 Rationale ................................ ................................ ................................ .......... 61 Experimental Design ................................ ................................ ........................ 62 Aim Three ................................ ................................ ................................ ............... 62 Objective ................................ ................................ ................................ .......... 62 Rationale ................................ ................................ ................................ .......... 62 Ex perimental Design ................................ ................................ ........................ 63 3 CHARACTERIZATION OF FORELIMB PLASTICITY FOLLOWING INCOMPLETE HIGH CERVICAL SPINAL CORD INJURY IN ADULT RATS ......... 64 Materials and Methods ................................ ................................ ............................ 67 Animals ................................ ................................ ................................ ............. 67 General Surgical Methods ................................ ................................ ................ 67 Spinal Cord Hemisection Injury ................................ ................................ ........ 68 Behavioral Testing of Forelimb Function ................................ .......................... 68 Limb use asymmetry (cylinder) test ................................ ........................... 69 Vermicelli pasta handling test ................................ ................................ .... 69 Forelimb locomotor scale (FLS) ................................ ................................ 69 Mus cle Protocols ................................ ................................ .............................. 70 Muscle tissue harvest ................................ ................................ ................ 70 Muscle immunohistochemistry ................................ ................................ ... 70 Muscle fiber size measurements ................................ ................................ 71 Anatomical Tracing Protocols ................................ ................................ ........... 71 Spinal Cord Histology ................................ ................................ ....................... 72 Data Analysis ................................ ................................ ................................ ... 74 Statistical Analysis ................................ ................................ ............................ 74 Results ................................ ................................ ................................ .................... 75 Anatomical Characterization of C2Hx Lesions ................................ ................. 75 Effect of C2Hx on Body Mass ................................ ................................ ........... 75 Effects of C2Hx on Upper E xtremity Function ................................ .................. 75 Effects of C2Hx on Upper Extremity Muscle Morphology ................................ 78

PAGE 8

8 Characterization of the Neuroanatomical Circuitry o f the ECRL in Uninjured Rats ................................ ................................ ................................ ............... 81 PRV Infection of Cervical Spinal Interneurons ................................ .................. 82 Control Experiments Demonstrate Extensor Carpi Radialis Longus dependent Second Order Labeling of Cervical Interneurons ........................ 83 Characterization of Supraspinal ECRL Circuitry in Uninjured Rats .................. 84 Changes in the Neuroanatomical Circuitry of the ECRL Muscle Following C2Hx ................................ ................................ ................................ ............. 84 Discussion ................................ ................................ ................................ .............. 85 Progressive Recovery of Ipsilateral Forelimb Function Following C2Hx .......... 85 ............... 86 Commenta ry on Methods ................................ ................................ ................. 91 Commentary on Injury Model for Studying Forelimb Function After cSCI ........ 94 Summary ................................ ................................ ................................ ................ 95 4 SEROTONERGIC INNERVATION OF PRE MOTOR CERVICAL INTERNEURONS IN ADULT RATS ................................ ................................ ..... 134 Materials and Methods ................................ ................................ .......................... 137 Animals ................................ ................................ ................................ ........... 137 Anatomical Tracing Protocols ................................ ................................ ......... 137 Spinal Cord Histology and Immunocytochemistry ................................ .......... 138 Data Analysis ................................ ................................ ................................ 139 Results ................................ ................................ ................................ .................. 139 Anatomical Identification of Pre Phrenic Interneurons Using Retrograde Tracing ................................ ................................ ................................ ........ 139 Serotonergic Innervation of Retrogradely Labeled Phrenic Motoneurons and Pre Phrenic Interneurons ................................ ................................ ............ 139 5 HT 2A Receptor Expression in Phrenic Motoneurons and Pre Motor Interneurons ................................ ................................ ................................ 139 5 HT 7 Receptor Expression in Phrenic Motoneurons and Pre Motor Interneurons ................................ ................................ ................................ 140 Discussion ................................ ................................ ................................ ............ 140 Summary ................................ ................................ ................................ .............. 144 5 CONTRIBUTION OF PROPRIOSPINAL NEURONS TO SPONTANEOUS FUNCTIONA L PLASTICITY FOLLOWING INCOMPLETE HIGH CERVICAL SPINAL CORD INJURY IN ADULT RATS ................................ ............................ 148 Materials and Methods ................................ ................................ .......................... 150 Animals ................................ ................................ ................................ ........... 150 General Surgical Methods ................................ ................................ .............. 151 Spinal Cord Hemisection Injury ................................ ................................ ...... 151 Excit otoxin Mediated Gray Matter Ablation ................................ .................... 152 Behavioral Testing of Forelimb Function ................................ ........................ 152 Limb Use Asymmetry (Cylinder) Test ................................ ...................... 152 Forelimb Locomotor Scale (FLS) ................................ ............................. 152

PAGE 9

9 Barometric Plethysmography ................................ ................................ ......... 153 Sp inal Cord Histology and Immunohistochemistry ................................ ......... 153 Data Analysis ................................ ................................ ................................ 154 Results ................................ ................................ ................................ .................. 155 Feasibility of Inducing Discrete Gray Matter Lesions ................................ ...... 155 Anatomical Characterization of C2Hx Lesions ................................ ............... 156 Anatomic al characterization of gray matter lesions ................................ ........ 156 Effect of C2Hx on Body Mass ................................ ................................ ......... 157 Effects of Gray Matter Deletion on Ventilation in Chronically Injured Rats ..... 157 Effects of Gray Matter Deletion on Ventilation in Chronically Injured Rats ..... 159 Effects of Gray Mat ter Deletion on Forelimb Function in Chronically Injured Rats ................................ ................................ ................................ ............. 159 Effects of Gray Matter Deletion on Upper Extremity Function in Chronically Injured Rats ................................ ................................ ................................ 160 Discussion ................................ ................................ ................................ ............ 160 Summary ................................ ................................ ................................ .............. 165 6 CONCLUSIONS AND FUTURE DIRECTIONS ................................ .................... 197 Characterization of Forelimb Plasticity Following Incomplete High Cervical Spinal Cord Injury in Adult Rats ................................ ................................ ......... 197 Serotonergic Innervation of Pre Motor Cervical Interneurons in Adult Rats .......... 198 Contribution of Propriospinal Neurons to Spontaneous Functional Plasticity Following Incomplete High Cervical Spinal Cord Injury in Adult Rats ................ 199 LIST OF REFERENCES ................................ ................................ ............................. 201 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 222

PAGE 10

10 LIST OF TABLES Table page 3 1 Cylinder test percentage of ipsilateral paw placements ................................ ... 125 3 2 Forelimb Locomotor Scale scoring rubric ................................ ......................... 126 3 3 Forelimb Locomotor Scale scores ................................ ................................ .... 127 3 4 Vermicelli pasta handling test atypical behaviors ................................ ............. 128 3 5 Vermice lli pasta handling test average number of paw adjustments ................ 129 3 6 Vermicelli pasta handling test average time to eat pasta ................................ 130 3 7 Vermicelli pasta handling test number of atypical behaviors per trial ............... 131 3 8 Average upper limb muscle wet weights ................................ .......................... 132 3 9 Avera ge extensor carpi radialis lon gus mus cle fiber cross sectional area ....... 133 5 1 Ventilation post C2Hx (pre KA) ................................ ................................ ........ 193 5 2 Cylinder test and Forelimb Locomotor Scale scores post C2Hx ....................... 194 5 3 Ventilation post kainic acid injection ................................ ................................ 195 5 4 Cylinder test and Forelimb Lo comotor Scale scores post kainic acid ............... 196

PAGE 11

11 LIST OF FIGURES Figure page 1 1 Schematic depiction of experimental model Lateral C2 Spinal Cord Hemisect ion ................................ ................................ ................................ ........ 54 1 2 Schematic depiction of the neural control of upper extremity motor function ...... 55 1 3 Schematic depiction of the neural co ntrol of breathing. ................................ ...... 56 3 1 H istological sections illustrating C2Hx lesions ................................ .................... 96 3 2 The impact of C2Hx on body mass ................................ ................................ ..... 97 3 3 The impact of C2Hx on ipsilateral upper extremity gross motor use The c ylinder test. ................................ ................................ ................................ ....... 98 3 4 The impact of C2Hx on ipsilateral locomotor fu nction FLS scores ..................... 99 3 5 E xamples of upper limb kinematics of during open field locomotion in an uninjured rat and at 1 and 16 weeks after C2Hx injury. ................................ ... 100 3 6 The impact of C2 Hx on number of paw adjustments Vermicelli pasta handling test ................................ ................................ ................................ ..... 101 3 7 The impact of C2Hx on the time to eat Vermicelli pasta handling test. ............. 102 3 8 The impact of C2Hx on number of atypical behaviors observed Vermicelli pasta handling test ................................ ................................ ........................... 103 3 9 Examples of normal and atyp ical behaviors demonstrated by rats during the Vermicelli Pasta Handling test. ................................ ................................ ......... 104 3 10 The impact of C2Hx on upper extremity muscle size ( wet weight ) ................... 105 3 11 Representative histological examples of Extensor Carpi Radialis Muscle immunostained with antibodies to dystrophin ................................ ................... 106 3 12 Representative histological examples of Extensor Carpi Radialis Muscle immunostained with antibodies to MHC type I ................................ .................. 107 3 13 E xamples of Extensor Carpi Radialis Muscle immunostained with antibodies to MHC type IIa. ................................ ................................ ................................ 108 3 14 Average muscle fiber cross sectional area of contralateral and ipsilateral Extensor Carpi Radialis Longus muscle across all muscle fibers. .................... 109

PAGE 12

12 3 15 Average muscle fiber cross sectional area of contralateral and ipsilateral Extensor Carpi Radialis Longus muscle type I muscle fibers. .......................... 110 3 16 Average muscle fiber cross sectional are a of Extensor Carpi Radialis Longus muscle type IIa fibers ................................ ................................ ........................ 111 3 17 Cervical spinal cord sections from uninjured rats, 24 hours following injection of PRV into the extensor carpi radialis longu s muscle.. ................................ .... 112 3 18 Cervical spinal cord sections from uninjured rats, 48 hours following injection of PRV into the extensor carpi radialis longus muscle. ................................ .... 113 3 19 Cervical spinal cord sections from uninjured rats, 72 hours following injection of PRV into the extensor carpi radialis longus muscle. ................................ .... 114 3 20 Cervical spin al cord sections from uninjured rats, 96 hours following injection of PRV into the extensor carpi radialis longus muscle. ................................ ..... 115 3 21 Cervical spinal cord sections from uninjured rats, 120 hours f ollowing injection of PRV into the extensor carpi radialis longus muscle. ...................... 116 3 22 High power images cervical spinal cord sections from uninjured and chronically injured rats, 96 hours followi ng injection of Pseudorabies virus into the extensor carpi radialis longus muscle ................................ .................. 117 3 23 Extensor carpi radialis longus circuitry in the cervical spinal cord .................... 118 3 24 Extensor carpi radialis longus circuitry in the medulla ................................ ...... 119 3 25 Extensor carpi radialis longus circuitry in the motor cortex. .............................. 120 3 26 Cervical spinal cord sections from uninjured rats with the radial nerve cut 72 hours following injection of PRV into the extensor carpi radialis longus muscle depict no labeling of sympathetic neurons in the ce rvical spinal cord. 121 3 27 Thoracic spinal cord sections from uninjured rats, 72 hours following injection of PRV into the extensor carpi radialis longus muscle depict sympathetic labelling ................................ ................................ ................................ ........... 122 3 28 Cervical spinal cord sections from rats, 16 weeks post C2Hx injury, 72 hours after injection of PRV into the extensor carpi radialis longus muscle ................ 123 3 29 Representative longitudinal (horizontal) sections through the cervical spinal cord of adult female Sprague Dawley rats, 16 weeks post C2Hx injury, 96 hours after injection of PRV into the extensor carpi radialis longus muscle ..... 124 4 1 Serotonergic immunoreactive projections and the phrenic motor circuitry.. ...... 145 4 2 5 HT 2A receptor immunoreactivit y and the phrenic motor circuitry.. .................. 146

PAGE 13

13 4 3 5 HT 7 receptor immunoreactivity in the cervical spinal cord. ............................ 147 5 1 Schematic depicti on of e xcitotoxin mediated ablation of cervical interneurons in chronically injured rats. ................................ ................................ ................. 167 5 2 Cervical spinal cord s ections from uninjured rats, 1 week after intraspinal injection of Kainic Acid depict effects kainic acid dose ................................ .... 168 5 3 Cervical spinal cord histology depicting effect of Kainic acid injections in chronically injured rats Example #1. ................................ ................................ 169 5 4 Cervical spinal cord histology depicting effect of Kainic acid injections in chronically injured rats Example #2 ................................ ................................ .. 170 5 5 Cervical spinal cord histology de picting effect of Kainic acid injections in chronically injured rats Example #3. ................................ ................................ 171 5 6 Cervical spinal cord histology depicting effect of Kainic acid injections in chronically injured rats Ex ample #4 ................................ ................................ .. 172 5 7 Cervical spinal cord histology depicting effect of Kainic acid injections in uninjured rats Example #1. ................................ ................................ ............... 173 5 8 C ervical spinal cord histology depicting effect of Kainic acid injections in uninjured rats Example #2 ................................ ................................ ................ 174 5 9 Cervical spinal cord histology depicting effect of Kainic acid injections in uninju red rats Example #3 ................................ ................................ ................ 175 5 10 Cervical spinal cord histology depicting effect of Kainic acid injections in uninjured rats Example #4. ................................ ................................ ............... 176 5 11 Cervical spinal cord histology depicting effect of s aline injections in chronically injured rats Example #1 ................................ ................................ .. 177 5 12 Cervical spinal cord histology depicting effect of s aline inject ions in uninjured rats Example #2 ................................ ................................ ............................... 178 5 13 Representative airflow traces showing the impact of kainic acid injection on ventilation in chronically injured rats ................................ ................................ 179 5 14 Ef fects of C2 hemisection injury on breathing frequency response to respiratory challenge. ................................ ................................ ....................... 180 5 15 Effects of C2 hemisection injury on tidal volume respo nse to respiratory challenge ................................ ................................ ................................ .......... 181 5 16 E ffects of C2 hemisection injury on minute ventilation response to respiratory challenge ................................ ................................ ................................ .......... 182

PAGE 14

14 5 17 Effects of kainic acid injection on breathing frequency response to respiratory challenge. ................................ ................................ ................................ ......... 183 5 18 Effects of kainic acid injection on tidal volume response to respiratory challenge. ................................ ................................ ................................ ......... 184 5 19 Effects of kainic acid injection on minute ventilation response to respiratory challenge ................................ ................................ ................................ .......... 185 5 20 Effects of kainic acid and saline injection in uninjured rats on change s in ventilat ory response s to respiratory challenge. ................................ ................ 186 5 21 Effects of C2 hemisection injury on upper limb gross motor use (Cylinder test) ................................ ................................ ................................ ................. 187 5 22 Effects of C2 hemisection injury (C2Hx) on forelimb locomotor function (FLS scores) ................................ ................................ ................................ ............. 188 5 23 Effects of kainic acid injecti on on upper limb gross motor use in chronically injured rats. ................................ ................................ ................................ ....... 189 5 24 Effects of kainic acid injection on forelimb locomotor function in chronically injured rats. ................................ ................................ ................................ ....... 190 5 25 Effects of kainic acid and saline injection on upper limb gross motor use and locomotor function in uninjured rats. ................................ ................................ 191 5 26 U pper limb locomotor kine matics prior to and after kainic acid injection ......... 192

PAGE 15

15 LIST OF ABBREVIATIONS 5 HT 5 hydroxytryptamine MN Alpha motoneuron BBB Basso Beattie Breshnahan Open Field Locomotion Scale BDNF Brain derived neurotrophic factor BIC Biceps brachii Bt C Bt Complex C2Hx C2 Hemisection cAMP Cyclic adenosine monophosphate CL Contralateral CSA Cross sectional area cSCI Cervical spin al cord injury CSPGs Chondroitin sulfate proteoglycans CT Chol era toxin B subunit DRC Dorsal respiratory column ECRL Extensor carpi radialis longus FCR Flexor carpi radialis FLS Forelimb Locomotor Scale GDNF Glial derived neurotrophic factor GI Gastrointestinal IL Ipsilateral i.p. Intraperitoneal K + Potassium KA Kainic acid MHC Myosin heavy ch ain

PAGE 16

16 Na + Sodium PBS Phosphate buffered saline PhMN Phrenic motoneuron PKC Protein kinase C Pre Bt C Pre Btzinger Complex PRV Pseudorabies virus ROS Reactive oxygen species SCI Spinal cord injury s.q. Subcutaneous TRI Triceps brachii VRC Ventral respiratory column

PAGE 17

17 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy SPINAL CORD INJURY AND PLASTICITY IN CERVICAL MOTOR SYSTEMS By Elisa J. Gonzalez Rothi May 2013 Chair: David D. Fuller Major: Rehabilitation Science Injury to the cervical spinal cord (cSCI) is often accompanied by impaired upper extremity and respiratory function. Though the potential for spontaneous recovery has been demonstrated, this is often limited. This dissertation presents original research exploring spontaneous plasticity and recovery in cer vical motor systems following experimental cSCI and explores potential anatomical substrates. We first characterized upper extremity motor recovery over several months following lateral C2 hemisection. This model has been used extensively to study plastici ty in the phrenic system. Early deficits followed by modest functional recovery were observed. Assessment of the neuroanatomical circuitry of the upper limb indicated a change in the distribution of pre motor interneurons, which we hypothesized were likely candidates for mediating functional recovery. Next we assessed the serotonergic innervation of cervical motor systems. S eroton in, which is correlated with motor function, has been shown to be reduced following SCI. S tudies have de monstrated that restorati on of serotonin to motoneuron pools improve s motor output however whether serotonin also innervates interneurons has not been investigated We demonstrated that serotonergic projections innervate

PAGE 18

18 phrenic motoneurons and pre motor interneurons and that 5 H T 2A and 5 HT 7 receptor subtypes are expressed on these neurons. The contribution of propriospinal interneurons to functional recovery (respiratory and upper extremity) following cervical hemisection was assessed. Early functional deficits were followed by spontaneous recovery and recent evidence (presented in this thesis) suggests this may be mediated by propriospinal interneurons. Focal ablation of propriospinal interneurons in the intermediate gray matter of the cervical cord of chronically injured rats attenuated upper extremity and respiratory functional recovery. In conclusion, the data presented here provide the following novel findings: 1) spontaneous recovery of upper extremity function occurs following cSCI and coincides with recruitment of pre mo tor interneurons 2) serotonin innervates cervical motoneuron and pre motor interneurons of the phrenic circuit, and 3) specific ablation of propriospinal cervical interneurons in the cervical cord further supports their role in mediating functional recover y. Therapeutic strategies that target this intraspinal network of interneurons and promote restoration of serotonergic innervation to motor circuitry may represent a promising avenue for optimizing functional recovery after SCI.

PAGE 19

19 CHAPTER 1 INTRODUCTION T he Concept of Plasticity In 1906, Santiago Ramon y Cajal was awarded the Nobel Prize in Medicine and Physiology for elucidating the intricacies of the nervous system. His work confirmed that the nervous system was made up of individual cells, neurons, whic h can act in concert with one another and communicate via synapses. Viewed by many as the father of modern neuroscience, Ram n y Cajal put forth significant contributions to the understanding of the human nervous system, and much of his work has stood the test of time, save one theory; Ram n y Cajal asserted that the neural connections within the these neural pathways were irreparable (Ramon y Cajal, 1913 1914) For nearly a century, this dictum of a fixed adult nervous system created the nihilistic belief in the mind of scientists and clinicians alike, that damaged nervous systems were damaged permanently, and that no intervention could alter this fate. Thus the over riding theme of neurorehabilitation and medicine for many years was to teach individuals to adapt to their fate and to compensate for the resulting impairments. Conversely, the concept of plasticity is not at all unfamiliar to those with an understanding of muscl e biology and physiology. Indeed there is a tremendous body of evidence demonstrating the immense plastic potential of skeletal muscle. Recent decades have realized a change in this pessimistic dogma, which has been so pervasive in the field of neuroscienc e, with the emergence of a significant body of research to support the notion of plasticity in the adult central nervous system. Furthermore, given the considerable interconnectedness between nerves and muscle, it is not surprising that plasticity observed in one system

PAGE 20

20 would also be evident to some extent in the other. Indeed, there is now considerable evidence that both skeletal muscle and the nervous system demonstrate tremendous adaptive plasticity in response to physiological perturbations such as inac tivity, injury, exercise, etc. P lasticity, for the purposes of the present work, can be delineated into three distinct, but related categories: 1) neuroplasticity, neural control system (morphology and/or funct ion) bas ed on experience (Mitchell and Johnson, 2003) ; 2) functional plasticity, defined as a persis tent change in motor function related to a defined behavior; and 3) muscle plasticity, defined as a change in whole muscle and/or myofiber morphology and/or phenot ype (Hutchinson et al., 2001) Here, we present a detailed review of spinal cord injury, with specific emphasis on the neural, functional, and muscular consequences that occur following injury to the cervical spinal cord. Spinal Cord Injury Spinal cord in jury (SCI) is a devastating neurological insult that leads to significant physical impairments including chronic paralysis and limitations in functional mobility. Motor vehicle accidents comprise the largest proportion of all SCIs (~40%), though in older a dults (>50), falls have become the most prevalent cause (Jackson et al., 2004, DeVivo and Chen, 2011) The socioeconomic burden of SCI is staggering, with an estimated 12,000 Americans affected annually and a surviving cohort of 250,000 individuals living with the chronic and disabling effects of SCI. Perhaps more concerning than the sheer number of individuals affected by SCI is the tremendous price tag associated with post injury medical and supportive care costs, which is estimated to exceed 10 billion dollars in the US alone. Such astounding demographics

PAGE 21

21 underscore the tremendous need for improved therapeutic management of spinal cord injury (Anderson, 2004, Anderson et al., 2005, DeVivo and Chen, 2011, NSCID, 2011) Pathophysiology of Spinal Cord Inju ry Traumatic injury to the spinal cord (SCI) initiates a cascade of events that is characterized by an initial primary injury response, a prolonged secondary injury phase, and finally stabilizing and reaching a chronic homeostasis (Tator, 1995, Tator and Koyanagi, 1997, Silver and Miller, 2004, Donnelley and Popovich, 2008, Rowland et al., 2008) Primary i njury The immediate effects of SCI include mechanical damage to axons, neurons, glia, and blood vessels in response to the initial physical trauma. Com mon mechanisms of traumatic SCI include physical stresses such as contusion, crushing, shearing, stabbing, and or stretching of the spinal cord, all of which can disrupt the structural integrity of the spinal column, resulting in damage to the spinal cord. Damage to neurons leads to immediate functional deficits related to the specific neurons that are affected (Tator, 1995, Hagg and Oudega, 2006, Belegu et al., 2007, Rowland et al., 2008) Secondary i njury Following the initial primary injury, a number of pathophysiological processes are initiated that evolve over a period of minutes, days, and weeks and can contribute to both preservation and/or exacerbation of further tissue damage. These processes include vascular dysfunction, edema, ischemia, electroly te imbalance, excitotoxicity, inflammatory processes, production of free radicals, and apoptosis. The early phase of secondary injury is generally considered to occur within the first few days following SCI

PAGE 22

22 and is characterized by continuing hemorrhage, in creasing edema, and inflammation and marks the onset of additional secondary injury processes including free radical production, ionic dysregulation, glutamate mediated excitotoxicity, and immune associated neurotoxicity that contribute to further axonal injury and cell death (Tator, 1991, Hagg and Oudega, 2006, Belegu et al., 2007, Rowland et al., 2008) Inflammatory response s Almost immediately after injury, a number of inflammatory responses are initiated. One factor contributing to the immediate rele ase of these inflammatory mediators is the increased permeability of the blood brain barrier that occurs in response of SCI (Horner et al., 1996, Donnelley and Popovich, 2008) Inflammation related responses include the activation and recruitment of a numb er of inflammatory cells, including microglia, leukocytes, neutrophils, monocytes, macrophages, and T cells (Popovich et al., 1997, Schnell et al., 1999a, Schnell et al., 1999b, Donnelley and Popovich, 2008) In addition, there is an immediate upregulation in the production of pro inflammatory cytokines such as Tumor Necrosis Factor alpha (TNF ) and Interleukin 1 beta (IL 1 ). Although the production of these cytokines is normal even in healthy nervous systems, the sustained upregulation of these substances can lead to severe neuroinflammation and can ultimately lead to further cell death (Lee et al., 2000, Hermann et al., 2001, Shamash et al., 2002) Indeed, a number of studies have implicated these pro inflammatory substances as playing a role in secondary tissue damage after SCI (Lacroix et al., 2002, Okada et al., 2004) In the early phases of SCI, neutrophils and monocytes are recruited to the site of injury. Studies have demonstrated that activation and recruitment of these inflammatory molecules leads to recruitment of cytokines and chemokines that are involved in

PAGE 23

23 mediating apoptotic cell death as well as contributing to vascular dysfunction and edema (Bareyre and Schwab, 2003) Excitotoxicity. Widespread neuronal depolarization leads to excessive release of excitatory amino acid neurotransmitters such as glutamate and aspartate. Release of excitatory neurotransmitters leads to hyperactivation of N methyl D aspartate (NMDA) receptors as well as alpha amino 3 hydroxy 5 methy 4 isoxazole propionic acid (AMPA)/kainite glutamate rece ptors in post synaptic neurons. Over activation of these rece ptors leads to an influx of calcium ions, which contributes to dysfunction of membrane bound sodium/potassium pumps. This leads to a massive influx of sodium and calcium ions and an outflow of potassium ions. This results in a substantial imbalance in the normal ionic homeostasis, which can lead to significant edema, loss of cell membrane integrity, and the production of protein kinases that can ultimately contribute to apoptosis and cell death (Choi, 1992, Matyja et al., 2005) Production of free r adical s Free radicals (also known as reactive oxygen species (ROS)), are highly toxic scavengers that are produced by cellular metabolism. After injury, tissue necrosis and recruitment of inflammatory cells such as neutrophils, macrophages, and microglia contrib ute to the increased formation of ROS and the normal antioxidant mechanisms responsible for offsetting the toxic effects of ROS are overcome. P roduction of free radicals contributes to lipid peroxidation, which plays a role in axonal disruption and the dea th of both neurons and glia. Lipid peroxidation leads to cell membrane damage, intracellular organelle dysfunction, and ultimately result s in cell lysis (Xiong et al., 2007) Further, production of ROS including superoxide and nitric oxide, which combine t o form peroxynitrite, (Colton and Gilbert, 1987, Liu et

PAGE 24

24 al., 2000, Chatzipanteli et al., 2002, Xiong et al., 2007) have been shown to induce cellular apoptosis and oxidation of proteins, lipids, and nucleic acids (Bao et al., 2003) Cell death and d emyelin ation. Secondary cell death after SCI may occur by necrosis or apoptosis (Beattie et al., 2002a, Beattie et al., 2002b) In addition to death of neurons, oligodendrocytes, which are highly sensitive to ischemic injury, readily undergo apoptosis following S CI (Crowe et al., 1997) Oligodendrocytes are also susceptible to excitotoxic cell death following injury, and the loss of oligodendrocytes can lead to axonal demyelination, and exacerbation of secondary cell death. Axonal Regeneration Following Spinal Co rd Injury Following injury to the adult mammalian spinal cord, axons fail to regrow and as a result, functional connectivity of the central nervous system is not fully restored. We now know that under certain conditions, axons of the adult central nervous system do in fact have the capacity to regenerate (David and Aguayo, 1981) however after injury there are a number of factors than limit this ability. One of the major limitations to axonal regeneration in the adult central nervous system seems to stem from the formation of the glial scar. The scar is composed of reactive astrocytes, microglia, oligodendrocyte precursor cells, macrophages, and extracellular matrix molecules (Fawcett and Asher, 1999) Formation of the glial scar begins in the subacute pha ses, in which astrocytes at the periphery of the lesion become hypertrophic and proliferative (a process called reactive gliosis). During this phase, a significant increase in the expression of the astrocytic intermediate filament glial fibrillary acidic p rotein is evident, and the astrocytes begin to form a compact and dense scar (Tator, 1995, Silver and Miller, 2004, Fitch and Silver, 2008, Rowland et al., 2008)

PAGE 25

25 Early studies indicated that this scar formed a mechanical barrier, through which axons wer e unable to traverse (Windle and Chambers, 1950) However, we now understand that the inability of axons to regrow after injury was not simply the result of a physical impediment, but in fact, partly due to factors inherent to the glial scar that are known to contribute to its inhibitory nature (Fitch and Silver, 2008, Silver, 2008) and to the development of an environment that is inhibitory to axonal growth and regeneration after injury. These factors include the presence of molecules such as myelin associ ated glycoproteins (MAGs) chon d roitin sulfate proteoglycans (CSPGs) and other inhibitory molecules (Fawcett and Asher, 1999) The extent to which endogenous inhibitory factors must be overcome such that long distance regeneration or regrowth of axons ac ross a lesion can occur, has become somewhat of a controversial topic in recent years. In fact, there is growing evidence to suggest that alternative strategies, including promotion of endogenous plasticity and the formation of novel synaptic relays, may i n fact provide more feasible therapeutic targets for optimizing functional recovery after spinal cord injury (Reier, 2004, Courtine et al., 2008b, Edgerton et al., 2008) Gray Versus White Matter Pathology Spinal cord injury results in damage to both gray and white matter. However, most experimental models of SCI have focused on the interruption of ascending and descending white matter pathways, with little attention to the role of gray matter. Indeed, damage to the white matter, which contains ascending a nd descending axonal tracts, has been shown to cause significant sensorimotor functional impairments of th e upper (Schrimsher and Reier, 1993, Onifer et al., 1997, Anderson et al., 2005, Onifer et al., 2005) and lower extremities (Magnuson et al., 1999, Ma gnuson et al., 2005, Courtine et

PAGE 26

26 al., 2008b) as well as the respiratory system (el Bohy et al., 1998, Doperalski et al., 2008, Fuller et al., 2008, Golder et al., 2011) in both humans as well as experimental animal models of SCI. Further evidence to sugge st the relative importance of white matter versus gray matter was implicated by Goldstein and colleagues in a case report of an individual who developed a post traumatic syrinx in the cervical spinal cord several years after suffering an incomplete spinal cord injury at T4. Upon his death, histological inspection of the spinal cord revealed substantial neuronal damage (gray matter destruction) that was not consistent with measures of upper extremity muscle function, which remained relatively preserved to a great extent (Goldstein et al., 1998) In addition, a number of studies have correlated the extent of residual function after spinal cord injury with the extent of axonal (white matter) tracts spared (Basso, 2000, Scholtes et al., 2011) Taken together, t hese studies might lead us to believe that the extent of white matter damage (or sparing) may be the primary factor contributing to functional outcomes after spinal cord injury (Anderson et al., 2005) Recently, increasing attention is being focused on th e relative importance of gray matter damage to functional outcomes after SCI (Magnuson et al., 1999, Hadi et al., 2000, Reier et al., 2002, Lane et al., 2008b) Similar to white matter damage, damage to the gray matter has also been shown to result in cons iderable functional deficits. For example, Hadi and colleagues used Kainic acid (KA) to induce excitotoxic focal lesions within the gray matter of the lumbar spinal cord. Injection of KA into the spinal cord resulted in neuronal death within the intermedi ate gray matter without damage to the surrounding white matter tracts or to the motoneuron pools. They hypothesized that such injections would interrupt the circuitry of the locomotor central pattern generator,

PAGE 27

27 leading to significant functional deficits. I ndeed, KA lesions targeted at the L2 segment resulted in extreme deficits in locomotor function. Interestingly, these deficits were observed in spite of the fact that the descending motor pathways were found to be intact (as assessed by measuring the laten cy of motor evoked potentials using transcranial magnetic stimulation ) (Hadi et al., 2000) The location of gray matter disruption seems to be an important factor in determining the functional consequences associated with injury. Indeed, injection of KA in to the lumbar region of the spinal cord (the suspected location of the locomotor central pattern generator circuitry) resulted in substantial damage to the gray matter as well as considerable functional (locomotor) deficits. These deficits were similar to those observed following contusion injuries to the thoracic cord, which have been shown to result in damage to both white and gray matter. Interestingly, KA injections to the thoracic gray matter at T9 also resulted in significant gray matter damage, howev er, locomotor function was minimally impaired (Magnuson et al., 1999) An understanding of the importance of local interneuronal circuitry is just beginning to be realized. Studies utilizing transneuronal tracing techniques have provided a mechanism by wh ich this circuitry can be identified and studied (Lane et al., 2008b) For example, recent studies have provided evidence of considerable remodeling within the local spinal cord circuitry after SCI (Fouad et al., 2001, Bareyre et al., 2004, Ballermann and Fouad, 2006, Courtine et al., 2008a, Fouad and Tse, 2008, Rosenzweig et al., 2010) Specifically Courtine and colleagues performed staggered lesions to the thoracic spinal cord in mice (at T7 and T12 on opposite sides) resulting in complete disruption of descending long tract axons. These injuries resulted in severe

PAGE 28

28 deficits in locomotor function. Substantial spontaneous recovery of stepping was observed by 4 weeks after injury. Retrograde tracers injected in the lumbar region indicated minimal labeling in supraspinal (brainstem) regions. By comparison, considerable labeling within thoracic propriospinal neurons was observed (~40% of control) suggesting that local intrinsic circuitry may be involved in this recovery. This hypothesis was further supported by the finding that focal lesions to the intervening thoracic gray matter completely abolished locomotor function. These findings lend support to the notion that gray matter may play an important role in functional recovery after SCI In particular, spinal g ray matter may provide an important anatomical substrate for plasticity within the neural circuitry involved in motor function (Bareyre et al., 2004, Courtine et al., 2008a) These results indicate that interruption of gray matter, including segmental a nd intersegmental interneuronal circuitry, can have significant functional consequences. In particular, the segmental location of injury appears to be an important factor in determining the extent of functional deficits related to the injury, as evident in studies comparing gray matter damage in thoracic versus lumbar segments. Specifically, neural circuit s involved in the control of a number of motor functions ( i.e. central pattern generators) have been hypothesized to reside in the cervical and lumbar reg ions of the spinal cord (Cowley and Schmidt, 1997) As a result, in addition to disruption of ascending and descending pathways related to white matter disruption, damage to the gray matter within these regions likely disrupts this regional circuitry, cont ributing to the significant functional impairment observed (Magnuson et al., 1999, Hadi et al., 2000, Courtine et al., 2008a) Further, studies demonstrating the importance of gray matter

PAGE 29

29 integrity to functional and anatomical plasticity after SCI provide evidence to suggest that the local neural circuitry of gray matter may provide an important substrate for neuroplasticity after spinal injury (Reier et al., 2002, Courtine et al., 2008a) While the majority of the current evidence regarding the importance of gray matter integrity after SCI has been focused primarily on hindlimb and locomotor function, similar findings might be expected in motor systems arising in the cervical region (respiratory and upper extremity motor systems ) of the spinal cord. Indeed, injuries to the cervical spinal cord result in respiratory and forelimb motor deficits that are analogous to hindlimb deficits following thoracic and lumbar spinal cord injury (Anderson et al., 2009b, Anderson et al., 2009c) Further, a number of investig ations have demonstrated the potential for some spontan eous functional recovery (plasticity) in these systems after incomplete injury, which may be mediated to some extent by the recruitment and plasticity within interneuronal circuitry (Fuller et al., 200 8, Stackhouse et al., 2008) C ervical Spinal Cord Injury Injuries to the cervical spinal cord comprise nearly half of the 250,000 cases of spinal cord injury in the US (Anderson, 2004, Anderson et al., 2005, NSCID, 2011) and are characterized by significa nt consequences including paralysis, impaired mobility, respiratory insufficiency, pain, and loss of functional independence (NSCID, 2011) Two of the most devastating consequences of cervical SCI are impaired respiratory and upper extremity function. Uppe r extremity consequences of cervical spinal cord injury Impaired upper extremity function is one of the major functional consequences of cervical SCI Importantly, the majority of individuals living with chronic tetraplegia

PAGE 30

30 indicate that recovery of upper extremity function is their highest priority, stating that improved arm and hand function would most enrich their quality of life (Anderson, 2004) Indeed, upper extremity function is essential to perform even simple tasks of daily life and even minor impr ovements in function may foster an improved sense of independence and autonomy (Anderson, 2004, Anderson et al., 2005, Anderson et al., 2009a) Respiratory consequences of cervical spinal cord injury In humans, high cervical SCI interrupts the rhythmic dr ive to the spinal respiratory motoneurons and results in paralysis of the respiratory muscles. Consequently, persons with such injuries demonstrate significant respiratory deficits, and usually require long term mechanical ventilatory support in order to s ustain life (Brown et al., 2006) In persons with incomplete SCI spontaneous respiration may be spared; however, they often exhibit a rapid and shallow pattern of breathing (i.e. their respiratory rate increases and tidal volume decreases) and they are re quire d to expend increased energy to maintain normal breathing. Additionally, it is quite common for individuals with cervical SCI to have an ineffective cough and difficulty clearing secretions which may predispos e these individuals to atelectasis, mucus retention, and respiratory infections and may ultimately lead to significant morbidity and even mortality (Fishburn et al., 1990, DeVivo et al., 1993, Schilero et al., 2009) In fact, r espiratory related complications represent the leading cause of morbi dity and mortality in persons with SCI (DeVivo et al., 1993, Winslow and Rozovsky, 2003, DeVivo and Chen, 2011) Experimental Models of cervical spinal cord injury While the detrimental effects of cSCI on UE function are well documented clinically, exper imental models of cSCI have not yet elucidated the underlying

PAGE 31

31 anatomical and physiological substrates. Further, studies of upper extremity function following experimental cervical SCI have been few in number. In contrast to the upper extremity, functional recovery within the phrenic (respiratory) motor system after experimental cSCI has been well characterized by our lab and others (Goshgarian et al., 1989, Golder et al., 2003, Fuller et al., 2006, Alilain et al., 2008, Doperalski et al., 2008, Fuller et al ., 2008, Lane et al., 2008a, Lane et al., 2008b, Fuller et al., 2009, Lane et al., 2009, Lane et al., 2012) Specifically, lateral spinal cord hemisection at C2 (C2H x ) is a well characterized a model of cervical SCI that disrupts descending brainstem proje ctions to the phrenic motoneuron pool (C3 C6 ; See Figure 1 1 for illustration) Consequently, this injury immediately silences ipsilateral phrenic nerve activity resulting in immediate ipsilateral diaphragm (and respiratory) dysfunction. Over the course of several weeks to months after injury, gradual, though incomplete, functional recovery occurs. This spontaneous recovery has been studied extensively, and is associated with activation of crossed phrenic pathways and recruitment of propriospinal interneuro ns. These spontaneous neuroplastic changes have been collectively termed the spontaneous crossed phrenic phenomenon (sCPP) (Moreno et al., 1992, Fuller et al., 2006, Huang and Goshgarian, 2009) Neural Control of Cervical Motor Function Injury to the cerv ical spinal cord often leads to significant deficits in both upper extremity and respiratory motor function (NSCID, 2011) These two motor systems are located within close proximity to one another within the cervical spinal cord (See Figure 1 1), though th ey are functionally disparate motor systems, with differing neural control systems (Figures 1 2 and 1 3 for review) In the following chapters, we explore the hypothesis that spinal plasticity is a robust phenomenon that likely occurs across both

PAGE 32

32 upper ext remity and respiratory motor systems. Here, we provide a detailed overview of the neural control of upper extremity and respiratory function. Neural Control of Upper Extremity Motor Function Here, we provide an overview of the neural control of upper extr emity functio n, which is also summarized in F igure 1 2. Supraspinal control of upper extremity function Corticospinal control of upper extremity function. Voluntary control of upper extremity function is primarily controlled by neurons located within the p rimary motor cortex (M1). The primary motor cortex is arranged in a topographic orientation, known as the motor homunculus. The area of the motor cortex innervating the muscles of the upper extremity comprises a relatively large portion of the motor homunc ulus and is located on the most rostral surface of the cortex. These neurons receive inputs from and project to supplementary and association motor areas, as well as the basal ganglia sensory cortex visual cortex, the cerebellum. These neurons transmit d escending signals via mono and poly synaptic (cortic o spinal) connections with upper extremity motoneurons and propriospinal interneurons within the cervical spinal cord and brainstem. Corticospinal projections descend in the anterior and lateral white mat ter tracts in humans, primates, and cats, and in the dorsal white matter tracts in rats and mice (Armand, 1982) Direct monosynaptic projections from the motor cortex to the motoneurons controlling muscles of the upper limb (in particular, distal muscles) h ave been demonstrated to be involved in skilled motor tasks, specifically tasks that require fine motor activity (Pettersson et al., 1997, Alstermark and Isa, 2002, Isa et al., 2007) In rats, few, if any, monosynaptic connections from the motor cortex to s pinal motoneurons have been demonstrated (Armand, 1982, Alstermark et al., 2004)

PAGE 33

33 Rubrospinal control of upper extremity function Neurons originating in the magnocellular red nucleus have extensive projections to spinal neurons. These neurons descend in the dorsolateral white matter tracts of the spinal cord in rats, and in humans. Dense terminations of rubrospinal projections have been demonstrated in the intermediate gray matter of the cervical spinal cord (laminae V VII) as well as projections to moto neurons of the upper extremity (McCurdy et al., 1987) Rubrospinal projections have been shown to play a role in the production of coordinated, whole arm movements. Specifically, these projections are believed to coordinate limb stability and reach accurac y during reach to grasp arm movements (Gibson et al., 1985, Levesque and Fabre Thorpe, 1990, van Kan and McCurdy, 2002) Reticulospinal control of upper extremity function Neurons arising in the reticular formation descend in the ventr o medial white matt er tracts in rat s, cats, primates and human s Reticulospina l neurons project primarily to segmental interneurons, though there is some evidence of monosynaptic projections to motoneurons (Holstege and Kuypers, 1982, Martin and R.P., 1985, Holstege and Kuy pers, 1987, Riddle et al., 2009) These neurons have been shown to branch extensively in the spinal cord, with a single axon making synaptic contacts with neuron pools in multiple spinal segments (Peterson et al., 1975, Matsuyama et al., 1997) Reticulospi nal influence on upper limb function has been shown to play a role in the coordination and control of proximal and axial musculature, and has been shown to be important to locomotor (Matsuyama and Drew, 2000) reaching (Schepens and Drew, 2004, 2006) and postural behaviors (Prentice and Drew, 2001, Schepens and Drew, 2004)

PAGE 34

34 Spinal Control of Upper Extremity Function Descending upper extremity tracts. Motor commands originating in supraspinal centers are transmitted via descending axonal projections onto sp inal interneurons and motoneurons, ultimately resulting in the activation of muscles of the upper extremity. Direct projections from the primary motor cortex to motoneurons of the forelimb are few in number in rodents, but have been shown to course along t he corticospinal tracts located in the dorsolateral white matter in humans, and the dorsomedial white matter columns in rats (Armand, 1982) Reticulospinal axons, carrying signals from the reticular nuclei in the brainstem to the forelimb motoneurons loca ted in the ventral gray matter cross at the level of the medullary pyramids and descend in the dorsolateral and ventromedial white matter tracts. Rubrospinal axons, originating in the red nucleus of the brainstem, travel in the dorsolateral white matter co lumns and project to interneurons and motoneurons in the spinal cord, and transmit information related to posture and orientation of the body in space (Alstermark et al., 1981b) Upper extremity motoneurons. The motoneurons innervating the muscles of the u pper extremity are distributed in continuous columns of cells within the ventral gray matter of the cervical and upper thoracic levels of the spinal cord (C2 T2) in both humans and rats (McKenna et al., 2000, Tosolini and Morris, 2012) These motoneurons r eceive mono and poly synaptic input from brainstem and cortical motor areas including the primary motor cortex, supplementary motor area, premotor cortex, red nucleus, vestibular and reticular nuclei, as well as inputs from subcortical and projection regi ons of the brain and afferent input from peripheral receptors. Axonal projections from forelimb motoneurons exit the spinal cord via the C2 T1 ventral roots

PAGE 35

35 and form a complex branching network of nerves known as the brachial plexus. The major branches ari sing from the brachial plexus to innervate the forelimb musculature include the median, ulnar, and radial nerves. The median nerve arises from the C5 to the C8 segments and innervates most of the distal flexor muscles of the upper extremity ( except flexor carpi ulnaris ). Specifically, t he median nerve innervates pronator teres, flexor carpi radialis, Palmaris longus, flexor digi t o r um superficialis, part of flexor digitorum profundus, flexor pollicis longus and pronator quadrates. The ulnar nerve arises from the C7 to the T1 spinal cord segments, and innervates the flexor carpi ulnaris, part of the flexor digitorum profundus, Palmaris brevis, the hypothenar muscles, the palmar and dorsal interossei, the third and fourth lu m bricals, and the adductor pollicis. The radial nerve arises from the C5 to T1 spinal cord segments, and innervates the triceps brachii, ancone u s, brachioradialis, extensor carpi radialis longus and brevis, the supinator, extensor digitorum, extensor digiti minimi, extensor carpi ulnaris, abd uctor pollicis longus, ext ensor pollicis brevis, extensor pollicis longus, and the extensor indicis muscles (Standring, 2009) Segmental and propriospinal interneurons Throughout the cervical and thoracic spinal cord are segmental and propriospinal inter neurons located in the dorsal and ventral horns as well as in the intermediate gray matter. Segmental interneurons are defined as short distance neurons that project to targets within the same spinal segment. Propriospinal are located within the spinal cor d and project to other spinal regions. These neurons can subdivided into short versus long propriospinal interneurons, based on the length of their axons. Short propriospinal interneurons typically span 1 6 spinal segments, whereas long propriospinal neuro ns project over

PAGE 36

36 greater than 6 spinal segments (Conta and Stelzner, 2009) The presence of propriospinal interneurons in the cervical spinal cord has been documented in a number of species, including cats, rats, and primates. Alstermark and colleagues dem onstrated that a population of propriospinal interneurons located in upper cervical segments (C3 C4) are involved in corticospinal tract dependent forelimb motor tasks (e.g. targeted reaching tasks). These neurons are commonly referred to as the C3 C4 prop riospinal system, and are involved in the polysynaptic transmission of corticospinal tract inputs to motoneurons innervating muscles of the distal upper extremities and digits. T hese interneurons have been shown to also convey polysynaptic r ubrospinal and r eticulospinal inputs to upper limb motoneurons. Propriospinal neurons relay descending motor commands from higher brain centers. In addition, propriospinal neurons receive inputs from peripheral afferents (mostly via segmental interneurons) which enables the integration of sensory inputs and motor commands Another target of cervical propriospinal neurons (long propriospinal interneurons ) are more caudal spinal segments. These connections have been shown to coordinate preparatory movements required in orde r to perform upper extremity tasks such as targeted reaching (Alstermark et al., 1981a, Alstermark et al., 1981b, Alstermark et al., 1984a, b, Alstermark et al., 1990, Alstermark et al., 1991, Alstermark et al., 2007) In addition, reciprocal connections b etween of cervical and lumbar motor circuits are mediated by long distance propriospinal neurons (Illert et al., 1977, Illert et al., 1978, Illert and Tanaka, 1978, Illert et al., 1981) Neural Control of Respiration Here, we provide an overview of the ne ural control of respiratory function, which is also summarized in F igure 1 3. The brainstem is the site of origin of respiratory

PAGE 37

37 rhythm in mammals (Feldman, 1986, Feldman et al., 2003) Premotor respiratory centers, responsible for the generation and maint enance of respiratory rhythm, are primarily located within functionally and anatomically distinct nuclei within the medulla and pons (Alheid and McCrimmon, 2008) Descending rhythmic drive is transmitted from these centers to spinal and cranial motoneurons that control respiratory muscles. Respiratory afferents convey inputs back to brainstem respiratory centers, facilitating feedback control of respiratory motor output. Neural control of respiration is summarized in Fig ure 1 3 Brainstem control of respira tion Medullary respiratory nuclei. The medulla contains a number of discrete groups of neurons that are involved in the neural control of breathing in mammals. These populations of neurons are involved in both the generation of respiratory rhythm, as well as the transmission of respiratory signals to motor nuclei located in the brainstem and spinal cord. The medullary respiratory nuclei include th e pre Btzinger complex, the Btzinger complex, and the ventral and dorsal respiratory columns. Pre Btzinger co mplex (pre BtC). The Pre BtC is located in the ventrolateral medulla. Sometimes considered an extension of the rostral ventral respiratory column, this distinct groups of neurons is located between the obex and the facial nucleus, caudal to the Btzinger Complex. Pre BtC neurons are primarily interneuronal and are connected to cranial motor neurons and para ambigual premotor neurons, which have descending projections to upper airway respiratory muscles and spinal motor neurons, respectively (Kalia, 1977, Merrill and Fedorko, 1984) The precise role of pre BtC neurons is not clear, however, they are considered essential for the generation of rhythmic respiratory drive (Lipski and Merrill, 1980) as they demonstrate intrinsic

PAGE 38

38 bursting that occurs in phase w ith the respiratory cycle (Feldman, 1986) Furthermore, lesions to the pre BtC have been shown to abolish rhythmic breathing in mammals (Ramirez et al., 1998) Thus, the pre BtC is ofte underlying respiratory rhythm (Smith et al., 1991) Btzinger complex (BtC). The BtC is l ocated rostral to the Pre BtC, and is comprised of neurons that are closely related to the expiratory phase of breathing (Ezure, 1990) BtC neurons are active during the expiratory phase of respiration, though it is unclear whether these neurons constitute an e xpiratory rhythm generator. Extensive inhibitory projections from the BtC to pre motor interneurons and respiratory neurons in the brainstem and spinal cord have been demonstrated, Thus, BtC neu rons are believed to play a role in active ly inhibit ing inspiratory motoneurons during expiration (Alheid and McCrimmon, 2008) Ventral respiratory column (VRC). The VRC is a population of pre motor and motor respiratory neurons located in the ventrolater al medulla of the brainstem The rostral part of the VRC (rVRC) extends from the level of the obex to the facial motor nucleus (Bianchi, 1971, Bianchi et al., 1973) and lies in close proximity to the nucleus ambiguus (Kalia, 1981) The caudal part of VRC (cVRC) extends from the level of the obex to the border between the spinal cord and medulla (Bianchi, 1971) and lies in close proximity to the nucleus retroambiguus (Merrill, 1975) The rVRC contains primarily inspiratory neurons (Bianchi, 1974) which cro ss midline rostral to obex and project to phrenic and intercostal motor nuclei in the spinal cord (Merrill, 1974) In addition to pre motor neurons the VRC includes laryngeal and

PAGE 39

39 pharyngeal motoneurons as well as propriobulbar neurons which coordinate the activity within upper airway muscles (Ellenberger and Feldman, 1990) The cVRC contains primarily expiratory neurons, which demonstrate increased firing rates as expiration progresses (Merrill, 1970) Projections from the cVRC cross the midline caudal to obex and project to expiratory intercostal and abdominal motoneurons in the contralateral spinal cord (Merrill, 1974) Dorsal respiratory column (DRC). The DRC, also referred to as the ventrolateral division of the Nucleus Tractus Solitarius, is locate d in the dorsal medulla (Baumgarten and Kanzow, 1958, Bastel and Lines, 1973, Alheid and McCrimmon, 2008) P rojections arising in the DRC terminate at various locations within medulla including the reticular formation, the ventral respiratory column, the n ucleus ambiguous, the hypoglossal nucleus, and the dorsal motor nucleus of vagus nerve. In addition, projections from the DRC have been shown to extend to the c ontralateral phrenic motoneuron (PhMN) pool in the spinal cord via the lateral funiculus (Berger et al., 1984, Otake et al., 1989) DRC neurons receive afferent input from pulmonary stretch receptors, carotid and aortic chemoreceptors, and C fiber nerve endings in the lung via the vagus nerve (Bianchi, 1971) In addition, DRC neurons receive input fr om the contralateral cVRC, contralateral pre Bt C and ipsilateral pontine respiratory centers (Kalia et al., 1979, Bystrzycka, 1980) The majority of DRC neurons are active during the inspiratory phase of respiration (Bastel, 1964) thus DRC neurons are b elieved to play a role in the coordination of inspiratory bursting via transmission of respiratory related information to other respiratory groups within the brainstem (Otake et al., 1989, Alheid and McCrimmon, 2008)

PAGE 40

40 Pontine respiratory nuclei. Respirato ry centers located within the dorsolateral pons, include the nucleus parabrachialis medialis and Klliker Fuse nucleus (Baxter and Olszewski, 1955) Neurons within pontine respiratory centers appear to be modulated by the respiratory cycle and are believed to play a role in the transition between respiratory phases (Douse and Duffin, 1993, Alheid et al., 2004) Cortical control of respiration Humans are capable of consciously manipulating their breathing pattern in order to perform complex behaviors such as speaking, eating, singing, holding their breath, etc. Projections arising in cerebral cortex are believed to coordinate changes in respiratory pattern in order to facilitate such complex behaviors. Cortical projections to brainstem respiratory centers ( Ramsay et al., 1993) and to spinal motoneurons (Rickard Bell et al., 1985, Shea, 1996, Corfield et al., 1998) have been identified. Spinal Respiratory Control Descending respiratory tracts Both involuntary (medullary) and voluntary (cortical) respiratory signals are relayed to motoneurons in the brainstem and spinal cord via white matter tracts. In humans as well as animals, involuntary rhythmic respiratory signals are transmitted via axons that origin ate in neurons in the medulla. These axons cross at the level of the brainstem and project to the ventrolateral column of the contralateral spinal cord (Gad and Marinesco, 1892, Davis and Plum, 1972) Within this column, the expiratory axons lie medial ly and inspiratory axons are located more laterally At cervical levels 25% of descending axons branch to make contacts with phrenic motoneurons, while the remain ing axons descend toward intercostal and abdominal motoneurons (Merrill, 1974) I nvoluntary non rhythmic (tonic) drive travels from medial reticular nuclei to the spinal segments via the ventral and ventrolateral

PAGE 41

41 columns of ipsilateral spinal cord (Pitts, 1940) Finally, descending spinal pathways associated w ith voluntary respiratory drive ( corticospinal tracts ) are located within the dorsolateral co lumn of the spinal cord in humans or the dorsomedial column in rats (Aminoff and Sears, 1971) Phrenic motoneurons (PhMNs). PhMNs innervate the diaphragm and thus, are the primary motoneurons involved in respiration in mammals. These motoneurons constitut e a narrow longitudinal column of motoneurons in the medial ventral horn of the cervical spinal cord (Duron et al., 1979) Th e PhMN pool extends from C3 C5 in humans C3 C 6 in rats, and C4 C6 in cats (Webber et al., 1979, Kuzuhara and Chou, 1980) an d contains about ~2 00 300 motoneurons per side (Furicchia and Goshgarian, 1987, Mantilla et al., 2009) Axons of PhMNs exit the spinal cord via the ventral roots on either side of the spinal cord to form the phrenic nerves, which each innervate one half of the diaphragm. Ph M Ns receive excitatory inputs during inspiration from inspiratory bulbospinal premotor neurons in the VRC and DRC (Berger, 1979) The bulbospinal pathways between pre motor medullary respiratory neurons and PhMNs are either monosynaptic o r polysynaptic, with synaptic relays formed with spinal respiratory interneurons (Cohen et al., 1974, Fedorko et al., 1981, Davies et al., 1985) PhMNs rhythmically activate the diaphragm via the phrenic nerves and demonstrate different firing patterns whi ch distinguish them as neurons that fire either or in inspiration (St John and Bartlett, 1979, Lee et al., 2009) Intercostal and Abdominal motoneurons. Intercostal motoneurons also play a role in respiratory function extend the entire leng th of the thoracic spinal cord and are located laterally in the ventral gray matter (Larnicol et al., 1982a, b) They receive

PAGE 42

42 mono and poly synaptic inputs from inspiratory and expiratory brainstem respiratory centers and innervate internal (expiratory) an d external (inspiratory) intercostal muscles (Duffin and Lipski, 1987) The abdominal motoneurons reside in the lower thoracic and upper lumbar spinal cord, and information regarding their morphological characteristics and organization are not well defined They receive synaptic input from pre motor neurons in the cVRC (Miller et al., 1985) and are involved in controlling the abdominal muscles during expiration (Fedorko and Merrill, 1984, Long and Duffin, 1986) Segmental and propriospinal interneurons Th e central respiratory drive to respiratory motoneurons is provided primarily via monosynaptic inputs from inspiratory bulbospinal neurons in the medullary respiratory column ( reviewed above) (Cohen et al., 1974, Hilaire and Monteau, 1976) There is, h oweve r, growing evidence for polysynaptic descending inputs to spinal respiratory motoneurons. Recent studies have demonstrated anatomical connectivity between descending brainstem projections and retrogradely labeled phrenic motoneurons and pre motor interneur ons (Dobbins and Feldman, 1994, Lane et al., 2008b) Retrograde transynaptic tracing has revealed pre phrenic interneurons in lamina e VII and X of the intermediate gray matter as well as in the dorsal horn of the cervical spinal cord. Furthermore, descend ing projections from the VRC were shown to project to retrogradely labeled pre phrenic interneurons indicating the potential role of these interneurons as synaptic relays between pre motor respiratory neurons in the brainstem and Ph MNs. In addition, dual labeling experiments demonstrated bilaterally projecting interneurons innervating the PhMN pools providing some evidence for a potential role in the integrating communication between the phrenic motoneuron pools as well as with other respiratory motor cir cuits (Lane et al., 2008b)

PAGE 43

43 Neurophysiological assessment of the relationship between firing of brainstem pre motoneurons and PhMN activity suggests the presence of both monosynaptic and poly synaptic connections to Ph MNs (Davies et al., 1985) Neuromodu latory Control of Respiration Serotonin, or 5 hydroxytryptamine (5 HT), is a monoaminergic neurotransmitter that also demonstrates neuromodulatory effects (Hochman et al., 2001) For example, 5 HT has been shown to modulate motoneuron excitability (Lindsay and Feldman, 1993) Neuromodulatory Projections to Respiratory Motoneurons. The majority of 5 HT projections to the spinal cord originate in the raphe nuclei, which are located around the midline of the medulla, pons, and midbrain. The majority of 5 HT projections to the spinal cord originate in the raphe nuclei, which are located around the midline of the medulla, pons, and midbrain. The raphe nuclei can be divided into two major subsections: the rostral nuclei, which are located in the midbrain and in the rostral pons, and the caudal nuclei, which are located in the caudal pons and the medulla. The rostral, or dorsal, nuclei of the raphe include the caudal linear nucleus, the dorsal raphe nucleus, the median raphe nucleus and the nucleus raphe pontis. P rojections from the rostral raphe nuclei project primarily innervate structures within the forebrain. The caudal, or ventral, raphe extends from the pons to the spinomedullary border and consists of the raphe pallidus, the raphe obscurus, the raphe magnus, the rostral ventrolateral medullary nucleus, and the lateral paragigantocellularis reticularis. Projections from the caudal raphe nuclei terminate primarily within the spinal cord as well as the brainstem. Respiratory motoneurons, PhMNs in particular, are highly

PAGE 44

44 innervated by 5 HT, receiving projections primarily for the raphe pallidus and raphe obscurus nuclei. (Steinbusch, 1981; Hochman et al., 2001). 5 HT Receptors. There are seven families of 5 HT receptors (5 HT 1 7 ), and 14 different receptor sub typ es (Hochman et al. 2001) 5 HT 1 receptors. The 5 HT 1 receptor family is comprised of 5 receptor subtypes (5 HT 1A,B,C, D,F ), which are G i/o protein coupled receptors that have been demonstrated in the spinal cord (primarily in the dorsal horn), as well as throughout the central nervous system and in blood vessels. 5 HT 1 receptors act to inhibit target neurons by decreasing cellular cyclic adenosine monophosphate ( cAMP ) (Hochman et al., 2001) 5 HT 2 receptors. The 5 HT 2 receptor family is comprised of thre e different receptor subtypes (5 HT 2A,B,C ) are G q protein coupled receptors that have been demonstrated in the spinal cord, blood vessels and the gastrointestinal ( GI ) tract (Hochman et al., 2001) In particular, 5 HT 2A receptors are expressed in the ventr al mo tor regions of the spinal cord, specifically on PhMNs, where they have been shown to play a role in regulating phrenic motoneuron excitability (MacFarlane et al ,. 2009, Fuller et al., 2005) 5 HT 2 receptors activate proteins leading to the formation o f inositol triphosphate, which leads to the activation of protein kinase C (PKC) Activation of 5 HT 2 receptors typically results in increased neuronal excitability via phosphorylation of glutamatergic synapses (Hochman et al., 2001) In addition, activati on of 5 HT 2 receptors also increases in persistent inward currents, which also influences neuronal excitability. 5 HT 3 receptors. 5 HT 3 receptors are ligand gated sodium ( Na + ) and potassium ( K + ) channel receptors that have been demonstrated in the spinal cord (primarily in the

PAGE 45

45 dorsal horn) and in the GI tract. 5 HT 3 receptors act to excite target neurons by fast cellular depolarization (Hochman et al., 2001).. 5 HT 4 receptors. 5 HT 4 receptors are G s protein coupled receptors that are located throughout the central nervous system and in the GI tract. 5 HT 4 receptors act to excite target neurons by increasing cellular cAMP (Hochman et al., 2001).. 5 HT 5 receptors. The 5 HT 5 receptor family are G i/o protein coupled receptors that are located throughout the c entral nervous system. 5 HT 5 receptors act to inhibit target neurons by decreasing cellular cAMP (Hochman et al., 2001).. 5 HT 6 receptors. The 5 HT 7 receptor family are G s protein coupled receptors that are located throughout the central nervous system. 5 HT 6 receptors act to excite target neurons by increasing cellular cAMP (Hochman et al., 2001). 5 HT 7 receptors. The 5 HT 7 receptor family are G s protein coupled receptors that have been demonstrated in the spinal cord (primarily in the ventral horns), bl ood vessels and the GI tract. 5 HT 7 receptors act to excite target neurons by increasing cellular cAMP (Hochman et al., 2001). Characterization of Functional Plasticity in Experimental SCI Assessment of Upper Extremity Function Gross motor function Assessm ent of gross motor function in rats is most commonly performed using the cylinder test, also known as the limb use asymmetry test. This test was originally designed for use in rodent models of stroke, but has since been applied to numerous models of nervou s system dysfunction and injury. The cylinder test has been used extensively to investigate left/right asymmetries in upper limb use during vertical exploration. Administration of this test entails that a rat be placed in a clear Plexiglas

PAGE 46

46 cylinder for a p eriod of five minutes, during which the number of paw placements on the cylinder wall are counted (Hua et al., 2001, DeBow et al., 2003) This test has also been used in cervical spinal cord injury models and has been shown to be an effective indicator for recovery of gross motor function (Gensel et al., 2006) Fine motor manipulation Numerous behavioral tests have been developed for assessing fine motor manipulation in rodents, including various versions of pasta manipulations (Allred et al., 2008, Tennant et al., 2010) skilled reaching (Montoya et al., 1991, Pagnussat Ade et al., 2009) and reach to grasp tasks (Stackhouse et al., 2008) The vermicelli pasta handling test is one such test, and was originally designed for use in rodent models of stroke. In thi s test, rodents are given seven centimeter lengths of pasta to eat. This test assesses several param e ters based on how the rat eats the pasta including the time it takes to eat the length of pasta, the number of adjustments made by each paw when eating the pasta, and the presence and extent of any abnormal behaviors observed while the animal is eating (Allred et al., 2008, Tennant et al., 2010) This test has been used in a number of neurological conditions, though its applicability in a cervical spinal cord injury model has not been thoroughly assessed (Khaing et al., 2012) Locomotor function Tests of both forelimb and hindlimb locomotion have been developed to investigate how injury to the nervous system may influence locomotor function. The most exte nsively used test of locomotor function in rodent spinal cord injury models is the Basso Beattie Breshnahan Open Field Locomotor Rating Scale (BBB) This test was originally developed to characterize locomotor deficits following thoracic contusion injuries in rats. This test has since been applied to a number of different species and

PAGE 47

47 injury models, however use ful in cervical models of spinal cord injury is limited, as it does not specifically assess forelimb deficits in locomotion (Basso et al., 1995, Metz et al., 1998, Magnuson et al., 1999, Basso, 2000, Metz et al., 2000, Ma et al., 2001) A number of forelimb specific locomotor tests have been developed or modified for use in cervical spinal cord injury models, including the Forelimb Locomotor Assess ment Scale (FLAS) (Anderson et al., 2009b) the Forelimb Locomotor Scale (FLS) (Sandrow et al., 2008) grid walking and horizontal ladder walking tests (Metz and Whishaw, 2002, 2009) as well as a modified version of the BBB which includes a forelimb spec ific scoring component (Martinez et al., 2009) The FLS is a locomotor test, which was developed by the Behavioral Analysis Core at Drexel University for the assessment of forelimb locomotor deficits following spinal cord injury. This test has been utilize d in a number of investigations, and has been shown to be a reliable indicator of upper extremity dysfunction and recovery after spinal injury (Sandrow et al., 2008, Khaing et al., 2012) Characterization of Ventilatory Function in Experimental SCI Asses sment of ventilatory function in rats can be performed using a variety of techniques. These techniques range from direct to indirect assessments of ventilatory function, and include both non invasive and invasive methods (Baekey et al., 2009) Ventilatory function Assessment of ventilatory function in rats is most commonly performed using whole body plethysmography (Baekey et al., 2009) Whole body plethysmography enables the non invasive quantitative measurement of respiratory ventilation. This method en tails placing an animal in a sealed chamber and measuring changes in pressures within the chamber. As the animal inspires and expires, the gas pressure

PAGE 48

48 inside the chamber increases and decreases, respectively (Stephenson and Gucciardi, 2002) This method f acilitates the quantification of ventilatory frequency as well as indirect measure ments of tidal volume and minute ventilation which are calculated using a complex algorithm, described by Drorbaugh and Fenn (Drorbaugh and Fenn, 1955, Jacky, 1978) In addi tion, a number of parameters relevant to the pattern of ventilation (inspiratory duration, expiratory duration, etc. ) can be assessed (Askanazi et al., 1980) Whole body plethysmography has been shown to be a valid and reliable measure for assessing respir atory function, and has been used in a number of experimental models of pathology and disease, including cervical SCI (Stephenson and Gucciardi, 2002) Respiratory electromyography Electromyographic (EMG) recordings of respiratory muscles including the d iaphragm, abdominals and intercostals have been used to study respiratory muscle activity in both anaesthetized and unanaesthetized, spontaneously breathing preparations (Dougherty et al., 2012, Lane et al., 2012, Nicaise et al., 2012) EMG recordings prov ide more specific information regarding the contribution of specific muscle groups to respiratory output than can be interpreted from plethysmography, though there are numerous technical considerations that must be taken into account when interpreting EMG data including electrode placement, electrical noise, cross talk from other muscle fibers and movement artifact (Merletti, 1999) Phrenic nerve recordings Phrenic neurograms have been extensively used to study respiratory motor output in a number of experi mental models of pathology and disease including spinal cord injury (Fuller et al., 2005, Fuller et al., 2006) Phrenic neurograms provide specific

PAGE 49

49 and easily interpretable information regarding the output of the phrenic nerve, however the interpretation of these results must take in to consideration that experimental preparations are conducted in anaesthetized, mechanically ventilated (and often vagotomized) preparations (Lee et al., 2010, Sandhu et al., 2010) Furthermore, phrenic neurograms are invasiv e, and are most often performed as terminal procedures, therefore repeated or long term measurements are not possible (Baekey et al., 2009) Interaction of Muscle and Nerve The relationship between muscles and the nervous system was first realized in 1791 when Luigi Galvani, an Italian physician, mistakenly discovered that electrical signals transmitted via nerves were capable of eliciting a muscular contraction. This discovery called into question the previous believe the muscles contracted by filling with fluid and introduced the notion of neuromuscular electrical signaling. In 1906, Santiago Ram n y Cajal was awarded the Nobel Prize for his work in characterizing the structure of the nervous system. His seminal work served to solidify neurons as the funct ional basis of the nervous system. Also in 1906, Dr. Charles Sherrington published a by which muscles are activated by neurons. He also made first reference to the ref er to an alpha motor neuron and all the muscle fibers innervated by it (Burke, 2007) The concept of the motor unit is of major importance to understanding the basic substrates underlying movement, as it describes the fundamental relationship between

PAGE 50

50 muscl e and nerves and defines the most basic unit required for movement (Burke, 2007) Components of the Motor Unit Alpha motoneurons ( MNs) innervate extrafusal skeletal muscles and are located in the ventral horn of the spinal cord. Axonal projections from MNs exit the spinal cord through the ventral roots and travel in peripheral nerves to synapse on their target muscle (Burke et al ., 1977, Westbury, 1982) Extrafusal skeletal muscle fibers demonstrate considerable heterogeneity of structure (morphology) and function (physiology), which has been shown to be closely associated with the morphological and physiological characteristics o f motoneuronal innervation. Skeletal muscle adaptations to spinal cord injury Morphological Adaptions Whole muscle atrophy is a major consequence of reduced neuromuscular activity foll owing SCI as evidenced by significantly decreased whole muscle mass beginning within days of injury and persisting weeks and even months after injury (Roy and Acosta, 1986, Landry et al., 2004, Liu et al., 2008, Biering Sorensen et al., 2009) The severit y of these adaptations depends on the severity of the spinal cord injury model, with complete transection or spinal cord isolation injuries demonstrating the most severe atrophic effects, followed by contusion injuries and other incomplete injuries. This a pparent atrophy is also evident at the level of the individual muscle fibers. Similar extent of muscle wasting and fiber atrophy is also apparent in humans with chronic SCI (Shah et al., 2006) In addition to a reduced proportion of lean muscle mass, neuro muscular inactivity following spinal cord also leads to increased

PAGE 51

51 intramuscular fat content within skel etal muscle (Edgerton et al., 2002, Elder et al., 2004) Furthermore there appears to be a spectrum of inactivity induced muscle fiber atrophy that pre ferentially affects slow fiber phenotypes. Muscles comprised of mostly slow muscle fibers demonstrate a greater decline in mass, and at the individual fiber lever, slow muscle fibers seem to atrophy to a greater extent that the faster fibers. Further, a s hift in the muscle fiber phenotype from slow to fast fibers is also evident, however, a general decline in fiber size is evident prior to phenotype transformations (Round et al., 1993) The muscles that appear to be m related atrophy and phenotype transitions are those that tend to be used most frequently under normal activity conditions. Specifically, the muscles that seem to be the most affected are muscles that demonstrate higher duty cycles (Edgerton et al., 2001, Edgerton et al., 2002, Harkema, 2008) such as postural or anti gravity muscles, such as the vastus lateralis, soleus, quadriceps, and gastrocnemius. These muscles tend to be comprised of mostly slow phenotype fibers, and therefore demonstrate the greatest muscle atrophy as well as a shift toward fast muscle phenotype (Grossman et al., 1998, Haddad et al., 2003, Kelley et al., 2006, Biering Sorensen et al., 2009, Liu et al., 2010) Physiological adaptations. Changes in the contractile properties of skeletal muscle adaptations again seem to preferentially affect slow phenotypes, with predominantly slow muscles, like the soleus demonstrating increases in specific tension and time to peak tension (Stevens et al., 2006) However if these adaptations are considered in reference to the slow to fast phenotype shift that occurs following chronic spinal cord

PAGE 52

52 injury, it is not surprising that these muscles demonstrate contractile properties more in accordance with faster muscle phenotypes (Edgerton et al., 2002) Morphologic adaptations. with a reduction in the number of motone urons within the motoneuron pool. (Bose et al., 2005). Morphological changes in MNs following chronic contusion spinal cord injury include increased somal area, perimeter, and diameter. The distribution of MNs based on somal area is also significantly altered by chronic spinal cord injury, with a larger proportion of large motoneurons and considerably fewer small and medium sized motoneurons as compared with uninjured animals (Ishihara et al., 1988, Ishihara et al., 1995, Bose et al., 2005, Matsumoto e t al., 2007, Roy et al., 2007) Changes in dendritic arborization are also a consequence of inactivity following spinal cord injury, as evidenced by fewer dendrites and decreased primary, secondary, and tertiary branches. Further, the primary branches of d endrites are longer and of larger diameter after chronic SCI (Bose et al., 2005) There is also evidence of expansion of the neuromuscular junction (Waerhaug and Lomo, 1994) Physiological adaptations. with altered motoneuron excitability and axonal conduction velocity (Bose et al., 2005) These adaptations are likely related to the increase in motoneuron size associated with ity of alpha that upregulation of serotonin sensitive postsynaptic sites on the MNs (Doly et al., 2004, Fuller et al., 2005, Ling, 2008, Mullner et al., 2008, Noga e t al., 2009)

PAGE 53

53 Furthermore, there is emerging evidence that both neural and muscular activity play a role in the regulation of trophic factors such as brain derived neurotrophic factor (BDNF), NT4/5 and glial derived neurotrophic factor (GDNF), all of which have the potential to lead to modulation of synaptic efficacy and/or modulating protein transcription and translation (Houenou et al., 1996, Kinkead et al., 1998, Fuller et al., 2003, Mantilla and Sieck, 2003)

PAGE 54

54 Figure 1 1 Schematic depiction of experimental model of cervical spinal cord injury Lat eral C2 Spinal Cord Hemisection ( C2Hx ) disrupts the descending inputs to phrenic and upper extremity motoneuron pools located in the cervical spinal cord, resulting in paralysis of affected muscle groups, ipsilateral to the injury.

PAGE 55

55 Figure 1 2 Schematic depiction of the neural control of upper extremity motor function. Motor commands originate in cortical and brainstem centers and are transmitted to motoneurons innervating postural and upper limb muscles. Afferent feedback is transmitted back to the spinal cord, brainstem, and higher brain centers where appropriate alterations in neural commands can be made to adjust motor output according to the task demands

PAGE 56

56 Figure 1 3 Schematic depiction of the neural control of breathing. Respiratory rhythm is generated in brainstem respiratory centers and transmitted to motoneurons in the brainstem and spinal cord innervating muscles of the upper airway, thorax and abdomen. Afferent and chemosensory feedback is transmitted back to the spinal cord, brainstem, and higher brain centers where appropriate alterations in neural commands can be made to adjust ventilation according to the task demands (Adapted from (Feldman, 1986) ).

PAGE 57

57 CHAPTER 2 OUTLINE OF EXPERIMENTS It is now recognized that the neuroplastic potential of the injured spinal cord offers novel opportunities for interventions designed to promote functional improvements vi a spared pathways and intraspinal circuits (Bareyre et al., 2004, Courtine et al., 2008a) Growing evidence suggests that there is also robust potential for spontaneous plasticity following injury to the adult spinal cord. This capacity for spontaneous pla sticity following incomplete spinal cord injury has been demonstrated in a number of experimental models (Bareyre et al., 2004, Ballermann and Fouad, 2006, Courtine et al., 2008a, Rosenzweig et al., 2010) Existing work, however, has focused primarily on r espiratory function and lower extremity function. The capacity for and relative extent of upper extremity plasticity following injury to the cervical spinal cord has not been carefully characterized. Furthermore, whether spontaneous functional recovery fol lowing spinal cord injury is reflected by changes in the underlying neural circuitry is a fundamental consideration, and with few exceptions, the neuroanatomical organization of specific intraspinal circuits has not been fully demonstrated (Lane et al., 20 08b) Accordingly, the major goals of the present work were to characterize the inherent neuroplastic potential of the spinal cord following injury with emphasis on the forelimb and respiratory motor circuits. The overriding hypothesis for this work is tha t plasticity within the cervical spinal cord occurs across multiple motor systems and that propriospinal interneurons are an important component of the recovery process. Aim 1 of this dissertation was designed to characterize the impact of incomplete cerv ical spinal cord injury on the upper extremities. Specific emphasis was placed on identifying the time course and extent of plasticity and recovery of upper extremity

PAGE 58

58 function, skeletal muscle morphology, and neuroanatomical circuitry. The specific hypothe ses being tested in Aim 1 were the following:1) following incomplete high cervical spinal cord injury, there is a progressive, but incomplete functional recovery of ipsilateral upper limb function; 2) that this time dependent recovery of ipsilateral upper extremity function is associated with similar changes in muscle morphology; and that 3) spontaneous upper extremity functional plasticity is associated with plasticity in the neuroanatomical circuitry controlling the upper limb. More specifically, we hypot hesized that spinal interneurons are anatomically positioned to serve as the primary substrate for neuroanatomical plasticity and functional recovery following incomplete cervical spinal cord injury. A battery of behavioral assessments were used to charact erize upper extremity function across a range of functional behaviors. Immunocytochemical techniques were employed to characterize changes in muscle fiber size and type that were occurring in parallel with upper extremity plasticity after chronic incomplet e cervical spinal cord injury. We also utilized transynaptic neuroanatomical tracing techniques to characterize the spinal network associated with a specific forelimb muscle in spinal intact and spinal cord injured rats. Serotonin has been shown to be a p otent modulator of motoneuron excitability and has been specifically implicated in mediating plasticity following spinal cord injury (Mitchell et al., 2001, Fuller et al., 2005, Courtine et al., 2009) Recent studies have demonstrated populations of propri ospinal interneurons are integrated with motor circuits and may be involved in mediating motor output (Lane et al., 2008b) Whether serotonin also modulates interneuronal activity has not been specifically investigated. A ccordingly, A im 2 tested the hypoth eses that spinal interneurons receive robust

PAGE 59

59 serotonergic innervation and explored specific serotonin receptor subtypes that may be expressed on these interneurons. Thus we used immunohistochemical techniques to characterize the serotonergic innervation of a population of interneurons that are believed to play a role in a defined model of spinal plasticity. Recent studies have highlighted the potential role of propriospinal interneurons in mediating motor function and plasticity following injury to the spi nal cord, however to what extent these interneurons participate in functional recovery has not been thoroughly investigated. Aim 3 tested the hypothesis that damage to the intermediate gray matter (interneurons) would attenuate functional plasticity observ ed following cervical spinal cord injury. Thus, we utilized exc itotoxic lesions to create focal lesions in the intermediate gray matter of chronically injured rats to assess the role of spinal interneurons in mediating upper extremity and ventilatory funct ional recovery in the weeks following cervical spinal cord injury In summary, these aims characterize the extent of spontaneous spinal plasticity in various motor systems and explore potential candidate substrates (propriospinal pre motor interneurons) t hat may mediate this plasticity. Overall Objectives 1. To determine the inherent capacity for functional, muscular, and neuroanatomical plasticity in the upper extremity following incomplete high cervical spinal cord injury. 2. To explore the serotonergic inne rvation of cervical motoneurons and pre motor interneurons associated with the phrenic motor circuitry 3. To examine the contribution of propriospinal interneurons to recovery of forelimb and respiratory function following cervical spinal cord injury.

PAGE 60

60 Aim O ne Objective Determine the impact of incomplete high cervical spinal cord injury on upper extremity functional plasticity. Rationale High cervical spinal cord injury is characterized by a disruption of the descending axonal projections innervating the uppe r extremity motoneuron pools, leading to decreased neural drive to the muscles of the upper limb, resulting in paralysis. While a number of investigations have demonstrated the potential for spontaneous functional recovery in other motor systems following C2Hx injury (Sandhu et al., 2009) it is not clear whether similar recovery occurs in the upper extremity. Further, the associated muscular and neural adaptations that correspond with upper extremity dysfunction following C2Hx injury have not been defined. Hypothesis I: Disruption of descending axonal projections innervating upper extremity motoneuron pools will result in significant functional deficits and associated muscular atrophy. Hypothesis II: Modest spontaneous recovery of upper extremity function a nd muscle fiber size will occur in the weeks months post injury. Hypothesis III: Recruitment of pre motor spinal interneurons into the neuroanatomical circuitry controlling the forelimb will correspond with improved upper extremity function following injur y. Experimental Design Adult female Sprague Dawley rats were received lateral C2 hemisection injuries and were allowed to recovery for 1 16 weeks. Forelimb function was assessed using

PAGE 61

61 behavioral tests examining a range of motor behaviors, including meas ures of gross upper limb use, fine motor manipulation, and forelimb function during locomotion. Assessments were conducted prior to, and at 1 2 4 6 8 12 14 and 16 weeks post cervical spinal cord injury Muscles of the forelimb were harvested in a subset of rats in order to assess changes in muscle morphology associated with spinal injury. In another subset of rats, the neuroanatomical circuitry controlling the extensor carpi radialis longus muscle was characterized using pseudorabies virus, a transynaptic retrograde neuroanatomical tracer. This circuitry was assessed in both uninjured rats, as well as in rats that were 16 weeks post injury, in order to assess changes in the neural circuitry controlling the muscles of the upper limb that coincid ed with observed functional recovery. At the pre defined experimental endpoint, were perfused and cervical spinal cords were dissected and processed for immunohistochemical analyses. Aim Two Objective Characterize the serotonergic innervation of phrenic mo toneurons and pre motor interneurons and explore the specific serotonin receptor subtypes that are present on these neurons. Rationale The concept that cervical interneurons can contribute to phrenic motor plasticity has not received formal evaluation. How ever, accumulating evidence suggests that cervical interneurons are synaptically coupled to motor neurons controlling the diaphragm (Lane et al., 2008b, Lane et al., 2009) Previous studies have shown that these cells may modulate phrenic output under cert ain conditions (Sandhu et al., 2009) Studies in the lumbar spinal cord show that spinal serotonin (5 HT) is a powerful

PAGE 62

62 regulator of propriospinal interneuron activity, and can even trigger locomotor output from hindlimb muscles (Ichiyama et al., 2008) Th e influence of 5 HT on cervical interneurons is of interest because spinal 5 HT receptor activation is both necessary and sufficient to trigger persistent increases in phrenic motor output. However, existing models of 5 HT dependent phrenic motor plasticit y (e.g., phrenic long term facilitation ), (MacFarlane et al., 2009, Wilkerson and Mitchell, 2009) focus on the impact of spinal 5 HT on motoneurons, with little focus on the contribution of pre phrenic interneurons to phrenic plasticity (Mitchell et al., 2 001, Baker Herman et al., 2004, MacFarlane et al., 2009, Dale Nagle et al., 2010c) Hypothesis I: Pre phrenic cervical interneurons are robustly innervated by 5 HT. Hypothesis II: Pre phrenic cervical interneurons express both 5 HT 2A and 5 HT 7 receptor s ubtypes. Experimental Design Pseudorabies virus was administered bilaterally to the diaphragm of adult Sprague Dawley rats in order to trace the phrenic motor circuitry. Dual labeling immunohistochemistry was conducted using antibodies to PRV and either 5 HT, 5 HT 2A or 5 HT 7 receptor subtypes. Aim Three Objective Examine the role of pre motor cervical interneurons in spontaneous functional plasticity occurring following incomplete cervical spinal cord injury in adult rats. Rationale Following C2Hx, ipsi lateral diaphragm function improves over weeks months post injury (Lane et al., 2009). This time dependent recovery of ipsilateral diaphragm

PAGE 63

63 activity following chronic C2H x has been attributed to the activation of descending motor pathways, which cross the spinal midline caudal to the injury. In addition, we have demonstrated a similar pattern of upper extremity functional recovery following chronic C2Hx in rats. Recent neuroanatomical data from our lab suggests that descending control of both phrenic and u pper limb motoneurons in the rat spinal cord also involves pre motor propriospinal cervical interneurons. We reasoned that if recovery of ipsilateral motor activity following chronic C2H x involves activation of propriospinal cervical interneurons, then sub sequent damage to these pre motor interneurons (gray matter injury) would significantly attenuate functional recovery. Hypothesis I: Recovery of respiratory function observed following chronic C2Hx injury will be significantly attenuated following focal d eletion of propriospinal interneurons in the intermediate gray matter of the cervical spinal cord. Hypothesis II: Recovery of upper extremity function observed following chronic C2Hx injury will be significantly attenuated following focal deletion of prop riospinal interneurons in the intermediate gray matter of the cervical spinal cord. Experimental De sign Adult female Sprague Dawley rats received lateral C2 Hx injuries and were allowed to recovery for 8 weeks. Focal lesions of the intermediate gray matte r were made via bilateral intraspinal injections of kainic acid injections from C4 C5. Weekly measures of ventilatory function, assessed by whole body plethysmography, and forelimb function, assessed by the forelimb locomotor scale and cylinder test, were conducted. Rats were perfused one week after kainic acid delivery and cervical spinal cords were dissected and processed for immunohistochemical analyses.

PAGE 64

64 CHAPTER 3 CHARA C TERIZATION OF FORELIMB PLASTICITY FOLLOWING INCOMPLETE HIGH CERVICAL SPINAL CORD INJ URY IN ADULT RATS Injuries to the cervical spinal cord comprise the greatest proport ion of all spinal cord injuries. One of the most devastating consequences of cervical spinal cord injury is impaired upper extremity function. Impaired upper extremity func tion results in an inability to perform even basic activities of daily living, and has been shown to contribute to reduced quality of life and development of major depressive disorder. Indeed, a survey conducted by Anderson and colleagues revealed that re covery of arm and hand function was of the highest priority for individuals with chronic tetraplegia secondary to cervical spinal cord injury (Anderson, 2004) Unfortunately, despite its significance, upper extremity dysfunction following spinal cord injur y has received relatively little attention, and experimental models of SCI have tended to focus on thoracic and lumbar models of spinal injury. Thus, there is a great need for the development and characterization of cervical models of spinal cord injury sp ecific to upper extremity function (Anderson et al., 2005) Hemisection from the midline to lateral edge of the cervical spinal cord has been used extensively to study respiratory plasticity following spinal cord injury (Lane et al., 2008a, Sandhu et al., 2009) The basic premise is that C2 hemisection (C2Hx) interrupts descending motor pathways from the brain and brainstem to cervical motoneurons located ipsilateral (IL) to the injury. Thus, the muscles ipsilateral to the side of injury are transiently par alyzed while contralateral (CL) activity persists. Interestingly, partial recovery of IL hemidiaphragm function has been shown to occur spontaneously over a period of weeks (Fuller et al., 2003, 2006; Golder and Mitchell, 2005; Vinit et al., 2007) to month s (Golder et al., 2001) after a C2Hx injury. This

PAGE 65

65 crossed intraspinal circuitry innervating the IL hemid iaphragm. This reorganization has been shown to include the activation of axon collaterals from intact, contralateral descending mono and poly synaptic projections to IL PhMNs (Goshgarian et al., 1991; Moreno et al., 1992, Bellingham, 1999; Yates et al., 1999 Lane, et al., 2008a ). This model has been extensively characterized and has provided a great deal of insight into the potential for spontaneous plasticity in the respiratory system. Importantly, however it is not clear how C2Hx injury may also impac t upper extremity function, despite the fact that the motoneurons controlling the forelimb are located in close proximity to the phrenic motoneuron pool (McKenna et al., 2000, Tosolini and Morris, 2012) Previous studies investigating upper extremity funct ion following experimental cervical spinal cord injury have typically utilized models of injury to the mid lower cervical levels (i.e. C3 C6) (Anderson et al, 2005, Anderson et al., 2009b, Anderson et al., 2009c, Khaing et al., 2012). For the most part, t hese studies have demonstrated a very limited potential for spontaneous functional recovery of the upper extremity after injury to the cervical spinal cord, unlike what has been shown in regards to the respiratory system. There are two potential reasons wh y this has been the case. First, motoneuron pools innervating the muscles of the upper extremity have been shown to extend from approximately C2 to T1. Thus injuries to mid lower cervical regions will likely result in some damage to the motoneuron pools, r esulting in lower motoneuron injuries, which may limit the potential for functional recovery. Another possibility is that there are, in fact differences in cervical motor systems, in regards to

PAGE 66

66 the potential for spontaneous plasticity. In this case, it is possible that the phrenic motor system is inherently more plastic than the upper extremity motor system, which would explain why limited functional recovery is observed in the upper extremity following injury. By investigating the potential for upper extre mity recovery using a model of high cervical spinal cord injury (C2Hx), it is possible to dennervate the motoneuron pools controlling the upper extremity, without concurrent damage to the motoneurons themselves, which will enable the study of the potential for plasticity with in this cervical motor system. Recent evidence has led to an increasing appreciation for the injured spinal inherent potential for neuroplasticity (Pearson, 2001; Bareyre et al., 2004; Edgerton et al., 2004; Ballermann and Fou ad, 2006). It is now recognized that interneurons can play an important role in mediating recovery of locomotor function after spinal cord injury by establishing intraspinal relay pathways (Bareyre et al., 2004; Courtine et al., 2008; Harkema, 2008). Howev er, with few exceptions, the intraspinal circuitry involved in the control of defined motor systems has not been extensively investigated (Lane et al., 2008a) In addition, the neural substrate s underlying functional recovery after cervical SCI are poorly defined A better understanding of the intraspinal circuitries associated with defined motor systems in the intact nervous system as well as following chronic SCI is important, as it will reveal the potential for neuroplasticity after SCI (Pearson, 2001; F ouad and Pearson, 2004) and will enable more thorough interpret ation of the outcomes of experimental interventions. Accordingly, our first goal was to determine the extent and time course of upper extremity functional and muscular plasticity following high cervical hemisection injury. In

PAGE 67

67 addition, we sought to characterize the intraspinal pre motor circuitry associated with a muscle of the upper extremity, the extensor carpi radialis longus muscle (ECRL), and to determine whether changes in this circuitry c orresponded with observed plasticity Materials and Methods All experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Florida and were conducted in accordance with NIH guidelines. Animals A total of 6 0 adult, female Sprague Dawley rats (2 00 25 0 g; ~ 16 weeks of age) were obtained from Harlan Laboratories Inc. (Indianapolis, IN, USA). Animals were housed in the Animal Care Facility at the McKnight Brain Institute at the University of Florida. Animals wer e assigned to one of two groups: Neuroanatomical Tracing Study or Muscle Study. Within the neuroanatomical tracing study, animals were assigned to control group or hemisection group. Within the control group, animals were assigned to either: 1) 24 hour con trol (n=2), 2) 48 hour control (n=4), 3) 72 hour control (n=8), 4) 96 hour control (n=8), or 5) 120 hour control (n=4). Within the chronic hemisection group (16 weeks post hemisection), animals were assigned to either 1) 72 hour hemisected (n=6) or 2) 96 h our hemisected (n=8). Within the muscle study, animals were assigned to one of four groups: 1) uninjured (n=7), 2) 1 week hemisected (n=6), 3) 2 week hemisected (n=7), or 4) 8 week hemisected (n=9). Animals in the chronic hemisection group (n=14) were anal yzed for forelimb function. General Surgical Methods Anesthesia and injury methods have been previously described (Doperalski et al., 2008, Fuller et al., 2008, Fuller et al., 2009) Briefly, rats were anesthetized by

PAGE 68

68 injection of xylazine (10mg/kg, s.q.) and ketamine (140 mg/kg, i.p., Fort Dodge Animal Health, IA, USA). Following completion of the surgical procedure, anesthesia was reversed via injection of yohimbine (1.2 mg/kg s.q.). Upon recovery, animals were given injections of buprenorphine (0.03 mg/k g s.q., Hospira, IL, USA) for analgesia and sterile lactated Ringers solution (5 ml s.q.) to prevent dehydration. Post surgical care included administration of buprenorphine (0.03 mg/kg, s.q.) for the initial 48 hours post injury and delivery of lactate Ri ngers solution (5ml/day, s.q.) and oral Nutri cal supplements (1 3 ml, Webster Veterinary, MA, USA) until adequate volitional drinking and eating resumed. Spinal Cord Hemisection Injury Injury methods have been previously described (Doperalski et al., 20 08, Fuller et al., 2008, Fuller et al., 2009) Briefly, a 1 inch midline dorsal incision was made from the base of the skull extending caudally to approximately the fourth cervical segment (C4). A laminectomy was performed at the second cervical segment (C 2) to expose the spinal cord. A small incision was the n made in the dura and a lateral hemisection performed on the left side of the spinal cord using a microscalpel, followed by gentle aspiration. Using this approach, the completeness of the lesion was re adily visible and the extent of the lesion was reproducible. The dura was then closed with interrupted 9 0 sutures and durafilm was placed over the dura. The overlying muscle was then sutured in layers and the skin was closed with stainless steel surgical wound clips. Behavioral Testing of Forelimb Function Prior to initiation of experimental testing, rats were handled for 3 5 minutes daily by lab personnel to familiarize them with test administrators.

PAGE 69

69 Limb use asymmetry (cylinder) test The cylinder test was conducted on awake, unrestrained animals. Testing consisted of a single trial in which rats were placed in a clear Plexiglas cylinder (20 cm in diameter, 20 cm high) for 5 minutes. Forelimb use was measured during vertical exploration as the number of individual and simultaneous forepaw contacts with the cylinder wall (Hua et al., 2001, DeBow et al., 2003, Gensel et al., 2006) Testing was videotaped for later quantification. Vermicelli pasta handling test Rats were given 7 cm lengths of uncooked verm icelli pasta, marked at 1.75cm intervals with a marker to facilitate visualization of paw and pasta movement. Prior to initiation of behavioral testing, all rats were given the vermicelli pasta pieces in their home cages in order to familiarize them with t he pasta handling. Testing consisted of 5 trials in which the rats were given a single piece of pasta per trial while still in the clear Plexiglas cylinder. Rats were food restricted overnight prior to testing. Trials were videotaped for later quantificati on. The primary method of quantification of the test involved counting the number of adjustments made by each forepaw as well as any asymmetry in the number adjustments made (left versus right), the time to eat the length of the pasta, and documentation o f any atypical behaviors. Adjustments were defined as any visually confirmed release and re grasp of the length of pasta (Allred et al., 2008, Tennant et al., 2010, Khaing et al., 2012) Forelimb l ocomotor s cale (FLS) Rats were placed in an open field pla stic enclosure measuring 2.5 ft. x 3 ft. and were observed for a period lasting no longer than 5 minutes. Trials were scored in real time by an examiner, and were also videotaped for later viewing and scoring. Scoring

PAGE 70

70 was based on the Forelimb Locomotor S cale (FLS) developed by the behavioral core facility at Drexel University, which was based on observed patterns of recovery in cervically injured rats (Sandrow et al., 2008) The FLS is 17 point scale that defines deficits based on range of motion, level o f weight support, and whether the paw is parallel to the body, similar to the hindlimb BBB rating scale (See Table 3 2) Muscle Protocols Muscle tissue harvest At the predefined experimental endpoint, the triceps (TRI) biceps (BI) and extensor carpi rad ialis longus (ECRL), and flexor carpi radialis (FCR) muscles were dissected, carefully rinsed in phosphate buffered saline (PBS) to remove excess blood, and snap frozen at resting length in isopentane, pre cooled in liquid nitrogen and stored at 80 o C. Mus cle immunohistochemistry Transverse cryostat sections (10 m) were prepared from the central portion (belly) of each muscle and mounted on SuperFrost Premium gelatin coated glass slides. Immunocytochemical reactions were performed with anti dystrophin and anti MHC antibodies at various dilutions. Rabbit anti dyst rophin (ThermoScientific, Waltham, MA, USA) was used to outline muscle fibers for quantification of cross sectional area. Two anti MHC monoclonal antibodies were selected on the basis of their reactivity toward adult MHC: A4 840 (1:15; Invitrogen Life Tech nologies, Grand Island, NY, USA) and SC 71 (1:50; Invitrogen Life Technologies, Grand Island, NY, USA). Sections were co incubated with rabbit anti dystrophin and the two anti MHC antibodies (4 o C) for two hours, followed by incubation with rhodamine conjug ated goat anti rabbit IgG (1:40; Invitrogen Life Technologies, Grand Island, NY, USA), Alexafluor 488 goat anti mouse

PAGE 71

71 IgG (1:333; Invitrogen Life Technologies, Grand Island, NY, USA), and Alexaflu o r 350 (goat anti mouse IgM (1:333; Invitrogen Life Technolo gies, Grand Island, NY, USA). Stained sections were covered with glass coverslips using Dako Fluorescent Mountin g Medium, and were stored at 4 o C to prevent fading. Stained cross sections were photographed at 10x magnification using a Leica microscope and d igital camera (Leica Microsystems, Solms, Germany). Muscle fiber size measurements Muscle fiber cross sectional area (CSA) was analyzed using LAS image analysis software. The average fiber CSA of fast and slow fibers was determined from a sample of 250 500 fibers. A region of the stained sections from each muscle was randomly selected for MHC composition analysis. The proportions of each fiber type were determined from a sample of 2 00 300 fibers across the entire section of each muscle. Anatomical Traci ng Protocols Recombinants of the Bartha strain of pseudorabies virus (PRV) or cholera toxin B subunit (CT B) 0.1% in distilled water were used as anatomical tracers to examine the neural circuitry associated with the ECRL muscle in cohort s of uninjured and injured rats. CT B is a monosynaptic tracer and thus, will only label those cells projecting to the site of the tracer application (i.e. ECRL) (Yates et al., 1999), whereas PRV is tran synaptically transported and will label the en tire motor circuitry over time. Propagation and culture methods for PRV have been extensively detailed (Lane et al., 2008b) The two PRV recombinants used in this study were PRV152 (8.0 9.9 x 108 pfu/ml) or PRV614 (2.0 x 108 pfu/ml). Motoneuron projections to the extensor carpi ra dialis were retrogradely labeled using PRV (10 l injections). A subset of animals also underwent concurrent unilateral or bilateral superior cervical ganglionectomy in order to

PAGE 72

72 mitigate the extent of pre sympathetic interneuron al labeling in the cervical spinal cord In addition, in a different subset the left radial nerve was severed prior to its innervation of the extensor carpi radialis muscle, which was then injected with PRV. This set of experiments was designed to determine whether any observed cervical labeling arose from labeling of pre sympat hetic neurons, as all labeling would be due to transmission of PRV via sympathetic innervation of blood vessels, not via retrograde infection from ECRL motoneurons. A small incision was made in the skin of the distal forelimb above the extensor carpi radia lis longus muscle in spinal intact and spinal injured rats. The skin and fascia overlying the ECRL were dissected to expose the ECRL muscle belly. 10 l of PRV was injected into the ECRL via Hamilton syringe (Hamilton Company, Reno, NV). Animals were left t o survive between 24 and 120 hours following tracer injection. A careful time course study was conducted in uninjured rats in order to determine the appropriate post injection time point for PRV incubation, in which primary ECRL motoneuron labeling and tra nsneuronal labeling of pre motor ECRL interneurons was observed. Spinal Cord Histology All C2Hx lesions were confirmed to extend to the spinal midline (anatomically complete) only if there was a complete absence of apparently healthy white matter in the ipsilateral spinal cord at the lesion epicenter as previously described (Doperalski et al., 2008, Fuller et al., 2008, Fuller et al., 2009, Sandhu et al., 2009) After the desired muscles were harvested, rats were euthanized by systemic perfusion with sali ne followed by 4% paraformaldehyde (Sigma, St. Louis, MO, USA). For lesion verification studies, the spinal cord was dissected and removed, embedded in paraffin, and 5 m

PAGE 73

73 transverse sections were prepared. Spinal cord sections were mounted on glass slides (Fisher Scientific, Pittsburgh, PA, USA), stained with Cresyl violet, and evaluated using light microscopy. A histological example of a C2Hx lesion is shown in Figure 3 1 Consistent with our previous publications, the apparent absence of healthy white matter in the ipsilateral spinal cord was taken as confirmation of an anatomically complete C2Hx (Doperalski et al., 2008, Fuller et al., 2008, Lane et al., 2008b, Fuller et al., 2009, Sandhu et al., 2009) For anatomical tracing studies, the cervical spinal cord was removed, and 40 m sections were made in the longitudinal plane using a vibratome. A subset of tissue was sectioned in the transverse plane (40 m vibratome sections) for assessment of distribution of labeled neurons in spinal cord cross section. Brainstem and brain tissue from a subset of animals was also sectioned in the transverse plane (40 m vibratome sections) for assessment of the distribution of supraspinal circuitry related to the ECRL muscle. For immunocytochemical analys e s of PRV or CTB labeling, longitudinal vibr atome sections were washed in PBS (0.1M, pH 7.4, 3 x 5 minutes), blocked against endogenous peroxidase activity (30% methanol, 0.6% hydrogen peroxide in 0.1 M PBS, incubated for 1 2 hours), rewashed in PBS, and blocked against nonspecific protein labeling (10% normal goat serum in 0.1 M PBS with 0.03% Triton X). Sections were then incubated at 4 o C overnight with primary antibodies against PRV (rabbit anti PRV; Rb133/134, raised against whole purified PRV particles that were acetone inactivated; 1:10,000; g enerously provided by Dr. Lynn Enquist). The following day, tissue was washed in PBS (0.1 M, 3 x 5 minutes), incubated for 2 hours at room temperature in a biotinylated secondary antibody (goat anti rabbit; Jackson Immunocytochemicals, West

PAGE 74

74 Grove, PA; 1:20 0) and rewashed in PBS (3 x 5 minutes). Sections were further incubated for 2 hours in an avidin biotin complex (ABC Elite Vectastain Kit; Vector Laboratories, Burlingame, CA), and given a third series of washes in PBS, and processed for antigen visualizat ion with diaminobenzidine (DAB; Sigma, St. Louis, MO). A subset of tissue sections were counterstained with Cresyl violet for visualization of neuronal cell bodies. Data Analysis Forelimb testing data were analyzed according to previously published guidel ines and were compared between groups. Statistical Analysis All statistical analyses were performed using SigmaStat statistical software (Sigma Stat, SPSS, Chicago, IL). Research hypotheses were tested at an alpha level of 0.05. T tests, as well as o ne an d t wo way analyses of variance (ANOVAs) were performed to determine differences between groups. For animals in the behavioral testing cohort, repeated measures (RM) one and two way ANOVAs were performed. Post hoc tests were performed using the Student Ne wman Keuls method to correct for multiple pair wise comparisons. One way RM ANOVAs were used to assess changes in body weight cylinder test scores, FLS test scores, and the time to eat and atypical behavior scores on the vermicelli pasta handling test. 2 way repeated measures ANOVA and the Student Newman Keuls post hoc test s were conducted for the number adjustments in the vermicelli pasta test. pre injury or the various post C2H x time points ) and factor 2 was the side of injury contralateral vs. ipsilateral ). Average muscle fiber size was calculated across the three muscle fiber types. One way, independent samples t tests were used

PAGE 75

75 to compare m uscle wet weight s and average fiber cross sectional area between groups. Statistical analyses were performed using commercially available software (Sigma Stat, SPSS, Chicago IL). All data are presented as the mean +/ standard error. A P value of <0.05 was considered statistically significant. Re sults Anatomical Characterization of C2Hx Lesions Spinal cord sections from rats receiving C2Hx injuries were visualized with light microscopy once stained for Cresyl violet. Anatomical completeness of C2Hx lesions were verified and confirmed to extend fro m the lateral border to the spinal midline (Figure 3 1). Animals that did not have a confirmed complete hemisection lesion were excluded from all analyses (n=2). Effect of C2Hx on Body Mass A time dependent change in body mass occurred following C2Hx ( F ig ure 3 2), similar to previous reports (Doperalski et al., 2008, Fuller et al., 2008, Fuller et al., 2009) At 1 and 2 weeks post C2HX, rats weighed less than they did prior to injury (P<0.05). By 4 weeks post injury, rats were of similar weight to pre inj ury. From 6 16 weeks post injury, rats weighed more than they did prior to injury (P<0.05). A one way ANOVA was performed on the subset of animals that were assigned to the muscle analysis study. The 1 week post injury group weighed less than control (P<0. 0001), but the 2 and 8 week post injury groups were no different from control. Effects of C2Hx on Upper Extremity Function To determine the extent and time course of C2Hx injury on forelimb function, a battery of functional assessments were performed pr ior to and at 1 2 4 6 8 10 12 14 and 16 weeks post C2Hx.

PAGE 76

76 Qua nt itative assessment of gross upper limb motor function was assessed using the cylinder (limb use asymmetry) test (Figure 3 3) One way repeated measures ANOVA revealed signific ant main effects of time on ipsilateral forelimb use and locomotor function (P<0.001 and P<0.001 respectively). Post hoc analyses revealed significant differences in ipsilateral forelimb use and locomotor function at multiple time points post injury (summa rized in Tabl e 3 1). There were significant differences in ipsilateral forelimb use (% of ipsilateral paw placements relative to total placements) occurring in the weeks post injury ( Figure 3 3, Tabl e 3 1). Specifically, o ne week following left lateral C2 Hx injury, a significant reduction in ipsilateral forelimb use was evident (P<0.001) Progressive increases in ipsilateral forelimb use were observed over several weeks, though ipsilateral forelimb use never returned to pre injury levels (P<0.001 at all ti me points compared to pre injury, Figure 3 3, Tabl e 3 1) Qua nt itative assessment of forelimb locomotion was investigated using the FLS (Table 3 2 for description) One way repeated measures ANOVA revealed significant main effects of time on locomotor func tion (P<0.001). Post hoc analyses revealed significant differences ipsilateral locomotor function at multiple time points post injury (summarized in Table 3 3, Fi gure 3 4). Specifically, locomotor function differed from pre injury at all post injury time p oints ( P<0.05 for all, see Table 3 3 for summary ). In addition, significant differences were revealed between 1 week post injury and 16 weeks post injury (P<0.05) and between 2 weeks and 16 weeks post injury (P<0.05). Figure 3 4, Table 3 3). These finding s were consistent with qualitative assessments of locomotor function (Figure 3 5), as uninjured rats demonstrated full range of motion, weight bearing on the plantar surface of the paw during stance, and adequate to

PAGE 77

77 clearance during swing. One week followi ng injury, rats demonstrated reduced range of motion at all joints of the upper limb during locomotion, as well as an inability to place or weight bear on the plantar surface of the limb and an inability to clear the toes during swing. 16 weeks after C2Hx injury, however, rats demonstrated improved toe clearance, and were able to place the limb and bear weight during stance, though some deficits persisted (lacked full toe clearance consistently, internal and external rotation of the limb, etc. ). Quantitati ve assessment of fine motor manipulation was assessed using the v ermicelli pasta handling test. A t wo way repeated measures ANOVA revealed a significant interaction between time and side on the average number of adjustments of the pasta (P<0.001). Post hoc analyses revealed significant differences ipsilateral adjustments at multiple time points post injury (summarized in Table 3 5, Fig ure 3 6). Specifically, the number of ipsilateral adjustments differed from contralateral at all post injury time points exc ept pre injury (P<0.05 for all, see Table 3 5 for summary). One way repeated measures ANOVA revealed significant main effects of time on the average time to eat the length of pasta (P=0.003). Post hoc analyses revealed significant differences in time to ea t pasta at multiple time points post injury (summarized in Table 3 6, Fig ure 3 7). Specifically, time to eat the pasta differed between 4 weeks and 12 weeks, and between 6 weeks and 12 weeks (P<0.05 for both, see Table 3 6 for summary). One way repeated me asures ANOVA revealed significant main effects of time on the average number of atypical behaviors observed per trial (P<0.001). Post hoc analyses revealed significant differences in the average number of atypical behaviors observed per trial at multiple t ime points post injury (summarized in

PAGE 78

78 Table 3 7, Fi gure 3 11). Specifically, the average number of atypical behaviors observed per trial (described in Table 3 4, Figure 3 9) differed between pre injury and all post injury time points ( P<0.05 for all, see F igure 3 11 and Table 3 7 for summary). Effects of C2Hx on Upper Extremity Muscle Morphology To determine the extent to which incomplete high cervical spinal cord injury impacted forelimb muscle size, muscle wet weights and average muscle fiber cross sect ional area were assessed. Independent samples t tests (one tailed) revealed that m uscles of the ipsilateral upper limb were smaller than muscles in the contralateral upper limb following C2Hx (Figure 3 10, Table 3 8) across all muscles evaluated (ECRL: P< 0.02; FCR: P<0.03; BIC: P<0.04; TRI: P<0.02). F urthermore, significant differences in ipsilateral and contralateral muscle size were evident at numerous time points in all four muscles evaluated (P<0.05). One tailed, independent samples t tests analyses were conducted to identify changes in muscle size at each of the time points investigated (Table 3 8 for summary, Figure 3 10). For the ipsilateral extensor carpi radialis muscle, significant differences were observed between control and 1 week post C2Hx (P<0.001), between control and 2 weeks post C2Hx (P<0.001), between 1 week and 2 weeks post C2Hx (P=0.03), between 1 week and 8 weeks post C2HX (P<0.001), and between 2 weeks and 8 weeks post C2Hx (P=0.005). Differences between control and 8 weeks post C2H x were not significant (P=0.11). For the contralateral extensor carpi radialis muscle, significant differences were observed between control and 1 week post C2Hx (P<0.001), between control and 2 weeks post C2Hx (P<0.05), between 1 week and 2 weeks post C2H x (P<0.001), between 1 week and 8 weeks post C2HX (P<0.001), and between 2 weeks

PAGE 79

79 and 8 weeks post C2Hx (P=0.001). Differences between control and 8 weeks post C2Hx were not significant (P=0.10 ) See table 3 8 for summary. For the ipsilateral triceps muscle significant differences were observed between control and 1 week post C2Hx (P<0.001), between control and 2 weeks post C2Hx (P=0.002), between 1 week and 2 weeks post C2Hx (P<0.001), between 1 week and 8 weeks post C2HX (P<0.001), and between 2 weeks and 8 weeks post C2Hx (P<0.001). Differences between control and 8 weeks post C2Hx were not significant (P=0.011). For the contralateral triceps muscle, significant differences were observed between control and 1 week post C2Hx (P=0.002), between 1 week and 2 weeks post C2Hx (P=0.001), between 1 week and 8 weeks post C2HX (P<0.001), and between 2 weeks and 8 weeks post C2Hx (P=0.002). Differences between control and 8 weeks post C2Hx and between control and 2 weeks post C2Hx were not significant (P=0.011and P= 0.002 respectively). See table 3 8 for summ ary. For the ipsilateral biceps muscle, significant differences were observed between control and 1 week post C2Hx (P<0.005), between control and 8 weeks post C2Hx (P<0.01), between 1 week and 2 weeks post C2Hx ( P=0.04), between 1 week and 8 weeks post C2HX (P<0.001), and between 2 weeks and 8 weeks post C2Hx (P=0.003). Differences between control and two weeks were not significant (P=0.15). For the contralateral biceps muscle, significant differences were observe d between control and 1 week post C2Hx (P=0.05), between control and 8 weeks post C2Hx (P=0.002), between 1 week and 2 weeks post C2Hx (P=0.002), between 1 week and 8 weeks post C2HX (P<0.001), and between 2 weeks and 8 weeks post C2Hx (P=0.003).

PAGE 80

80 Differenc es between control and two weeks were not significant (P=0.18).See table 3 8 for summary. For the ipsilateral flexor carpi radialis muscle, significant differences were observed between control and 1 week post C2Hx (P=.01), between 1 week and 8 weeks post C2Hx (P=0.014), and between 2 weeks and 8 weeks post C2Hx (P=0.03). No significant differences were found between control and 2 weeks post C2Hx (P=0.08), between 1 week and 2 weeks post C2Hx (P=.38) and between control and 8 week post C2Hx (P=0.2). For the contralateral flexor carpi radialis muscle, significant differences were observed between 2 weeks and 8 weeks post C2Hx (P=0.03) and between control and 8 week post C2Hx (P=0.008). No significant differences were found between control and 2 weeks post C2H x (P=0.26), between 1 week and 2 weeks post C2Hx (P=.39), between control and 1 week post C2Hx (P=.21), between 1 week and 8 weeks post C2Hx (P=0.08). Se e table 3 8 for summary. Representative examples of cross sections through the ECRL muscle are depicte d in Figures 3 11, 3 12, and 3 13. Mean ipsilateral ECRL muscle fiber cross sectional area across all muscle fibers (Figures 3 11 and 3 14, Table 3 9) was smaller than contralateral ECRL muscle fibers at 2 weeks post C2Hx (P=0.007). Analysis of type I musc le fiber size (Figures 3 12 and 3 15, Table 3 9) revealed significant differences between ipsilateral and contralateral ECRL fibers at 2 weeks post C2Hx (P=0.04). Analysis of type IIa muscle fiber size (Figures 3 13 and 3 16, Table 3 9) did not reveal sign ificant differences between ipsilateral and contralateral ECRL fibers at 2 weeks post C2Hx (P=0.07). Significant differences in ipsilateral ECRL (Table 3 9) muscle fiber size were observed between control and 2 weeks post C2Hx across all

PAGE 81

81 fibers (P=0.02), a s well as across type I fibers (P=0.004), and type IIa fibers (P=0.008). Differences in ipsilateral ECRL CSA were also observed between 1 and 2 weeks post C2Hx in type I muscle fibers (P=0.03). In addition, differences in ipsilateral ECRL muscle fiber CSA were observed between control and 8 weeks post C2Hx across type I and type IIa muscle fibers (P=0.03 P=0.04 respectively). Significant differences in contralateral ECRL muscle fiber size (Table 3 9) were observed between control and 8 weeks post C2Hx acros s type I fibers (P=0.006), and type IIa fibers (P=0.04). Characterization of the Neuroanatomical Circuitry of the ECRL in Uninjured Rats No labeling of ECRL circuitry was evident 24 hours following administration of PRV to the left ECRL (Figure 3 17). On ly 1 PRV infected ECRL motoneurons was observed in the ipsilateral ventral horn of the spinal cord 48 hours following administration of PRV to the left ECRL ( Fi gure 3 18), indicating that at 48 hours, primary motoneuron infection was in its earliest stages There was no evidence of bilateral ECRL motoneuron pool labeling at any survival interval. 72 hours following application of PRV to the left ECRL, the number of infected ECRL motoneurons increased significantly as compared to that seen at 48 hours post a pplication of PRV (Figure 3 19). N o evidence of glial cell infection was observed at this time However, by 96 hours, there was a noticeable infiltration of mononuclear cells around infected ECRL motoneurons (Figure 3 20). Evidence suggestive of early lysi s of infected motoneurons was observed at this time point. By 120 hours, extensive labeling was seen extending throughout the cervical spinal cord (Figure 3 21). 96 hours after PRV delivery, ECRL motoneuron labeling appeared equally robust, and there appe ared to be less variability in motoneuron labeling between animals as compared to 72 hours. 96 hours after PRV delivery, there was considerable variability in the extent of pre motor interneuronal

PAGE 82

82 labeling, which limited our ability to distinguish between secondary, tertiary, etc. infection. Therefore, quantitative findings were limited to the 72 hour survival interval PRV Infection of Cervical Spinal Interneurons At both 72 and 96 hours after delivery of PRV, transynaptic infection of pre motor cervical interneurons was observed primarily in lamina VII, near the laminae VII X border, dorsal to the central canal in lamina X, and in the dorsal horns. While labeling of ECRL motoneurons was only observed ipsilateral to the side of injection, second order cell s were bilaterally distributed throughout the cervical spinal cord, although the majority of labeling was observed ipsilateral to the infected ECRL nucleus at this time point (Figures 3 19 and 3 22). Most cervical interneuron labeling was largely restrict ed to the level of the ECRL motoneuron pool. There were, however, some interneurons labeled as far rostrally as C1, as well as labeling observed several segments caudal to the ECRL motoneuron pool, though these neurons were fewer in number (Figures 3 19 an d 3 22). ECRL motoneuron diameter ranged from 100 150 m. Pre motor neurons around the central canal and in the dorsal horn were relatively smaller on average, ranging in size from 40 50 m. Designation of these second/third order infected cells as putativ e interneurons is consistent with their size range and their gray matter locations. Further confirmation that these were second/third order infected cells, was supported by the observation that they were infected at a later time point, evidenced by the lac k of mononuclear (glial) cell accumulation that was described around ECRL motoneurons ( Figure 3 22). The Rexed laminar distribution of cells was confirmed from transverse tissue sections (F igure 3 23). Serial horizontal (longitudinal) tissue sections

PAGE 83

83 demon strated the relative number and rostral caudal distribution of PRV labeled cells which spanned the entire length of the cervical spinal cord (Figure 3 17 through 3 21). Examination of thoracic sections from all animals revealed significant labeling of sym pathetic pre ganglionic neurons in the inter o mediolateral gray matter of the upper thoracic cord, ipsilateral to the limb injected (Figure 3 27). Control Experiments Demonstrate Extensor Carpi Radialis Longus dependent Second Order Labeling of Cervical Int erneurons The second order (n o n motoneuron) labeling observed at 72+ hours suggested the presence of a population of pre motor interneurons. To confirm that these were, in fact pre motor second order interneurons, experiments were performed in which both P RV and CT were simultaneously injected into the left ECRL (not shown) In this case, neurons that were labeled with both PRV and CT labeling would be considered ECRL motoneurons, while only pre motor interneurons would be labeled with PRV alone, as PRV is a tra nsynaptic tracer, while CT is monosynaptic. Dual retrograde labeling of the ECRL motoneuron pool was clearly evident at 96 hours, but none of the second order PRV p ositive cells labeled with CT Additional control experiments were conducted to elimin ate the possib ility of labeling of putative ECRL interneurons from non ECRL motoneuron sources. First, unilateral as well as bilateral superior cervical ganglionectomies (ipsilateral and contralateral) to the PRV labeled left ECRL were performed in orde r to factor out any possible contribution to our original labeling results from sympathetic innervation of blood vessels into the ECRL. After these lesions, significant labeling of neurons was observed in the ipsilateral intermediolateral cell column in th e thoracic cord as well as labeling of interneurons in the thoracic and cervical cord (as described above). To verify

PAGE 84

84 that cervical neurons were not infected by diffusion of virus to non ECRL structures, and to confirm that the presence of sympathetic labe ling was not confounding our interpretation that interneuronal labeling in the cervical spinal cord was due solely to delivery of PRV to the ECRL, the left radial nerve was cut prior to PRV delivery in 4 animals. With this approach, no PRV infected cells w ere detected at cervical levels (Figure 3 26) However, sympathetic labeling of thoracic neurons in the intermediolateral gray matter was still evident. Even at late post infection intervals (96 hours), where significant labeling of sympathetic neurons in the thoracic cord was evident, no labeling in the cervical cord (pre motor to the sympathetic labeling in the thoracic cord) was observed. Thus it is unlikely that cervical interneurons were infected secondary to sympathetic nervous system circuitry, rathe r, it is more likely that labeling of cervical interneurons originated from primary (first order) infection of ECRL motoneurons Characterization of Supraspinal ECRL Circuitry in Uninjured Rats The supraspinal distribution of PRV labeled neurons associate d with the ECRL was characterized in a subset of animals. Transverse sections through the brain revealed minimal cortical labeling 96 hours after injection of PRV to the left ECRL. By 120 hours, bilateral labeling was observed in layer V of the motor cort ex (Figure 3 25). Transverse sections through the brainstem revealed bilateral labeling within the reticular and raphe nuclei 96 hours after injection of PRV (Figure 3 24) Changes in the Neuroanatomical Circuitry of the ECRL Muscle Following C2Hx To dete rmine if changes in ipsilateral forelimb function were related to changes in the neuroanatomical circuitry controlling the ECRL, we assessed the distribution of PRV labeling in rats that were 16 weeks post C2Hx. On average, fewer motoneurons were

PAGE 85

85 labeled c ompared with uninjured animals at both 72 and 96 hour time points (Figures 3 28 and 3 29 compared to 3 19 and 3 20, see 3 22 for higher power comparison). In fact, at 72 hours post PRV, no labeling was observed in chronically injured rats. 96 hours after i njection of PRV, bilaterally distributed pre motor interneurons were observed throughout the cervical spinal cord. Similar to uninjured animals, interneuronal labeling was observed within Rexed laminae VII and X, as well as in the dorsal horns. In contrast to uninjured animals, 96 hours after injection of PRV, the greatest density of interneuronal labeled was caudal to injury, but rostral to the motoneuron pool (Figure 3 29) Discussion Th is study provides the first comprehensive characterization of the fu nctional, muscular and neuroanatomical sequelae following lateral C2Hx in rats. These data demonstrate a robust potential for spontaneous upper extremity plasticity, which has previously been demonstrated in studies of the phrenic motor system. Furthermore the results presented here are the first to characterize neuroanatomical changes in a defined intraspinal network following chronic spinal cord injury and provide evidence of a potential anatomical substrate mediating upper limb functional recovery and p lasticity Progressive Recovery of Ipsilateral Forelimb Function Following C2Hx Modest spontaneous recovery of ipsilateral forelimb function occurred over the weeks and months following lateral C2Hx injury. This recovery appeared to plateau by 2 months p ost injury. These data suggest a similar extent and time course of recovery to that observed in respiratory recovery following lateral C2 hemisection injury (Golder et al., 2003, Fuller et al., 2006, Fuller et al., 2008, Fuller et al., 2009) Modest recove ry of upper extremity function occurs in the weeks and months post injury. Interestingly,

PAGE 86

86 recovery appears to be most evident in gross motor behaviors, with chronic deficits persisting in more fine motor behaviors, specifically, toe clearance and paw rota tion during locomotion. Furthermore, assessment of fine motor manipulation using the vermicelli pasta handling test revealed that rats demonstrated a remarkable ability to perform the task, even after injury. Rats developed numerous alternative strategies to accomplish the task, which made quantification of results challenging (discussed below). It has been well documented that in rodents, the majority of descending supraspinal inputs to u pper extremity motoneurons are polysynaptic relays through segmental interneurons. This organization within the neural circuitry controlling the upper limbs is logical, considering the capacity for the range of complex behaviors that are possible (i.e. int erlimb coordination for reaching, grasping, locomotion, etc ; intralimb coordination for bimanual tasks such as feeding, object manipulation and locomotion). These interneurons provide the common pathway for coordination between agonist muscles, antagonist muscles, and limbs in order to facilitate efficient performance of such behaviors. In addition, there is evidence to suggest that these interneurons may be a part of a spinal pattern generation network involved in the control of locomotion. The present st udy provides further evidence for the existence of these interneurons and characterizes the distribution of these interneurons within the neuroanatomical circuitry controlling the upper limb. One possibility is that these neurons may represent a possible anatomical of a significant body of work in the respiratory system, which has characterized a similar

PAGE 87

87 pattern of functional recovery of respiratory function in the wee ks and months following (Fuller et al., 2003, Fuller et al., 2006, Moreno et al., 1992, Porter, 1895) The basic premise of the CPP is that lateral C2Hx injury disrupts the descending bul bospinal inputs to the phrenic motoneuron pool ipsilateral to the injury, and that, over a period of several weeks, previously latent, crossed pathways are activated, resulting in restoration of ipsilateral motor output. The present study demonstrated a si milar pattern of upper extremity functional recovery occurring in the weeks post injury. In addition, a population of pre motor cervical interneurons was identified. Interestingly, in chronically injured rats, a change in the distribution of pre motor cerv ical interneurons was evident, such that a relative increase in interneuronal labeling was observed caudal to the hemisection injury in chronically injured rats. These results provide evidence for a possible neuroanatomical substrate that may be related to recovery of upper extremity function following hemisection injury, though the functional significance of these interneurons however is not clear. A recent study by Courtine et al. (2008 ) investigated the potential role of propriospinal interneurons in m ediating functional recovery following thoracic spinal cord injury. In this study, staggered thoracic hemisection lesions were performed on opposite sides of the spinal cord (left sided hemisection at T7 and right sided hemisection at T12). These lesions e ssentially severed all descending long tract projections innervating motoneuron pools in the lumbar spinal cord of mice. However, because the lesions were separated by several spinal segments, a zone of intact spinal cord was spared between them, in which local, short distance spinal circuitry remained intact. When the

PAGE 88

88 hemisections were performed several weeks apart, locomotor function recovered over a period of several weeks. However, if the hemisections were performed simultaneously, functional recovery d id not occur. Retrograde tracing from L1 L2 indicated no differences in supraspinal labeling between staggered and simultaneously hemisected groups that could explain differences in functional recovery (e.g. spared or regenerated supraspinal projections we re not responsible for recovery observed in the staggered hemisection group). There were, however, a greater proportion of retrogradely labeled propriospinal neurons located between T8 and T10 in the staggered group as compared to the simultaneous group. T hese results were consistent with a time dependent reorganization of short distance, intraspinal circuitry that served to bypass the staggered lesions. Furthermore, delivery of NMDA to the intervening spinal cord between the hemisections resulted in select ive lesions to propriospinal interneurons and significantly attenuated recovered locomotor function bilaterally. The authors posit that these data confirm that the locomotor recovery that occurred following the staggered hemisection could not have been the result of long distance regrowth of supraspinal axons, but rather, short distance propriospinal interneurons located between the lesions created relays by which transmission of descending neural drive was re established. A similar study conducted by Cowl ey and colleagues (2008) using an isolated neonatal rat spinal cord preparation, demonstrated that following staggered lateral hemisections, locomotor like activity could still be elicited in the lumbar spinal cord with stimulation of the brainstem. These results are consistent with the notion that descending signals could still be transmitted to motoneurons below an injury via spared

PAGE 89

89 intraspinal circuits (propriospinal interneurons) located within the region between lesions (Cowley et al., 2008, 2010). Whe ther a similar potential for intraspinal reorganization exists within the cervical spinal cord is an intriguing possibility. The results of the present study are consistent with this possibility, and the relative increase in interneuronal labeling caudal t o the injury may represent a possible neuroanatomical substrate by which this reorganization may be taking place. The ability to systematically assess this potential in vivo using methods like those employed by Courtine, however, is technically challengin g in regards to the cervical spinal cord. First, the ability to create staggered hemisections far enough apart to spare intrinsic spinal circuits is challenging in the cervical cord. Second, the phrenic motor system, which supplies innervation to the diaph ragm (the primary muscle of respiration) is located in the mid cervical spinal cord, performing simultaneous hemisections would likely result in an inability to maintain adequate ventilation to sustain life. Lastly, the cervical spinal cord, unlike the tho racic spinal cord, contains numerous motoneuron pools As such, lesions to this cervical spinal cord, (even if they are quite small), would likely result in some degree of disruption to motoneuron pools This would complicate interpretation of results (e.g are deficits related to the severing of projections innervating motoneurons vs. damage to motoneurons themselves ? ). Thus, whether the apparent reorganization within the ECRL circuitry that appears to coincide with functional recovery in the present stud y is indeed associated this recovery remains to be seen. Furthermore, the mechanisms underlying functional recovery associated with reorganization of intraspinal circuits have not been specifically investigated. Two potential mechanisms by which this may o ccur include plasticity

PAGE 90

90 within neuroanatomical circuits and synaptic plasticity. The first involves sprouting of axon collaterals and/or expansion of dendritic fields (Raineteau and Schwab, 2001). In the present study, the apparent change in the distributi on of interneuronal labeling pre motor to ECRL motoneurons may provide some evidence for this, as one possible mechanism by which this distribution may be altered is sprouting of axon collaterals from descending motor tracts such as the corticospinal and/o r Reticulospinal tracts (Bareyre et al., 2004, Ballermann and Fouad, 2006) The second potential mechanism underlying reorganization of intraspinal circuitry involves alterations in the strength of existing neural circuits possibly mediated by changes in neurotransmitter release or post synaptic receptor density. For example, serotonin has been shown to be associated with motoneuron excitability. Following spinal cord injury, serotonergic projections innervating motoneuron pools below the injury are signi ficantly reduced. Interestingly, recovery of motor function has been shown to correlate with restoration of serotonin to the spinal cord (Golder and Mitchell, 2005a) Synaptic plasticity within propriospinal neurons has not been extensively studied. Furthe rmore, whether spinal cord injury alters neuromodulatory inputs to/post synaptic receptor density on propriospinal interneurons in the cervical spinal cord is not known. Future studies to assess whether changes in the distribution of pre motor ECRL interne uronal labeling should investigate changes in the dendritic arborization of neurons caudal to the injury, as well as sprouting within spared descending pathways following C2Hx injury. Additionally, investigation of changes in post synaptic receptors, (suc h as serotonin or glutamate receptors) within the ECRL circuitry may reveal

PAGE 91

91 evidence of potential synaptic plasticity that may underscore observed functional recovery. Commentary on Methods Here we provide a brief discussion regarding the assessment of fo relimb function and the interpretation of results in regards to recovery of upper extremity function in the months following C2Hx injury. As with the majority of functional assessments of spontaneous movements in the upper extremity, tests are subject to a number of issues that can make interpretation of results difficult if taken at face value. The first issue relates to test selection. Tests should be selected based upon the behavior of interest. Upper extremity function, even in rodents can be assessed a cross a wide range of behaviors including gross motor function, fine motor manipulation, reaching and grasping behaviors, general locomotor function and complex locomotor function. A number of different assessments have been developed to assess each of the se behaviors, and the selection of the appropriate test can be quite challenging. First, some tests require pre training. Pre training can be as simple as familiarizing the animal to being handled, or as complicated as training the animal to want to eat a pellet (reward) or length of pasta, to be comfortable in tight enclosures, to reach and grasp a pellet, to run across a cat walk or stair case, to not turn away from a camera when being filmed, etc. The ability to train animals can be influenced by the tes t environment, the time of day, the species and strain of the animals being used, handedness, etc. In addition, for some tasks, some animals do not ever become acclimated to performing the task of interest, thus must be excluded from analysis, which raises questions regarding when to include and exclude animals from a study. Another issue related to pre training has to do with the time it takes for an animal to be fully trained. Long

PAGE 92

92 training requirements for a given test may influence whether or not experi menters choose to use it. In addition, determination of when an animal has been trained can be difficult. In some cases, a plateau is evident, while in other tests, the training itself can in fact influence the outcomes on the test, complicating interpreta tion of subsequent results. In addition to initial test selection, whether or not a test can be administered on repeated occasions is not always clear. For example, if be being tested on repeated occasions, an animal improves functional abilities simply b ecause they are learning to perform the task better, interpretation of results becomes difficult, as it is difficult to determine whether functional improvements are related to spontaneous functional recovery versus motor learning. Lastly, assessment of u pper extremity function can be quite complicated, as most functional tasks of the upper extremity involve complex coordination between multiple muscles and multiple joints within a limb, as well as the ability to stabilize the limb appropriately in order t o perform the desired task as well as coordination between limbs. Furthermore, one of the goals of using standardized assessments of functional abilities is to facilitate comparison across groups of animals or across different time points. However, in appl ying a quantification rubric to any given test, limits the ability to assess subjective measures related to the task being assessed. For example, the vermicelli pasta test entails the quantification of the time it takes to eat a length of pasta, the number of left and right adjustments made on the pasta, and the number of defined atypical behaviors that were observed in a given trial. In the present study, rats demonstrated a remarkable ability to perform the pasta eating task. Interestingly, they

PAGE 93

93 were able to accomplish the task by using a wide array of different movement strategies that were not able to be quantified using the rubric defined by the test. Thus, while scores on the test may have indicated a lack of recovery (specifically related to the time it takes to eat the pasta), it was evident to the rater that over the period of weeks and months post injury, rats were actually modifying the way they were performing the test. This highlights the importance of selecting tests that will be sensitive to fu nctional changes in order to provide the most accurate assessments. Interestingly, although differences in muscle fiber size between limbs were noted, they were not as robust as might have been expected, given the severity of the injury. One potential exp lanation for this finding may have to do with the muscle studied. For these experiments, the ECRL muscle was selected, primarily based upon its size as well as the observation that distal deficits were most prominent following C2Hx injury. However, had we selected a muscle that was more involved in weight bearing activities, such as the triceps, perhaps observed differences between ipsilateral and contralateral muscle may have been more pronounced. Importantly, there were very few published reports investig ating muscle biology in the upper extremity in rodents, therefore several muscles of the upper limb were also harvested and processed for quantification in both uninjured and injured rats, and quantification is ongoing. Another important consideration is that following injury, the majority of rats are quite lethargic and sedentary in the days following injury, which may contribute significantly to the extent of muscle atrophy observed. Relative to the findings of the present study, it is possible, that the lack of activity that occurs in the days following injury, as well as the generalized weight loss that occurs in the first few weeks after

PAGE 94

94 injury, may have led to changes (atrophy) in not just the ipsilateral limb, but the contralateral limb as well. In f act, the majority of upper extremity behaviors demonstrated in rats involve bi manual limb use (such as locomotion, grooming, eating, etc. ), therefore, the fact that animals are relatively sedentary during the days post injury, may have also led to some at rophy in the contralateral limb as well. Future studies investigating markers of muscle atrophy should also consider this possibility and investigate whether there is evidence of muscle atrophy occurring across the contralateral limb, but also in other mus cle groups that would be seemingly less affected by the injury (such as the hindlimb). Commentary on Injury Model for Studying Forelimb Function After cSCI A discussion regarding the choice of injury model utilized in this study is warranted as there are i mportant issues that should be taken into consideration when interpreting these data. In humans, the majority of cSCIs occur at mid cervical levels and are generally the result of combined contusive and compressive forces. In contrast, the present study us ed a high (C2) hemisection injury. While this may seem counter intuitive in regards to the development of clinically relevant models of cSCI, this model actually provides a number of benefits. Firstly, C2Hx injures result in robust functional deficits that are highly reproducible. Second, the goal of the present study was to investigate whether functional plasticity observed in respiratory motor systems following hemisection injury was also evident in other cervical motor systems. For this reason, we select ed a model of injury that has demonstrated the potential for robust plasticity that cervical motor systems in more clinically relevant models of cervical spinal cord injury are necessary.

PAGE 95

95 Summary Significant impairments in upper extremity function occur immediately following incomplete high cervical hemisection injury. Modest s pontaneous functional recovery occurs in the weeks months following injury. This early dysfu nction and s ubsequent s pontaneous recovery are mirrored by changes in upper limb muscle morphology, characterized by initial reduction and subsequent recovery of muscle size. Retrograde neuroanatomical tracing of the spinal motor circuitry controlling the extensor carpi radialis longus muscle in uninjured rats revealed primary (motoneuron) labeling in the ventrolateral gray matter extending from ~C4 C6. Secondary and tertiary (pre motor interneuronal) labeling was observed bilaterally, extending throughout the cervical spinal cord, primarily in laminae VII and X, as well as in the dorsal horn. Characterization of ECRL motor circuitry following C2 hemisection revealed a bilaterally distributed population of interneurons, caudal to the injury and rostral to th e motoneuron pool, which extends for approximately 1 2 segments. These interneurons may provide an anatomical substrate mediating the observed functional and muscular plasticity.

PAGE 96

96 Figure 3 1. Representative histological sections illustrating C2Hx le sions. These 40um transverse sections were taken from the second cervical segment (C2) of Sprague Dawley rats 1 week ( A ), 2 weeks ( B ), and 8 weeks post injury (C) and were stained with Cresyl violet. The absence of healthy white and gray matter in the ips ilateral spinal cord suggests anatomically complete C2Hx lesions. CC: central canal; DH: dorsal horn; VH: ventral horn. Scale Bar: 200um.

PAGE 97

97 Figure 3 2. The impact of C2Hx on body mass A time dependent change in body mass was observed following C2Hx inju ry, characterized by initial weight loss followed by progressive recovery over the first four weeks after injury. Values are mean + SE using One way ANOVA. P<0.05. *significantly different from pre injury.

PAGE 98

98 Figure 3 3. The impact of C2Hx on ipsilater al upper extremity gross motor use during vertical exploration. Ipsilateral upper extremity use was determined using the Cylinder test. Ipsilateral paw placements observed during a 5 minute period of cylinder exploration were calculated and represented as the percentage of ipsilateral placements relative to the total number of placements on the cylinder wall. Initial deficits in upper limb were observed, followed by modest spontaneous recovery of gross motor function. Values are mean + SE using One way RM A NOVA. P<0.05. *significantly different from pre injury. # significantly different from 1 week post C2Hx. @ significantly different from 2 weeks post C2Hx. & significantly different from 4 weeks post C2Hx.

PAGE 99

99 Figure 3 4. The impact of C2Hx on ipsilateral lo comotor function. FLS scores were determined in uninjured rats and at 1 2 4 6 8 10 12 14 and 16 weeks post C2Hx injury. Initial deficits in ipsilateral locomotor function were observed. Modest spontaneous recovery of ipsilateral function wa s observed over the course of 16 weeks post injury Values are mean + SE using One way RM ANOVA. P<0.05. *significantly different from pre injury. # significantly different from 1 week post C2Hx. @ significantly different from 2 weeks post C2Hx

PAGE 100

100 Figure 3 5. The impact of C2Hx on ipsilateral upper extremity function during locomotion. Representative examples of ipsilateral upper limb kinematics of during open field locomotion in an uninjured rat and at 1 week post C2Hx injury and 16 weeks after C2Hx inju ry. Uninjured rats demonstrated full range of motion, weight bearing on the plantar surface of the paw during stance, and adequate to clearance during swing. Significantly impaired ipsilateral upper extremity locomotor function was evident one week after injury, characterized by reduced range of motion at all joints of the upper limb during locomotion, as well as an inability to place or weight bear on the plantar surface of the limb and an inability to clear the toes during swing. Moderate recovery of ips ilateral upper limb locomotion was observed over 16 weeks post injury, however, as rats demonstrated improved toe clearance, and were able to place the limb and bear weight during stance, though some deficits persisted (lacked full toe clearance consistent ly, internal and external rotation of the limb, etc. ).

PAGE 101

101 Figure 3 6. The impact of C2Hx on number of paw adjustments during a fine motor manipulation task Vermicelli pasta handling test. Adjustments made by the ipsilateral and contralateral paw on the p iece of pasta during a trial were counted. The average number of placements per trial demonstrates a dramatic reduction in ipsilateral paw contacts on the piece of pasta after injury. Throughout the course of 16 weeks post injury, rats do not regain use of the ipsilateral paw for grasping the pasta. Values are mean + SE using One way RM ANOVA. P<0.05. % significantly different from contralateral. *significantly different from pre injury.

PAGE 102

102 Figure 3 7. The impact of C2Hx on the time to eat Vermicelli pas ta handling test. The time it took for an animal to eat the entire piece of pasta during a trial was calculated. The average time to eat the pasta varied considerably between animals and between testing sessions, which limits interpretation of this test re lated to the extent of functional recovery. Values are mean + SE using one way RM ANOVA. P<0.05. & significantly different from 4 weeks post C2Hx. $ significantly different from 6 weeks post C2Hx.

PAGE 103

103 Figure 3 8. The impact of C2Hx on number of atypical b ehaviors observed during a fine motor manipulation task Vermicelli pasta handling test. Adjustments made by the ipsilateral and contralateral paw on the piece of pasta during a trial were counted. The average number of placements per trial demonstrates a d ramatic reduction in ipsilateral paw contacts on the piece of pasta after injury. Throughout the course of 16 weeks post injury, rats do not regain use of the ipsilateral paw for grasping the pasta. Values are mean + SE using One way RM ANOVA. P<0.05. *si gnificantly different from pre injury.

PAGE 104

104 Figure 3 9. Examples of normal and atypical behaviors demonstrated by rats during the Vermicelli Pasta Handling test following hemisection injury. A) Example of normal pasta eating behavior. B E) Examples of t h e most common atypical behaviors, which included B) tilted head, C&E) hunched posture, and B&D) failure to contact the length of pasta. Atypical behaviors were observed frequently after injury, and may represent compensatory strategies for accomplishing th e pasta eating task.

PAGE 105

105 Figure 3 10. The impact of C2Hx on upper extremity muscle size. Ipsilateral and contralateral muscle wet weights from A) extensor carpi radialis longus, B) flexor carpi radialis C) biceps brachii, and D) triceps brachii muscle s. Wet weights were measured in uninjured rats, as well as from rats that were 1 2 and 8 weeks post C2Hx injury. Ipsilateral muscles were significantly smaller at 1 2 and 8 weeks post injury. Values are means + SE using independent samples t tests. P <0.05. % significantly different from contralateral. *significantly different from pre injury. # significantly different from 1 week post C2Hx. @ significantly different from 2 weeks post C2Hx.

PAGE 106

10 6 Figure 3 11. Representative histological examples of Exten sor Carpi Radialis Muscle immunostained with dystrophin. Cross sections (10 micron thick) were taken from mid muscle belly of c ontralateral ( A, C, E, G ) and ipsilateral ( B,D,F,H ) Extensor Carpi Radialis muscle s of uninjured rats ( A&B ), and rats that were 1 week ( C&D ), 2 weeks ( E&F ), and 8 weeks ( G&H ) post C2Hx injury. Sections were labeled with antibodies to dystrophin (red) to outline the muscle fiber membrane. This approach enables quantification of muscle f iber cross sectional area

PAGE 107

107 Figure 3 12 Rep resentative histological examples of Extensor Carpi Radialis Muscle immunostained with antibodies to MHC type I. Cross sections (10 micron thick) were taken from mid muscle belly of c ontralateral ( A,C,E&G ) and ipsilateral ( B,D,F&H ) from uninjured rats ( A&B ), and rats that were 1 week ( C&D ), 2 weeks ( E&F ), and 8 weeks ( G&H ) post C2Hx injury. Sections were labeled with antibodies to MHC type I ( blue ) to identify type I muscle fiber s

PAGE 108

108 Figure 3 13 Representative histological examples of Extensor Carpi Ra dialis Muscle immunostained with antibodies to MHC type IIa. Cross sections (10 micron thick) were taken from mid muscle belly of c ontralateral ( A,C,E&G ) and ipsilateral ( B,D,F&H ) from uninjured rats ( A&B ), and rats that were 1 week ( C&D ), 2 weeks ( E&F ), a nd 8 weeks ( G&H ) post C2Hx injury. Sections were labeled with antibodies to MHC type IIa ( green ) to identify the type IIa muscle fiber s

PAGE 109

109 Figure 3 14 Average muscle fiber cross sectional area of contralateral and ipsilateral Extensor Carpi Radialis Lo ngus muscle across all muscle fibers Cross sectional area was calculated from 250 500 fibers from the contralateral and ipsilateral ECRL muscles from adult, female Sprague Dawley rats. Muscles were harvested from uninjured rats, and from rats 1 2 and 8 weeks post C2Hx Values are means + SE using independent samples t tests. P<0.05. % significantly different from contralateral. *significantly different from pre injury

PAGE 110

110 Figure 3 15 Average muscle fiber cross sectional area of contralateral and ipsilateral Extensor Carpi Radialis Longus muscle in type I muscle fibers Cross sectional area was calculated from 250 500 fibers from the contralateral and ipsilateral ECRL muscles from adult, female Sprague Dawley rats. Muscles were harvested from uninj ured rats, and from rats 1 2 and 8 weeks post C2Hx Values are means + SE using independent samples t tests. P<0.05. % significantly different from contralateral. *significantly different from pre injury. # significantly different from 1 week post C2Hx.

PAGE 111

111 Figure 3 16 Average muscle fiber cross sectional area of contralateral and ipsilateral Extensor Carpi Radialis Longus muscle in type IIa fibers Cross sectional area was calculated from 250 500 fibers from the contralateral and ipsilateral ECRL m uscles from adult, female Sprague Dawley rats. Muscles were harvested from uninjured rats, and from rats 1 2 and 8 weeks post C2Hx. Values are means + SE using independent samples t tests. P<0.05. % significantly different from contralateral. *signifi cantly different from pre injury.

PAGE 112

112 Figure 3 17 Representative longitudinal (horizontal) sections through the cervical spinal cord of uninjured adult female Sprague Dawley rats, 24 hours following injection of Ps e u dorabies virus (PRV) into the left ex tensor carpi radialis longus (ECRL) muscle. Sections have been immunolabeled for the presence of PRV and demonstrate no apparent labeling in the dorsal horn ( A ), the intermediate gray matter ( B ), or the ventral horn (C) of the cervical spinal cord at this time point post injection. Scale bar is 1mm.

PAGE 113

113 Figure 3 18 Representative longitudinal (horizontal) sections through the cervical spinal cord of uninjured adult female Sprague Dawley rats, 48 hours followin g injection of Ps e u dorabies virus (PRV) into the left extensor carpi radialis longus (ECRL) muscle. Sections have been immunolabeled for the presence of PRV and demonstrate no apparent labeling in the dorsal horn ( A ), the intermediate gray matter ( B ), or the ventral horn (C) of the cervical spinal co rd at this time point post injection. Scale bar is 1mm.

PAGE 114

114 Figure 3 19 Representative longitudinal (horizontal) sections through the cervical spinal cord of uninjured adult female Sprague Dawley rats, 72 h ours following injection of Ps e u dorabies virus (PRV) into the left extensor carpi radialis longus (ECRL) muscle. Sections have been immunolabeled for the presence of PRV and demonstrate ECRL motoneuron labeling in the ventral horn ( C ), as well as modest interneuronal labeling in the intermediate gray m atter ( B ) and the dorsal horn (A) of the cervical spinal cord at this time point post injection. Scale bar is 1mm.

PAGE 115

115 Figure 3 20 Representative longitudinal (horizontal) sections through the cervical spinal cord of uninjured adult female Sprague Dawl ey rats, 96 hours following injection of Ps e u dorabies virus (PRV) into the left extensor carpi radialis longus (ECRL) muscle. Sections have been immunolabeled for the presence of PRV and demonstrate ECRL motoneuron labeling in the ventral horn ( C blue arro ws ), as well as considerable interneuronal labeling (green arrows) in the intermediate gray matter ( B ) and the dorsal horn ( A) of the cervical spinal cord at this time point post injection. Scale bar is 1mm.

PAGE 116

116 Figure 3 21. Representative longitudinal (horizontal) sections through the cervical spinal cord of uninjured adult female Sprague Dawley rats, 120 hours following injection of Pseudorabies virus (PRV) into the left extensor carpi radialis longus (ECRL) muscle. Sections have been immunolabeled for the presence of PRV and demonstrate ECRL motoneuron labeling in the ventral horn ( C ), as well as extensive pre motor interneuronal labeling in the intermediate gray matter ( B ) and in the dorsal horn (A) of the cervical spinal cord at this time point post injection. Scale bar is 1mm.

PAGE 117

117 Figure 3 22. High power images of longitudinal (horizontal) sections through the cervical spinal cord of uninjured and injured adult female Sprague Dawley rats, 96 hours following injection of Pseudorabies virus (PRV) into the left extensor carpi radialis longus (ECRL) muscle. Sections have been immunolabeled for the presence of PRV and demonstrate ECRL motoneuron labeling in the ventral horn (C&F), as well as extensive pre motor interneuronal labeling in the intermedia te gray matter (B&E) and in the dorsal horn (A&D) of the cervical spinal cord at this time point post injection. As compared to uninjured controls, fewer motoneurons were labeled at this time point in injured animals, though a relative increase in interneu ronal labeling was observed caudal to the injury, but rostral to the motoneuron pool. Scale bar is 1mm.

PAGE 118

118 Figure 3 23. Extensor carpi radialis longus circuitry in the cervical spinal cord. A) Schematic diagram (camera lucida) and B&C) high power image s of transverse sections through the C4 cervical spinal cord of uninjured adult female Sprague Dawley rats, 96 hours following injection of Pseudorabies virus (PRV) into the left extensor carpi radialis longus (ECRL) muscle. Sections have been immunolabele d for the presence of PRV and demonstrate ECRL labeling in the B) ventral horn as well as pre motor labeling in C) laminae VII and X (around the central canal) and in the dorsal horns. Scale bar=200 m.

PAGE 119

119 Figure 3 24. Extensor carpi radialis longus circuitry in the medulla. A) Schematic diagram (camera lucida) and B&C) high power images of transverse sections through the brainstem of uninjured adult female Sprague Dawley rats, 96 hours following injection of Pseudorabies virus (PRV) into the left extensor carpi radialis longus (ECRL) muscle. Sections have been immunolabeled for the presence of PRV and demonstrate extensive labeling in the B) lateral reticular and C) medullary raphe nuclei. Scale b ar=200 m.

PAGE 120

120 Figure 3 25. Extensor carpi radialis longus circuitry in the motor cortex. A) Schematic diagram (camera lucida) and B&C) high power images of transverse sections through the brain of uninjured adult female Sprague Dawley rats, 96 120 hours foll owing injection of Pseudorabies virus (PRV) into the left extensor carpi radialis longus (ECRL) muscle. Sections have been immunolabeled for the presence of PRV and demonstrate ECRL labeling in layer V of the motor cortex at B) 96 hours post injection and c) 120 hours post injection Scale bar=200 m.

PAGE 121

121 Figure 3 26 Representative longitudinal (horizontal) sections through the cervical spinal cord of uninjured adult female Sprague Dawley rats, 72 hours following injection of Ps e u dorabies virus (PRV) into the left extensor carpi radialis longus (ECRL) muscle. Prior to injection of PRV, the left radial nerve was cut to prevent retrograde labeling via ECRL motoneurons. Sections have been immunolabeled for the presence of PRV and demonstrate no apparent labeling in the dorsal horn ( A ), the intermed iate gray matter ( B ), or the ventral horn (C) of the cervical spinal cord following radial nerve section as compared to labeling observed in rats with intact radial nerves at this time point post injection. Scale bar is 1mm.

PAGE 122

122 Figure 3 27 Representat ive longitudinal (horizontal) sections through the thoracic spinal cord of uninjured adult female Sprague Dawley rats, 72 hours following injection of Pseudorabies virus (PRV) into the left extensor carpi radialis longus (ECRL) muscle. Sections have been i mmunolabeled for the presence of PRV A) L ow power and B&C) high power images demonstrate the distribution of sympathetic pre ganglionic labeling in the intermediolateral gray matter of the thoracic spinal cord Scale bars are 1mm ( A ) and 100um ( B&C ).

PAGE 123

123 Figure 3 28. Representative longitudinal (horizontal) sections through the cervical spinal cord of adult female Sprague Dawley rats, 16 weeks post C2Hx injury, 72 hours after injection of Pseudorabies virus (PRV) into the left extensor carpi radialis lo ngus (ECRL) muscle. Sections have been immunolabeled for the presence of PRV and demonstrate minimal motor neuron labeling ventral horn at this time post PRV (C) No labeling was observed in the dorsal horn ( A ) or in the intermediate gray matter ( B ) at thi s time point post injection. As compared to uninjured controls, the presence of PRV labeling at this time point was dramatically reduced. Scale bar is 1mm.

PAGE 124

124 Figure 3 29. Representative longitudinal (horizontal) sections through the cervical spina l cord of adult female Sprague Dawley rats, 16 weeks post C2Hx injury, 96 hours after injection of Pseudorabies virus (PRV) into the left extensor carpi radialis longus (ECRL) muscle. Sections have been immunolabeled for the presence of PRV and demonstrate motoneuron labeling (blue arrows) in the ventral horn at this time post PRV (C). Modest interneuronal labeling (green arrow) was observed in the dorsal horn (A) with considerable labeling in the intermediate gray matter (B). As compared to uninjured contr ols, the first order PRV labeling at this time point was reduced, though a population of interneurons caudal to the injury, but rostral to the motoneuron pool was identified (B). Scale bar is 1mm.

PAGE 125

125 Table 3 1. Cylinder t est Percentage of i psilateral paw p lacements Time post i njury % i psilateral p aw p lacements Pre injury 49 + 1 1 week 7 + 1 a 2 weeks 15 + 2 ab 4 weeks 20 + 2 ab 6 weeks 24 + 2 ab 8 weeks 27 + 3 abc 10 weeks 26 + 3 abc 12 weeks 26 + 2 abc 14 weeks 32 + 3 abcd 16 weeks 33 + 3 abcd Values are mean + SE using 2 way RM ANOVA. P<0.05, a = significantly different from pre injury, b = significantly different from 1 week post C2Hx, c =significantly different from 2 weeks post C2Hx, d = significantly different from 4 weeks post C2Hx.

PAGE 126

126 Table 3 2. Forelimb L ocomotor S cale (FLS) scoring rubric FLS s core Behavioral d escription 0 No movements of the forelimb (shoulder, elbow or wrist joints) 1 Slight movements of one or two joints of the forelimb 2 Extensive movement of one joint and slight movement of another joint o f the forelimb 3 Slight movement of all three joints of the forelimb 4 Extensive movement of one joint and slight movement of two joints of the forelimb 5 Extensive movement of two joints and slight movement of one joint of the forelimb 6 Extensive mov ement of all three joints of the forelimb 7 Plantar placement of the forelimb with no weight support 8 Dorsal stepping only 9 Dorsal stepping and/or occasional plantar stepping 10 Frequent plantar stepping 11 Continuous plantar stepping 12 Continuous plantar stepping with paw position rotated (either at initial contact, lift off, or both) 13 Continuous plantar stepping with paw position parallel (either at initial contact, lift off, or both) 14 Continuous plantar stepping with paw position rotated ( either at initial contact, lift off, or both) and occasional toe clearance 15 Continuous plantar stepping with paw position parallel (either at initial contact, lift off, or both) and occasional toe clearance 16 Continuous plantar stepping with paw posit ion rotated (either at initial contact, lift off, or both) and continuous toe clearance 17 Continuous plantar stepping with paw position parallel (either at initial contact, lift off, or both) and continuous toe clearance Scoring rubric for Forelimb Loco motor Scale. Numerical score values are given based on defined behavioral criteria ( Adapted from ( Sandrow et. al, 2008 ) ).

PAGE 127

127 Table 3 3. Forelimb Locomotor Scale (FLS) scores Time post i njury FLS s core Pre injury 17.0 + 0.0 1 week 4.7 + 0.5 a 2 weeks 7.1 + 0.4 a 4 weeks 8.6 + 0.2 a 6 weeks 8.1 + 0.2 a 8 weeks 9.5 + 0.2 ab 10 weeks 9.8 + 0.2 abc 12 weeks 9.9 + 0.2 abc 14 weeks 10.0 + 0.2 abc 16 weeks 10.3 + 0.3 bc Values are mean + SE using 2 way RM ANOVA. P<0.05, a = significantly different from uninjured, b = significantly dif ferent from 1 week post C2Hx, c =significantly different from 2 weeks post C2Hx.

PAGE 128

128 Table 3 4. Vermicelli p asta h andling t est a typical b ehaviors Atypical b ehavior Behavioral d escription a. Paws together when long b. Guide and grasp switch c. Failure to c ontact d. Drop e. Paws apart when short f. Mouth pulling g. Hunched/abnormal posture h. Iron grip i. Guide around grasp j. Angling with head tilt Definitions of atypical behaviors observed during the Vermicelli Pasta Handling Test ( Adapted from ( Al lred et al., 2008, Tennant et al., 2010 ) ).

PAGE 129

129 Table 3 5. Vermicelli p asta h andling t est average n umber of paw a djustments Time post injury # of c ontralateral a djustments # of i psilateral a djustments Pre injury 18 + 2 18 + 1 1 week 13 + 4 0 + 0* a 2 weeks 13 + 1 0 + 0* a 4 weeks 12 + 1 0 + 0* a 6 weeks 12 + 1 0 + 0* a 8 weeks 13 + 4 1 + 1* a 10 weeks 13 + 1 1 + 1* a 12 weeks 12 + 1 1 + 0* a 14 weeks 12 + 1 0 + 0* a 16 weeks 12 + 1 0 + 0* a Values are mean + SE using 2 way RM ANOVA. P<0.05, *= significantly different from contralateral, a = sign ificantly different from pre injury.

PAGE 130

130 Table 3 6. Vermicelli p asta h andling t est a verage t ime to e at pasta Time post i njury Average t ime t o e at (sec onds) Pre injury 19.92 + 2.28 1 week 29.06 + 3.99 2 weeks 21.73 + 2.01 4 weeks 18.20 + 1.47 6 weeks 17.54 + 3.1 1 8 weeks 23.57 + 2.27 10 weeks 22.85 + 4.27 12 weeks 30.49 + 2.29 de 14 weeks 29.12 + 3.47 16 weeks 24.31 + 2.39 Values are mean + SE using 2 way RM ANOVA. P<0.05, a = significantly different from pre injury, b = significantly different from 1 week post C2Hx, c =significantly different from 2 weeks post C2Hx, d = significantly different from 4 weeks post C2Hx.

PAGE 131

131 Table 3 7. Vermicelli p asta h andling t est number of a typical b ehaviors p er t rial Time post i njury Average # of a typical b ehaviors i dentified p er t rial P re injury 0 + 0 1 week 4 + 0 a 2 weeks 3 + 0 a 4 weeks 3 + 0 a 6 weeks 3 + 0 a 8 weeks 3 + 0 a 10 weeks 3 + 0 a 12 weeks 3 + 0 a 14 weeks 3 + 0 a 16 weeks 3 + 0 a Values are mean + SE using 2 way RM ANOVA. *P<0.05, a = significantly different from pre inju ry, b = significantly different from 1 week post C2Hx, c =significantly different from 2 weeks post C2Hx, d = significantly different from 4 weeks.

PAGE 132

132 Table 3 8 Average u pper l imb m uscle wet w eights Muscle U ninjured muscle weight (g) 1 w ee k post C2Hx muscle weight (g) 2 w ee ks post C2Hx muscle weight (g) 8 w ee ks post C2Hx muscle weight (g) Contralateral ECRL 22. 2 + 1.3 18.9 + 0.7 a 21. 1 + 1.1 ab 23. 0 + 1.1 bc Ipsilateral ECRL 21.9 + 1.3 18.4 + 0.6* a 19. 3 + 1.1* ab 21. 1 + 1.3 bc Contralateral FCR 9.1 + 0. 9 9. 7 + 1. 4 9. 5 + 1. 2 10. 6 + 1. 1 ac Ipsilateral FCR 9. 1 + 0. 5 8. 3 + 0. 5 a 8. 5 + 0. 8 9. 5 + 1. 1* bc Contralateral BIC 17. 5 + 1.0 16. 5 + 0.8 a 18. 0 + 0.8 b 19. 9 + 1.5 abc Ipsilateral BIC 17. 6 + 0.9 15. 2 + 1.8* a 16. 8 + 1.6* b 19. 4 + 1.7 abc Contralateral TRI 128. 0 + 7.5 117. 5 + 3.5 a 127. 9 + 6.3 b 140. 8 + 8.9 abc Ipsilateral TRI 130. 0 + 5.4 106. 3 + 4.9* a 118. 2 + 7.2* ab 131. 6 + 7.4* bc Values are mean muscle weights (g) + SD using one tailed, independent samples t tests *P<0.05 difference from CL; a P<0.05 difference from uninjured, b P<0.05 difference from 1 week post C2Hx, c P<0.05 di fference from 2 weeks post C2Hx ECRL extensor carpi radialis longus, FCR flexor carpi radialis, BIC biceps brachii, TRI triceps brachii.

PAGE 133

133 Table 3 9 Average extensor carpi radialis longus m uscle f iber c ross s ectional a rea (CSA) Uninjured muscle fiber CSA 1 w ee k post C2Hx muscle fiber CSA 2 w ee ks post C2Hx muscle fiber CSA 8 w ee ks post C2Hx muscle fiber CSA Contralateral a ll f ibers 1162.0 + 38.5 1140.6 + 39.4 1145.7 + 35.8 1058.8 + 35.1 Ipsilateral all fibers 1103.6 + 36.6 1002.7 + 27.5 939.2 + 25.5 a 991.1 + 30 .1 Contralateral type I f ibers 1059.6 + 59.9 1038.7 + 67.9 964.8 + 55.3 922.3 + 54.9 a Ipsilateral type I f ibers 1012.4 + 62.4 986.7 + 76.4 790. 2 + 44.7 ab 806.8 + 46.0 a Contralateral Type IIa Fibers 1124.8 + 30.7 1030.6 + 72.5 1015.5 + 29.3 964.3 + 28.3 a Ipsilateral type IIa f ibers 1057.4 + 31.0 938.5 + 27.9 867.0 + 23.3 a 879.2 + 27.2 a Values are mean fiber CSA (um 2 ) + SD using 2 way ANOVA. *P<0.05 difference from CL; a P<0.05 difference from uninjured, b P<0.05 difference from 1 week post C2Hx.

PAGE 134

134 CH APTER 4 SEROTONERGIC INNERVATION OF PRE MOTOR CERVICAL INTERNEURONS IN ADULT RATS Serotonin (5 HT) is a monoaminergic neurotransmitter, found throughout the central nervous system. The primary source of 5 HT is the raphe nuclei, which a re found in the midline of the brainstem. It is well established that 5 HT is a potent modulator of motor activity within the brainstem and spinal cord, and this occurs via descending pathways originating the caudal raphe nuclei. These nuclei include the raphe pallidus, the raphe obscurus, the raphe magnus, the rostral ventrolateral medulla, and the lateral paragigantocellularis reticularis (Hochman et al., 2001) The caudal raphe nuclei have descending 5 HT immunoreactive projections terminating in the ventral horn of the spinal cord, innervating spinal motoneurons (Steinbusch, 1981, Alvarez et al., 1998) Release of 5 HT from these projections acts to modulate motoneuron excitability (Roberts et al., 1988, Jacobs and Fornal, 1993, White et al., 1996) F or example, activation of 5 HT 2A receptors depolarizes motoneurons towards the action potential threshold by 5 HT increasing persistent inward Ca 2+ currents, which results in a sustained depolarization and an amplification of synaptic inputs to motoneurons (Li et al., 2007, Heckman et al., 2008) The association between 5 HT and motor function has been demonstrated in a variety of conditions, including healthy and diseased/injured states, and across a number of motor systems. Jacobs and colleagues have pr oposed the overall hypothesis that release of 5 HT in the spinal cord acts to increase excitability of motoneurons via activation of certain 5 HT receptor subtypes (5 HT 2 and 5 HT 7 receptors). Conversely, release of 5 HT in the region of the dorsal horn of the spinal

PAGE 135

135 cord activates different 5 HT receptor subtypes that act to inhibit excitability of sensory neurons (Jacobs and Fornal, 1993) Serotonin plays a profound role in the neural regulation of breathing (Holtman et al., 1984, Holtman et al., 1986, Lalley, 1986a, b, Holtman and Dick, 1987, Zhan et al., 1989, Pilowsky et al., 1990) Plasticity within the phrenic motor system has been extensively studied, (Mitchell et al., 2001, Fuller et al., 2003, Baker Herman et al., 2004, Fuller et al., 2005, Fulle r et al., 2006, Lane et al., 2008a, Sandhu et al., 2009, Dale Nagle et al., 2010a, Dale Nagle et al., 2010b, Lee et al., 2010) and 5 HT has been specifically implicated in playing a major role in phrenic motoneuron plasticity Serotonergic projections ar ising in the brainstem have been shown to inner vate the phrenic motoneuron pool, and the role of serotonin in phrenic motor function has been the subject of much investigation F or example, activation of spinal 5 HT receptors has been shown to induce long term facilitation (LTF) of phrenic motor output (Mitchell et al., 2001, Baker Herman and Mitchell, 2002) Mitchell and colleagues have proposed a detailed mechanistic model of phrenic long term facilitation, and this model focuses on the role of 5 HT in m odulating phrenic motor neurons. Specifically, they have proposed that activation of the 5 HT 2A receptor subtype, which is found on phrenic motoneurons (MacFarlane et al ,. 2009) leads to activation of protein kinase C (PKC), which initiates synthesis of n ew BDNF. BDNF activates TrkB receptors which activates ERK MAP kinase (pERK), which is hypothesized to phosphorylate glutamate receptors, thus leading to increased synaptic efficacy (Dale Nagle et al ., 2010). Most investigators have concluded that the primary control of PhMNs arises from a monosynaptic connection arising in the brainstem respiratory groups. However, a

PAGE 136

136 growing body of literature indicates that cervical interneurons are also an integral component of the phrenic neural circuitry (Lane et a l., 2008b) Recent evidence suggests that these interneurons may play a role in controlling phrenic motor output, particularly after injuries to the cervical spinal cord. Although there have been numerous studies investigating mechanisms underlying plast icity within the phrenic system, the majority of studies have focused solely on phrenic motoneurons. Consequently, very little is known about the expression of plasticity within cervical interneurons. Furthermore, the potential for 5 HT to modulate pre mot or cervical interneuron activity has not been clearly described. In fact, whether pre motor interneurons are even innervated by 5 HT projections is not known. In addition, there are numerous 5 HT receptor subtypes (Hochman et al., 2001) however, the speci fic subtypes expressed on pre phrenic cervical interneurons, has not been investigated The literature indicates that spinal activation of 5 HT 2A and 5 HT 7 receptors can profoundly influence phrenic output and in particular the expression of phrenic motor plasticity (Zhou and Goshgarian, 2000, Basura et al., 2001, Fuller et al., 2005, Hoffman and Mitchell, 2011) Accordingly, our primary purpose was to determine if cervical interneurons, which are synaptically coupled to PhMNs, are innervated by 5 HT immu noreactive projections. Based on literature in other motor systems, we hypothesized that retrogradely identified pre phrenic cervical interneurons would be robustly innervated by 5 HT immunoreactive projections. In addition, we hypothesized that phrenic mo toneurons and pre phrenic interneurons would express 5 HT 2A and 5 HT 7 receptor subtype immunoreactivity.

PAGE 137

137 Materials and Methods All experimental procedures were approved by the Institutional Animal Care and Use Committee at the University of Florida. Anim als A total of 4 adult male Sprague Dawley rats obtained from Harlan Laboratories Inc. (Indianapolis, IN, USA) were used in this study Anatomical Tracing Protocols A recombinant of the Bartha strain of pseudorabies virus (PRV) was used as an anatomical t racer to examine the neural circuitry associated with the diaphragm muscle of uninjured adult male Sprague Dawley rats. As PRV is transynaptically transported, it will infect the entire phrenic circuitry if given enough time. The PRV recombinant used in th is study was PRV 614 (2.0 x 108pfu/ml), which was engineered to express a monomeric red fluorescent protein. Details regarding the preparation of PRV have been previously described (Banfield et al., 2003; Lane et al., 2008b). Phrenic motoneuron projections to the diaphragm muscle were retrogradely labeled using PRV 614 (50ul per hemidiaphragm). An incision was made along the linea alba, and the skin and abdominal muscles were retracted to expose the abdominal surface of the diaphragm. Tracer was administere d to both the left and right sides of the diaphragm by topical application as previously described (Lane et al., 2008c). Animals were left to survive for 72 hours following tracer application. This time course was based on previously published reports (Lan e et al., 2008b). At this time point, primary phrenic motoneuron labeling was observed, as was transneuronal labeling of pre motor interneurons.

PAGE 138

138 Spinal Cord Histology and Immunocytochemistry 3 days following administration of PRV to the diaphragm, rats w ere euthanized by systemic perfusion of saline followed by 4% paraformaldehyde (Sigma, St. Louis, MO, USA). The cervical spinal cord was removed, and 40um sections were made in the transverse plane using a vibratome. Tissue sections were processed for immu nodetection of 1) 5 HT and PRV, 2) 5 HT 2A receptor and PRV, and 3) 5 HT 7 receptor. For tissue processed for dual labeling (5 HT/PRV and 5 HT 2A receptor/PRV), sections were washed in PBS (0.1 M, pH 7.4, 3 x 5 minutes), blocked against endogenous peroxidase activity (30% methanol, 0.06% hydrogen peroxide in 0.1 M PBS, incubated for 1 hour), given a second set of washes with PBS (3 x 5 minutes) and blocked agains t nonspecific protein labeling (10% normal donkey serum in 0.1 M PBS, incubated for 1 hour). Sectio ns were then incubated with primary antibodies to either 5 HT & PRV ( 5 HT 1:500; AbCam; PRV 1:10,000; generously provided by Dr. Lynn Enquist ) or 5 HT2A receptor & PRV ( 5 HT 2A 1:50; Novus Biologicals, Littleton, CO; PRV 1:10,000; generously provided by Dr Lynn Enquist ) with 10% normal donkey serum and 0.03% Triton X for 48 hours at 4 o C. Following incubation with primary antibodies, sections were washed in PBS (0.1 M, 3 x 5 minutes), and incubated for 1 hour in fluorescent secondary antibodies (AlexaFluor 488, donkey anti goat, Invitrogen Molecular Probes & Life Technologies, 1:1000; AlexaFluor 594, donkey anti rabbit, Invitrogen Molecular Probes & Life Technologies, 1:1000). Sections were washed again in PBS (0.1 M, 3 x 5 minutes), mounted on glass slides (Fisher), and coverslipped for fluorescent microscopy (Dako).

PAGE 139

139 Data Analysis Fluorescent sections were visualized using fluorescent microscopy techniques and overlay images from dual labeling experiments were created using Adobe Photoshop CS6 (Adobe Systems Incorporated, San Jose, CA). Results Anatomical Identification of Pre Phrenic Interneurons Using Retrograde Tracing Retrogradely labeled phrenic motoneurons (Psuedorabies virus immuno postitive) were identified in clusters in the ventral gray matter in t ransverse spinal cord sections between C3 C5/6 (Figure 4 1). Serotonergic Innervation of Retrogradely Labeled Phrenic Motoneurons and Pre Phrenic Interneurons R epresentative transverse spinal cord section s identifying PRV positive phrenic motoneurons and p re motor interneurons co labeled with antibodies to 5 HT are shown in Figure 5 1 In all animals assessed, robust 5 HT immunoreactivity in the immediate vicinity of phrenic motoneurons was observed. The proximity of 5 HT positive projections to phrenic mot oneurons demonstrates evidence for both axo somal as well axo axonal contacts with phrenic motoneurons In addition, pre phrenic cervical interneurons were also retrogradely labeled with PRV (Figure 4 1) also in close proximity to 5 HT positive projections 5 HT 2A Receptor Expression in Phrenic Motoneurons and Pre Motor Interneurons R epresentative 40 m transverse spinal cord section s identifying PRV positive phrenic motoneurons and pre motor interneurons co labeled with antibodies to the 5 HT 2A receptor subtype are shown in Figure 5 2 In all animals assessed, robust expression of 5 HT 2A receptor immu noreactivity on retrogradely labeled phrenic

PAGE 140

140 motoneurons was observed. In addition, 5 HT 2A re ceptors were also expressed on retrogradely labeled pre phrenic cervical interneurons located in laminae VII and X. 5 HT 2A receptor labeling was localized on the s oma of both motoneurons and interneurons (Figure 4 2). 5 HT 7 Receptor Expression in Phrenic Motoneurons and Pre Motor Interneurons R epresentative 40 m transverse spinal cord sections from the mid cervical spinal cord labeled with antibodies to the 5 HT 7 receptor subtype are shown in Figure 5 3. In all animals assessed, the presence of 5 HT 7 receptor immunoreactivity on putative motoneurons located in the region of the phrenic nucleus was observed. In addition, 5 HT 7 re ceptor immunoreactivity was detected on interneurons located in the intermediate gray matter in laminae VII and X in the same region where pre phrenic interneurons have been shown to exi st (Lane et al., 2008b) 5 HT 7 receptor labeling was localized on the soma of neurons (Figure 4 3) Discussion This study provides the first anatomical evidence that pre phrenic cervical interneurons are innervated by 5 HT projections. Furthermore, these d ata confirm the presence of both 5 HT 2A and 5 HT 7 receptor immunoreactivity on these interneurons, which supports our working hypothesis that serotonin may play a role in modulation of interneuronal activity, which may ultimately influence the expression o f spinal plasticity. Interneurons in the intermediate gray matter were shown to be in close proximity to 5 HT immunoreactive projections. Serotonin has been shown to be a potent modulator of neuronal function in the central nervous system (Hochman, et al. 2001, Jacobs and Fornal 1993) The importance of serotonin to motor function is particularly evident following spinal cord injury. For example, reduced bioavailability of serotonin

PAGE 141

141 caudal to SCI is associated with reduced motor output in both respiratory and locomotor systems. Furthermore, recovery of functional abilities after SCI is correlated with restoration of spinal serotonin to motoneuron pools below the level of injury (Hadley et al., 1999, Golder and Mitchell, 2005b) For example, Golder and Mitc hell observed that the relative amplitude of inspiratory phrenic motor bursting following cervical SCI was highly correlated with the intensity of serotonergic immunostaining on and around phrenic motoneurons. Furthermore, f o llowing cervical spinal cord in jury, application of 5 HT synthesis inhibitors or 5 HT antagonists impairs phrenic output (Hadley et al., 1999) Conversely, delivery of 5 HT precursor s or 5 HT agonists has been shown to induce and enhance phrenic motoneuron firing (Zhou and Goshgarian, 2 000) Our data confirm robust 5 HT immunoreactivity in the cervical spinal cord, in close proximity to PhMNs, as well as pre phrenic interneurons. Importantly no prior work has examined the potential for 5 HT to modulate cervical interneuron activity and the possibility that cervical interneurons are part of the substrate for inducing or maintaining phrenic motor plasticity has not been extensively tested. Importantly, existing models of 5 HT dependent phrenic motor plasticity have focused exclu sively on the impact of spinal 5 HT on motoneurons (Zhou and Goshgarian, 2000, Golder and Mitchell, 2005b, Mitchell et al., 2001, Baker Herman and Mitchell, 2002, Dale Nagle et al, 2010a) The concept that cervical interneurons can contribute to phrenic motor plasti city, either in health or disease has not received formal evaluation, and may ultimately prove critical for the future development of therapeutic interventions S tudies of the lumbar spinal cord show that spinal 5 HT is a powerful

PAGE 142

142 regulator of propriospin al interneuron activity, and can even trigger locomotor output from hindlimb muscles (Courtine et al., 2008a) Little is kn own about the specifics of how 5 HT is involved in the recovery process, other than the fact that it is important. For example, the re are numerous subtypes of 5 HT receptors (Hochman et al., 2001) but the specific subtypes contributing to motor recovery after chronic cervical SCI have not been extensively investigated. The 5 HT 2 receptor subtypes are strong candidates for regulating 5 HT dependent phrenic motor plasticity. 5 HT 2 receptors are G protein coupled metabotropic receptors that are associated with many forms of neuroplasticity. Growing evidence indicates that the 5 HT 2A receptor subtype is a major regulator of 5 HT induced c hanges in phrenic motor output. Administration of a 5 HT 2A receptor agonists has been shown to initiate phrenic bursting acutely after experimental cervical SCI. In addition, upregulation of 5 HT 2A receptors on and around phrenic motoneurons has been demon strated 2 weeks after experimental SCI (Fuller et al., 2005) Previous studies have demonstrated that phrenic motoneurons express 5 HT 2A receptors, however, wheth er pre phrenic cervical interneurons also express 5 HT 2A receptors has not been demonstrated. The results presented here confirm that pre phrenic cervical interneurons do, in fact, express 5 HT 2A receptors. The 5 HT 7 receptor family is another potential candidate involved in regulating 5 HT dependent phrenic motor plasticity. 5 HT 7 receptors are G protein coupled metabotropic receptors that are associated with many forms of neuroplasticity. Growing evidence indicates that the 5 HT 7 receptors also play a role in the induction and maintenance of long lasting facilitation of phrenic motor output. Adm inistration of a 5

PAGE 143

143 HT 7 receptor agonists has been shown to initiate robust phrenic motor facilitation, an effect that was abolished by administration of a 5 HT 7 receptor antagonist (Hoffman and Mitchell, 2011) Whether phrenic motoneurons and pre phrenic cervical interneurons also express 5 HT 7 receptors has not been previously demonstrated. The results presented here confirm that 5 HT 7 receptors are located on neurons in the regions where phrenic motoneurons and cervical interneurons are located. The res ults presented here highlight the lack of consideration of cervical interneurons in existing models of respiratory plasticity. Accordingly, these data provide the rationale for future studies investigating how 5 HT activation via the various 5 HT receptor subtypes modulates interneuronal activity and ultimately how this influences spinal plasticity. In future studies, we will be testing the hypothesis that 5 HT can modulate cervical interneuron bursting after cervical SCI. In our opinion, this is an import ant question for several reasons. First, existing models of 5 HT dependent phrenic motor plasticity (e.g., phrenic long term facilitation LTF) have focused exclusively on the impact of spinal 5 HT on motoneurons. The concept that cervical interneurons can contribute to phrenic motor plasticity, either in health or disease (e.g. SCI) has not received formal evaluation, and may ultimately prove critical for future therapeutic designs. Second, prior studies of the lumbar spinal cord show that spinal 5 HT is a powerful regulator of propriospinal interneuron activity, and can even trigger locomotor output from hindlimb muscles. Consistent with studies of the lumbar spinal cord, these data confirm that pre phrenic cervical interneurons are innervated by 5 HT.

PAGE 144

144 Our results highlight the importance of considering how 5 HT may modulate interneuronal function and how this may ultimately influence motor output and expression of plasticity Summary Propriospinal/pre motor interneurons have been shown to be integrated with the motor circuitry controlling the diaphragm. Understanding the role of these interneurons in mediating respiratory activity has become a topic of great interest. Serotonin has been shown to be a key component mediating motor function and has been shown to innervate phrenic motor neurons. The results of the present study demonstrate for the first time that 5 HT pr ojections are colocalized with r etrogradely labeled pre phrenic cervical interneurons in the uninjured spinal cord. Within the ventral horns of the cervical spinal cord, robust 5 HT 2A receptor immunoreactivity was observed on phrenic moto neurons 5 HT 2A receptor immunoreactivity was also observed on pre phrenic cervical interneurons in laminae VII and X. In addition robust 5 HT 7 immunoreactivity was observed on putative phrenic motoneurons in the ventral horn, as well as on interneurons in the intermediate gray matter in the region where pre phrenic cervical interneurons have been demonstrated.

PAGE 145

145 Figure 4 1 Serotonergic immunoreactive projec tions and the phrenic motor circuitry. Representative transverse sections through the cervical spinal cord of uninjured adult Sprague Dawley rats, 72 hours following application of Pseudorabies virus (PRV) to the diaphragm. Sections have been immunolabeled for the presence of PRV (red) and 5 HT (green). Low power (A) and high power (B&C) images demonstrate the co localization of 5 HT immunoreactivity in the immediate vicinity of phrenic motoneurons in the ventral horn (B) and pre phrenic interneurons in lam inae VII and X (C).

PAGE 146

146 Figure 4 2 5 HT 2A receptor immunoreactivity and the phrenic motor circuitry. Representative transverse sections through the cervical spinal cord of uninjured adult Sprague Dawley rats, 72 hours following application of Pseudorabi es virus (PRV) to the diaphragm. Sections have been immunolabeled for the presence of PRV (red) and 5 HT 2A receptors (green). High power images demonstrate the apparent co localization of 5 HT 2A receptor immunoreactivity on phrenic motoneurons in the ventr al horn (A,C,E) and on pre phrenic interneurons in laminae VII and X (B,D,F).

PAGE 147

147 Figure 4 3 5 HT 7 receptor immunoreactivity in the cervical spinal cord. Representative transverse sections through the cervical spinal cord of uninjured adult Sprague Dawle y rats. Sections have been immunolabeled for the presence of 5 HT 7 receptors (green). Low power (A) and high power (B&C) images demonstrate 5 HT 7 receptor immunoreactivity in the region where phrenic motoneurons in the ventral horn (B) and pre phrenic inte rneurons in laminae VII and X (C) are located.

PAGE 148

148 CH APTER 5 CONTRIBUTION OF PROPRIOSPINAL NEURONS TO SPONTANEOUS FUNCTIONAL PLASTICITY FOLLOWING INCOMPLETE HIGH CERVICAL SPINAL CORD INJURY IN ADULT RATS Networks of propriospinal interneurons have been shown to be integrated with the motor circuitry controlling diaphragm upper extremities and lower extremities (Alstermark et al., 1981a, Alstermark et al., 1981b, Alstermark et al., 1984a, b, Alstermark et al., 1990, Alstermark et al., 1999, Bareyre et al., 20 04, Alstermark et al., 2007, Lane et al., 2008b) Previous work from our group has identified populations of propriospinal cervical interneurons located within the intermediate gray matter of the cervical spinal cord that innervate both forelimb (above Cha pter 3) and phrenic motoneuron pools (Lane et al., 2008b) The functional significance of these interneurons, however, has not been extensively investigated. Lateral spinal cord hemisection at C2 has been used extensively to study respiratory motor plastic ity following spinal cord injury (SCI) (Goshgarian, 2003, Fuller et al., 2005b, Lane et al., 2008a). The basic premise is that C2 hemisection (C2Hx) interrupts descending motor pathways from the brain and brainstem to cervical motoneurons located ipsilater al (IL) to the injury. Thus, the muscles ipsilateral to the side of injury are transiently paralyzed while contralateral activity persists. Modest recovery of ipsilateral motor function has been demonstrated in the weeks and months after injury. Recent evi dence (presented in chapter 3) indicates a similar pattern of dysfunction and spontaneous recovery occurs in the upper extremity following C2Hx injury. We speculate that pre motor propriospinal cervical interneurons are anatomically positioned to serve as a synaptic relay for descending inputs to re innervate ipsilateral upper extremity and phrenic motoneuron pools following injury. While definitive evidence of a role for propriospinal cervical

PAGE 149

149 interneurons in functional recovery after cSCI is lacking, the re is strong evidence in other motor systems (e.g. locomotor) that cervical interneurons can promote functional recovery (Bareyre et al., 2004) A recent study by Courtine et al. (2008) investigated the potential role of propriospinal interneurons in medi ating functional recovery following spinal cord injury. In this study, staggered thoracic hemisection lesions were performed in mice on opposite sides of the spinal cord, essentially severing all descending projections innervating motoneuron pools in the l umbar spinal cord. A zone of intact spinal cord spanning several segments was spared between the lesions, in which local, short distance spinal circuitry remained intact. If the hemisections were also staggered in time, locomotor function recovered over a period of weeks. However, if the hemisections were performed simultaneously, no functional recovery was observed Retrograde tracing from L1 L2 indicated no differences in supraspinal labeling between groups to suggest that spared or regenerated supraspina l projections might be responsible for recovery observed in the staggered hemisection group. There were, however, a greater proportion of labeled propriospinal neurons located in the thoracic cord of the staggered group, consistent with a time dependent re organization of short distance, intraspinal circuitry that served to bypass the staggered lesions. Furthermore, excitotoxic lesions administered to the spinal cord between the lesions abolished recovered locomotor function. The authors hypothesized that lo comotor recovery that occurred in the staggered group was mediated by short distance propriospinal interneuronal circuits located between the lesions that served as relays by which transmission of descending neural drive was re established (Courtine et al., 2008a)

PAGE 150

150 Whether a similar potential for intraspinal reorganization exists within the cervical spinal cord has not been formally evaluated. The results presented in chapter three of this dissertation demonstrate a relative increase in interneuronal labeli ng caudal to injury in chronically injured rats, which may represent a possible neuroanatomical substrate by which intraspinal reorganization (and possibly functional recovery) may be taking place. The hypothesis of the present study was that tim e depende nt recovery of ipsilateral forelimb and respiratory function following chronic C2H x may be mediated, at least in part, by pre motor propriospinal cervical interneurons located in the intermediate gray matter of the cervical spinal cord. The aims of this in itial study were : 1) to test the feasibility of specifically ablating cervical interneurons in the intermediate gray matter of chronically injured rats ; and 2) to investigate the functional significance of cervical interneurons to ventilatory and upper ext remity motor plasticity and functional recovery following chronic spinal cord injury. Materials and Methods All experimental procedures were approved by the Institutional Animal Care and Use Committee at the University of Florida. Animals A total of 1 3 S prague Dawley rats were obtained from Harlan Laboratories Inc. (Indianapolis, IN, USA). Animals were randomly assigned to one of three groups: 1) C2Hx+bilateral C4 5 kainic acid injection (n=8), 2) bilateral C4 5 kainic acid injection (n= 4 ), and 3) Saline vehicle control (n=2).

PAGE 151

151 General Surgical Methods Anesthesia and injury methods have been previously described (Doperalski et al., 2008, Fuller et al., 2008, Fuller et al., 2009) Briefly, rats were anesthetized by injection of xylazine (10mg/kg, s.q.) and ketamine (140 mg/kg, i.p., Fort Dodge Animal Health, IA, USA). Following completion of the surgical procedure, anesthesia was reversed via injection of yohimbine (1.2 mg/kg s.q.). Upon recovery, animals were given injections of buprenorphine (0.03 mg/kg s. q., Hospira, IL, USA) for analgesia and sterile lactated Ringers solution (5 ml s.q.) to prevent dehydration. Post surgical care included administration of buprenorphine (0.03 mg/kg, s.q.) for the initial 48 hours post injury and delivery of lactate Ringer s solution (5ml/day, s.q.) and oral Nutri cal supplements (1 3 ml, Webster Veterinary, MA, USA) until adequate volitional drinking and eating resumed. Spinal Cord Hemisection Injury Injury methods have been previously described (Doperalski et al., 2008, Fuller et al., 2008, Fuller et al., 2009) Briefly, a 1 inch midline dorsal incision was made from the base of the skull extending caudally to approximately the fourth cervical segment (C4). A laminectomy was performed at the second cervical segment (C2) t o expose the spinal cord. A small incision was them made in the dura and a lateral hemisection performed on the left side of the spinal cord using a microscalpel, followed by gentle aspiration. Using this approach, the completeness of the lesion was readil y visible and the extent of the lesion was reproducible. The dura was then closed with interrupted 10 0 sutures and durafilm was placed over the dura. The overlying muscle was then sutured in layers and the skin was closed with stainless steel surgical wou nd clips.

PAGE 152

152 Excitotoxin Mediated Gray Matter Ablation Rats were anesthetized by injection of xylazine (10mg/kg, s.q.) and ketamine (140 mg/kg, i.p., Fort Dodge Animal Health, IA, USA). The spinal cord was exposed at the fourth and fifth cervical levels (C 4) via a dorsal laminectomy approach. Six to create discrete gray matter lesions. 3 injections were performed on either side of the spinal cord (0.5mm to left or right o f midline). Injections were spaced 1.5mm apart (rostro caudal) and were made at a 20 o angle and a depth of 1.0 1.25 mm from the dorsal surface of the spinal cord. (Figure 5 1) Behavioral Testing of Forelimb Function Prior to initiation of experimental tes ting, rats were handled for 3 5 minutes daily by lab personnel to familiarize them with test administrators. Limb Use Asymmetry (Cylinder) Test The cylinder test was conducted on awake, unrestrained animals. Testing consisted of a single trial in which ra ts were placed in a clear Plexiglas cylinder (20 cm in diameter, 20 cm high) for 5 minutes. Forelimb use was measured during vertical exploration as the number of individual and simultaneous forepaw contacts with the cylinder wall. Testing was videotaped f or later quantification. Forelimb Locomotor Scale (FLS) Rats were placed in an open field plastic enclosure measuring 2.5 ft. x 3 ft. and were observed for a period lasting no longer than 5 minutes. Trials were scored in real time by an examiner, and were also videotaped for later viewing and scoring. Scoring was based on the Forelimb Locomotor Scale (FLS) developed by the behavioral core facility at Drexel University, which was based on observed patterns of recovery in

PAGE 153

153 cervically injured rats (Sandrow et al., 2008) The FLS is 17 point scale that defines deficits based on range of motion, level of weight support, and whether the paw is parallel to the body, similar to the hindlimb BBB rating scale. Barometric Plethysmography Whole body plethysmography (Bux co Inc., Wilmington, NC, USA ) was used to obtain measures of breathing in unanaesthetized rats prior to injury, and at 3 days, 1 2 3 4 6 and 8 weeks post injury. For rats that received spinal injections, plethysmography was performed at 3 days an d 1 week after injection. The plethysmography system was calibrated by injecting known volumes of air into a Plexiglas recording chamber using 5 and 50 ml syringes. The chamber pressure, temperature, and humidity, as well as the rectal temperature of the r at were used in the Drorbaugh and Fenn equation to calculate respiratory parameters including breathing frequency (f; breaths/minute), tidal volume ( VT, ml/breath), minute ventilation (VE, ml/minute), peak inspiratory flow, peak expiratory flow were extrac ted. Baseline recordings were made for 60 minutes while the Plexiglas chamber was flushed with 21% O2 (balance N2, 2 L/minute). Subsequently, rats underwent a 10 minute hypercapnic respiratory challenge (7% CO2, 21% O2, balance N2, 2 L/minute). Mean values for analyses were obtained from a 10 minute period during baseline and from the last 2 minutes of the hypercapnic challenge. Spinal Cord Histology and Immunohistochemistry All C2Hx lesions were confirmed to extend to the spinal midline as previously descr ibed (Doperalski et al., 2008, Fuller et al., 2008, Fuller et al., 2009, Sandhu et al., 2009) Kainic acid lesions were also assessed and the extent of gray v ersus white matter damage was characterized. At the pre determined termination point of the study,

PAGE 154

154 rats were euthanized by systemic perfusion of saline followed by 4% paraformaldehyde (Sigma, St. Louis, MO, USA). The cervical spinal cord was removed, and processed for paraffin embedding. 5 um sections were made in the transverse plane using a microtome Tissue sections were mounted on glass slides (Fisher Scientific, Pittsburgh, PA, USA), stained with Cresyl violet, and sections from the C2 level were evaluated for lesion completeness by light microscopy. Consistent with our previous publications the ap parent absence of healthy white matter in the ipsilateral C2 spinal cord was taken as confirmation of an anatomically complete C2Hx injury (Doperalski et al., 2008, Fuller et al., 2008, Fuller et al., 2009) Tissue sections at the level of the kainic acid injections were stained with Cresyl violet and were evaluated using light microscopy. Data Analysis Plethysmography data were analyzed in 10 second bins per our previously published reports (Fuller et al., 2006, Fuller et al., 2008) For the baseline condi tion, data represent the average of consecutive bins over a stable 10 minute period just prior to hypercapnic challenge. For the hypercapnic condition, we report the average of the last 2 minutes of exposure. Statistical analyses were performed using comm ercially available software (Sigma Stat Statistical Analysis Software Suite, Chicago, IL). All data are presented as the mean +/ standard error. A P value of <0.05 was considered statistically significant. Our intent was to test the impact of focal kainic acid injections on ventilation during quiet breathing and a brief respiratory challenge accomplished b y raising the inspired CO2 (i.e. hypercapnia). Prior studies from our laboratory have clearly documented the impact of C2H x on ventilation in unrestraine d unanaesthetized rats using barometric plethysmography. These studi es have established that the C2 H x injury causes an

PAGE 155

155 immediate reduction in inspiratory tidal volume, and that there is a persistent deficit in the respiratory response to hypercapnic challe nge following the injury. Thus, our intent here was not to document the impact of the initial C2H x lesion on breathing, but rather we were focused our statistical analysis on the question of whether or not focal ablation of gray matter in the cervical cor d would impact breathing and upper extremity function following chronic C2H x Therefore, initial studies were done in which ventilation and forelimb function were assessed for eight weeks post C2H x The specific purpose of these initial studies was to con firm the prior work and to document when the ventilatory assess the impact of the kainic acid injection only after the spontaneous recovery and/or compensation processe s were no longer leading to progressive increases in upper extremity function or in minute ventilation or other associated ventilatory parameters. Statistical analysis of ventilation and its parameters using two way repeated measures ANOVA confirm that by six weeks post C2H x the rats had reached a plateau in their functional recovery. Accordingly, 8 weeks after C2Hx injury, rats were then given focal injections of kainic acid into the cervical spinal cord at eight weeks post C2H x Ventilation and forelim b motor function were both characterized at three days and seven days post kainic acid injection. For these initial studies, the two post kainic acid data points (3 days and 7 days post KA) were statistically compared to values obtained at 8 weeks post C2H x Results Feasibility of Inducing Discrete Gray Matter Lesions Preliminary investigations were aimed at determining the feasibility of inducing discrete and localized lesions in the intermediate gray matter of the cervical spinal cord.

PAGE 156

156 Initial pilot e xperiments were conducted in control animals to determin e the appropriate injection parameters, including the appropriate dose, Kainic Acid concentration, and depth/location of injection (F igure 5 2 ). Initial studies in injured animals were aimed at determ ining whether injection of kainic acid to chronically injured animals was feasible. Of eight chronically injured rats injected with Kainic Acid, three did not survive the first 24 hours following injection. Anatomical Characterization of C2Hx Lesions Spin al cord sections from rats receiving C2Hx injuries were visualized with light microscopy once stained for Cresyl violet. Anatomical completeness of C2Hx lesions were verified in transverse sections from the C2 level and confirmed to extend from the lateral border to the spinal midline Anatomical characterization of gray matter lesions Spinal cord sections from animals receiving C2Hx+KA injections (Figures 5 3 through 5 6 ), KA injections alone (Figures 5 7 through 5 10 ), or saline injections (F igures 5 1 1 through 5 12 ) were visualized with light microscopy once processed for Cresyl violet Characterization of white (and gray) matter damage was conducted on cross sections (5 micron thick; B G) approximately 240 microns apart through the C4 C5 cervical spin al cord Schematic diagrams were compiled for each animal to illustrate the regions of tissue pathology associated with C2Hx lesions as well as KA associated excitotoxic lesion s There was a great deal of variability in the extent of tissue damage followin g K A injections G ray matter pathology was observed in the intermediate (IMG) and ventral regions of the gray matter of the spinal cord in animals injected with KA. The extent of gray matter damage varied between animals, though across all animals lateral motoneuron pools located in the ventral horns appeared spared. Specifically, minimal

PAGE 157

157 gray matter damage was observed in the region of putative phrenic and ECRL motoneuron pools. Gray matter lesions were characterized by cystic cavitations with necrotic neu rons and cellular debris surrounding the lesion. KA associated white matter damage was quite variable. In some cases, modest white matter disruption was detectable in the dorsal columns, possibly associated with needle tracts. In some cases t here is variab le disruption of ventral and ventromedial white matter in the region of the reticulospinal and vestibulospinal tracts. C2Hx associated pathology was evident in the white matter of the ipsilateral dorsal columns, as well as in the lateral and ventral tracts Lesions within white matter tracts were characterized by axonal degeneration Minimal damage to gray and white matter was observed in saline injected controls. Effect of C2Hx on Body Mass A time dependent change in body mass occurred following C2Hx (not shown) similar to previous reports (F uller 2006, Doperalski 2008, fuller 2008, fuller 2009). At 1 and 2 weeks post C2HX, rats weighed less than they did prior to injury (P<0.05). By 4 weeks post injury, rats were of similar weight to pre injury. From 4 8 weeks post injury, rats weighed more than they did prior to injury (P<0.05). Effects of Gray Matter Deletion on Ventilation in Chronically Injured Rats Before describing the impact of gray matter ablation, we first provide a brief overview of the effect s of C2Hx on ventilatory function. Ventilatory function was assessed using barometric plethysmography prior to injury, and at 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, and 8 weeks post C2Hx injury. Representative air flow traces are demonstrated in Figure 5 13. Two way repeated measures analysis of variance (ANOVA) revealed a significant interaction between time and condition on breathing frequency (P<0.001). Post hoc analyses revealed significant differences in

PAGE 158

158 breathing frequency between baseli ne and hypercapnic challenge at all time points (P<0.05 for all). Furthermore, significant differences in baseline breathing frequency and breathing frequency during hypercapnic challenge were noted at numerous time points post injury (summarized in Table 5 1). One way ANOVA revealed a significant effect of time on change in breathing frequency in response to hypercapnic challenge (P<0.05). Post hoc analyses revealed significant differences in the change in breathing frequency (Figure 5 14) in response to c hallenge were revealed between pre injury and three days post injury (P<0.01) as well as at a number of other time points (Summarized in Table 5 1, Figure 5 14). One way ANOVAs revealed significant effects of time on change in tidal volume and minute vent ilation in response to hypercapnic challenge (P=0.015 and P<0.001 respectively). Post hoc analyses revealed significant differences in the change in tidal volume (Figure 5 15) in response to challenge were revealed between pre injury and three days post in jury (P=0.018). Significant differences in change in tidal volume were not observed at any other time points (Summarized in Table 5 1, Figure 5 16). Significant differences in the change in minute ventilation (Figure 5 16) in response to challenge were rev ealed between pre injury at 3 days post injury (P<0.001), as well as at a number of other time points (Summarized in Table 5 1, Figure 5 15). Importantly, no differences in change in breathing frequency, tidal volume or minute ventilation in response to c hallenge were revealed between 4 and 8 weeks post injury, confirming that stabilization of ventilatory changes in response to injury had taken place prior to injection of kainic acid (5 14 through 5 16).

PAGE 159

159 Effects of Gray Matter Deletion on Ventilation in Chronically Injured Rats To determine whether lesions to the intermediate gray matter between C4 and C5 were associated with impaired ventilatory function, barometric plethysmography was performed at 3 and 7 days post injection of Kainic Acid Representa tive examples of airflow signals from a chronically injured rat prior to and 3 days after delivery of kainic acid to the intermediate gray matter are depicted in Figure 5 12. One way ANOVAs revealed significant effects of time post injection on change in breathing frequency (5 17), tidal volume (5 18) and minute ventilation (5 19) in response to hypercapnic challenge (P=0.026, P=0.033 and P<0.003 respectively). Post hoc analyses revealed significant differences in the change in breathing frequency, tidal v olume, and minute ventilation in response to challenge between pre injection (i.e. 8 weeks post C2Hx) and 3 days post injection (P=0.033, P=.038, and P=0.004 respectively). By 7 days post injection, no differences in response to challenge were evident (dat a summarized in Table 5 3, Figures 5 17 through 5 19). In control experiments where either kainic acid or saline injections were performed in uninjured animals, no differences in ventilatory function were observed (F igure 5 20 ). Effects of Gray Matter Dele tion on Forelimb Function in Chronically Injured Rats Before describing the impact of gray matter ablation, we first provide a brief overview of the effects of C2Hx on upper extremity function. Upper extremity function was assessed using the cylinder (Figu re 5 21) and FLS tests (Figure 5 22), which were conducted prior to injury, and at 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, and 8 weeks post C2Hx injury. One way analyses of variance (ANOVAs) revealed significant main effects of time on ipsilat eral forelimb use and locomotor function (P<0.001 and P<0.001 respectively). Post hoc analyses revealed significant differences

PAGE 160

160 ipsilateral forelimb use and locomotor function at multiple time points post injury (summarized in Table 5 2). Forelimb function al recovery had stabilized by the time kainic acid injections were performed 8 weeks post C2Hx (Figure 5 21 through 5 22). Effects of Gray Matter Deletion on Upper Extremity Function in Chronically Injured Rats To determine whether lesions to the intermedi ate gray matter between C4 and C5 were associated with impaired upper extremity function, the cylinder test (Figure 5 23) and FLS test (Figure 5 24) were performed at 3 and 7 days post injection of Kainic Acid (or sham saline injection). Representative ex amples of left upper extremity locomotor kinematics in a chronically injured rat prior to and 3 days after delivery of Kainic acid to the intermediate gray matter at C4 C5 are depicted in Figure 5 26. One way analyses of variance (ANOVAs) revealed no diff erences in ipsilateral forelimb using following Kainic acid injection in chronically injured rats, though there was a trend towards reduced ipsilateral limb use following injection (P=0.091). Significant effects of time post injection on forelimb locomotor function were revealed (P=0.003). Post hoc analyses revealed significant differences in forelimb locomotor function at 3 and 7 days following kainic acid injection (P=0.003 and P=0.009 respectively) (data summarized in Table 5 4, Figure 5 23 through 5 24 ). In control experiments where either kainic acid or saline injections were performed in uninjured animals, no differences in ipsilateral forelimb use or locomotor function were observed (Figure 5 25). Discussion This study provides evidence to support th e feasibility of specifically ablating populations of spinal neurons in order to assess the contribution of these neurons to motor function and recovery. Specifically, using intraspinal delivery of Kainic Acid to

PAGE 161

161 mid cervical intermediate gray matter, we w ere able to create relatively discrete lesions to the intermediate gray matter between C4 and C5 of the spinal cord without concurrent disruption of nearby motoneuron pools. In addition, these studies also demonstrated the feasibility of using this techni que in chronically injured rats in order to probe the contribution of certain populations of neurons to spontaneous functional recovery that occurs in the weeks and months post injury. A major consideration that arose when designing this study was whether delivery of kainic acid to the intermediate gray matter in chronically injured rats would result in an inability to maintain adequate ventilation for maintenance of life. Interestingly, of the eight chronically injured rats that were injected with kainic a cid, only five survived the initial 24 hours after injection. Whether this low rate of survival indicates that these interneurons are critical for maintenance of ventilation following chronic hemisection injury is not clear. These pilot data are intriguing as they begin to probe potential anatomical substrates underlying plasticity within the spinal cord. Further investigation is necessary in order to fully assess the contribution of cervical interneurons to ventilatory and upper extremity function in both healthy and chronically injured animals. Consistent with our working hypothes i s, ablation of a bilateral population of cervical interneurons between C4 and C5 acutely resulted in a dramatic attenuation of both upper limb and respiratory functional gains t hat had occurr ed over the course of 8 weeks following C2 hemisection injury The possibility that cervical interneurons (propriospinal neurons) may provide a potential anatomical substrate for spontaneous intraspinal plasticity and functional recovery has become a topic of much investigation. Indeed, recent studies have demonstrated the potential for sprouting of descending

PAGE 162

162 tracts onto propriospinal neurons located in the thoracic cord, which serve to bypass an injury (Bareyer et al ., 2004; Courtine et al., 2008; Rosenweig et al., 2010; Ballerman et al. 2006). Examples of such plasticity have been demonstrated in a number of descending motor tracts, including the corticospinal and reticulospinal tracts. Ballermann and colleagues demonstrated that 42 days af ter thoracic lateral hemisection, increased sprouting of BDA labeled Reticulospinal axons was observed caudal to the injury. In addition, the authors found that sprouting of Reticulospinal axons was correlated with recovered locomotor function as measured by the BBB and horizontal ladder tests. Based on the histology from some of the animals in the present study, it is interesting to speculate that perhaps some of the observed deficits upper extremity deficits may have been related to the damage we observe d in the ventrolateral white matter tracts following kainic acid injection. Based on numerous studies indicating a role for Reticulospinal fibers in locomotion and posture, it is not unlikely that damage within these tracts, and not necessarily simply dama ge to cervical interneurons, may underlie some of the functional deficits we observed. It is important to note however that if this were entirely the case, we might expect to see some evidence of upper limb functional deficits in the uninjured animals that received kainic acid injection and also presented with some damage in the region of the Reticulospinal tract. (Ballermann et al., 1996). Further evidence for the relative importance of cervical interneurons is based on findings from a number of recent stu dies which have indicated that damage to cervical gray matter from more clinically relevant contusion injuries, results in a distinct pattern of ventilatory deficits (Lane et al., 2012 ). Specifically, these injuries are characterized by

PAGE 163

163 minimal deficits in baseline ventilatory function but significantly blunted ability to respond to respiratory challenge (Lane et al., 2012). These findings are in contrast to studies investigating primarily white matter type injuries (such as C2HX injuries), in which baseli ne ventilation is significantly impaired after injury, with a characteristic rapid, shallow breathing pattern (Sandhu et al ., 2009; Doperalski et al ., 2008; Fuller et al. deficit following the original hemisection injury. However, this dysfunction is somewhat recovered by eight weeks post injury, and what we observed following the kainic acid injections was a pattern of ventilatory dysfunction that was very similar to the matter significantly attenuated after injection. These results are consistent with what we might expect by specifically targeting gray matter in the cervical spinal cor d. Interestingly, we observed these findings even when the gray matter damage does not appear to directly influence phrenic motoneurons. Interestingly, one week after kainic acid injection, significant functional recovery of ventilation was observed. One potential explanation for this recovery is that this apparent recovery is actually underscored by compensation within other motor systems or within intraspinal networks. This phenomenon is similar to what is observed in the early phases of recovery follow ing lateral C2 hemisection injury, when acutely following injury, deficits in ventilatory function (as assessed by plethysmography) is significantly impaired. In addition, phrenic neurograms indicate that early after C2Hx the ipsilateral phrenic nerve is s ilent By two weeks post C2Hx injury, ventilatory function has improved significantly, though phrenic neurograms of the ipsilateral phrenic nerve

PAGE 164

164 indicate persistent deficits in phrenic motor output. This apparent recovery of ventilatory function despite continued ipsilateral phrenic output has been attributed to compensation by the contralateral hemidiaphragm as well as recruitment of accessory respiratory muscles. Interestingly, the extent of upper extremity functional recovery one week after kainic acid injection is not nearly as robust as observed ventilatory recovery. One possibility is that cervical interneurons play a more significant role in mediating recovery of upper extremity function after injury, thus ablation of these interneurons results in m ore profound, persistent deficits. Alternatively, this finding may reflect the complex nature of upper extremity function, as multiple muscle groups and motor pools are involved in coordinating multi joint movement patterns of the upper limb. It is more li kely, that these interneuron pools play a role in the coordination of complex movements, and that disruption of this circuit has a greater functional impact on complex versus single joint movements. Commentary on M ethods Unfortunately, in addition to foc al lesions of the intermediate gray matter following delivery of kainic acid, some degree of white matter damage appeared to result in almost all cases. Whether damage to white matter tracts contributed to observed deficits in ventilatory and upper extremi ty function is not clear though there did not appear to be major deficits in baseline breathing frequency, which are characteristic damage to white matter tracts (Lane et al., 2012). Furthermore the fact that control animals injected with kainic acid did not forelimb deficits 3 and 7 days post injection as compared to chronically injured rats, suggests that damage to white matter alone is not sufficient to cause such deficits.

PAGE 165

165 It is possible that some of the white matter damage in the region of the dors al columns may be attributable to the course of the needle tracts. Damage to the white matter tracts in the ventromedial spinal cord may be related to inaccurate depth of injection or possibly the spread of kainic acid into the ventromedial white matter. T hus, these data highlight the technical considerations that must be considered in future experiments regarding the delivery of kainic acid to the spinal cord. Additional studies will need to further explore the appropriate dosage and concentration of kain ic acid, the determination of the exact stereotaxic coordinates for targeting the desired region within the spinal cord, and parameters specific to the injection procedure itself (duration and speed of injection, needle dwell time, etc. ) (Magnusson et al ., 1996) These data support our working hypothesis that pre motor cervical interneuron s may be ideally positioned to serve as synaptic relays, mediating spontaneous recovery and plasticity following injury. In addition these data highlight the immense pot ential for endogenous plasticity within the spinal cord, even after injury. Summary Previous work from our group (Lane et al., 2008b) has demonstrated the presence of a population of propriospinal interneurons in the intermediate gray matter of the cervica l spinal cord that innervate phrenic and upper extremity motoneurons. The specific role these interneurons play in mediating recovery of forelimb and respiratory function following incomplete high cervical spinal cord injury has not been specifically teste d. Our working hypothesis is that this population of interneurons represents part of the substrate mediating functional recovery of both respiratory and upper extremity function following chronic incomplete cervical SCI. The aim of the present study was to determine if focal ablation of intermediate cervical gray matter would attenuate

PAGE 166

166 functional gains occurring in the weeks and months following experimental SCI (lateral C2 hemisection). Over a period of 8 weeks following injury, upper extremity and ventila tory function were assessed weekly. Once functional gains had plateaued, bilateral intraspinal (C4 C5) injections of Kainic Acid (KA) were used to create focal lesions of intermediate gray matter. At both three and seven days following delivery of KA, bot h respiratory and upper extremity functional gains were dramatically attenuated. Histological evaluation of cervical spinal cords indicated discrete bilateral lesions to the intermediate gray matter without concurrent damage to the nearby motoneuron pools. These preliminary data are consistent with our working hypothesis, and ongoing studies are investigating the potential mechanisms by which these circuits may modulate motor recovery and plasticity.

PAGE 167

167 Figure 5 1 Schematic depiction of ex perimental mod el of excitotoxin mediated ablation of cervical interneurons in chronically injured rats, 8 weeks post C2 Spinal Cord Hemisection. Lateral C2Hx injury disrupts descending inputs to upper extremity and phrenic motoneuron pools ipsilateral to the side of in jury, resulting in paralysis of ipsilateral musculature. Moderate spontaneous recovery of upper extremity and ventilatory function is observed over a period of weeks following injury. Propriospinal interneurons in the intermediate gray matter of the cervi cal spinal cord are hypothesized to contribute to this observed functional recovery. Application of kainic acid to the intermediate gray matter of the cervical spinal cord results in excitotoxicity and cell death of propriospinal interneurons in the cervic al spinal cord.

PAGE 168

168 Figure 5 2 Representative transverse sections through the cervical spinal cord of adult Sprague Dawley rats, 1 week after intraspinal injection of Kainic Acid (1mM). Sections have been stained with Hematoxylin and Eosin. L ow power image s demonstrate the cell death and tissue damage at the site of injection, and depict differences between a 0.5uL dos e of KA (A) and a 1.0uL dose (B) of kainic acid

PAGE 169

169 Figure 5 3 Kainic acid injections in chronically injured rats Example #1. Schematic diagram (A) and representative cross sections (B G) through the cervical spinal cord of a chronically injured adult Sprague Dawley rat (Example #1), 1 week after intraspinal injection of Kainic Acid (1mM). Bilateral injections of 1uL Kainic Acid (1mM) were made between C4 C5, 0.5mm to either side of midline (3 per side, 1.5mm apart) at a depth of 1mm from the dorsal surface of the spinal cord. 5 micron sections have been Cresyl violet and demonstrate significant damage to the intermediate gray matter, witho ut concurrent damage to the motoneuron pools in the ventral horns. No KA related damage to the white matter damage was observed.

PAGE 170

170 Figure 5 4 Kainic acid injections in chronically injured rats Example #2. Schematic diagram (A) and representative cross sections (B G) through the cervical spinal cord of a chronically injured adult Sprague Dawley rat (Example #2), 1 week after intraspinal injection of Kainic Acid (1mM). Bilateral injections of 1uL Kainic Acid (1mM) were made between C4 C5, 0.5mm to either side of midline (3 per side, 1.5mm apart) at a depth of 1mm from the dorsal surface of the spinal cord. 5 micron sections have been Cresyl violet and demonstrate significant damage to the intermediate gray matter, without concurrent damage to the motoneuro n pools in the ventral horns. Modest KA related damage to the white matter damage was observed.

PAGE 171

171 Figure 5 5 Kainic acid injections in chronically injured rats Example #3. Schematic diagram (A) and representative cross sections (B G) through the cervi cal spinal cord of a chronically injured adult Sprague Dawley rat (Example #3), 1 week after intraspinal injection of Kainic Acid (1mM). Bilateral injections of 1uL Kainic Acid (1mM) were made between C4 C5, 0.5mm to either side of midline (3 per side, 1.5 mm apart) at a depth of 1mm from the dorsal surface of the spinal cord. 5 micron sections have been Cresyl violet and demonstrate significant damage to the intermediate gray matter, without concurrent damage to the motoneuron pools in the ventral horns. Mo dest KA related damage to the white matter damage was observed.

PAGE 172

172 Figure 5 6 Kainic acid injections in chronically injured rats Example #4. Schematic diagram (A) and representative cross sections (B G) through the cervical spinal cord of a chronically injured adult Sprague Dawley rat (Example #4), 1 week after intraspinal injection of Kainic Acid (1mM). Bilateral injections of 1uL Kainic Acid (1mM) were made between C4 C5, 0.5mm to either side of midline (3 per side, 1.5mm apart) at a depth of 1mm from the dorsal surface of the spinal cord. 5 micron sections have been Cresyl violet and demonstrate significant damage to the intermediate gray matter, without concurrent damage to the motoneuron pools in the ventral horns. Modest KA related damage to the whi te matter damage was observed.

PAGE 173

173 Figure 5 7 Kainic acid injections in uninjured rats Example #1. Schematic diagram (A) and representative cross sections (B G) through the cervical spinal cord of an uninjured adult Sprague Dawley rat (Example #1), 1 wee k after intraspinal injection of Kainic Acid (1mM). Bilateral injections of 1uL Kainic Acid (1mM) were made between C4 C5, 0.5mm to either side of midline (3 per side, 1.5mm apart) at a depth of 1mm from the dorsal surface of the spinal cord. 5 micron sect ions have been Cresyl violet and demonstrate significant damage to the intermediate gray matter, without concurrent damage to the motoneuron pools in the ventral horns. Damage to the ventral white matter damage was also observed.

PAGE 174

174 Figure 5 8 Kainic a cid injections in uninjured rats Example #2. Schematic diagram (A) and representative cross sections (B G) through the cervical spinal cord of an uninjured adult Sprague Dawley rat (Example #2), 1 week after intraspinal injection of Kainic Acid (1mM). Bilat eral injections of 1uL Kainic Acid (1mM) were made between C4 C5, 0.5mm to either side of midline (3 per side, 1.5mm apart) at a depth of 1mm from the dorsal surface of the spinal cord. 5 micron sections have been Cresyl violet and demonstrate significant damage to the intermediate gray matter, without concurrent damage to the motoneuron pools in the ventral horns. Damage to the ventral white matter damage was also observed.

PAGE 175

175 Figure 5 9 Kainic acid injections in uninjured rats Example #3. Schematic dia gram (A) and representative cross sections (B G) through the cervical spinal cord of an uninjured adult Sprague Dawley rats (Example #3), 1 week after intraspinal injection of Kainic Acid (1mM). Bilateral injections of 1uL Kainic Acid (1mM) were made betwe en C4 C5, 0.5mm to either side of midline (3 per side, 1.5mm apart) at a depth of 1mm from the dorsal surface of the spinal cord. 5 micron sections have been Cresyl violet and demonstrate significant damage to the intermediate gray matter, without concurre nt damage to the motoneuron pools in the ventral horns. Damage to the ventral white matter damage was also observed.

PAGE 176

176 Figure 5 10 Kainic acid injections in uninjured rats Example #4. Schematic diagram (A) and representative cross sections (B G) throu gh the cervical spinal cord of an uninjured adult Sprague Dawley rats (Example # 4 ), 1 week after intraspinal injection of kainic acid. Bilateral injections of 1uL Kainic Acid (1mM) were made between C4 C5, 0.5mm to either side of midline (3 per side, 1.5mm apart) at a depth of 1mm from the dorsal surface of the spinal cord. 5 micron sections have been Cresyl violet and demonstrate significant damage to the intermediate gray matter, without concurrent damage to the motoneuron pools in the ventral horns. Dama ge to the ventral white matter damage was also observed.

PAGE 177

177 Figure 5 11 Saline injections in chronically injured rats Example #1. Schematic diagram (A) and representative cross sections (B G) through the cervical spinal cord of an uninjured adult Spragu e Dawley rats (Example #2), 1 week after intraspinal injection of saline. Bilateral injections of 1uL saline were made between C4 C5, 0.5mm to either side of midline (3 per side, 1.5mm apart) at a depth of 1mm from the dorsal surface of the spinal cord. 5 micron sections have been Cresyl violet and demonstrate minimal damage to gray/white matter.

PAGE 178

178 Figure 5 12 Saline injections in uninjured rats Example #2. Schematic diagram (A) and representative cross sections (B G) through the cervical spinal cord o f an uninjured adult Sprague Dawley rats (Example #2), 1 week after intraspinal injection of saline. Bilateral injections of 1uL saline were made between C4 C5, 0.5mm to either side of midline (3 per side, 1.5mm apart) at a depth of 1mm from the dorsal sur face of the spinal cord. 5 micron sections have been Cresyl violet and demonstrate minimal damage to gray/white matter.

PAGE 179

179 Figure 5 13 Representative airflow traces showing the impact of kainic acid injection on ventilation in chronically injured rats. 8 weeks after receiving left lateral C2Hx injuries, kainic acid was injected into the intermediate gray matter between C4 C5. A&B) Representative traces from an animal, 8 weeks post hemisection (prior to administration of kainic acid ) during baseline (A) and hypercapnic (B) breathing conditions C&D) Representative airflow traces from the same rat, 3 days post kainic acid injection during baseline (C) and hypercapnic (D) breathing conditions A blunted ventilatory response to hypercapnic challenge was obse rved following kainic acid injection in chronically injured rats Animals were unable to increase ventilation in response to respiratory challenge.

PAGE 180

180 Figure 5 14 Effects of C2 hemisection injury (C2Hx) on breathing frequency response to respiratory cha llenge. Increase in breathing frequency in response to hypercapnic challenge relative to baseline breathing frequency in unanaesthetized rats prior to, and at 3 days, 1 2 3 4 6 and 8 weeks post C2Hx. Values are mean + SE using one way RM ANOVA. P <0.05, = significantly different from pre injury, # = significantly different from 3 days post C2Hx, @ =significantly different from 1 week post C2Hx.

PAGE 181

181 Figure 5 15 Effects of C2 hemisection injury (C2Hx) on tidal volume response to respiratory challeng e. Increase in tidal volume in response to hypercapnic challenge relative to baseline tidal volume in unanaesthetized rats prior to, and at 3 days, 1 2 3 4 6 and 8 weeks post C2Hx. Values are mean + SE using one way RM ANOVA. P<0.05, = significa ntly different from pre injury.

PAGE 182

182 Figure 5 16 Effects of C2 hemisection injury (C2Hx) on minute ventilation response to respiratory challenge. Increase in minute ventilation in response to hypercapnic challenge relative to minute ventilation during bas eline breathing in unanaesthetized rats prior to, and at 3 days, 1 2 3 4 6 and 8 weeks post C2Hx. Values are mean + SE using one way RM ANOVA. P<0.05, = significantly different from pre injury, # = significantly different from 3 days post C2Hx, @ =significantly different from 1 week post C2Hx.

PAGE 183

183 Figure 5 17 Effects of kainic acid injection (KA) on breathing frequency response to respiratory challenge. Change in breathing frequency in response to hypercapnic challenge relative to baseline breath ing frequency in unanaesthetized rats prior to, and at 3 and 7 days post KA injection. Values are mean + SE using one way RM ANOVA. P<0.05, = significantly different from pre KA.

PAGE 184

184 Figure 5 18 Effects of kainic acid injection (KA) on tidal volume r esponse to respiratory challenge. Change in tidal volume in response to hypercapnic challenge relative to baseline breathing frequency in unanaesthetized rats prior to, and at 3 and 7 days post KA injection Values are mean + SE using one way RM ANOVA. P <0.05, = significantly different from pre KA.

PAGE 185

185 Figure 5 19 Effects of kainic acid injection (KA) on minute ventilation response to respiratory challenge. Change in tidal volume in response to hypercapnic challenge relative to baseline breathing frequ ency in unanaesthetized rats prior to, and at 3 and 7 days post KA injection Values are mean + SE using one way RM ANOVA. P<0.05, = significantly different from pre KA, # = significantly different from 3 days post KA.

PAGE 186

186 Figure 5 20 Effects of kaini c acid (KA) and saline injection in uninjured rats on the change in A) breathing frequency, B) tidal volume, and C) minute ventilation in response to respiratory (hypercapnic) challenge Data represent ventilatory parameters from rats prior to and at 3 and 7 days post injection No obvious deficits were observed with kainic acid or saline injection into control animals.

PAGE 187

187 Figure 5 21 Effects of C2 hemisection injury (C2Hx) on ipsilateral upper limb use. The cylinder test was administered to rats p rior to, and at 3 days, 1 2 3 4 6 and 8 weeks post C2Hx. Ipsilateral upper limb use was measured as the percentage ipsilateral paw placements relative to the total number of placements during a period of 5 minutes of vertical exploration. Initial deficits in upper limb use were observed, followed by modest spontaneous recovery in the weeks post injury. Recovery of ipsilateral upper extremity use had plateaued by 8 weeks post injury. Values are mean + SE using one way RM ANOVA. P<0.05, = significa ntly different from pre injury, # = significantly different from 1 week post C2Hx.

PAGE 188

188 Figure 5 22 Effects of C2 hemisection injury (C2Hx) on forelimb locomotor function. FLS scores were determined in rats prior to, and at 1 2 3 4 6 and 8 wee ks post C2Hx Initial deficits in upper limb locomotor function were observed, followed by modest spontaneous recovery in the weeks post injury. Recovery of ipsilateral upper extremity locomotor function had plateaued by 8 weeks post injury. Values are mea n + SE using one way RM ANOVA. P<0.05, = significantly different from pre injury, # = significantly different from 1 week post C2Hx, @ =significantly different from 2 weeks post C2Hx, @ = significantly different from 3 weeks post C2Hx, & = significantly diffe rent from 3 weeks post C2Hx, $ = significantly different from 4 weeks post C2Hx, % = significantly different from 6 weeks post C2Hx.

PAGE 189

189 Figure 5 23 Effects of bilateral C4 C5 kainic acid injections on ipsilateral forelimb use in rats with chronic C2 hemisection injury (C2Hx) I psilateral paw placements (relative to the total number of placements) on the cylinder wall during vertical exploration were recorded in rats, 8 weeks post C2Hx and 3 and 7 days after bilateral intraspinal injections of kainic acid to the intermed iate gray matter between C4 C5. Values are mean + SE using one way RM ANOVA. P<0.05.

PAGE 190

190 Figure 5 24 Effects of bilateral C4 C5 kainic acid injections on ipsilateral forelimb locomotor function in rats with chronic C2 hemisection i njury (C2Hx) I psilateral locomotor function as measured by the FLS was recorded in rats, 8 weeks post C2Hx and 3 and 7 days after bilateral intraspinal injections of kainic acid to the intermediate gray matter between C4 C5. Values are mean + SE using on e way RM ANOVA. P<0.05, = significantly different from pre injury.

PAGE 191

191 Figure 5 2 5 Effects of kainic acid (KA) and saline injection on A) upper limb gross motor use and B) locomotor function in uninjured rats prior to and at 3 and 7 days post injec tion. No deficits in upper extremity function were observed following either kainic acid or saline injections. Values are mean + SE.

PAGE 192

192 Figure 5 26 Ipsilateral upper limb locomotor kinematics prior to administration of kainic acid (i.e.8 post C2Hx) ( A ) and 3 days post KA ( B ). Still frames from videotaped Forelimb Locomotor Scale (FLS) testing. Prior to delivery of kainic acid, rats demonstrated modest spontaneous recovery of ipsilateral upper limb locomotor function characterized by adequate ra nge of motion throughout the gait cycle at all joints of the limb. Palmar placement during stance was evident by this time point and parallel paw position was observed throughout stance. 1 week after bilateral C4 C5 kainic acid injections in rats with chro nic C2 hemisection injury (C2Hx) rats demonstrated reduced range of motion throughout the gait cycle at all joints of the limb, in particular at the distal (wrist) joint. In addition, palmar placement during stance was absent and no toe clearance was obse rved in swing.

PAGE 193

193 Table 5 1. Ventilation p ost C2Hx ( p re KA) Time p ost C2Hx Frequency (% of baseline) Tidal v olume (% of baseline) Minute v entilation (% of baseline) Pre i njury 249.95 + 3.92 208.20 + 10.13 517.86 + 38.30 3 days 143.03 + 9.29 a 155.57 + 10 .23 a 222.11 + 18.66 a 1 week 182.52 + 7.88 173.08 + 8.03 308.40 + 19.55 a 2 weeks 237.74 + 19.12 b 190.65 + 16.60 419.61 + 22.57 b 3 weeks 221.70 + 12.95 b 193.30 + 8.29 416.86 + 23.23 b 4 weeks 237.07 + 14.02 b 178.70 + 8.76 409.05 + 32.31 b 6 weeks 286.73 + 23.73 bc 168.44 + 9.63 450.01 + 32.78 bc 8 weeks 259.15 + 29.83 bc 170.21 + 5.00 408.15 + 36.25 b Values are mean + SE using o ne way RM ANOVA. *P<0.05, a = significantly different from pre injury, b = significantly different from 3 days post C2Hx, c =signific antly different from 1 week post C2Hx.

PAGE 194

194 Table 5 2. Cylinder t est and F orelimb Locomotor Scale (FLS) scores p ost C2Hx Time p ost C2Hx Cylinder (% ipsilateral placements) FLS score Pre injury 50.34 + 1.52 17 + 0 1 week 16.54 + 8.19 a 7 + 1.08 a 2 weeks 18 .41 + 4.55 a 8 + 0.23 ab 3 weeks 20.58 + 3.22 ab 8.33 + 0.24 ab 4 weeks 29.65 + 5.64 ab 8.67 + 0.37 ab 6 weeks 30.23 + 2.74 ab 9.38 + 0.42 abc 8 weeks 30.78 + 4.24 ab 10. 86 + 0.55 abcdef Values are mean + SE using one way RM ANOVA. *P<0.05, a = significantly d ifferent from pre injury, b = significantly different from 1 week post C2Hx, c =significantly different from 2 weeks post C2Hx, d = significantly different from 3 weeks post C2Hx, e = significantly different from 4 weeks post C2Hx, f = significantly different f rom 4 weeks post C2Hx.

PAGE 195

195 Table 5 3. Ventilation post k ainic a cid injection Time post KA Frequency (% of baseline) Tidal v olume (% of baseline) Minute v entilation (% of baseline) Pre KA (8 wks post C2Hx) 259.15 + 31.23 170.21 + 6.49 408.15 + 34.68 a 3 days 167.08 + 14.07 a 136.04 + 4.84 a 227.50 + 20.23 a 7 days 247.60 + 17.85 160.30 + 14.62 376.53 + 27.57 b Values are mean + SE using One way RM ANOVA. *P<0.05, a = significantly different from pre KA, b = significantly different from 3 days post KA.

PAGE 196

196 Table 5 4. Cylinder t est and F orelimb L ocomotor S cale scores p ost kainic a cid Time post KA Cylinder (% ipsilateral placements) FLS scores Pre KA (8 wk post C2Hx) 30.78 + 4.24 10.86 + 0.55 3 days 6.67 + 6.67 6.4 + 0.87 a 7 days 13.93 + 7.46 7.4 + 0.87 a Values are mean + SE using One way RM ANOVA. *P<0.05, a = significantly different from pre KA.

PAGE 197

197 CHAPTER 6 CONCLUSIONS AND FUTURE DIRECTIONS Characterization of Forelimb Plasticity Following Incomplete High Cervical Spinal Cord Injury in Adult Rats I njuries to the cervical spinal cord comprise the largest proportion of all spinal cord injuries Recovery of arm and hand function has been cited as the highest priority for individuals living with chronic tetraplegia. While there is some evidence to sugge st that some degree of spontaneous recovery is possible following incomplete injuries to the cervical spinal cord, very little is known about the specific substrates mediating functional recovery and plasticity, particularly in reference to upper extremity functional recovery. The aims of the present study were to determine the time course and extent of upper extremity functional and muscular recovery following lateral C2 spinal cord hemisection, and to investigate changes in the neuroanatomical circuitry t hat may coincide with this recovery. In a time course study, chances in upper limb function were assessed across a range of functional behaviors. Significant functional impairments resulted following injury, followed by modest spontaneous functional recove ry. Similar reductions in whole muscle size and muscle fiber size were evident early post injury, and appeared to resolve by 8 weeks post injury. The neuroanatomical circuitry controlling the extensor carpi radialis muscle was characterized in uninjured ra ts using retrograde transynaptic tracing techniques. Changes in the distribution of pre motor cervical interneurons were observed in chronically injured rats. In a s eries of dual immunocytochemical labeling experiments, the distribution of retrogradely lab eled phrenic motoneurons and pre motor interneurons was investigated. These data are consistent with the hypothesis that propriospinal interneurons located in the cervical spinal cord may serve as a potential substrate by which spontaneous plasticity withi n

PAGE 198

198 intraspinal circuits may occur following spinal injury. O ngoing studies are investigating the role of these interneurons in mediating functional plasticity after injury. Serotonergic Innervation of Pre Motor Cervical Interneurons in Adult Rats Serotoni n is an important neuromodulator that has been shown to play a role in mediating motoneuron excitability and plasticity in phrenic motor systems. Previous work from our group (Lane et al., 2008b) has demonstrated the presence of a population of propriospin al interneurons in the intermediate gray matter of the cervical spinal cord that innervate phrenic motoneurons Whether these interneurons are innervated by 5 HT has not been specifically tested. Our working hypothesis is that serotonin plays an important role in modulating motor function and plasticity and that 5 HT innervation of pre motor interneurons represents an important part of the neural substrate mediating plasticity in the phrenic motor system. The aims of the present study were to determine the extent to which pre phrenic cervical interneurons are innervated by 5 HT as well as to identify whether specific 5 HT receptor subtypes may be expressed on these neurons. In a series of dual immunocytochemical labeling experiments, the presence of serotone rgic projections and the expression of serotonin receptor subtypes on retrogradely labeled phrenic motoneurons and pre phrenic interneurons were investigated. Histological evaluation revealed robust immunoreactivity of serotonergic projections in close pr oximity to phrenic motoneurons and pre phrenic interneurons in laminae VII and X. In addition, both 5 HT 2A and 5 HT 7 receptor subtypes were expressed on putative phrenic motoneurons and pre motor interneurons. These data are consistent with our working hyp othesis, and ongoing studies are investigating the potential mechanisms by which serotonin modulates respiratory activity and plasticity in both injured and uninjured nervous systems.

PAGE 199

199 Contribution of Propriospinal Neurons to Spontaneous Functional Plastic ity Following Incomplete High Cervical Spinal Cord Injury in Adult Rats P ropriospinal interneurons in the intermediate gray matter of the cervical spinal cord have been shown to innervate both phrenic and upper extremity motoneurons. Recent studies have d emonstrated that propriospinal interneurons may be involved in mediating spontaneous functional recovery following thoracic injury, however whether or not propriospinal interneurons may play mediate functional plasticity in the cervical spinal cord not bee n specifically tested. Our working hypothesis is that this population of interneurons represents part of the substrate mediating functional recovery of both respiratory and upper extremity function following chronic incomplete cervical SCI. The aim of the present study was to determine if focal ablation of neurons in the intermediate cervical gray matter would attenuate functional gains occurring in the weeks and months following experimental SCI (lateral C2 hemisection). Over a period of 8 weeks following injury, upper extremity and ventilatory function were assessed weekly. Once functional gains had plateaued, bilateral intraspinal (C4 C5) injections of Kainic Acid (KA) were used to create focal lesions of intermediate gray matter. Three days following de livery of KA, both respiratory and upper extremity functional gains were dramatically attenuated. By 7 days post injection, ventilatory function had somewhat recovered, though deficits in upper extremity function persisted. Histological evaluation of cervi cal spinal cords indicated discrete bilateral lesions to the intermediate gray matter without concurrent damage to the nearby motoneuron pools. These preliminary data are consistent with our working hypothesis, and ongoing studies are further investigating the role of propriospinal interneurons within intraspinal circuits and the potential for motor recovery and plasticity.

PAGE 200

200 In summary, rats express a modest capacity for spontaneous functional and muscular plasticity following C2Hx. A potential anatomical s ubstrate for this spontaneous recovery exists in the form of propriospinal interneurons located in the intermediate gray matter of the cervical spinal cord. Innervation of motoneuron pools by serotonin has been shown to correlate with to the extent of func tional recovery after spinal cord injury. Evidence now suggests that 5 HT also innervates pre motor interneurons and the extent to which 5 HT modulates interneuronal circuitry should also be considered. Furthermore, specific ablation of pre motor, proprio spinal interneurons results in attenuation of recovered motor function. Future studies exploring more specifically how these propriospinal interneurons mediate motor function in both uninjured and injured nervous systems are necessary and will highlight th e potential for targeting these circuits with therapeutic interventions to enhance neuroplasticity and optimized functional recovery after spinal cord injury.

PAGE 201

201 LIST OF REFERENCES Alheid GF, McCrimmon DR (2008) The chemical neuroanatomy of breathing. Resp iratory Physiology & Neurobiology. Alheid GF, Milsom WK, McCrimmon DR (2004) Pontine influences on breathing: an overview. Respir Physiol Neurobiol 143:105 114. Alilain WJ, Li X, Horn KP, Dhingra R, Dick TE, Herlitze S, Silver J (2008) Light induced rescue of breathing after spinal cord injury. J Neurosci 28:11862 11870. Allred RP, Adkins DL, Woodlee MT, Husbands LC, Maldonado MA, Kane JR, Schallert T, Jones TA (2008) The vermicelli handling test: a simple quantitative measure of dextrous forepaw function i n rats. J Neurosci Methods 170:229 244. Alstermark B, Isa T (2002) Premotoneuronal and direct corticomotoneuronal control in the cat and macque monkey. Adv Exp Med Biol 508:281 297. Alstermark B, Isa T, Ohki Y, Saito Y (1999) Disynaptic pyramidal excitatio n in forelimb motoneurons mediated via C3 C4 propriospinal neurons in the Macaca fuscata. J Neurophysiol 82:3580 3585. Alstermark B, Isa T, Pettersson LG, Sasaki S (2007) The C3 C4 propriospinal system in the cat and monkey: a spinal pre motoneuronal centr e for voluntary motor control. Acta Physiol 189:123 140. Alstermark B, Isa T, Tantisira B (1990) Projection from excitatory C3 C4 propriospinal neurones to spinocerebellar and spinoreticular neurones in the C6 Th1 segments of the cat. Neurosci Res 8:124 1 30. Alstermark B, Isa T, Tantisira B (1991) Pyramidal excitation in long propriospinal neurones in the cervical segments of the cat. Exp Brain Res 84:569 582. Alstermark B, Lindstrom S, Lundberg A, Sybirska E (1981a) Integration in descending motor pathway s controlling the forelimb in the cat. 8. Ascending projection to the lateral reticular nucleus from C3 C4 propriospinal also projecting to forelimb motoneurones. Exp Brain Res 42:282 298. Alstermark B, Lundberg A, Norrsell U, Sybirska E (1981b) Integrat ion in descending motor pathways controlling the forelimb in the cat. 9. Differential behavior defects after spinal cord lesions interrupting defined pathways from higher centers to motoneurones. Exp Brain Res 42:299 318. Alstermark B, Lundberg A, Sasaki S (1984a) Integration in descending motor pathways controlling the forelimb in the cat. 10. Inhibitory pathways to forelimb motoneurons via C3 4 propriospinal neurones. Exp Brain Res 56:279 292.

PAGE 202

202 Alstermark B, Lundberg A, Sasaki S (1984b) Integration in de scending motor pathways controlling the forelimb in the cat. 11. Inhibitory pathways from higher motor centers and forelimb afferents to C3 C4 propriospinal neurones. Exp Brain Res 56:293 307. Alstermark B, Ogawa J, Isa T (2004) Lack of monosynaptic cortic omotoneuronal EPSPs in rats; disynaptic EPSPs mediated via reticulospinal neurons and polysynaptic EPSPs via segmental interneurons. J Neurophysiol 91:1832 1839. Alvarez F, Pearson J, Harrington D, Dewey D, Torbeck L, Fyffe R (1998) Distribution of 5 hydro xytryptamine immunoreactive boutons on alpha motoneurons in the lumbar spinal cord of adult cats. J Comp Neurol 393. Aminoff MJ, Sears TA (1971) Spinal integration of segmental, cortical, and breathing inputs to thoracic respiratory motoneurones. J Physiol 215:557 575. Anderson KD (2004) Targeting recovery: priorities of the spinal cord injured population. J Neurotrauma 21:1371 1383. Anderson KD, Friden J, Lieber RL (2009a) Acceptable benefits and risks associated with surgically improving arm function in i ndividuals living with cervical spinal cord injury. Spinal Cord 47:334 338. Anderson KD, Gunawan A, Steward O (2005) Quantitative assessment of forelimb motor function after cervical spinal cord injury in rats: relationship to the corticospinal tract. Exp Neurol 194:161 174. Anderson KD, Sharp KG, Hofstadter M, Irvine KA, Murray M, Steward O (2009b) Forelimb locomotor assessment scale (FLAS): novel assessment of forelimb dysfunction after cervical spinal cord injury. Exp Neurol 220:23 33. Anderson KD, Sharp KG, Steward O (2009c) Bilateral cervical contusion spinal cord injury in rats. Exp Neurol 220:9 22. Armand J (1982) The origin, course, and terminations of corticospinal fibers in various mammals. Prog Brain Res 57:329 360. Askanazi J, Silverberg PA, Fost er RJ, Hyman AI, Milic Emili J, Kinney JM (1980) Effects of respiratory apparatus on breathing pattern. J Appl Physiol 48:577 580. Baekey DM, Feng P, Decker MJ, Strohl KP (2009) Breathing and sleep: Measurement methods, genetic influences, and developmenta l impacts. ILAR 50:248 261. Baker Herman TL, Fuller DD, Bavis RW, Zabka AG, Golder FJ, Doperalski NJ, Johnson RA, Watters JJ, Mitchell GS (2004) BDNF is necessary and sufficient for spinal respiratory plasticity following intermittent hypoxia. Nature Neuro science 7:48 55.

PAGE 203

203 Baker Herman TL, Mitchell GS (2002) Phrenic long term facilitation requires spinal serotonin receptor activation and protein synthesis. J Neurosci 22:6239 6246. Ballermann M, Fouad K (2006) Spontaneous locomotor recovery in spinal cord inj ured rats is accompanied by anatomical plasticity of reticulospinal fibers. Eur J Neurosci 23:1988 1996. Bao F, DeWitt DS, Prough DS, Liu D (2003) Peroxynitrite generated in the rat spinal cord induces oxidation and nitration of proteins: reduction by Mn ( III) tetrakis (4 benzoic acid) porphyrin. J Neurosci Res 71:220 227. Bareyre FM, Kerschensteiner M, Raineteau O, Mettenleiter TC, Weinmann O, Schwab ME (2004) The injured spinal cord spontaneously forms a new intraspinal circuit in adult rats. Nature Neuro science 7:269 277. Bareyre FM, Schwab ME (2003) Inflammation, degeneration and regeneration in the injured spinal cord: insights from DNA microarrays. Trends Neurosci 26:555 563. Basso DM (2000) Neuroanatomical substrates of functional recovery after exper imental spinal cord injury: implications of basic science research for human spinal cord injury. Phys Ther 80:808 817. Basso DM, Beattie MS, Breshnahan JC (1995) A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma 12:1 21. Bastel HL (1964) Localization of bulbar respiratory center by microelectrode sounding. Exp Neurol 9:410 426. Bastel HL, Lines AJ (1973) Bulbar respiratory neurons participating in the sniff reflex in the cat. Exp Neurol 39:469 481. Basura GJ, Zhou SY, Walker PD, Goshgarian HG (2001) Distribution of serotonin 2A and 2Creceptor mRNA expression in the cervical ventral horn and phrenic motoneurons following spinal cord hemisection. Exp Neurol 169:255 263. Baumgarten RV, Kanzow E (1958) The interaction of two types of inspiratory neurons in the region of the tractus solitarius of the cat. Arch Ital Biol 96:361 373. Baxter DW, Olszewski J (1955) Respiratory responses evoked by electrical stimulation of pons and mesencephalon. J Neurophysiol 18:276 287. Be attie MS, Harrington AW, Lee R, Kim JY, Boyce SL, Longo FM, Bresnahan JC, Hempstead BL, Yoon SO (2002a) ProNGF induces p75 mediated death of oligodendrocytes following spinal cord injury. Neuron 36:375 386. Beattie MS, Hermann GE, Rogers RC, Bresnahan JC ( 2002b) Cell death in models of spinal cord injury. Prog Brain Res 137:37 47.

PAGE 204

204 Belegu V, Oudega M, Gary DS, McDonald JW (2007) Restoring function after spinal cord injury: promoting spontaneous regeneration with stem cells and activity based therapies. Neuro surgery Clinics of North America 18:143 168. Bellingham MC (1999) Synaptic inhibition of cat phrenic motoneurons by intercostal nerve stimulation. J Neurophysiol 82:1224 1232. Berger AJ (1979) Phrenic motoneurons in the cat: subpopulations and nature of re spiratory drive potentials. J Neurophysiol 42:76 90. Berger AJ, Cameron WE, Averill DB, Kramis RC, Binder MD (1984) Spatial distributions of phrenic and medial gastrocnemius motoneurons in the cat spinal cord. Exp Neurol 86:559 575. Bianchi AL (1971) [Loca lization and study of respiratory medullary neurons using antidromic stimulation of the spinal cord or vagus]. J Physiol (Paris) 63:5 40. Bianchi AL (1974) Modalites de decharge et properties anatomofonctionelles des neurones respiratoires bulbaries. J Phy siol (Paris) 64:555 587. Bianchi AL, Dussardier M, Barillot JC, Planche D (1973) Anatomical and functional heterogeneity of the medullary respiratory neurons. Acta Neurobiol Exp 33:319 328. Biering Sorensen B, Kristensen IB, Kjaer M, Biering Sorensen F (20 09) Muscle after spinal cord injury. Muscle & Nerve 40:499 519. Bose P, Parmer R, Reier PJ, Thompson FJ (2005) Morphological changes of the soleus motoneuron pool in chronic midthoracic contused rats. Exp Neurol 191:13 23. Brown R, DiMarco AF, Hoit JD, Gar shick E (2006) Respiratory dysfunction and management in spinal cord injury. Respir Care 51:853 870. Burke RE (2007) Sir Charles Sherrington's The integrative action of the nervous system: a centenary appreciation. Brain 130:887 894. Burke RE, Strick P, Ka nda K, Kim CC, Walmsley B (1977) Anatomy of medial gastrocnemius and soleus motor nuclei in cat spinal cord. J Neurophysiol 40:667 680. Bystrzycka EK (1980) Afferent projections to the dorsal and ventral respiratory nuclei in the medulla oblongata of the c at studied bu the horseradish peroxidase technique. Brain Res 185:59 66. Chatzipanteli K, Garcia R, Marcillo AE, Loor KE, Kraydieh S, Dietrich WD (2002) Temporal and segmental distribution of constitutive and inducible nitric oxide synthases after traumati c spinal cord injury: effect of aminoguanidine treatment. J Neurotrauma 19:639 651.

PAGE 205

205 Choi DW (1992) Excitotoxic cell death. J Neurobiol 23:1261 1276. Cohen MI, Piercey MF, Gootman PM, Wolotsky P (1974) Synaptic connections between medullary inspiratory neur ons and phrenic motoneurons as revealed by cross correlation. Brain Res 81:319 324. Colton CA, Gilbert DL (1987) Production of superoxide anions by a CNS macrophage, the microglia. FEBS Lett 223:284 288. Conta AC, Stelzner DJ (2009) The propriospinal syste m. In: The Spinal Cord(Paxinos, G. et al., eds), pp 180 190 London: Academic Press. Corfield DR, Murphy K, Guz A (1998) Does the motor cortical control of the diaphragm 'bypass' the brainstem respiratory centers in man? Respir Physiol Neurobiol 114:109 117 Courtine G, Gerasimenko Y, van den Brand R, Yew A, Musienko P, Zhong H, Song B, Ao Y, Ichiyama RM, Lavrov I, Roy RR, Sofroniew MV, Edgerton VR (2009) Transformation of nonfunctional spinal circuits into functional states after the loss of brain input. Na ture Neuroscience 12:1333 1342. Courtine G, Song B, Roy RR, Zhong H, Herrmann JE, Ao Y, Qi J, Edgerton VR, Sofroniew MV (2008a) Recovery of supraspinal control of stepping via indirect propriospinal relay connections after spinal cord injury. Nature Medici ne 14:69 74. Courtine G, Song B, Roy RR, Zhong H, Herrmann JE, Ao Y, Qi J, Edgerton VR, Sofroniew MV (2008b) Recovery of supraspinal control of stepping via indirect propriospinal relay connections after spinal cord injury. Nat Med 14:69 74. Cowley KC, Sch midt BJ (1997) Regional distribution of the locomotor pattern generating network in the neonatal rat spinal cord. J Neurophysiol 77:247 259. Crowe MJ, Bresnahan JC, Shuman SL, Masters JN, Beattie MS (1997) Apoptosis and delayed degeneration after spinal co rd injury in rats and monkeys. Nat Med 3:73 76. Dale Nagle EA, Hoffman MS, MacFarlane PM, Mitchell GS (2010a) Multiple pathways to long lasting phrenic motor facilitation. New Frontiers in Respiratory Control 225 230. Dale Nagle EA, Hoffman MS, MacFarlane PM, Satriotomo I, Lovett Barr MR, Vinit S, Mitchell GS (2010b) Spinal plasticity following intermittent hypoxia: implications for spinal injury. Annals of the New York Academy of Sciences 1198:252 259. Dale Nagle EA, Hoffman MS, MacFarlane PM, Satriotomo I Lovett Barr MR, Vinit S, Mitchell GS (2010c) Spinal plasticity following intermittent hypoxia: implications for spinal injury. Ann N Y Acad Sci 1198:252 259.

PAGE 206

206 David S, Aguayo AJ (1981) Axonal elongation into peripheral nervous system "bridges" after centr al nervous system injury in adult rats. Science 214:931 933. Davies JG, Kirkwood PA, Sears TA (1985) The detection of monosynaptic connexions from inspiratory bulbospinal neurones to inspiratory motoneurones in the cat. J Physiol 368:33 62. Davis JN, Plum F (1972) Separation of descending spinal pathways to respiratory motoneurons. Exp Neurol 35:78 94. DeBow SB, Davies MLA, Clarke HL, Colbourne F (2003) Constraint induced movement therapy and rehabilitation exercises lessen motor deficits and volume of brai n injury after striatal hemorrhagic stroke in rats. Stroke 34:1021 1026. DeVivo MJ, Black KJ, Stover SL (1993) Causes of death during the first 12 years after spinal cord injury. Arch Phys Med Rehabil 74:248 254. DeVivo MJ, Chen Y (2011) Trends in new inju ries, prevalent cases, and aging with spinal cord injury. Arch Phys Med Rehabil 92:332 338. Dobbins EG, Feldman JL (1994) Brainstem network controlling descending drive to phrenic motoneurons in the rat. J Comp Neurol 347:64 86. Doly S, Madeira A, Fischer J, Brisorgueil M J, Daval G, Bernard R, Verge D, Conrath M (2004) The 5 HT2A receptor is widely distributed in the rat spinal cord and mainly localized at the plasma membrane of postsynaptic neurons. The Journal of Comparative Neurology 472:496 511. Donnel ley DJ, Popovich PG (2008) Inflammation and its role in neuroprotection, axonal regeneration and functional recovery after spinal cord injury. Experimental Neurology 209:378 388. Doperalski NJ, Sandhu MS, Bavis RW, Reier PJ, Fuller DD (2008) Ventilation an d phrenic output following high cervical spinal hemisection in male vs. female rats. Respiratory Physiology & Neurobiology 162:160 167. Dougherty BJ, Lee KZ, Gonzalez Rothi EJ, Lane MA, Reier PJ, Fuller DD (2012) Recovery of inspiratory intercostal muscle activity following high cervical hemisection. Respiratory Physiology & Neurobiology 183:186 192. Douse MA, Duffin J (1993) Axonal projections and synaptic connections of C2 segment expiratory interneurons in the cat. J Physiol 470:431 444. Drorbaugh JE, Fe nn WO (1955) A barometric method for measuring ventilation in infants. Pediatrics 16:81 89.

PAGE 207

207 Duffin J, Lipski J (1987) Monosynaptic excitation of thoracic motoneurones by inspiratory neurones of the nucleus tractus solitarius in the cat. J Physiol 390:415 4 31. Duron B, Marlot D, Larnicol N, Jung Caillol MC, Macron JM (1979) Somatotopy in the phrenic motor nucleus of the cat as revealed by retrograde transport of horseradish peroxidase. Neurosci Lett 14:159 163. Edgerton VR, Courtine G, Gerasimenko YP, Lavrov I, Ichiyama RM, Fong AJ, Cai LL, Otoshi CK, Tillakaratne NJ, Burdick JW, Roy RR (2008) Training locomotor networks. Brain Res Rev 57:241 254. Edgerton VR, de Leon RD, Harkema SJ, Hodgson JA, London N, Reinkensmeyer DJ, Roy RR, Talmadge RJ, Tillakaratne NJ Timoszyk W, Tobin A (2001) Retraining the injured spinal cord. J Physiol 533:15 22. Edgerton VR, Roy RR, Allen DL, Monti RJ (2002) Adaptations in skeletal muscle disuse or decreased use atrophy. Am J Phys Med Rehabil 81:S127 S147. E l Bohy AA, Schrimsher GW, Reier PJ, Goshgarian HG (1998) Quantitative assessment of respiratory function following contusion injury of the cervical spinal cord. Exp Neurol 150:143 152. Elder CP, Apple DF, Bickel SC, Meyer RA, Dudley GA (2004) Intramuscular fat and glucose into lerance after spinal cord injury A cross sectional study. Spinal Cord 42:711 716. Ellenberger HH, Feldman JL (1990) Subnuclear organization of the lateral tegmental field of the rat. I: Nucleus ambiguus and ventral respiratory group. J Comp Neurol 294:202 211. Ezure K (1990) Synaptic connections between medullary respiratory neurons and considerations on the genesis of respiratory rhythm. Prog Neurobiol 35:429 450. Fawcett JW, Asher RA (1999) The glial scar and central nervous system repair. Brain Res Bull 49:377 391. Fedorko L, Merrill EG (1984) Axonal projections from the rostral expiratory neurones of the Botzinger complex to medulla and spinal cord in the cat. J Physiol 350:487 496. Fedorko L, Merrill EG, Lipski J (1981) Two descending medullary inspira tory pathways to phrenic motoneurones. Neurosci Lett 43:285. Feldman JL (1986) Neurophysiology of breathing in mammals. In: Handbook of Physiology, vol. The Nervous System (Bloom, F. E., ed), pp 463 524: APS.

PAGE 208

208 Feldman JL, Mitchell GS, Nattie EE (2003) Breat hing: rhythmicity, plasticity, chemosensitivity. Annu Rev Neurosci 26:239 266. Fishburn MJ, Marino RJ, Ditunno JF (1990) Atelectasis and pneumonia in acute spinal cord injury. Arch Phys Med Rehabil 71:197 200. Fitch MT, Silver J (2008) CNS injury, glial sc ars, and inflammation: Inhibitory extracellular matrices and regeneration failure. Experimental Neurology 209:294 301. Fouad K, Pearson K (2004) Restoring walking after spinal cord injury. Prog Neurobiol 73: 107 126. Fouad K, Pedersen V, Schwab ME, Bromsam le C (2001) Cervical sprouting of corticospinal fibers after thoracic spinal cord injury accompanies shifts in evoked motor responses. Curr Biol 11:1766 1770. Fouad K, Tse A (2008) Adaptive changes in the injured spinal cord and their role in promoting fun ctional recovery. Neurological Research 30:17 27. Fuller DD, Baker Herman TL, Golder FJ, Doperalski NJ, Watters JJ, Mitchell GS (2005) Cervical spinal cord injury upregulates ventral spinal 5 HT2A receptors. J Neurotrauma 22:203 213. Fuller DD, Doperalski NJ, Dougherty BJ, Sandhu MS, Bolser DC, Reier PJ (2008) Modest spontaneous recovery of ventilation following chronic high cervical hemisection in rats. Exp Neurol 211:97 106. Fuller DD, Golder FJ, Olson EB, Jr., Mitchell GS (2006) Recovery of phrenic activ ity and ventilation after cervical spinal hemisection in rats. Journal of Applied Physiology 100:800 806. Fuller DD, Johnson SM, Olson EB, Jr. (2003) Synaptic pathways to phrenic motoneurons are enhanced by chronic intermittent hypoxia after cervical spina l cord injury. The Journal of Neuroscience 23:2993 3000. Fuller DD, Sandhu MS, Doperalski NJ, Lane MA, White TE, Bishop MD, Reier PJ (2009) Graded unilateral cervical spinal cord injury and respiratory motor recovery. Respir Physiol Neurobiol 165:245 253. Furicchia JV, Goshgarian HG (1987) Dendritic organization of phrenic motoneurons in the adult rat. Exp Neurol 96:621 634. Gad J, Marinesco G (1892) Recherches experimentales sur le center respiratoire bulbarie. C R Acad Sci Paris 115:444 447.

PAGE 209

209 Gensel JC, To var A, Hamers FPT, Deibert RJ, Beattie MS, Breshnahan JC (2006) Behavioral and histological characterization of unilateral cervical spinal cord contusion injury in rats. journal of Neurotrauma 23:36 54. Gibson AR, Houk JC, Kohlerman NJ (1985) Magnocellular red nucleus activity during different types of limb movement in the macaque monkey. J Physiol 358:527 549. Golder FJ, Reier PJ, Bolser DC (2001) Altered respiratory motor drive after spinal cord injury: supraspinal and bilateral effects of a unilateral le sion. J Neurosci. 21: 8680 8689. Golder FJ, Fuller DD, Davenport PW, Johnson RD, Reier PJ, Bolser DC (2003) Respiratory motor recovery after unilateral spinal cord injury: Eliminating crossed phrenic activity decreases tidal volume and increases contralate ral respiratory motor output. The Journal of Neuroscience 23:2494 2501. Golder FJ, Fuller DD, Lovett Barr MR, Vinit S, Resnick DK, Mitchell GS (2011) Breathing patterns after mid cervical spinal contusion in rats. Exp Neurol 231:97 103. Golder FJ, Mitchell GS (2005a) Spinal synaptic enhancement with acute intermittent hypoxia improves respiratory functino after chronic cervical spinal cord injury. The Journal of Neuroscience 25:2925 2932. Golder FJ, Mitchell GS (2005b) Spinal synaptic enhancement with acute intermittent hypoxia improves respiratory function after chronic cervical spinal cord injury. The Journal of Neuroscience 25:2925 2932. Goldstein B, Hammond MC, Stiens SA, Little JW (1998) Posttraumatic syringomyelia: profound neuronal loss, yet preserved function. Arch Phys Med Rehabil 79:107 112. Goshgarian HG, Yu X J, Rafols JA (1989) Neuronal and glial changes in the rat phrenic nucleus occuring within hours after spinal cord injury. The Journal of Comparative Neurology 284:519 533. Goshgarian HG (1981 ) The role of cervical afferent nerve fiber inhibition of the crossed phrenic phenomenon. Exp Neurol 72:211 225. Grossman EJ, Roy RR, Talmadge RJ, Zhong H, Edgerton VR (1998) Effects of inactivity on myosin heavy chain composition and size of rat soleus fi bers. Muscle & Nerve 21:375 389. Haddad F, Roy RR, Zhong H, Edgerton VR, Baldwin KM (2003) Atrophy responses to muscle inactivity. II. Molecular markers of protein deficits. J Appl Physiol 95.

PAGE 210

210 Hadi B, Zhang YP, Burke DA, Shields CB, Magnuson DS (2000) Last ing paraplegia caused by loss of lumbar spinal cord interneurons in rats: no direct correlation with motor neuron loss. J Neurosurg 93:266 275. Hadley SD, Walker PD, Goshgarian HG (1999) Effects of serotonin synthesis inhibitor p CPA on the expression of t he crossed phrenic phenomenon 4 h following C2 spinal cord hemisection. Exp Neurol 160:479 488. Hagg T, Oudega M (2006) Degenerative and spontaneous regenerative processes after spinal cord injury. journal of Neurotrauma 23:264 280. Harkema SJ (2008) Plast icity of interneuronal networks of the functionally isolated human spinal cord. Brain Research Reviews 57:255 264. Heckman CJ, Johnson M, Mottram C, Schuster J (2008) Persistent inward currents in spinal motoneurons and their influence on human motoneuron firing patterns. Neuroscientist 14:264 275. Hermann GE, Rogers RC, Bresnahan JC, Beattie MS (2001) Tumor necrosis factor alpha induces cFOS and strongly potentiates glutamate mediated cell death in the rat spinal cord. Neurobiol Dis 8:590 599. Hilaire G, M onteau R (1976) Connections between inspiratory medullary neurons and phrenic or intercostal motoneurones. J Physiol (Paris) 72:987 1000. Hochman S, Garraway SM, Machacek DW, Shay BL (2001) 5 HT receptors and neuromodulatory control of spinal cord function : CRC Press. Hoffman MS, Mitchell GS (2011) Spinal 5 HT7 receptor activation induces long lasting phrenic motor facilitation. J Physiol 589:1397 1407. Holstege G, Kuypers HG (1982) The anatomy of brain stem pathways to the spinal cord in cat. A labeled ami no acid tracing study. Prog Brain Res 57:145 175. Holstege G, Kuypers HG (1987) Brainstem projections to spinal motoneurons: an update. Neuroscience 23:809 821. Holtman JR, Dick TE (1987) Serotonin mediated excitation of recurrent laryngeal and phrenic mot oneurons evoked by stimulation of the raphe obscurus. Brain Res 417:12 20. Holtman JR, Dick TE, Berger AJ (1986) Involvement of serotonin in the excitation of phrenic motoneurons evoked by stimulation of the raphe obscurus. J Neurosci 6:1185 1193. Holtman JR, Norman WP, Gillis RA (1984) Projections from the raphe nuceli to the phrenic motor nucelus in the cat. Neurosci Lett 44:105 111.

PAGE 211

211 Horner PJ, Popovich PG, Mullin BB, Stokes BT (1996) A quantitative spatial analysis of the blood spinal cord barrier. II. P ermeability after intraspinal fetal transplantation. Exp Neurol 142:226 243. Houenou LJ, Oppenheim RW, Li L, Lo AC, Prevette D (1996) Regulation of spinal motoneuron survival by GDNF during deveopment and following injury. Cell Tissue Res 286:219 223. Hua Y, Schallert T, Keep RF, Wu J, Hoff JT, Xi G (2001) Behavioral tests after intracerebral hemorrhage in the rat. Stroke 33:2478 2484. Huang Y, Goshgarian HG (2009) Identification of the neural pathway underlying spontaneous crossed phrenic activity in neona tal rats. Journal of Neuroscience 163:1109 1118. Hutchinson KJ, Linderman JK, Basso DM (2001) Skeletal muscle adaptations following spinal cord contusion injury in rat and the relationship to locomotor function: A time course study. Journal of Neurotrauma 18:1075 1089. Ichiyama RM, Courtine G, Gerasimenko YP, Yang GJ, van den Brand R, Lavrov IA, Zhong H, Roy RR, Edgerton VR (2008) Step training reinforces specific spinal locomotor circuitry in adult spinal rats. J Neurosci 28:7370 7375. Illert M, Jankowska E, Lundberg A, Odutola A (1981) Integration in descending motor pathways controlling the forelimb in the cat. 7. Effects from the reticular formation on C3 C4 propriospinal neurones. Exp Brain Res 42:269 281. Illert M, Lundberg A, Padel Y, Tanaka R (1978) Integration in descending motor pathways controlling the forelimb in the cat. 5. Properties of and monosynaptic excitatory convergence on C3 C4 propriospinal neurones. Exp Brain Res 33:101 130. Illert M, Lundberg A, Tanaka R (1977) Integration in descendi ng motor pathways controlling the forelimb in the cat. 3. Convergence on propriosponal neurones transmitting disynaptic excitation from the corticospinal tract and other descending tracts. Exp Brain Res 29:323 246. Illert M, Tanaka R (1978) Integration in descending motor pathways controlling the forelimb in the cat. 4. Corticospinal inhibition of forelimb motoneurones mediated by short propriospinal neurones. Exp Brain Res 31:131 141. Isa T, Ohki Y, Alstermark B, Pettersson LG, Sasaki S (2007) Direct and indirect cortico motoneuronal pathways and control of hand/arm movements. Physiology 22:145 152. Ishihara A, Naitoh H, Araki H, Nishihira Y (1988) Soma size and oxidative enzyme activity of motoneurons supplying the fast and slow twitch muscles in the rat Brain Res 446:195 198.

PAGE 212

212 Ishihara A, Roy RR, Edgerton VR (1995) Succinate dehydrogenase activity and soma size of motoneurons innervating different portions of the rat tibialis anterior. J Neurosci 68:813 822. Jackson AB, Dijkers M, DeVivo MJ, Poczatek RB (2004) A demographic profile of new traumatic spinal cord injuries: change and stability over 30 years. Arch Phys Med Rehabil 85:1740 1748. Jacky JP (1978) A plethysmograph for long term measurements of ventilation in unrestrained animals. J Appl Physiol 4 5:644 647. Jacobs BL, Fornal CA (1993) 5 HT and motor control: a hypothesis. Trends Neurosci 16:346 352. Kalia M (1977) Neuroanatomical organization of the respiratory centers. Fed Proc 36:2405 2411. Kalia M (1981) Anatomical organization of central respir atory neurons. Annu Rev Physiol 43:105 120. Kalia M, Feldman JL, Cohen MI (1979) Afferent projections to the inspiratory neuronal region of the ventrolateral nucleus of the tractus solitarious in the cat. Brain Res 171:135 141. Kelley MD, Nim S, Rosseau G, Fowles JR, Murphy RJL (2006) Early effects of spinal cord transection on skeletal muscle properties. Journal of Applied Physiology Nutrition and Metabolism 31:398 406. Khaing ZZ, Geissler SA, Jiang S, Milman BD, Aguilar SV, Schmidt SE (2012) Assessing for elimb function after unilateral cervical spinal cord injury: Novel forelimb tasks predict lesion severity and recovery. J Neurotrauma 29:488 498. Kinkead R, Zhan W Z, Prakash YS, Bach KB, Sieck GC, Mitchell GS (1998) Cervical dorsal rhizotomy enhances sero tonergic innervation of phrenic motoneurons and serotonin dependent long term facilitation of respiratory motor output in rats. The Journal of Neuroscience 18:8436 8443. Kuzuhara S, Chou SM (1980) Localization of the phrenic nucleus in the rat: a HRP study Neurosci Lett 16:119 124. Lacroix S, Chang L, Rose John S, Tuszynski MH (2002) Delivery of hyper interleukin 6 to the injured spinal cord increases neutrophil and macrophage infiltration and inhibits axonal growth. J Comp Neurol 454:213 228. Lalley PM (1 986a) Responses of phrenic motoneurons of the cat to stimulation of medullary raphe nuclei. J Physiol 380:349 371.

PAGE 213

213 Lalley PM (1986b) Serotonergic and non serotonergic responses of phrenic motoneurones to raphe stimulation in the cat. J Physiol 380:373 385. Landry E, Frenette J, Guertin PA (2004) Body weight, limb size, and muscular properties of early paraplegic mice. J Neurotrauma 21:1008 1016. Lane MA, Fuller DD, White TE, Reier PJ (2008a) Respiratory neuroplasticity and cervical spinal cord injury: trans lational perspectives. Trends Neurosci 31:538 547. Lane MA, Lee KZ, Fuller DD, Reier PJ (2009) Spinal circuitry and respiratory recovery following spinal cord injury. Respir Physiol Neurobiol 169:123 132. Lane MA, Lee KZ, Salazar K, O'Steen BE, Bloom DC, F uller DD, Reier PJ (2012) Respiratory function following bilateral mid cervical contusion injury in the adult rat. Exp Neurol 235:197 210. Lane MA, White TE, Coutts MA, Jones AL, Sandhu MS, Bloom DC, Bolser DC, Yates BJ, Fuller DD, Reier PJ (2008b) Cervica l prephrenic interneurons in the normal and lesioned spinal cord of the adult rat. J Comp Neurol 511:692 709. Larnicol N, Rose D, Marlot D, Duron B (1982a) Anatomical organization of cat intercostal motor nuclei as demonstrated by HRP retrograde labeling. J Physiol (Paris) 78:198 206. Larnicol N, Rose D, Marlot D, Duron B (1982b) Spinal localization of the intercostal motoneurones innervating the upper thoracic spaces. Neurosci Lett 31:13 18. Lee KZ, Reier PJ, Fuller DD (2009) Phrenic motoneuron discharge p atterns during hypoxia induced short term potentiation in rats. J Neurophysiol 102:2184 2193. Lee KZ, Sandhu MS, Dougherty BJ, Fuller DD, Reier PJ (2010) Influence of vagal afferents on supraspinal and spinal respiratory activity following cervical spinal cord injury in rats. J Appl Physiol 109:377 387. Lee YB, Yune TY, Baik SY, Shin YH, Du S, Rhim H, Lee EB, Kim YC, Shin ML, Markelonis GJ, Oh TH (2000) Role of tumor necrosis factor alpha in neuronal and glial apoptosis after spinal cord injury. Exp Neurol 166:190 195. Levesque F, Fabre Thorpe M (1990) Motor deficit induced by red nucleus lesion: reappraisal using kainic acid destructions. Exp Brain Res 81:191 198. Li X, Murray K, Harvey PJ, Ballou EW, Bennett DJ (2007) Serotonin facilitates a persistent cal cium current in motoneurons of rats with and without chronic spinal cord injury. J Neurophysiol 97:1236 1246. Lindsay A, Feldman JL (1993) Modulation of respiratory activity of neonatal rat phrenic motoneurones by serotonin. J Physiol 461:213 233.

PAGE 214

214 Ling L ( 2008) Serotonin and NMDA receptors in respiratory long term facilitation. Respiratory Physiology & Neurobiology. Lipski J, Merrill EG (1980) Electrophysiological demonstration of the porjection from expiratory neurones in rostral medulla to contralateral d orsal respiratory group. Brain Res 197:521 524. Liu D, Ling X, Wen J, Liu J (2000) The role of reactive nitrogen species in secondary spinal cord injury: formation of nitric oxide, peroxynitrite, and nitrated protein. J Neurochem 75:2144 2154. Liu M, Bose P, Walter GA, Thompson FJ, Vandenborne K (2008) A longitudinal study of skeletal muscle following spinal cord injury and locomotor training. Spinal Cord 46:488 493. Liu M, Stevens Lapsley JE, Jayaraman A, Ye F, Conover C, Walter GA, Bose P, Thompson FJ, Bo rst SE, Vandenborne K (2010) Impact of treadmill locomotor training on skeletal mucsle IGF1 and myogenic regulatory factors in spinal cord injured rats European Journal of Applied Physiology 109:709 720. Long S, Duffin J (1986) The neuronal determinants of respiratory rhythm. Prog Neurobiol 27:101 182. Ma M, Basso DM, Walters P, Stokes BT, Jakeman LB (2001) Behavioral and histological outcomes following graded spinal cord contusion injury in the C57Bl6 mouse. Exp Neurol 169:239 254. MacFarlane PM, Vinit S, Mitchell GS (2009) Serotonin 2A and 2B receptor induced phrenic motor facilitation: differential requirement for spinal NADPH oxidase activity. Neuroscience 178:45 55. Magnuson DS, Lovett R, Coffee C, Gray R, Han Y, Zhang YP, Burke DA (2005) Functional con sequences of lumbar spinal cord contusion injuries in the adult rat. J Neurotrauma 22:529 543. Magnuson DS, Trinder TC, Zhang YP, Burke D, Morassutti DJ, Shields CB (1999) Comparing deficits following excitotoxic and contusion injuries in the thoracic and lumbar spinal cord of the adult rat. Exp Neurol 156:191 204. Mantilla CB, Sieck GC (2003) Mechanisms underlying motor unit plasticity in the respiratory system. Journal of Applied Physiology 94:1230 1241. Mantilla CB, Zhan WZ, Sieck GC (2009) Retrograde la beling of phrenic motoneurons by intrapleural injection. J Neurosci Methods 182:244 249. Martin GF, R.P. V (1985) Spinal projections of the gigantocellular reticular formation in the rat. Evidence for projections from different areas to laminae I and II an d lamina IX. Exp Brain Res 58:154 162.

PAGE 215

215 Martinez M, Brezun JM, Bonnier L, Xerri C (2009) A new rating scale for open field evaluation fo behavioral recovery after cervical spinal cord injury in rats. J Neurotrauma 1043 1053. Matsumoto A, Nagatomo F, Mori A, Ohira Y, Ishihara A (2007) Cell size and oxidative enzyme activity of rat biceps brachii and triceps brachii muscles. Journal of Phyiological Sciences 57:311 316. Matsuyama K, Drew T (2000) Vestibulospinal and reticulospinal neuronal activity during locom otion in the intact cat. 1. Walking on a level surface. J Neurophysiol 84:2237 2256. Matsuyama K, Takakusaki K, Nakajima K, More S (1997) Multi segmental innervation of single pontine reticulospinal axons in the cervico thoracic region of the cat: anterogr ade PHA L tracing study. J Comp Neurol 377:234 250. Matyja E, Naganska E, Taraszewska A, Rafalowska J (2005) The mode of spinal motor neurons degeneration in a model of slow glutamate excitotoxicity in vitro. Folia Neuropathol 43:7 13. McCurdy ML, Hansama DI, Houk JC, Gibson AR (1987) Selective projections from the cat red nucleus to digit motoneurons. J Comp Neurol 265:367 379. McKenna JE, Prusky GT, Whishaw IQ (2000) Cervical motoneuron topography reflects the proximodistal organization of muscles and mov ements of the rat forelimb: a retrograde carbocyanine dye analysis. J Comp Neurol 419:286 296. Merletti R (1999) Standards for reporting EMG data. J Electromyography and Kines 1999:III IV. Merrill EG (1970) The lateral respiratory neurones of the medulla: their associations with nucleus ambiguus, nucleus retroambigualis, the spinal accessory nucleus, and the spinal cord. Brain Res 24:11 28. Merrill EG (1974) Finding a respiratory function for the medullary respiratory neurons. In: Essays on the Nervous Syst em(Bellairs, R. and Gray, E. G., eds), pp 451 486 New York: Oxford University Press (Clarendon). Merrill EG (1975) Preliminary studies on nucleus retroambigualis nucleus of the solitary tract interactions in cats. J Physiol 244:54P 55P. Merrill EG, Fedorko L (1984) Monosynaptic inhibition of phrenic motoneurons: a long descending projection from Botzinger neurons. J Neurosci 4:2350 2353. Metz GA, Dietz V, Schwab ME, Fouad K (1998) The effects of unilateral pyramidal tract section on hindlimb motor performan ce in the rat. Behav Brain Res 96:37 46.

PAGE 216

216 Metz GA, Merkler D, Dietz V, Schwab ME, Fouad K (2000) Efficient testing of motor function in spinal cord injured rats. Brain Res 883:165 177. Metz GA, Whishaw IQ (2002) Cortical and subcortical lesions impair skill ed walking in the ladder rung walking test: a new task to evaluate fore and hindlimb stepping, placing and coordination. J Neurosci Methods 115:169 179. Metz GA, Whishaw IQ (2009) The ladder rung walking task: a scoring system and its practical applicatio n. J Vis Exp. Miller AD, Ezure K, Suzuki I (1985) Control of abdominal muscles by brainstem respiratory neurons in the cat. J Neurophysiol 54:155 167. Mitchell GS, Baker TL, Nanda SA, Fuller DD, Zabka AG, Hodgeman BA, Bavis RW, Mack KJ, Olson EB, Jr. (2001 ) Intermittent hypoxia and respiratory plasticity. Journal of Applied Physiology 90:2466 2475. Mitchell GS, Johnson SM (2003) Neuroplasticity in respiratory motor control. Journal of Applied Physiology 94:358 374. Montoya CP, Campbell Hope LJ, Pemberton KD Dunnett SB (1991) The "staircase test": a measure of independent forelimb reaching and grasping abilities in rats. J Neurosci Methods 36:219 228. Moreno DE, Yu X J, Goshgarian HG (1992) Identification of the axon pathways which mediate functional recover y of a paralyzed hemidiaphragm following spinal cord hemisection in the adult rat. Experimental Neurology 116:219 228. Mullner A, Gonzenbach RR, Weinmann O, Schnell L, Liebscher T, Schwab ME (2008) Lamina specific restoration of serotonergic projections af ter Nogo A antibody treatment of spinal cord injury in rats. European Journal of Neuroscience 27:326 333. Nicaise C, Putatunda R, Hala TJ, Regan KA, Frank DM, Brion JP, Leroy K, Pochet R, Wright MC, Lepore AC (2012) Degeneration of phrenic motor neurons in duces long term diaphragm deficits following mid cervical contusion in mice. J Neurotrauma 29:2748 2760. Noga BR, Johnson DMG, Riesgo MI, Pinzon A (2009) Locomotor activated neurons of the cat. I. Serotonergic innervation and co localization of 5 HT7, 5 HT 2A, and 5 HT1A receptors in the thoraco lumbar spinal cord. J Neurophysiol 102:1560 1576. NSCID NSCID (2011) National Spinal Cord Injury Database.

PAGE 217

217 Okada S, Nakamura M, Mikami Y, Shimazaki T, Mihara M, Ohsugi Y, Iwamoto Y, Yoshizaki K, Kishimoto T, Toyama Y Okano H (2004) Blockade of interleukin 6 receptor suppresses reactive astrogliosis and ameliorates functional recovery in experimental spinal cord injury. J Neurosci Res 76:265 276. Onifer SM, Rodriguez JF, Santiago DI, Benitez JC, Kim DT, Brunschwig JP, Pacheco JT, Perrone JV, Llorente O, Hesse DH, Martinez Arizala A (1997) Cervical spinal cord injury in the adult rat: assessment of forelimb dysfunction. Restor Neurol Neurosci 11:211 223. Onifer SM, Zhang YP, Burke DA, Brooks DL, Decker JA, McClure NJ, F loyd AR, Hall J, Proffitt BL, Shields CB, Magnuson DS (2005) Adult rat forelimb dysfunction after dorsal cervical spinal cord injury. Exp Neurol 192:25 38. Otake K, Sasaki H, Ezure K, Manabe M (1989) Axonal trajectory and terminal distribution of inspirato ry neurons of the dorsal respiratory groups in the cat medulla. J Comp Neurol 286:218 230. Pagnussat Ade S, Michaelsen SM, Achaval M, Netto CA (2009) Skilled forelimb reaching in Wistar rats: evaluation by means of Montoya staircase test. J Neurosci Method s 177:115 121. Pearson KG (2001) Could enhanced reflex function contribute to improving locomotion after spinal cord repair? J Physiol 533:75 81. Peterson BW, Maunz RA, Pitts NG, Mackel RG (1975) Patterns of projection and branching of reticulospinal neuro ns. Exp Brain Res 23:333 351. Pettersson LG, Lundberg A, Alstermark B, Isa T, Tantisira B (1997) Effect of spinal cord lesions on forelimn target reaching and on visually guided switching of target reaching in the cat. Neurosci Res 29:241 256. Pilowsky PM, de Castro CD, Llewellyn Smith I, Lipski J, Voss MD (1990) Serotonin immunoreactive boutons make synapses with feline phrenic motoneurons. j Neurosci 10:1091 1098. Pitts RF (1940) The respiratory center and its descending pathways. J Comp Neurol 72:605 62 5. Popovich PG, Wei P, Stokes BT (1997) Cellular inflammatory response after spinal cord injury in Sprague Dawley and Lewis rats. J Comp Neurol 377:443 464. Porter WT (1895) The path of respiratory impulse from the bulb to the phrenic nuclei. J Physiol 17: 455 485. Prentice SD, Drew T (2001) Contributions of the reticulospinal system to the postural adjustments occuring during voluntary gait modifications. J Neurophysiol 85:679 698.

PAGE 218

218 Ramirez JM, Schwarzachwer SW, Pierrefiche O, Olivera BM, Richter DW (1998) Selective lesioning of the cat Pre Botzinger complex in vivo eliminates breathing but not gasping. J Physiol 507:895 907. Ramon y Cajal S (1913 1914) Estudios sobre la Degeneracion y Regeneracion del Sistema Nervioso. Madrid: Oxford University Press. Ramsa y SC, Adams L, Murphy K, Corfield DR, Grootoonk S, Bailey DL, Frackowiak RS, Guz A (1993) Regional cerebral blood flow during volitional expiration in man: a comparison with volitional inspiration. J Physiol 461:85 101. Reier PJ (2004) Cellular transplanta tion strategies for spinal cord injury and translational neurobiology. NeuroRx 1:424 451. Reier PJ, Golder FJ, Bolser DC, Hubscher C, Johnson R, Schrimsher GW, Velardo MJ (2002) Gray matter repair in the cervical spinal cord. Prog Brain Res 137:49 70. Rick ard Bell GC, Bystrzycka EK, Nail BS (1985) Cells of origin of corticospinal projections to phrenic and thoracic respiratory motoneurones in the cat as shown by retrograde transport of HRP. Brain Res Bull 14:39 47. Riddle CN, Edgley SA, Baker SN (2009) Dire ct and indirect connections with upper limb motoneurons from the primate reticulospinal tract. J Neurosci 29:4993 4999. Roberts M, Davies M, Girdlestone D, Foster G (1988) Effects of 5 hydroxytryptamine agonists and antagonists on the responses of rat spin al motoneurones to raphe obscurus stimulation. Br J Pharmacol 95. Rosenzweig ES, Courtine G, Jindrich DL, Brock JH, Ferguson AR, Strand SC, Nout YS, Roy RR, Miller DM, Beattie MS, Havton LA, Breshnahan JC, Edgerton VR, Tuszynski MH (2010) Extensive spontan eous plasticity of corticospinal projections after primate spinal cord injury. Nat Neurosci 13:1505 1510. Round JM, Barr FMD, Moffat B, Jones DA (1993) Fibre areas and histochemical fibre types in the quadriceps muscle of paraplegic subjects. J Neurol Sci 116:207 211. Rowland JW, Hawryluk GW, Kwon B, Fehlings MG (2008) Current status of acute spinal cord injury pathophysiology and emerging therapies: promise on the horizon. Neurosurg Focus 25:E2. Roy RR, Acosta L, Jr. (1986) Fiber type and fiber size chang es in selected thigh muscles siz months after low thoracic spinal cord transection in adult cats: Exercise effects. Exp Neurol 92:675 685. Roy RR, Matsumoto A, Zhong H, Ishihara A, Edgerton VR (2007) Rat alpha and gamma motoneuron soma size and succinate dehydrogenase activity are independent of neuromuscular activity level. Muscle & Nerve 36:234 241.

PAGE 219

219 Sandhu MS, Dougherty BJ, Lane MA, Bolser DC, Kirkwood PA, Reier PJ, Fuller DD (2009) Respiratory recovery following high cervical hemisection. Respiratory Ph ysiology & Neurobiology 169:94 101. Sandhu MS, Lee KZ, Fregosi RF, Fuller DD (2010) Phrenicotomy alters long term facilitation following intermittent hypoxia in anesthetized rats. J Appl Physiol 109:279 287. Sandrow HR, Shumsky JS, Amin A, Houle JD (2008) Aspiration of a cervical spinal contusion injury in preparation for delayed peripheral nerve grafting does not impair forelimb behavior or axon regeneration. Exp Neurol 210:489 500. Schepens B, Drew T (2004) Independent and convergent signals from the pont omedullary reticular formation contribute to the control of posture and movemnt during reaching in the cat. J Neurophysiol 92:2217 2238. Schepens B, Drew T (2006) Descending signal s from the pontomedullary reticular formation are bilateral, asymmetric, and gated during reaching movements in the cat. J Neurophysiol 96:2229 2252. Schilero GJ, Spungen AM, Bauman WA, Radulovic M, Lesser M (2009) Pulmonary function and spinal cord injury Respir Physiol Neurobiol 166:129 141. Schnell L, Fearn S, Klassen H, Schwab ME, Perry VH (1999a) Acute inflammatory responses to mechanical lesions in the CNS: differences between brain and spinal cord. Eur J Neurosci 11:3648 3658. Schnell L, Fearn S, Sc hwab ME, Perry VH, Anthony DC (1999b) Cytokine induced acute inflammation in the brain and spinal cord. J Neuropathol Exp Neurol 58:245 254. Scholtes F, Theunissen E, Phan Ba R, Adriaensens P, Brook G, Franzen R, Gelan J, Schoenen J, Martin D (2011) Post m ortem assessment of rat spinal cord injury and white matter sparing using inversion recovery supported proton density magnetic resonance imaging. Spinal Cord 49:345 351. Schrimsher GW, Reier PJ (1993) Forelimb motor performance following dorsal column, dor solateral funiculi, or ventrolateral funiculi lesions of the cervical spinal cord in the rat. Exp Neurol 120:264 276. Shah PK, Stevens JE, Gregory CM, Pathare NC, Jayaraman A, Bickel SC, Bowden M, Behrman AL, Walter GA, Dudley GA, Vandenborne K (2006) Lowe r extremity muscle cross sectional area after incomplete spinal cord injury. Arch Phys Med Rehabil 87:772 778. Shamash S, Reichert F, Rotshenker S (2002) The cytokine network of Wallerian degeneration: tumor necrosis factor alpha, interleukin 1alpha, and i nterleukin 1beta. J Neurosci 22:3052 3060.

PAGE 220

220 Shea SA (1996) Behavioural and arousal related influences on breathing in humans. Exp Physiol 81:1 26. Silver J (2008) Special issue: spinal cord regeneration and repair. Exp Neurol 209:293. Silver J, Miller JH (2 004) Regeneration beyond the glial scar. Nat Rev Neurosci 5:146 156. Smith JC, Ellenberger HH, Ballanyi K, Richter DW, Feldman JL (1991) Pre Botzinger complex: a brainstem region that may generate respiratory rhythm in mammals. Science 254:726 729. St John WM, Bartlett D (1979) Comparison of phrenic motoneuron responses to hypercapnia and isocapnia hypoxia. J Appl Physiol 46:1096 1102. Stackhouse SK, Murray M, Shumsky JS (2008) Effect of cervical dorsolateral funiculotomy on reach to grasp function in the r at. J Neurotrauma 25:1039 1047. Standring S (2009) Gray's Anatomy: Elsevier. Steinbusch HW (1981) Distribution of serotonin immunoreactivity in the central nervous system of the rat cell bodies and terminals. J Neurosci 6:557 618. Stephenson R, Gucciardi EJ (2002) Theoretical and practical considerations in the application of whole body plethysmography to sleep research. Eur J Appl Physiol 87:207 219. Stevens JE, Liu M, Bose P, O'Steen WA, Thompson FJ, Anderson DK, Vandenborne K (2006) Changes in soleus mu scle function and fiber morphology with one week of locomotor training in spinal cord contusion injured rats. journal of Neurotrauma 23:1671 1681. Tator CH (1991) Review of experimental spinal cord injury with emphasis on the local and systemic circulatory effects. Neurochirurgie 37:291 302. Tator CH (1995) Update on the pathophysiology and pathology of acute spinal cord injury. Brain Pathol 5:407 413. Tator CH, Koyanagi I (1997) Vascular mechanisms in the pathophysiology of human spinal cord injury. J Neur osurg 86:483 492. Tennant KA, Asay AL, Allred RP, Ozburn AR, Kleim JA, Jones TA (2010) The vermicelli and capellini handling tests: simple quantitative measures of dextrous forepaw function in rats and mice. J Vis Exp 41. Tosolini AP, Morris R (2012) Spati al characterization of the motor neuron columns supplying the rat forelimb. Neuroscience 200:19 30.

PAGE 221

221 van Kan PLE, McCurdy ML (2002) Discharce of primate magnocellular red nucleus neurons during reachg to grasp in different spatial locations. Exp Brain Res 1 42:151 157. Vinit S, Gauthier P, Stamegna JC, Kastner A (2006) High cervical lateral spinal cord injury results in long term ipsilateral hemidiaphragm paralysis. J Neurotrauma 23:1137 1146. Waerhaug O, Lomo T (1994) Factors causing different properties at neuromuscular junctions in fast and slow rat skeletal muscles. Anat Embryol 190:113 125. Webber CL, Wurster RD, Chung JM (1979) Cat phrenic nucleus architecture as revealed by horseradish peroxidase mapping. Exp Brain Res 35:395 406. Westbury DR (1982) A c omparison of the structures of alpha and gamma spinal motoneurones of the cat. J Physiol 325:79 91. White SR, Fung SJ, Jackson DA, Imel KM (1996) Serotonin, norepinepherine, and associated neuropeptides: effects on somatic motoneuron excitability. Prog Bra in Res 107:183 199. Wilkerson JER, Mitchell GS (2009) Daily intermittent hypoxia augments spinal BDNF levels, ERK phospohrylation and respiratory long term facilitation. Experimental Neurology 217:116 123. Windle WF, Chambers WW (1950) Regeneration in the spinal cord of the cat and dog. J Comp Neurol 93:241 257. Winslow C, Rozovsky J (2003) Effect of spinal cord injury on the respiratory system. Am J Phys Med Rehabil 82:803 814. Xiong Y, Rabchevsky AG, Hall ED (2007) Role of peroxynitrite in secondary oxida tive damage after spinal cord injury. J Neurochem 100:639 649. Yates BJ, Smail JA, Stocker SD, Card JP (1999) Transneuronal tracing of pathways controlling activity of diaphragm motoneurons in the ferret. Neuroscience 90: 1501 1513. Zhan W Z, Ellenberger H H, Feldman JL (1989) Monoaminergic and GABAergic terminations in phrenic nucleus of rat identified by immunohistochemical labeling. Neuroscience 31:105 113. Zhou SY, Goshgarian HG (2000) 5 Hydroxytryptophan induced respiratory recovery after cervical spina l cord hemisection in rats. J Appl Physiol 89:1528 1536.

PAGE 222

222 BIOGRAPHICAL SKETCH Elisa Janine Gonzalez Rothi was born in Gainesville, Florida in 1981. She graduated from Buchholz High School in 2000 and attended the University of Florida, where she completed a Bach elor of Science (BS) degree in p sychology in 2004. Subsequently, she attended the University of Miami where she received her Doctor Physical Therapy (DPT) degree. Elisa then worked full time as a physical therapist in acute inpatient and critical c are settings for a year and a half before returning to full time Rehabilitation Sciences Doctoral (RSD) Program at the University of Florida in 2008 and graduated in May 2013 with her Ph.D.