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Respiratory Motor Plasticity and Cervical Spinal Cord Injury

Permanent Link: http://ufdc.ufl.edu/UFE0043635/00001

Material Information

Title: Respiratory Motor Plasticity and Cervical Spinal Cord Injury
Physical Description: 1 online resource (209 p.)
Language: english
Creator: Dougherty, Brendan J
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: plasticity -- respiration -- sci
Neuroscience (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Traumatic injury to the cervical spinal cord is often accompanied by respiratory dysfunction. This results from direct damage to respiratory-related neurons and interruption of bulbospinal respiratory pathways. Injuries above the fourth cervical segment (C4) in particular may directly impact control of the diaphragm muscle, necessitating mechanical ventilation for survival. Thus, finding ways to enhance recovery of breathing function following spinal cord injury (SCI) is an important clinical priority. This dissertation presents original research exploring spontaneous respiratory recovery in rats following experimental cervical SCI and treatments to augment this recovery. We first investigated the spontaneous "crossed-phrenic phenomenon" (CPP) and its contributions to respiratory recovery in the initial days to several weeks following unilateral cervical SCI (i.e. hemisection). Commonly studied as a model of evoked neuroplasticity in the respiratory system, the role of the CPP in spontaneous recovery from SCI is less understood. In this study we developed a method to quantify the contribution of the spontaneous CPP to tidal volume recovery by cutting the phrenic nerve in spontaneously breathing, anesthetized rats. Our results suggest that the CPP may play an important role in normal breathing function in the weeks following hemisection. However, the relative contribution of the CPP is similar during both normal breathing and during respiratory challenge with hypercapnia, an unexpected result. This led to a follow-up study examining the role of intercostal (IC) muscles in spontaneous respiratory recovery following cervical hemisection. It is established that IC muscles play an active role in normal breathing and can be recruited during periods of high respiratory demand (e.g. vigorous exercise, hypoxia, hypercapnia). Yet, little is known about how the activity in these muscles changes in response to cervical SCI. Thus, we utilized electromyography (EMG) to analyze variations in IC muscle activity and retrograde anatomical tracers to examine changes in IC neural organization in the weeks to months following cervical hemisection. Our results demonstrated that IC muscles, particularly rostral ICs (i.e. between the first few ribs), displayed substantial spontaneous recovery following cervical hemisection. In addition, this recovery may have been mediated by recruitment of cervical and thoracic interneurons promoting progressive improvements in tidal volume. Our research focus then shifted from plasticity aiding spontaneous respiratory recovery to treatments intended to enhance it. Specifically, Chapter 5 describes a cell-based transplantation method designed to augment functional contributions of the CPP to respiratory recovery following cervical hemisection. Instead of directly altering neural regeneration, reorganization or sprouting in bulbospinal respiratory pathways, we aimed to resupply respiratory motoneurons with serotonin, an important neuromodulator lost following injury. Serotonin has the ability to increase the excitability of motoneurons, making it possible for them to fire action potentials in response to lesser synaptic input. Therefore, we hypothesized that enhancing serotonergic inputs to respiratory motoneurons below a cervical hemisection would facilitate greater activation of the CPP and improved respiratory recovery. Serotonin-expressing cells dissected from embryonic rat brainstems were transplanted intraspinally below a cervical hemisection in rats. After 6 weeks, immunohistochemistry experiments indicated that surviving grafts appeared to enhance serotonin near respiratory motoneurons. This increased serotonergic innervation resulted in augmented ipsilateral phrenic nerve activity and the production of larger tidal volumes under normal and challenged breathing conditions. These results represent a proof-of-principle that long-term serotonin supplementation to the region of respiratory motoneurons may facilitate respiratory recovery following cervical SCI. In conclusion, the data presented in this dissertation provide the following novel findings: 1) rats utilize neural pathways associated with the CPP to spontaneously enhance tidal volume following cervical hemisection. However, utilization appears to plateau after 2 weeks and is not dependent on respiratory drive, 2) progressive increases in tidal volume observed between 2 and 8 weeks post hemisection are likely facilitated by ipsilateral inspiratory IC muscles that spontaneously recover function by 2 weeks post-hemisection, and 3) re-establishment of serotonin innervation to phrenic motoneurons via cell transplantation is associated with enhanced motor output and improved tidal volume following cervical hemisection. Accordingly, chronic serotonin supplementation may represent a promising SCI therapeutic strategy.
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 Brendan J Dougherty.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Fuller, David D.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043635:00001

Permanent Link: http://ufdc.ufl.edu/UFE0043635/00001

Material Information

Title: Respiratory Motor Plasticity and Cervical Spinal Cord Injury
Physical Description: 1 online resource (209 p.)
Language: english
Creator: Dougherty, Brendan J
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: plasticity -- respiration -- sci
Neuroscience (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Traumatic injury to the cervical spinal cord is often accompanied by respiratory dysfunction. This results from direct damage to respiratory-related neurons and interruption of bulbospinal respiratory pathways. Injuries above the fourth cervical segment (C4) in particular may directly impact control of the diaphragm muscle, necessitating mechanical ventilation for survival. Thus, finding ways to enhance recovery of breathing function following spinal cord injury (SCI) is an important clinical priority. This dissertation presents original research exploring spontaneous respiratory recovery in rats following experimental cervical SCI and treatments to augment this recovery. We first investigated the spontaneous "crossed-phrenic phenomenon" (CPP) and its contributions to respiratory recovery in the initial days to several weeks following unilateral cervical SCI (i.e. hemisection). Commonly studied as a model of evoked neuroplasticity in the respiratory system, the role of the CPP in spontaneous recovery from SCI is less understood. In this study we developed a method to quantify the contribution of the spontaneous CPP to tidal volume recovery by cutting the phrenic nerve in spontaneously breathing, anesthetized rats. Our results suggest that the CPP may play an important role in normal breathing function in the weeks following hemisection. However, the relative contribution of the CPP is similar during both normal breathing and during respiratory challenge with hypercapnia, an unexpected result. This led to a follow-up study examining the role of intercostal (IC) muscles in spontaneous respiratory recovery following cervical hemisection. It is established that IC muscles play an active role in normal breathing and can be recruited during periods of high respiratory demand (e.g. vigorous exercise, hypoxia, hypercapnia). Yet, little is known about how the activity in these muscles changes in response to cervical SCI. Thus, we utilized electromyography (EMG) to analyze variations in IC muscle activity and retrograde anatomical tracers to examine changes in IC neural organization in the weeks to months following cervical hemisection. Our results demonstrated that IC muscles, particularly rostral ICs (i.e. between the first few ribs), displayed substantial spontaneous recovery following cervical hemisection. In addition, this recovery may have been mediated by recruitment of cervical and thoracic interneurons promoting progressive improvements in tidal volume. Our research focus then shifted from plasticity aiding spontaneous respiratory recovery to treatments intended to enhance it. Specifically, Chapter 5 describes a cell-based transplantation method designed to augment functional contributions of the CPP to respiratory recovery following cervical hemisection. Instead of directly altering neural regeneration, reorganization or sprouting in bulbospinal respiratory pathways, we aimed to resupply respiratory motoneurons with serotonin, an important neuromodulator lost following injury. Serotonin has the ability to increase the excitability of motoneurons, making it possible for them to fire action potentials in response to lesser synaptic input. Therefore, we hypothesized that enhancing serotonergic inputs to respiratory motoneurons below a cervical hemisection would facilitate greater activation of the CPP and improved respiratory recovery. Serotonin-expressing cells dissected from embryonic rat brainstems were transplanted intraspinally below a cervical hemisection in rats. After 6 weeks, immunohistochemistry experiments indicated that surviving grafts appeared to enhance serotonin near respiratory motoneurons. This increased serotonergic innervation resulted in augmented ipsilateral phrenic nerve activity and the production of larger tidal volumes under normal and challenged breathing conditions. These results represent a proof-of-principle that long-term serotonin supplementation to the region of respiratory motoneurons may facilitate respiratory recovery following cervical SCI. In conclusion, the data presented in this dissertation provide the following novel findings: 1) rats utilize neural pathways associated with the CPP to spontaneously enhance tidal volume following cervical hemisection. However, utilization appears to plateau after 2 weeks and is not dependent on respiratory drive, 2) progressive increases in tidal volume observed between 2 and 8 weeks post hemisection are likely facilitated by ipsilateral inspiratory IC muscles that spontaneously recover function by 2 weeks post-hemisection, and 3) re-establishment of serotonin innervation to phrenic motoneurons via cell transplantation is associated with enhanced motor output and improved tidal volume following cervical hemisection. Accordingly, chronic serotonin supplementation may represent a promising SCI therapeutic strategy.
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 Brendan J Dougherty.
Thesis: Thesis (Ph.D.)--University of Florida, 2011.
Local: Adviser: Fuller, David D.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043635:00001


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1 RESPIRATORY MOTOR PLASTICITY AND CERVICAL SPINAL CORD INJURY By BRENDAN J. DOUGHERTY A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011

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2 2011 Brendan J. Dougherty

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3 To Mackenzie

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4 ACKNOWLEDGMENTS I would like to thank all of the colleagues friends and family who made this journey a memorable one In particular, I want to ex press my deepest gratitude to my mentor Dr. David Fuller for his guidance and friendship during my time as a graduate student and Dr. Paul Reier for his support and encouragement dating back to before my arrival at the University of Flo rida I have been fo rtunate to work with an incredible group of people in the Fulle r and Reier Labs over the years but I want to specifically acknowledge Dr Milap Sandhu, my long time colleague and friend for all of his assistance and advice Also, many thanks go to Dr. Kun Ze Lee for bringing a new level of drive and excitement to the lab and for his helpful suggestions during the preparation of this dissertation In addition, I would like to thank Doperalski for teaching me rodent surgery Dr. Mich ael Lane for his friendship and e xpertise in neuroanatomy and Dr. Heather Ross for her mentorship in all things related to cell culture and stem cell biology. There are countless others who have impacted my life and professional development over the years them for fear of forgettin g someone. Please know that you are all appreciated. Finally I want to thank my parents for letting me find my own way, my wife Mackenzie for being my perfect partner, and my daughter Keagan for keeping me grounded and laughing. I love you all very much.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURES ................................ ................................ ................................ ........ 10 ABSTRACT ................................ ................................ ................................ ................... 12 CHAPTER 1 LITERATURE REVIEW ................................ ................................ .......................... 15 Neural Control of Respiration ................................ ................................ .................. 15 Medullary Respiratory Centers ................................ ................................ ......... 15 Pre Btzinger complex (preBtC) ................................ .............................. 15 B tzinger complex (B tC) ................................ ................................ .......... 16 Dorsal respiratory column (DRC) ................................ ............................... 17 Ventral respiratory column (VRC) ................................ .............................. 17 Retrotrapezoid nucleus/parafacial respiratory group ................................ .. 18 Pontine Respiratory Centers ................................ ................................ ............ 19 Cortical Respiratory Influence ................................ ................................ .......... 19 Spinal Respiratory Rhythm ................................ ................................ ............... 20 Spin al Cord Respiratory System ................................ ................................ ...... 21 Descending respiratory pathways ................................ .............................. 21 Phrenic motor nucleus ................................ ................................ ............... 21 Intercostal motor nucleus ................................ ................................ ........... 22 Propriospinal interneurons ................................ ................................ ......... 23 Respiratory Neuromodulation ................................ ................................ ................. 24 5 HT Receptors ................................ ................................ ................................ 25 5 HT 1 receptors ................................ ................................ .......................... 26 5 HT 2 receptors ................................ ................................ .......................... 26 5 HT 4/7 receptors ................................ ................................ ........................ 27 Raph Nuclei ................................ ................................ ................................ .... 28 Rostral raph nuclei ................................ ................................ ................... 28 Ventral raph nuclei ................................ ................................ ................... 29 Spinal Cord Injury (SCI) ................................ ................................ .......................... 29 Respiratory Consequences of SCI ................................ ................................ ... 31 Models of Cervical SCI ................................ ................................ ..................... 31 Respiratory Related Spinal Neuroplasticity ................................ ............................. 33 The Crossed Phrenic Phenomenon ................................ ................................ 33 CPP induction ................................ ................................ ............................ 34 Spontaneous CPP expression ................................ ................................ ... 34 Mechanisms of CPP expression ................................ ................................ 35

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6 Functional relevance of the CPP ................................ ................................ 36 Plasticity in Respiratory Intercosta ls ................................ ................................ 36 Improving SCI Related Respiratory Dysfunction ................................ ..................... 37 Clinical Modes of Respiratory Therapy ................................ ............................. 37 Inspiratory muscle strength training ................................ ........................... 38 Electrical activation of respiratory muscles ................................ ................ 38 Experime ntal Models for Respiratory Therapy ................................ ................. 39 Intermittent hypoxia ................................ ................................ .................... 39 Cell transplantation ................................ ................................ .................... 40 Serotonin cell transplants ................................ ................................ ........... 43 2 OUTLINE OF EXPERIMENTS ................................ ................................ ................ 48 Aim One ................................ ................................ ................................ .................. 49 Objective ................................ ................................ ................................ .......... 49 Rationale ................................ ................................ ................................ .......... 49 Hypothesis 1 ................................ ................................ ................................ ..... 50 Hypothesis 2 ................................ ................................ ................................ ..... 50 Experimental Design ................................ ................................ ........................ 50 Aim Two ................................ ................................ ................................ .................. 50 Objectiv e ................................ ................................ ................................ .......... 50 Rationale ................................ ................................ ................................ .......... 51 Hypothesis ................................ ................................ ................................ ........ 51 Experimental Design ................................ ................................ ........................ 51 Aim Three ................................ ................................ ................................ ............... 51 Objective ................................ ................................ ................................ .......... 51 Rationale ................................ ................................ ................................ .......... 52 Hypothesis 1 ................................ ................................ ................................ ..... 52 Hypothesis 2 ................................ ................................ ................................ ..... 52 Experimental Design ................................ ................................ ........................ 52 3 THE CONTRIBUTION OF THE SPONTANEOUS CROSSED PHRENIC PHENOMENON TO INSPIRATORY TIDAL VOLUME IN SPONTANEOUSLY BREATHING RATS ................................ ................................ ................................ 54 Materials and Methods ................................ ................................ ............................ 56 Animals ................................ ................................ ................................ ............. 56 Spinal Cord Injury ................................ ................................ ............................. 56 Experimental Preparation ................................ ................................ ................. 57 Experimental Protocols ................................ ................................ ..................... 58 Spinal Cord Histology ................................ ................................ ....................... 59 Data Analysis ................................ ................................ ................................ ... 59 Results ................................ ................................ ................................ .................... 61 Effect of C2HS on Ventilation ................................ ................................ ........... 61 Immediate Impact of Ipsilateral Phren icotomy ................................ .................. 63 Effect of Phrenicotomy on Subsequent Hypercapnic Ventilatory Responses ... 65 Effect of Phrenicotomy on Spontaneous Augme nted Breaths .......................... 65

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7 Effect of Phrenicotomy on Contralateral Respiratory Muscle EMG Activity ...... 66 Discussion ................................ ................................ ................................ .............. 66 Commentary on Methods and Diaphragm Biomechanics ................................ 67 The Contribution of the sCPP to Tidal Volume Following C2HS ...................... 68 Compensation Following Phrenicotomy ................................ ........................... 71 Axotomy and Phrenic Afferent Neurons ................................ ........................... 72 Summary ................................ ................................ ................................ ................ 74 4 SPONTANEOUS RECOVERY OF INSPIRATORY INTERCOSTALS FOLLOWING HIGH CERVICAL HEMISECTION IN RATS ................................ ..... 89 Materials and Methods ................................ ................................ ............................ 91 Animals ................................ ................................ ................................ ............. 91 Spinal Cord Injury ................................ ................................ ............................. 91 Experimental Preparation ................................ ................................ ................. 92 Experimental Protocols ................................ ................................ ..................... 93 Anatomical Tracing Protocols ................................ ................................ ........... 94 Spinal Cord Histology an d Immunohistochemistry ................................ ........... 94 Data Analysis ................................ ................................ ................................ ... 96 Results ................................ ................................ ................................ .................... 97 Effect of C2HS on Ventilation ................................ ................................ ........... 98 Progressive Changes in Rostral Intercostal EMG Activity Following C2HS ..... 99 Impact of Ipsilateral Phrenicot omy on Ventilation ................................ ........... 100 Immediate Impact of Ipsilateral Phrenicotomy on Intercostal EMG Activity .... 101 Neural Circuitry of the Rost ral Inspiratory Intercostals ................................ ... 102 Caudal Intercostal EMG Activity Following Ipsilateral Phrenicotomy .............. 103 Discussion ................................ ................................ ................................ ............ 107 Commentary on Methods and Intercostal Biomechanics ............................... 108 Progressive Recovery of Ipsilateral Rostral Intercostals Following C2HS ...... 110 The Effect of Acute Ipsilateral Phrenicotomy on Intercostal Motor Activity ..... 112 Intercos .......... 113 Commentary on Observed Effects of Phrenicotomy on Caudal Intercostal Activity ................................ ................................ ................................ ......... 114 Summary ................................ ................................ ................................ .............. 11 5 5 TRANSPLANTATION OF EMBRYONIC MEDULLARY RAPH CELLS ENHANCES TIDAL VOLUME AND PHRENIC BURSTING FOLLOWING C2 HEMISECTION IN RATS ................................ ................................ ...................... 135 Materials an d Methods ................................ ................................ .......................... 138 Animals ................................ ................................ ................................ ........... 138 Spinal Cord Injury ................................ ................................ ........................... 138 Cell Suspension s ................................ ................................ ............................ 139 Cell Transplantation ................................ ................................ ....................... 140 Barometric Plethysmography ................................ ................................ ......... 140 Ne urophysiology Preparation ................................ ................................ ......... 141 V T measurement in spontaneously breathing rats ................................ .... 141

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8 Phrenic nerve recording in mechanically ventila ted rats .......................... 143 Spinal Cord Histology and Immunohistochemistry ................................ ......... 144 Data Analysis ................................ ................................ ................................ 145 Results ................................ ................................ ................................ .................. 147 Anatomical Characterization of Cell Transplants ................................ ............ 147 Effects of Cell Transplantation on Ventilation (Unanesthetized Rats) ............ 148 Effects of Cell Transplantation on Tidal Volume (Anesthetized Rats) ............ 149 Effects of Cell Transplantati on on Phrenic Motor Output ................................ 150 The Effects of Ketanserin on Phrenic Activity Following Cell Transplantation 151 Discussion ................................ ................................ ................................ ............ 152 RN Transplants Enhance 5 HT Innervation to Target Spinal Regions ........... 152 Enhanced Inspiratory Tidal Volume Following RN Transplantati on ................ 155 RN Transplants Augment Ipsilateral Phrenic Nerve Activity ........................... 155 Effect of Acute Phrenicotomy on V T Following RN Transplanta tion ................ 158 Summary ................................ ................................ ................................ .............. 160 6 CONCLUSIONS AND FUTURE DIRECTIONS ................................ .................... 179 The Contribution of the Spontaneous Crossed Phrenic Phenomenon to Inspiratory Tidal Volume in Spontaneously Breathing Rats ............................... 179 Spontaneous Recovery of Inspiratory Intercostals Following High Cerv ical Hemisection in Rats ................................ ................................ ........................... 180 Transplantation of Embryonic Medullary Raph Cells Enhances Tidal Volume and Phrenic Bursting Following C2 Hemisection in Rats ................................ ... 182 LIST OF REFERENCES ................................ ................................ ............................. 185 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 209

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9 LIST OF TABLES Table page 1 1 Overview of 5 HT receptor families. ................................ ................................ ... 47 3 1 Age and weight values for all experimental groups.. ................................ .......... 85 3 2 B lood gas and mean arterial blood pressure (MAP) values taken during baseline ventilation. ................................ ................................ ............................ 85 3 3 Respiratory parameters during pre phrenicotomy baseline (BL) and hypercapnic respiratory challe nge (CO 2 ). ................................ ........................... 86 3 4 Respiratory parameters pre and post ipsilateral phrenicotomy (PhrX) during baseline ventilation. ................................ ................................ ............................ 87 3 5 Respi ratory parameters pre and post ipsilateral phrenicotomy (PhrX) during hypercapnic respiratory challenge. ................................ ................................ ..... 88 4 1 Age and weight values for spinal intact control rats and rats 1 3 days, 2 wks, and 8 wks post C2HS.. ................................ ................................ ..................... 132 4 2 Blood gas and mean arterial blood pressure (MAP) values taken during baseline ventilation. ................................ ................................ .......................... 132 4 3 Respiratory parameters during pre phrenicotomy (phrX) baseline (BL) and hypercapnic respiratory challenge (CO 2 ). ................................ ......................... 133 4 4 Respiratory parameters during hypercapnic respiratory challenge. .................. 134 5 1 Age and weight values for rats receiving delayed (7 days) cell transplants ...... 177 5 2 Blood gas and mean arterial blood pressure (MAP) values taken during baseline ventilation in spontaneously breathing, anesthetized rats.. ................ 177 5 3 Blood gas and mean arterial blood pressure (MAP) values taken during baseline ventilatio n in anesthetized, mechanically ventilated rats. ................... 178

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10 LIST OF FIGURES Figure page 1 1 Schematic illustration of the neural control of breathing. ................................ .... 44 1 2 Neuromodulatory inputs to the brainstem respiratory network. .......................... 45 1 3 A representative histological section depicting a C2HS l esion. .......................... 46 3 1 A representative histological section illustrating a C2HS lesion. ......................... 76 3 2 Representative examples of V T during spon taneous breathing at baseline and hypercapnic respiratory challenge ................................ ............................... 77 3 3 Representative airflow traces showing the impact of phrenicotomy on V T ......... 78 3 4 The effect of phrenicotomy on V T during baseline breathing and hypercapnic respiratory challenge. ................................ ................................ ......................... 79 3 5 The immediate (next breath) decline in V T following phrenicot omy.. .................. 80 3 6 The impact of phrenicotomy on the hypercapnic ventilatory response.. ............. 81 3 7 The impact of phrenicotomy on the volum e of spontaneous augmented breaths (AB).. ................................ ................................ ................................ ..... 82 3 8 Example of contralateral intercostal EMG (first intercostal space) recorded at 8wks post C2HS.. ................................ ................................ ............................... 83 3 9 Examples of contralateral diaphragm EMG (4 days [A] and 2 weeks [B]) following C2HS. ................................ ................................ ................................ .. 84 4 1 A representative histological section illustrating a C2HS lesion. ....................... 117 4 2 Representative examples of rostral inspiratory IC EMG during poikilocapnic baseline and hypercapnic respiratory challenge.. ................................ ............. 118 4 3 The effect of C2HS on rostral IC EMG amplitude (arbitrary units, a.u.) during baseline breathing and hypercapnic respiratory challenge .............................. 119 4 4 The relative activation of rostral ipsilateral (IL) versus contralateral (CL) inspiratory intercostals following C2HS. ................................ ........................... 120 4 5 The effect of phrenicotomy on rostral IC EMG. ................................ ................. 121 4 6 Relative change in IC EMG amplitude following phrenicotomy.. ....................... 122 4 7 Retrograde anatomical tracing of inspiratory intercostals. ................................ 123

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11 4 8 Example of caudal IC EMG decline with acute phrenicotomy.. ........................ 124 4 9 Example of IC EMG activity following contralateral phrenicotomy. ................... 125 4 10 The effects of lidocaine application to the ipsilateral phrenic nerve on IC EMG activity.. ................................ ................................ ................................ ............ 126 4 11 Example of caudal intercostal EMG responses to phrenic nerve sti mulation. .. 127 4 12 Example of caudal IC EMG response to phrenic stimulation in follow up studies.. ................................ ................................ ................................ ............ 128 4 13 Effects of capsaicin on caudal intercostal EMG activity. ................................ ... 129 4 14 Effects of ipsilateral dorsal rhizotomy on phrenicotomy induced intercostal EMG activity. ................................ ................................ ................................ .... 130 4 15 Evoked potentials measured in the caudal ipsilateral intercostal EMG signal.. 131 5 1 Dissection of the embryonic raph nucleus. ................................ ..................... 162 5 2 A representative histological section depicting a C2HS lesion.. ....................... 163 5 3 Representative sections illustrating cell transplants at C3. ............................... 164 5 4 Representative sections illustrating 5 HT immunodetection at C4. ................... 165 5 5 Effects of cell transplantation on ventilation in unanesthetized rats. ................. 166 5 6 Effect of acute phrenicotomy on tidal volume in anesthetized rats. .................. 167 5 7 Examples of phrenic motor output recorded in anestheti zed rats.. ................... 168 5 8 The effect of hypoxia on bilateral phrenic motor output. ................................ ... 169 5 9 The effect of ketanserin on phrenic output. ................................ ...................... 170 5 10 The effect of ketanserin on phrenic burst amplitudes. ................................ ...... 171 5 11 Example of Immunohistochemical staining for 5 HT (Rat F1 1) ......................... 172 5 12 Example of Immunohistochemical staining for 5 HT (Rat E) ............................ 173 5 13 Example of Immunohistochemical staining for 5 HT ( Rat G) ............................ 174 5 14 Example of Immunohistochemical staining for 5 HT (Rat 6.3) .......................... 175 5 15 Example of NT2.19 cells.. ................................ ................................ ................. 176

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12 Abstract of Dissertation Presented to the Graduate School of the University of Florida in in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy RESPIRATORY MOTOR PLASTICITY AND CERVICAL SPIN AL CORD INJURY By Brendan J. Dougherty December 2011 Chair: David D. Fuller Major: Medical Sciences -Neuroscience Traumatic injury to the cervical spinal cord is often accompanied by respiratory dysfunction. This results from direct damage to respiratory related neurons and interruption of bulbospinal respiratory pathways Injuries above the fourth cervical segment ( C4 ) in particular may directly impact control of the diaphragm muscle, necessitating mechanical ventilation for survival. Thus, finding ways to enhance recove ry of breathing function following spinal cord injury (SCI) is an important clinical priority. This dissertation presents original research exploring spontaneous respiratory recovery in rats following experimental cervical SCI and treatments to augment thi s recovery. We first investigate d its contributions to respiratory recovery in the initial days to several weeks f ollowing unilateral cervical SCI (i.e. hemisection) Commonly studied as a model of evo ked neuroplasticity in the respiratory system, the role of the CPP in spontaneous recovery from SCI is less understood. In this study we developed a method to quantify the contrib ution of the spontaneous CPP to tidal volume recovery by cutting the phrenic nerve in spontaneously breathing, anesthetized rats Our results suggest that the CPP

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13 may play a n important role in normal breathing function in the weeks following hemisection However the relative contribution of the CPP is similar during both normal br eathing and during respiratory challenge with hypercapnia an unexpected result. This led to a follow up study examining the role of intercostal (IC) muscles in spontaneous respiratory recovery following cervical hemisection It is established that IC mu scles play an active role in normal breathing and can be recruited during periods of high respiratory demand (e.g. vigorous exercise, hypoxia and hypercapnia ). Yet, little is known about how the activity in these muscles changes in response to cervical SCI Thus, we utilized electromyography (EMG) to analyze variations in IC muscle activity and retrograde anatomical tracers to examine changes in IC neural organization in the weeks to months following cervical hemisection Our results demonstrated that IC mu scle s, particularly rostral ICs (i.e. between the first few ribs ) displayed substantial spontaneous recovery following cervical hemisection In addition, t his recovery may have been mediated by recruitment of cerv ical and thoracic interneurons promoting p rogressive improvements in tidal volume Our research focus then shifted from plasticity aiding spontaneous respiratory recovery to treatment s intended to enhance it Specifically, C hapter 5 describes a cell based transplantation method designed to augme nt functional contribution s of the CPP to respiratory recovery following cervical hemisection Instead of directly altering neural regeneration, reorganization or sprouting in bulbospinal respiratory pathways, we aim ed to resupply respiratory motoneurons w ith serotonin, an important neuromodulator lost following injury Serotonin has the ability to increase the excitability of motoneurons making it possible for them to fire action potentials in response to lesser synaptic input.

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14 Therefore, w e hypothesized that enhancing serotonergic inputs to respiratory motoneurons below a cervical hemisection would facilitate greater activation of the CPP and improved respiratory recovery. Serotonin expressing cells dissected from embryonic rat brainstems were transplante d intraspinally below a cervical hemisection in rats. After 6 weeks, immunohistochemistry experiments indicated that surviving grafts appeared to enhance serotonin near respiratory motoneurons This increased serotonergic innervation resulted in augmented ipsilateral phrenic nerve activity and the production of larger tidal volumes under normal and challenged breathing conditions. These results represent a proof of principle that long term serotonin supplementation to the region of respiratory motoneurons m ay facilitate respiratory recovery following cervical SCI. In conclusion the data presented in this dissertation provide the following novel findings: 1) rats utilize neural pathways associated with the CPP to spontaneously enhance tidal volume following cervical hemisection. However, utilization appears to plateau after 2 weeks and is not dependent on respiratory drive, 2) p rogressive increases in tid al volume observed between 2 and 8 weeks post hemisection are likely facilitated by ipsilateral inspirator y IC muscles that spontaneously recover function by 2 weeks post hemisection and 3) re establishment of serotonin innervation to phrenic motoneurons via cell transplantation is associated with enhanced motor output and improved tidal volume following cerv ical hemi section. Accordingly chronic serotonin supplementation may represent a promising SCI therapeutic strategy.

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15 CHAPTER 1 LITERATURE REVIEW Neural Control of Respiration For mammals, breathing originates in the brainstem (Porter, 1895; Rekling & Feldman, 1998; Feldman et al. 2003) The complex network dynamics associated with initiation and maintenance of this automatic behavi or are yet to be fully realized. H owever, our basic understanding of brainstem resp iratory centers has become less ambiguous (Alheid & McCrimmon, 2008) in the medulla and pons transmit rhythmic neural signals to cranial and spinal motor nuclei controlling respiratory mus cles. In turn, afferent modulation of respiratory drive is conveyed back to the brainstem, allowing adaptation of the respiratory rhythm to internal and external stimuli. A schematic illustration of the respiratory neural control system is shown in Figure 1 1 Medullary Respiratory C enters The medulla is often considered the most essential part of the brain. It contains the neural areas controlling heart rate and respiratory function and acts as an integration and relay station between the brain and spin al cord. In the context of neural control of breathing, t he mammalian medulla contains discrete collections of neurons important for both generation of respiratory rhythm (e.g. Pre B tzinger complex ) and transmission of pre motor respiratory signals to cra nial and spinal motor nuclei (e.g. dorsal an d ventral respiratory columns). Pre Btzinger complex (preBtC) The preBtC is a distinct grouping of neurons located ventral to the nucleus ambiguus, midway between the facial nucleus and the obex, caudal to t he Btzinger

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16 com plex and rostral to the rostral ventral respiratory column (rVRC) (Rekling & Feldman, 1998) Functionally, preBtC neurons spontaneously burst in phase with respiration ( Feldman 1986) and are necessary for generation of respiratory related neural activity in in vitro slice pre parations (Smith et al. 1991) In addi tion, disruption of the preBtC in vivo severely alters breathing in adult mammals (Ramirez et al. 1998; Solomon et al. 1999; Wenninger et al. 2004; Tan et al. 2008) Therefore, the preBtC has been postulated to contain the pacemaker neurons responsible for respiratory rhythmogenesis (Smith et al. 1991; Rekling & Feldman, 1998) PreBtC neurons are uniquely propriobulbar (Guyenet et al. 2002; Feldman et al. 2003) with connections to both cranial nerve nuclei (Koizumi et al. 2008) and pre motor nuclei that innervate spinal respiratory centers (Merrill & Fedorko, 1984) B tzinger c omplex (B tC) Located just rostral to the preB tC, the B tC contains neurons primarily involved with the ex piratory phase of breathing (Ezure, 1990; Jiang & Lipski, 1990) Although it is unknown whether B tC neurons represent a true expiratory rhythm generator, extensive bulbospinal connectivity underscores their vital r ole in orchestration of regular respiratory patterning (Smith et al. 2009) Specifically, B tC neurons active during expiration provide widespread inhibitory influence to inspiratory pre motor neurons throughout the ventral respiratory column (see below) and to brainstem respiratory motoneurons (Jiang & Lipski, 1990) In addition, B tC projections have been shown as far caudle as phrenic motoneurons (PMNs) (Tian et al. 1998) T here t hey are believed to assist with active inhibition of inspiratory motoneurons during expiration (Alheid & McCrimmon, 2008)

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17 Dorsal respiratory c olumn (DRC) Concentrated in the ventrolateral Nucleus of the Solitary Tract (NTS), neurons comprisi ng the DRC fire action potentials chiefly in phase with inspiration (Alheid & McCrimmon, 2008) Though closely approximating neurons within the NTS that receive afferent feedback from the lungs and peripheral chemoreceptors, the phase locked r espiratory bursting in DRC neurons appears to be independent of afferent input (Alheid & McCrimmon, 2008) Projections of DRC neurons reach multiple regions within the medulla including the medullary reticular formation, medial portions of the NTS, ventral respiratory column and hypoglossal nucleus (Berger et al. 1984; Otake et al. 1989) Spinal projections from DRC neurons contact PMNs in the contralateral cervical cord (Berger et al. 1984; Otake et al. 1989) as well as interneurons in the rostral cervical segments (C1 C2) (Lipski & Duffin, 1986; Lane et al. 2008b) The exact function of DRC neurons is unknown, though the diverse medullary and spinal connectivity may suggest a role in coordination of inspiratory bursting among medullary respiratory centers and spinal motor neurons (Otake et al. 1989; Alheid & McCrimmon, 2008) V entral respiratory c olumn (VRC) Spanning the entire ventrolateral medulla, the VRC contains pre motor neurons active during both inspiratory and expiratory phases of respiration. As such, the VRC is separated into functional groups based on neuronal firing characteristics (Ezure et al. 1988; Sun et al. 1998) Generally, the rostral portion of the VRC (rVRC) contains neurons involved with shaping the inspiratory phase of respiration (Bianchi, 1974) while caudle VRC (cVRC) neurons shape the expiratory phase (Ezure et al. 1988; Iscoe, 1998) Receiv ing afferent connections from the preB tC, subsets of rVRC neurons monosynaptically innervate phrenic and intercostal motoneurons in the spinal cord to

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18 facilitate inspiratory activation of the diaphragm and inspiratory intercostals (Merrill & Fedorko, 1984; Dobbins & Feldman, 1994; Iscoe, 1998) In addition, propriobulbar connections to pharyngeal and laryngeal motoneurons in the medulla activate upper airway muscles during inspiration (Dobbins & Feldman, 1994) Propriobulbar inputs related to expiration, in particular from the BtC, converge in the cVRC (Smith e t al. 2009) The integration of these signals forms the functional counterpart to the inspiratory rVRC. Excitatory bulbospinal connections from the cVRC activate expiratory pump muscles via connections to expiratory intercostal and abdominal motoneurons and actively inhibit PMNs during expiration (Ezure et al. 1988; Iscoe, 1998) Within the brainstem, cVRC projections are primarily inhibitory to inspirator y related VRC neurons though excitatory terminations on l aryngeal motoneurons have been demonstrated (Boers et al. 2002) Retrotrapezoid nucleus/parafacial respiratory group An additional collection of respiratory related neurons, the retrotrapezoid nucleus (RTN) is fou nd just below the facial nucleus overlapping a portion of the BtC (Alheid & McCrimmon, 2008) RTN neurons are sensitive to changes in arterial CO 2 and receive afferent projections from peripheral O 2 chemoreceptors via the NTS (Smith et al. 2009) Thus the RTN likely functions as an important medullary chemosensitive region (Mulkey et al. 2004; Guyenet et al. 2005) Excitatory connections from the RTN project to neurons throughout the VRC and modulate their activity based on metabolic status (Nattie, 1999; Mulkey et al. 2004; Guyenet et al. 2005) In neo natal in vitro preparations, neurons analogous to the RTN identified as the parafacial respiratory group exhibit rhythmic pre inspiratory burst characteristics (Onimaru et al. 1988; Onimaru & Homma, 2003; Onimaru et al. 2006) and were thus hypothesized to be

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19 drivers of the preB tC (Onimaru et al. 2006) However, difficulty in locating neurons with similar firing characteristics in the adult, in vivo prep made this hypothesis controversial (Fortuna et al. 2008) An alternative theory suggests that neurons within the parafacial respiratory group represent an expiratory rhythm generator functionally opposed to the preB tC inspi ratory rhythm generator (Feldman & Del Negro, 2006) Pontine R espiratory C enters The VRC appears to extend rostrally into the dorsolateral pons as a continuous neuronal grouping (Alheid et al. 2004) Several respiratory related nuclei are present along this corridor (Alheid & McCrimmon, 2008) but their functional role in rhythm generation or modulation remains largely unknown. Retrograde tracing stud ies have identified efferent synaptic projections from the pontine K lliker Fuse (KF) nucleus and parabrachial complex to the VRC (Smith et al. 1989; Dobbins & Feldman, 1994; Alheid et al. 2002; Alheid et al. 2004 ) In addition, descending projections to cranial (facial and hypoglossal) and spinal motor nuclei originate in the KF nucleus (Ezure & Tanaka, 2006; Yokota et al. 2007) Most neurons in these regions have tonic f iring characteristics that are modulated by the respiratory cycle (Jiang et al. 2004; Song et al. 2006) suggesting a role in respiratory phase transitions (Cohen, 1979; St John, 1998; Alheid et al. 2004) Cortical Respiratory I nfluence Humans can consciously alter their pattern of breathing to complete complex motor tasks like speaking, eating and holding breath under water. Projections from the cerebral cortex to brainst em (Bassal et al. 1981; Bassal & Bianchi, 1982) and spinal neurons (Rikard Bell et al. 1985a) l ikely facilitate these highly coordinated behaviors. In fact, cortical projections to intercostal motoneurons (Rikard B ell et al. 1985b) and

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20 phrenic motoneurons (Shea, 1996) have been described, suggesting direct cortical activation is po ssible. However, the location (brainstem vs. spinal cord) where signals are integrated for completion of behavioral tasks remains unknown (Butler, 2007) Spinal Resp iratory Rhythm A group of propriospinal, respiratory neurons in the upper cervical (C1/C2) cord of cats (Aoki et al. 1980) and rodents (Lipski & Duffin, 1986; Lane et al. 2008b) receive respiratory input from medullary respiratory centers and project excitatory inputs onto ipsilateral phrenic motoneurons (Tian & Duffin, 1996) These neurons, termed u pper c ervical i nspiratory n eurons (UCINs, (Kobayashi et al. 2010) ) have been hypothesized to contribute to spinal respiratory rhythm generation (Kobayashi et al. 2010) Indeed, neurophysiological evidence for spontaneous, spinal respiratory rhythm associated with UCINs has been shown in isolated slice preparations in neonatal mice (Kobayashi et al. 2010) From these findings, it has been proposed that a respiratory neuronal circuit, consisting of putative UCINs, under certain conditions, has the po tential for spontaneously generating a respiratory rhythm ( Feldman 1986; Kobayashi et al. 2010) Though compelling as a working hypothesis, further studies will be necessary to support spinal respiratory rhythm ge neration in adult animals or humans. Documented reports of s pontaneous, spinal respiratory output in spinalized animals, are sparse and come with significant methodological concerns ( Feldman 1986) For example, Coglianese et al. (1977) showed spontaneous, r hythmic phrenic activity following C1/C2 transection in dogs following administration of Doxapram HCL (Coglianese et al. 1977) However, i n this study, some respiratory movements would have continued following injury (e.g. from the sternocleidomastoid innervated by cranial nerves) since the dogs were not paralyzed (Coglianese et al. 1977) As a result residual respiratory

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21 movements could have periodically deformed the thorax and abdo minal cavity, activating afferent pathways and providing input to the spinal cord. Such inputs may activate phrenic or I intercostal motoneurons, accounting for the observed phrenic activity ( Feldman 1986) Therefore, the significance of observed spinal resp iratory rhythms under non paralyzed conditions must be interpreted with caution. It appears more likely, that the combined anatomical and physiological characteristics of UCINs point to a function in non respiratory motor behaviors like vomiting (Nonaka & Miller, 1991) Other potential roles for these neurons are discussed below. Spinal Cord Respiratory S ystem Descending respiratory pathways Respiratory drive is transmitted from cortical (voluntary) and ponto medullary (involuntary) centers to spinal motoneurons for activation of respiratory musculature. Direc t projections to phrenic and intercostal nuclei from the cerebral cortex appear to course along corticospinal and corticorubral tracts located in the dorsolateral white matter in humans and dorsomedial column in rats (Aminoff & Sears, 1971) Bulbospinal axons carrying rhythmic respiratory signals cross at the level of the brainstem and project to motoneurons in the contrala teral ventral grey matter through the dorsolateral and ventromedial white matter (Davis & Plum, 1972; Lipski et al. 1994; Fuller et al. 2009) Phrenic motor nucleus Containing motor neurons innervating the diaphr agm, the phrenic nucleus forms a continuous column of cells within the ventral gray matter of cervical levels 3 5 (C3 C5) in humans and cervical levels 3 6 (C3 C6) in rats (Webber, 1979; Kuzuhara & Chou, 1980; Lane e t al. 2008b) and mice (Qiu et al. 2010) These motor neurons receive

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22 mono and poly synaptic respiratory input from brainstem respiratory centers including the VRC, DRC and pontine respiratory nuclei (Berger, 1979) The combined axonal projections from phrenic motoneurons comprise the bilateral phrenic nerves supplying the diaphragm. Excitation of phrenic motoneurons leads to diaphrag m contraction and the initiation of the inspiratory phase of breathing. Active inhibition of phrenic motoneurons allows for cessation of inspiration, diaphragm relaxation and an expiratory breath (Richter, 1982) Add itional inputs onto phrenic motoneurons include neuromodulatory inputs and segmental reflexes. Phrenic motoneurons receive neuromodulatory input from brainstem neuromodulatory centers a llow ing for adaptation of their membrane properties and flexibility in output responses (Dahlstrom et al. 1965; Rajaofetra et al. 1989) Examples of neuromodulatory inputs include dopamine, norepinepherine (NE) and serotonin (5 hydroxytryptamine, 5 HT). The phrenic nerves also carry afferent information from the diaphragm back to the phrenic circuitry. These afferents come in the form of small diameter c fibers from pain and chemical receptors (Jammes et al. 1986) and larger my e linated or unmy e linat ed axons from stretch receptors and golgi tendon organs (Road, 1990) Activation of these afferent pathways affects phrenic motoneuron activity, though the extent o f which remains unknown. Suggestion of ipsilateral and contralateral inhibition of phrenic motor output from phrenic afferent activity has been reported (Goshgarian, 1981; Speck & Revelette, 1987a; Sandhu et al. 200 9b) Intercostal motor nucleus The moto neurons controlling both inspiratory intercostal muscles and expiratory intercostal muscles reside in the lateral ventral horn of the thoracic spinal cord (T1 11)

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23 (Monteau & Hilaire, 1991) They receive descending respiratory drive similar to phrenic motoneurons with activation of external intercostals for inspiratory activity and internal intercostals for initiation of expiratory activities. The intercostals als o serve dual purposes in postural control and receive tonic motor drive from motor centers associated with postural control and integration of movements (Hudson et al. 2010) Further, there appears to be preferential activation of certain segments of the intercostal muscles during respiratory activity. Namely, a rostral to caudle and dorsal to ventral activation gradient is described in dogs (De Troyer et al. 2005) and humans (Butler & Gandevia, 2008) hinting at the relative impo rtance of these regions in respiratory control. Propriospinal interneurons Throughout the cervical and thoracic spinal cord are propriospinal interneurons (INs) in the dorsal and ventral horns and surrounding the central canal (Dobbins & Feldman, 1994; Lane et al. 2008b) These cells receive direct respiratory projections from brainstem centers like the VRG and DRG and pro ject to phrenic and intercostal motoneurons (Lane et al 2008b) Though it is compelling to speculate on their relative importance to coordinated respiratory activity, only recently have their appearance and functional relevance been probed. A population of these INs reside in the highest cervical segments ( C1 2) and have been shown to connect with respiratory neurons in the brainstem (Lipski & Duffin, 1986) Their location apart from primary spinal motor centers suggests an integrative function, but the potential dua l capacity for these INs to facilitate a bulbospinal synaptic relay around cervical SCIs has also been considere d (Lane et al. 2008b; Lane et al. 2009) With connections to the phrenic motor nucleus, additional cervical INs can be found in the area surrounding the central canal, and in

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24 both dorsal and ventral horns. Retrogr ade, transynaptic tracing techniques and neurophysiological cross correlation studies suggest these cervical INs may subserve bulbospinal relays around cervical SCI lesions (Lane et al. 2009; Sandhu et al. 2009a) For example, Lane et al. (2008) used both mono and polysynaptic retrograde tracers (Cholera toxin B and pseudorabies Virus [PRV] respectively) in combination with an anterograde tracer (mini ruby) injected into the VRC to show a substantial population of cervical pre phrenic INs in the normal and injured (C2HS) cord ideally located in the gray matter (Laminae VII and X) to transmit bulbospinal inputs to phrenic motoneurons ipsilateral to a C2HS (Lane et al. 2008b) Thus, these cervical INs may constitute a reasonable therapeutic target to augment respiratory outcomes following cervical SCI. Respiratory Neuromodulation Activity of respiratory neurons in the brainstem and spinal cord can be altered by a wide range o f chemical neuromodulators. A neuromodulator is most commonly defined as a substance that alters the response of a target neuron to the traditional neurotransmitters without directly leading to depolarization or hyperpolarization (Hodges & Richerson, 2008) For example, many receptors to neuromodulators are G protein coupled receptors As such, their activation may initiate intra cellular cascades leading to increased expression of AMPA receptor s or phosphorylation of existing glutamatergic synapses. Both would lead to enhanced glutamate sensitivity. Figure 1 2 illustrates the extensive neuromodulatory influences acting on the brainstem respiratory network. Perhaps the most studied of the neurom odulators in respiratory control is 5 HT This primitive bioamine projects from a small cluster of brainstem neurons to the entire neuroaxis, influencing behavioral activity across all species of mammals (Hodges & Richerson, 2008) via classical synaptic mechanisms and non synaptic paracrine

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25 mechanisms ( i.e en passant or volume transmission) (Liposits et al. 1987; DeFelipe & Jones, 1988; McCrimmon, 1995) The relatively constant output of 5 HT neurons within the brainstem during wakefulness has been suggested to contribute to a tonic drive to breath, while reductions in 5 HT neuronal output during sleep results in normal decreases in ventilation (Hodges & Richerson, 2008) This would suggest an overall excitatory influence of 5 HT on respiratory control. However, the neuromodulatory effect of 5 HT on target neurons can be excitatory or inhibit ory depending on the specific receptors expressed. 5 HT R eceptors As a result of SCI, 5 HT innervation to spinal motoneuron pools may be disrupted, altering motoneuron membrane properties (Hochman S et al. 2001) and changing 5 HT receptor expression (Fuller et al. 2005) In Chapter 5, we explore the hypothesis that long term res toration of 5 HT to phrenic motoneurons below a C2HS using cell transplants of embryonic raph (see below) cells would lead to improved respiratory motor output and ventilation. We consider the effects of these transplants in terms of 5 HT receptor express ion and activation on phrenic motoneurons. As such, an understanding of 5 HT receptors and their function in the spinal cord is prerequisite to interpretation of these results. Here we provide a detailed overview of 5 HT receptors with emphasis on their ro les in respiratory control. There are seven major 5 HT receptor families (5 HT 1 7 ) and at least 14 distinct receptor subtypes encompassing splice variants and post translational modification s (Hochman S et al. 2001; Andrade et al. 2011) 5 HT receptors can be expressed both pre and post synaptically and in extra synaptic regions (Hochman S et al. 2001; Hodges & Richerson, 2008) Table 1 1 provides an overview of all 5 HT re ceptor

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26 families. Though not all receptor subtypes have been described in relation to breathing, the 5 HT 1, 2, 4 and 7 receptors appear particularly important for respiratory control. 5 HT 1 receptors The 5 HT 1 receptor family contains five unique receptors (5 HT 1A,B,D,E and F ) sharing common structure and function (A ndrade et al. 2011) G protein coupled 5 HT 1 receptors activate G i /G o proteins inhibiting cAMP formation, generally leading to inhibition of neuronal firing (Bayliss et al. 1995; Bayliss et al. 1997) Locate d throughout the neuraxis, including the NTS and hypoglossal nuclei (Okabe et al. 1997) it is hypothesized that the highest concentration of 5 HT 1 receptors appear on the dendrites and soma of 5 HT neurons themselves, making the 5 HT 1 a natural autoreceptor (Sotelo et al. 1 990) In the spinal cord, 5 HT 1 receptors are most heavily concentrated in the dorsal horn where they play a n important role in first order processing of sensory and reflex information (Hochman S et al. 2001) 5 HT 2 receptors Three 5 HT 2 receptors subtypes (5 HT 2A,B and C ) couple to Gq/G11 proteins to generate inositol trisphosphate (IP3) and activate protein kinase C (Conn & Sanders Bush, 1986; Sanders Bush & Conn, 1986) This generally results in increased neuronal excitability through multiple mechanisms including phosporylation of existing glutamatergic synapses (Fuller et al. 2000; Bocchiaro & Feldman, 2004; McGuire et al. 2008) and enhancement of persistent sodium currents (Pena & Ramirez, 2002; Heckman et al. 2008) 5 HT 2 receptors are found throughout the brain stem respiratory network where they play an important role in respiratory rhythm generation (Pena & Ramirez, 2002; Gunther et al. 2006) and are highly expressed in the ventral motor regions of the spinal cord (Fuller et al. 2005) where they are involved with regulation of

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27 phrenic motor neuron excitability. 5 HT 2 receptors are additionally important for spinal motor plasticity following stimulation or inj ury. For example, activation of the 5 HT 2A receptor on phrenic motoneurons following acute, intermittent hypoxia results in a cascade of intracellular events leading to production and release of brain derived neurotrophic factor (BDNF), activation of Trk B receptors, and phosphorylation of existing glutamatergic synapses (Baker Herman et al. 2004; Baker Herman et al. 2010; Dale Nagle et al. 2010; Hoffman & Mitchell, 2011) The end result is the prolonged enhanceme term (Mitchell et al. 2001; Mahamed & Mitchell, 2007) In addition, recent reports suggest that loss of serotonergic input to lumbar motoneurons followin g thoracic SCI results in conformational changes to 5 HT 2 C receptors leading to their constitutive activation (Murray et al. 2010) The on going spontaneous activation of these receptors represents a form of neurop lasticity that restor es excitability to lumbar motoneurons This excitability, however, may underlie progressive muscle spasms experienced by humans following SCI (Murray et al. 2010) Similar conformational change s in 5 HT 2 receptors on phrenic or intercostal motoneurons have not been observed, but may be important components of spontaneous respiratory recovery following cervical SCI. 5 HT 4/7 receptors Information regarding 5 HT 4 and 7 receptors related to central respiratory control is just beginning to emerge (Richter, 1982; Hodges & Richerson, 2008; Manzke et al. 2008) In general, these receptor families act through Gs proteins to increase cAMP production (Andrade et al. 2011) causing an increase in neuronal excitability. Both 5 HT 4 and 7 receptors are located in the preB tC where they may play a role in the formation of the respiratory network (Manzke et al. 2008) In the spinal cord, 5 HT 7

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28 receptors are expressed on spinal motor neuron s (Doly et al. 2005) and have been implicated in phrenic motor plasticity (McGuire et al. 2004; Zhang et al. 2004) In fact, agonists to the 5 HT 7 receptor cause long lasting facilitation of phrenic motor output (Hoffman & Mitchell, 201 1) Raph Nuclei The origins of most 5 HT projections arise from the raph nuclei in the midline of the medulla, pons and midbrain. First described by Dahlstrom and Fuxe (Dahlstrom & Fuxe, 1964; Dahlstrom et al. 1 965) the raph nuclei can be subdivided into two major subsections: the rostral nuclei residing in the midbrain and rostral pons projecting to the forebrain and the caudle nuclei with major projections to the spinal cord (Tork, 1990) Bu lbar projections arise from both of these regions, though serotonergic innervation of brainstem respiratory centers chiefly arise from the medullary raph nuclei. Rostral raph nuclei The rostral portion s of the serotonergic raph nuclei reside in the mid bra in and rostral pons and include : the caudle linear nucleus, dorsal raph nucleus, median raph nucleus, and nucleus raph pontis (Tork, 1990) In general, projections from these areas travel rostrally to supply 5 HT to structures of the forebrain (Parnavelas & Papadopoulos, 1989) The dorsal raph nucleus is the largest and most well defined of the rostral raph nuclei. A large portion of dorsal raph 5 HT projections target the amygdala where they contribute to regulation of emotional states and memory formation (Ma et al. 1991) As a result, the dorsal raph nucleus is likely the primary location of effect for selective serotonin reuptake inhibitors (SSRIs) used as anti depressants (Briley & Moret, 1993) Additional areas of receiving 5 HT projections from the r ostral

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29 raph nuclei include the olfactory bulb, basal ganglia and other limbic structures (Steinbusch, 1981) Ventral raph nuclei The ventral raph nuclei includes: raph pallidus, raph obscures, raph magnus rostral ventrolateral medulla and the lateral paragigantocellularis reticularis (Hochman S et al. 2001) These nuclei extend from the pontine tegmentum to the spinomedullary border (Tork, 1990) Significa nt for respiratory control, the medullary portions of the ventral raph project 5 HT to all areas of the central respiratory network. In addition, nearly all 5 HT found in the spinal cord descends from the ventral raph nuclei (Steinbusch, 1981; Skagerberg & Bjorklund, 1985; Jacobs & Azmitia, 1992) T rk (1990) describes the 5 work of fibers certain spinal regions receive a higher density of 5 HT projections including lamina I of the dorsal horn, the gray matter adjacent to the central canal, the ventral horn motor nuclei, and the intermediolateral cell column of the thoracic cord (Bowker et al. 1982) Also, particularly strong 5 HT innervation is present around phrenic motoneurons (Holtman et al. 1984; Lipski et al. 1994) arising mainly from raph pallidus and obscurus (Bowker et al. 1982) Spinal Cord I njury (SCI) Traumatic injury to the sp inal cord may result in catastrophic functional loss, the extent of which is dependent upon injury location and severity. Disruption of ascending and descending white matter tracts disconnects motor neurons from brainstem and cortical centers. This, c ombin ed with injury induced loss of motor neurons, results in loss of function caudal to the injury site. As such, SCI in more rostral spinal segments corresponds with more substantial functional impairment.

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30 The functional deficits associated with SCI result b oth from the primary insult and progressive secondary damage. Vascular hemorrhage, cell death, demylination, and edema may continue for days or weeks post injury (Tator, 1995; Dumont et al. 2001; Kwon et al. 2004; Silver & Miller, 2004; Donnelly & Popovich, 2008) Isolation of the area from spared spinal tissue, but also effectively blunts axonal regrowth through the area (Tator, 1995; Dumont et al. 2001; Kwon et al. 2004; Norenberg et al. 2004; Silver & Miller, 2004; Fitch & Silver, 2008; Rowland et al. 2008) Prevention of secondary damage and modulation of the inhibitory glial s car remain active research targets for SCI researchers. There are estimated 12,000 new cases of SCI each year in the United States ( National Spinal Cord Injury Statistical Center 2011 ) and many thousand more worldwide (DeVivo & Chen, 2011) Common causes of SCI as well as the nature of the injuries and SCI patient demographics have remained stable over the past 30 years (Jackson et al. 2004) However, as the general age of the population continues to increase, the average age of individuals sustaining SCIs has risen concurrently, up 9% National Spinal Cord Injury Statistical Center 2011 ). Motor vehicle accidents (~40%) continue to be the most prevent cause of SCI in the United States and worldwide (DeVivo & Chen, 2011) though as age advances over 50, falls become the leading source (Jackson et al. 2004) In addition, males a ccount for over 80% of new SCIs annually, a trend that has remained relatively stable over time (DeVivo & Chen, 2011)

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31 Respiratory C onsequences of SCI The majority (~40%) of traumatic SCIs occur to one of the ei ght cervical spinal segments (C1 C8). When the injury is sustained rostral to the fourth cervical segment (C4), significant respiratory effects are seen. Progressively more rostral injuries above C4 usually result in the need for mechanical ventilation (MV ) to sustain life (Brown et al. 2006) This reliance on MV is associated with pulmonary fibrosis, pneumonia, aspiration and sepsis (Gutierrez et al. 2003) Luckily, patients on MV are frequently able to be weaned from ventilatory assistance and regain adequate alveolar ventilation albeit with altered breathing patterns (increased frequency and decreased respiratory volume) (Loveridge et al. 1992) This recovery of function is possible due to the incomplete respiratory centers to the phrenic motor nucleus. Breathing continues to b e diffic ult for these patients however, and involves increased neural drive to remaining diaphragm motor neurons and increased energy expenditure for breathing. Thus, even with recovery of ventilatory function, patients are at increased risk of respiratory related complications. In fact, respiratory complications are the most common cause of death in patients following spinal cord injury (Winslow et al. 2002; Winslow & Rozovsky, 2003; DeVivo & Chen, 2011) Models of C ervical SCI In order to identify treatments and rehabilitation strategies for improving respiratory function following cervical SCI, clinically relevant animal models of SCI are needed (Lane et al. 2008a) Currently, several such injury models are used. To represent common contusive injuries in humans, rodent midline and lateral contusion SCI model s use weights to impact the exposed spinal cord (Rosenberg & Wrathall, 1997; el Bohy et

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32 al. 1998; Teng et al. 1999; Lane et al. 2008a; Golder et al. 2011) These injuries lead to cavitations within spinal white and grey matter affecting either descending bulbospinal pathways or phrenic motoneurons or both (Lane et al. 2008a; Golder et al. 2011) The respiratory implications of experimental contusion injuries vary dependi ng on location and severity. For example, lateralized mid cervical (C4 C5) contusion injuries 10 g weight dropped from 12.5 mm 10 g weight dropped from 25 mm ) result in injury sizes ranging from 2% 28% of total spinal cross sectional area (Golder et al. 2011) and respiratory deficits proportional t o injury severity (Golder et al. 2011) Thus, this model may mimic some aspects of respiratory imp airment seen in humans following cervical SCI (W inslow & Rozovsky, 2003) However, extensive variation in tissue sparing from animal to animal in these models may present difficult interpretational considerations. Surgical spinal hemi section lesions extending from midline to the lateral edge allow fo r a more consistent lesion affecting defined motoneuron groups. Hemi section at the second cervical segment (C2HS, Fig 1 3 ) interrupts descending bulbospinal input to contr lesion is acute silencing of the ipsilateral phrenic nerve and paralysis of one side of the diaphragm (Goshgarian, 2003) Though perhaps not representative of common SCIs in humans, the C2HS provides an important proof of principle model for study of respiratory neuroplasticity following high cervical SCI. All experiments presented in this dissertation utilize the C2HS model of cervical SCI.

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33 Respiratory Related Spinal N europlasticity Plastici ty is a persistent change in a neural control system (morphology and/or function) based on prior experience (Mitchell & Johnson, 2003) Respiratory related plasticity is an important characteristic allowing for behavioral adapt ations across a wide variety of short term (e.g. arousal state, altitude changes) and long term (e.g. pregnancy, weight fluctuations) conditions. The morphological or functional changes associated with respiratory plasticity can occur at any level of the r espiratory hierarchy. In the C2HS model of cervical SCI, multiple mammalian species exhibit a form of spinal neuroplasticity termed the crossed phrenic phenomenon (Goshgarian, 2003) The Crossed Phrenic P henomenon Disruption of unilateral descending respiratory bulbospina l input to phrenic motoneurons following C2HS results in paralysis of the IL diaphragm. However, the phrenic motor system is capable of considerable neuroplasticity that results in partial restoration of IL phrenic output as early as 2 weeks post injury (Golder & Mitchell, 2005; Fuller et al. 2006; Fuller et al. 2008) This experimental model for studying plasticity and functional recovery of respiration has been termed the crossed phrenic phenomenon (CPP) (Rosenblueth & Ortiz, 1936; Goshgarian, 2003) The neuroanatomical substrate for the CPP is accepted to be a descending bulbospinal pathway that crosses the midline at the spinal level synapsing on motor neurons in the CL v entral horn below a C2HS lesion. In the uninjured rat, these pathways are presumed to be primarily inactive during normal ventilation (Lewis & Brookhart, 1951; Goshgarian, 2003) Conversely, following C2HS, crossed phrenic pathways are able to restore partial hemi diaphragm contraction (Goshgarian, 2009) and partial phrenic motor output (Fuller et al. 2008)

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34 CPP induction In 1895, Porter first demonstrated restoration of IL hemidiaphragm contraction (i.e. CPP) following cervical hemilesion and sectioning of the CL phrenic nerve (Porter, 1895) These inducible characteristics of neuroplasticity within the phrenic system lead to the hypothesis that CPP expression was related to the intensity of central respiratory discharge (Lewis & Brookhart, 1951) The CPP can further be elicited by respiratory therapies like chronic intermittent hypoxia (Fuller et al. 2003) pharmacological treatments such as theophylline (Kajana & Goshgarian, 2008) and chronic cervical sensory denervation (Fuller et al. 2002) Additionally, the induced recovery of respira tory functioning via the CPP can be seen with administration of specific 5 HT receptor agonists (Ling et al. 1994; Zhou et al. 2001b; Fuller et al. 2005) and activation of light sensitive channel rhodopsins tran sduced into the phrenic motor pool (Alilain et al. 2008) Spontaneous CPP expression P artial recovery of IL phrenic functioning below a C2HS (Nantwi et al. 1999; Fuller et a l. 2008) over weeks to months is observed in rats allowed to recover spontaneously ( i.e. with no specific therapeutic intervention) This phenomenon points to a natural capacity for CPP facilitated recovery of diaphragm functioning. Few studies have expl ored the spontaneous crossed phrenic phenomenon (sCPP) in detail, but initial observations were made as far back as 1940 (Pitts, 1940) The post injury time period for unmasking the sCPP varies. Some reports suggest that the sCPP is not revealed in the first 4 weeks post C2HS, is partially apparent by 8 weeks and is fully present 12 16 weeks post injury (Nantwi et al. 1999; Alilain & Goshgarian, 2008) However, others report IL phrenic bursting by 2 weeks post C2HS (Fuller et al. 2003) Regardless,

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35 evidence has consistently supporte d the notion that the effectiveness of the sCPP depends on level of respiratory drive (Golder et al. 2003; Fuller et al. 2008) As such, minimal contributions of the sCPP to recovery of eupneic breathing have been hypothesized (Golder et al. 2003; Fuller et al. 2006; Fuller et al. 2008) Mechanisms of CPP expression A combination of synaptic remodeling, neuromodulatory recovery and respiratory drive trigger the expression of the CPP. Goshgarian and collegues demonstrated remodeling of synaptic inputs to phrenic motoneurons following C2HS facilitating CPP activation (Goshgarian & Rafols, 1984; Sperry & Goshgarian, 1993) Specifically retraction of astrocytic processes separating phrenic dendrites increased the size and number of synapses onto phrenic motoneurons (Goshgarian, 2009) In addition, decreased phrenic motor neuron size below a C2HS has been suggested (Mantilla & Sieck, 2003) S mal ler motoneuron size may effectively augment neuronal activation (Henneman, 1957) facilitating activation of IL PMNs acros s the CPP. ( i.e. the resultant neural output for a given level of input) (McCrimmon, 1995) Accordingly, the magnitude of IL phrenic burstin g over time following C2HS correlates with recovery of 5 HT in the vicinity of PMNs (Golder & Mitchell, 2005) Further, acute elevation of 5 HT leads to premature CPP induction following C2HS (Zhou & Goshgarian, 2000) and 5 HT depletion prevents C2HS induced morphological changes (Hadley et al. 1999a) Indeed, these studies suggest that 5 HT, likely acting via the 5 HT 2 A receptor on phrenic motoneurons, must be present in sufficient quantity for the induction of crossed phrenic activity (Zimmer et al. 2008)

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36 Another 5 HT mechanism of facilitated CPP activity may come from activation of 5 HT 1 A receptors on sensory neurons in the ce rvical dorsal horn. Application of 5 HT 1 A agonists to the dorsal horn of C2HS rats increased IL phrenic output and activated crossed phrenic pathways (Zimmer & Goshgarian, 2006) Since activation of 5 HT 1 A receptors hyperpolarizes sensory neurons (Hains et al. 2003) this result suggests the chronic inhibi tion of PMNs by sensory neurons in the dorsal horn. Funct ional r elevance of the CPP The extent to which increased phrenic motor output via crossed phrenic pathways increases functional capacity following C2HS is not clear (Zimmer et al. 2008) The prevailing hypothesis based on recurrent findings suggests that phrenic motor recovery associated with the spon taneous CPP does not contribute to increased eupneic tidal volume (Golder et al. 2003; Fuller et al. 2006) Instead, activation of the CPP occurs only under conditions of severe respiratory stress (e.g. asphyxia) or during augmented (Golder et al. 2003; Fuller et al. 2008) However, methodological constraints in these studies prevented direct definition of crossed phrenic co ntributions to inspiratory volu mes following C2HS. In chapter 3 we establish a novel method of quantifying the role of the spontaneous CPP to inspiratory motor recovery following C2HS. Plasticity in Respiratory I ntercostals Surprisingly little is known re garding the precise role of inspiratory intercostal (IC) muscles in recovery of ventilation following cervical SCI. What is known, however, is the ability of the IC muscles to exhibit neuroplasticity in response to respiratory stimuli. Fregosi and Mitchell report ed long lasting (60 90 minutes) enhanced IC output following intermittent stimulation (i.e. long term facilitation, see below) (Fregosi & Mitchell, 1994)

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37 The magnitude of this plasticity appears to be greater than that seen in the phrenic motor system, yet requires 5 HT, suggesting a common mechanism (Fregosi & Mitchell, 1994) Additionally, propriospinal interneurons within the thoracic cord (Kirkwood et al. 1988) and connecting phrenic and intercostal circuits (Lane et al. 2008b) may facilitate plasticity and functional recovery following SCI (Kirkwood et al. 1984) In chapter 4 we explore spontaneous recovery of the inspiratory intercostals following C2HS and co recovery. Improving SCI R elated Respiratory Dysfunction All traumatic spinal cord injuries to the cervical spine impact the respiratory system. Indeed, even SCIs to the thorac produce large inspiratory volumes needed to cough, exposing them to increased risk for respiratory infection and increased mortality (Sheel et al. 2008; Jefferson et al 2010) Further, experimental evidence suggests spontaneous recovery of inspiratory motor performance is incomplete following cervical SCI (Fuller et al. 2008) Thus, maximizing treatments to enhance respiratory recovery following SCI remains an important clinical and experimental objective. Clinical Modes of Respiratory T herapy Of primary clinical signifi cance is establishing methods to free SCI patients from prolonged mechanical ventilation ( MV ) Though vital to sustaining life in people with high cervical SCIs, MV is associated with weakening of inspiratory musculature and increased infection risk (Gutierrez et al. 2003) .Therefore, non invasive therapies aimed at enhancing strength in preserved respiratory musculature and long term substitutes for mechanical ventilation are frequently considered.

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38 Inspiratory muscle strength training Since the majority of SCIs are functionally incomplete, strengthening of inspiratory muscles with re spiratory resistive loads may provide the musculoskeletal adaptation necessary for breathing independent of MV (Martin et al. 2011) Fur ther, improved inspiratory muscle strength may improve cough, maximize exercise ventilation and decrease dyspnea in SCI patients (Sheel et al. 2008) Devices for inspiratory muscle strength training are relatively inexpensive, non invasive and treatment sessions can be completed bedside in combination with MV making them attractive treatment options (Geddes et al. 2005) However, though promising as an adjunct therapy for respiratory dysfunction following SCI, there is currently insufficient evidence to strongly support inspir atory muscle strength training as the primary means of improving pulmonary function or ventilatory responses in individuals with SCI (Brooks et al. 2005) Electrical activation of respiratory muscle s The use of external electrical stimulation to activate residual respiratory musculature is gaining acceptance as an alternative to MV. Bilateral phrenic nerve pacing, a clinical modality developed by Glenn and colleagues can partially restore diaphragm fu nctioning in high tertraplegics, allowing for permanent removal of MV support (Glenn, 1980; Glenn et al. 1980; Glenn et al. 1984) Alternative respiratory muscle pacing paradigms include chronic intramuscular st imulation (Nochomovitz et al. 1983) and the recent development of direct spinal stimulation for activation of inspiratory intercostals (DiMarco & Kowalski, 2010) However, high initial costs, and prerequisite invasive surgery for electrode placement may be deterrents for already compromised SCI patients (DiMarco, 2005) I n addition, high tertraplegics sustaining damage to

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39 phrenic motor pools, or phrenic rootlets are not candidates for conventional phrenic pacing. Experimental Models for R espiratory T herapy A number of experimental models targeting enhanced respiratory fun ctioning following SCI are currently under investigation. These include respiratory specific training interventions and cell based therapeutics. The rationale for respiratory specific training is augmentation of endogenous neuroplasticity within surviving respiratory circuits to enhance functional recovery. This may involve increasing respiratory drive in specific patterns to elicit cellular and molecular effects, as with intermittent hypoxia (Dale Nagle et al. 2010) Rationales for cell based therapies to enhance function following SCI generally fall into two primary categories: 1) reconnection of ascending and descending white matter pathways via axonal regeneration, or 2) replacement of lost or damaged cells (neur ons and glia). A third category, restoring specific neuromodulatory transmitters (e.g. serotonin) below a SCI to take advantage of intrinsic spinal capabilities, is an additional target for cell based therapies (Orsa l et al. 2002; Reier, 2004) Intermittent hypoxia Intermittent hypoxia (IH) increases phrenic motor output via spinal plasticity, and therefore may be harnessed as a future therapy to restore breathing capacity following SCI (Mitchell, 2007; Dale Nagle et al. 2010) The time course of IH treatment varies from a few bouts within a single treatment session (acute IH) to repetitive episodes, 8 12 hours per day, over several days or weeks (chronic IH) (Dale Nagle et al. 2010) Acute intermittent h ypoxia (aIH). The phrenic motor system responds to aIH with progressively enhanced respiratory motor output, termed phrenic long term

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40 facilitation (pLTF) (Bach & Mitchell, 1996; Mahamed & Mitchell, 2007) This model of neuroplasticity appears not only in normal (i.e. spinal intact) rats, but rats receiving C2HS lesions as well (Fuller et al. 2000; Dopera lski & Fuller, 2006) As such, aIH may be useful as a daily therapeutic intervention to enhance the functional capacity of spared respiratory pathways following SCI (Fuller et al. 2003) IH induced pLTF requires activation of 5 HT2 A receptors on PMNs for its induction, but not maintenance (Fuller et al. 2001; Baker Herman & Mitchell, 2002) Interestingly, though 5 HT2 A receptors are upregulated following C2HS (Fuller et al. 2005) aIH induced pLTF does n ot appear in the ipsilateral phrenic nerve until weeks to months post injury (Golder & Mitchell, 2005) The sharp reduction in 5 HT innervation to the ipsilateral phrenic motor nucleus following C2HS may be one limiting factor (Golder & Mitchell, 2005) Chronic intermittent hypoxia (cIH). Initially designed as a model of chronic, obstructive sleep apn ea, cIH can facilitate plasticity at multiple levels of the respiratory control system (Dale Nagle et al. 2010) Indeed, more robust protocols of IH as with cIH may overcome the loss of 5 HT innervation to PMNs in the acute stages of SCI recovery, strengthening the CPP (Fuller et al. 2003) However, potential benefits of cIH and future translation into clinical practice are likely negated by deleterious side effects including hypertension (Fletcher et al. 1992) impaired baroreflex control of heart rate (Gu et al. 2007) neurocognitive deficits (Row, 2007) and metabolic syndrome (Tasali & Ip, 2008) Cell transplantation Though the rationales for cell based therapies appear direct and achievable, practical issues related to complex SCI biology have prevented significant clinical advancement in this direction. Nevertheless, cell transplantation approach es to spinal

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41 cord injury remain feasible components to future multimodal SCI therapies (Reier, 2004) As such, efforts have been made to identify appropriate candidate cells to maximize functional improvement following SCI. Schwann cells. Schwann c ells (SC) express characteristics favorable to axonal regeneration in the peripheral nervous system including neurotrophic, extra cellular matrix, and cell adhesion properties (Hall, 1978; Bray et al. 1981) Theref ore, they make attractive candidate cells for restoration of long white matter tracts through the injured spinal cord. Peripheral nerve grafts comprised mostly of growth promoting SCs are also able to support long distance growth of central nervous system axons from brainstem (David & Aguayo, 1981) and spinal cord (David & Aguayo, 1985) However, few instances of functional recovery related to SC grafts have been reported in animal models. This is due to the limited ability of regenerating axons to establish connectivity beyond the supportive SC environment (David & Aguayo, 1981) Olefactory ensheathing cells (OEC) A cell type with similar growth promoting characteristics as SCs is OECs. These cells encase unmylinated axons of the olefactory tract against the non permissive CNS environment, supporting their elongation (Doucette, 19 90) Since OECs reside normally within the CNS, they may possess unique capacity to integrate with central nervous system tissue, overcoming a major obstacle for axonal growth beyond the astrocytic scar. Additional self regenerating capacity and character istic motility along CNS white matter make the future application of OECs in a comprehensive SCI therapeutic treatment an exciting possibility. However, procurement of autologous OECs for human SCI transplantation will be challenging given their location w ithin the olefactory bulb.

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42 Bone marrow stromal cells. Bone marrow stromal cells are pleuripotent stem cells with the ability to differentiate into multiple cell lineages, including neural cells (Woodbury et al. 20 00) They are appealing candidates for autologous grafting because of the relatively ease of their procurement, ability to expand in vitro, and flexible modes of delivery (Jendelova et al. 2004) In rat models of SCI, bone marrow stromal cells injected either intravenously or directly into the injured spinal cord facilitate remyelination of spinal axons (Akiyama et al. 2002) and promote functional recovery (Reier, 200 4) Unfortunately, the underlying mechanisms by which these cells exert their functional effect remains poorly understood. Embryonic spinal tissue. Most ambitious and concurrently challenging of the cell based therapeutic options is spinal circuit rebuil ding using embryonic spinal tissue. This transplant model encompasses not only the facilitation of white matter growth frequently dismissed therapeutic target (Reier, 2004) The developing spinal cord provides neuronal and glial progenitor cells capable of robust growth within the milieu of an injured spinal cord. Thus, grafted tissue has been shown to robustly grow to fill SCI lesion cavities (Bregman & Reier, 1986; Reier et al. 1988; Reier et al. 1992) In addition, functional improvements in locomotion (Kunkel Bagden & Bregman, 1990; Howland et al. 1995) and ventilation (White et al. 2010) have been attributed to embryonic cell transplants T he safety and efficacy of embryonic spinal tissue transplants have been shown in human trials of severe and progressive spinal injury (Thompson et al. 2001; Wirth et al. 2001) as well However, procurement of embryonic tissue is limited by significant moral and ethical considerations as well as

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43 fluctuating federal regulations, making it an unlikely candidate for long term human therapeutic use. S erotonin c ell t ransplants An alternative goal for cell based therapies is restitution of specific neurotransmitters to neural targets below a SCI to facilitate intrinsic spinal capabilities. For example, the lumbar locom otor circuit in rats can initiate reciprocal stepping movements in the absence of supraspinal drive with appropriate afferent input (Rossignol et al. 1996; Rossignol, 2000) in the presence of 5 HT (Jordan, 1998; Feraboli Lohnherr et al. 1999) Therefore, cell transplants with the capability of restoring 5 HT chronically to these circuits were developed using embryonic 5 HT neurons micro dissected from the developing raph nu clei (see above). Early studies illustrated that raph cells transplanted into the sublesional spinal cord survived for long periods and reinnervated main spinal targets (Bjorklund et al. 1986; Privat et al. 1986; Privat et al. 1989; Rajaofetra et al. 1992) in similar 5 HT patterns as spinal intact rats (Steinbusch, 1981; Ridet et al. 1993) Further, functional locomotor recovery returned in spinally transected rats after receiving raph transplants (Ribotta et al. 2000) Though the phrenic motor system may not represent a true central pattern generator like the lumbar locomotor circuit, the importance of 5 HT to functional respir atory recovery following SCI is analogous. Cell transplants of embryonic 5 HT cells in a model of cervical SCI have yet to be undertaken. In chapter 5 we investigate the effects of raph cell transplants in a rodent C2HS model to facilitate functional res piratory recovery in the context of the spontaneous CPP.

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44 Figure 1 1. Schematic illustration of the neural control of breathing. Brainstem respiratory centers provide neural input to cranial motoneurons innervating upper airway muscles a nd spinal motoneurons controlling thoracoabdominal muscles. Activation of respiratory muscles leads to ventilation which maintains appropriate concentrations of oxygen (O 2 ) and carbon dioxide (CO 2 ) in the blood. Changes in these tightly regulated blood var iables are sensed by chemoreceptors and transmitted back to the respiratory control centers where appropriate adaptations in neural output can be made. Additionally, the respiratory controller adjusts its neural output in response to mechanoreceptor affere nt feedback and descending neural input from cortical and subcortical areas. P a O 2 ; partial pressure of oxygen in arterial blood. Adapted from Feldman 1986.

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45 Figure 1 2. Neuromodulatory inputs to the brainstem respiratory network. The res piratory network receives e xtensive neuromodulatory input from various sources. Shown are neuromodulators known to play a key role in the neural control of breathing. H owever, it is likely that the catalog of neuromodulators known to influence respiratory function is far from complete. Adapted from Doi and Ramirez, 2008. Abbreviations : NTS; nucleus of the solitary tract, PAG; periaquedutal gray LC; locus ceruleus SST; somatostatin CCK; cholecystokinin SP; substance P, NE; norepinephrine, Ach; a cetylchol ine DA; dopamine, 5 HT; 5 hydoxytryptamine.

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46 Figure 1 3. A representative histological section depicting a C2HS lesion. This example was taken from cervical segment C2 6 weeks following injury. An anatomically complete lesion is defined as the absence of gray matter (GM) and white matter (WM) on the side of injury ( i.e ipsilateral; IL). CL; contralateral (opposite to injury).

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47 Table 1 1. Overview of 5 HT receptor families. Family Type Subtypes Distribution Neural effect Mechanism 5 H T 1 G i /G o protein coupled 5 HT 1A,B,C,D,F Spinal dorsal horn 5 HT neurons throughout CNS Blood vessels Inhibition cAMP 5 HT 2 G q /G 11 protein coupled 5 HT 2A,B,C Spinal v entral horn s moo t h muscle blood vessels GI Trac t Excitation 3 and DAG 5 HT 3 Ligand gated Na + and K + cation channel n/a Superficial spinal dorsal horn GI Tract Excitation fast cellular depolarization 5 HT 4 G s protein coupled n/a CNS GI Tra ct Excitation cAMP 5 HT 5 G i /G o pr otein coupled n/a CNS Inhibition cAMP 5 HT 6 G s protein coupled n/a CNS Excitation cAMP 5 HT 7 G s protein coupled n/a Spinal v entr al ho r n blood vessel s GI Tract Excitation cAMP

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48 CHAPTER 2 OUTLINE O F EXPERIMENTS The overall goals of this thesis are to gain a better understanding of the mechanisms underlying respiratory recovery following cervical SCI, and to determine if intra spinal transplantation of serotonergic neurons could be a viable method fo r enhancing respiratory recovery. Here we provide a brief overview of the experiments conducted for this PhD dissertation. Adult rats will exhibit a degree of spontaneous respiratory recovery following experimental cervical spinal cord injury. In the C2 hemisection model, interruption of primary bulbospinal pathways to phrenic motoneurons leads to temporary hemidiaphragm paralysis. Partial recovery of phrenic motor output ipsilateral to the lesion occurs over time by way of a network of axons that project across the spinal midline c audal to C2. This recovery has phenomenon (CPP). Despite extensive research dedicated to neuroplasticity associated with the CPP, its significance (i.e., functional contribution) to recovery of spontaneous breathing remains uncertain. Thus, Aim 1 of this dissertation was designed to test the prevailing hypotheses that neural activity associated with the spontaneous CPP makes a significant contribution to chronic tidal volume recovery and that t his functional contribution increases in parallel with respiratory drive in a spontaneously breathing rat. Aim 2 explored the role of the intercostal muscles in respiratory recovery following SCI. We reasoned that progressive, spontaneous tidal volume re covery seen in the weeks to months following experimental cervical SCI was unlikely the result of CPP related plasticity alone, but also from plasticity in neural pathways activating accessory muscles of inspiration. Therefore, using electromyography (EMG) we explored the

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49 spontaneous return of muscle activity in inspiratory intercostal muscles. We hypothesized that intercostal muscles, particularly ipsilateral to a C2 hemisection lesion, would display enhanced activity to compensate for loss of ipsilateral diaphragm function. The final Aim investigated the use of embryonic brainstem cells to promote ventilatory recovery following cervical SCI. Prior work has established conclusively that serotonin can promote respiratory motor recovery following SCI. Acco rdingly, we tested the hypothesis that embryonic medullary serotonergic (raph) cells transplanted b elow a C2 hemisection would survive and reinnervate phrenic motoneurons with serotonin. We reasoned that enhanced serotonergic availability would lead to au gmentation of phrenic motor output and enhanced ventilatory recovery. Together, the studies conducted for this doctoral dissertation investigated neural activity associated with spontaneous ventilatory recovery after cervical SCI as well as methods to boos t this recovery. Below the objectives, rationale and experiments associated with Aims 1 3 are summarized Aim One Objective Determine the contribution of neural signals associated with the spontaneous CPP to tidal volume following C2 hemisection. Rational e Modest, yet incomplete recovery of inspiratory tidal volume is observed in rats following C2 hemisection. This recovery has been linked to plasticity associated with the

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50 crosse d phrenic neural activity to tidal volume following C2 hemisection in spontaneously breathing rats is unknown. Hypothesis 1 The functional significance of the spontaneous crossed phrenic pathway increases in a time dependent manner over weeks to months po st C2 hemisection. Hypothesis 2 Neural activity associated with the crossed phrenic pathway makes more significant contributions to tidal volume when respiratory drive is increased Experimental D esign Respiratory airflow was measured using pneumotachograp hy in anesthetized, spontaneously breathing adult Sprague Dawley rats under urethane anesthesia. Ipsilateral phrenicotomy during either baseline (inspired O 2 fraction = 0.50) or hypercapnic respiratory challenge (7% CO 2 21% O 2 balance N 2 ) was utilized to asses s immediate declines in integrated inspiratory airflow signals (i.e. tidal volume) as an index of neural contribution. Changes in tidal volume were expressed in ml/breath, as well as % decline from pre phrenicotomy values. Data from rats 1 3 days (n = 10), 2 weeks (n = 9) or 8 weeks (n = 9) post C2 hemisection were analyzed for comparison to spinal intact, age matched control rats (n = 9). Aim Two Objective Assess spontaneous recovery in inspiratory intercostal muscle activity and examine changes i n intercostal neural circuitry following C2 hemisection.

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51 Rationale Despite an apparent plateau in the relative contribution of crossed phrenic neural activity to tidal volume by two weeks following C2 hemisection, gross tidal volume continues to improve, suggesting parallel plasticity in other spinal respiratory circuits. Inspiratory intercostal muscles have demonstrated robust neuroplasticity in response to respiratory challenge, yet few studies have assessed recovery of intercostal activity or changes in intercostal neural connectivity following cervical spinal cord injury. Hypothesis Progressive enhancement of inspiratory intercostal activity measured with electromyography (EMG), will continue for months following C2 hemisection and contribute to on go ing tidal volume improvements. Experimental D esign Bilateral intercostal neurograms were recorded 1 3 days (n = 4), 2 weeks (n = 9) or 8 weeks (n = 7) following C2 hemisection in urethane anesthetized, spontaneously breathing Sprague Dawley rats. Integr ated burst amplitudes were compared to spinal intact control rats (n = 7) as an index of spontaneous recovery following C2 hemisection. In a separate cohort of uninjured animals (n=2) retrograde anatomical tracers (cholera toxin and pseudo rabies virus) were co injected into rostral external intercostal muscles to map motoneuron and pre motor interneuron circuitry. Aim Three Objective Examine the feasibility of serotonin replacement therapy using embryonic cell transplants to enhance ventilatory recove ry following C2 hemisection.

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52 Rationale Serotonin is a potent neuromodulator of motoneuron function at all levels of the spinal cord. The C2 hemisection model of cervical spinal cord injury not only interrupts primary bulbospinal motor drive to phrenic neur ons, but disrupts serotonergic innervation as well. This alteration in normal neuromodulatory input may play a role in the delayed activation of crossed phrenic motor circuits and ipsilateral phrenic motor recovery following C2 hemisection. In fact, amplit ude of ipsilateral phrenic bursts following C2 hemisection strongly correlates with quantity of serotonin present in the vicinity of phrenic motoneurons. Hypothesis 1 Transplants of embryonic serotonin cells into the spinal cord below a C2 hemisection lesi on will survive for up to 6 weeks and enhance serotonergic innervation to the area of ipsilateral phrenic motoneurons. Hypothesis 2 Enhanced serotonergic innervation to ipsilateral phrenic motoneurons in rats receiving serotonin cell transplants will be a ssociated with increased phrenic burst amplitudes and higher tidal volume production compared with control rats receiving either sham or non serotonergic cell transplants. Experimental D esign One week following C2 hemisection adult male Sprague Dawley rat s received intraspinal (C3) allografts of embryonic ( gestational day 14; E14) brainstem tissue contai ning serotonergic raph cells. Control transplants consisted of cell culture media (i.e. sham) or dissociated fetal spinal cords. Six weeks post transplant ation, ventilation was assessed in unanesthetized rats using plethysmography and in anesthetized rats

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53 using pneumotachograpy. Phrenic neural output was measured under ventilated, paralyzed and vagotomized conditions Rats were perfused at the end of each e xperiment and cervical spinal cords were dissected for immunohistochemical analysis.

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54 CHAPTER 3 THE CONTRIBUTION OF THE SPONTANEOUS CROS SED PHRENIC PHENOME NON TO INSPIRATORY T IDAL VOLUME IN SPONTANEOUSLY BREATHING RATS Severing ipsilateral bulbospinal inpu ts to phrenic motoneurons (PMNs) via lateral hemisection of the C2 spinal cord (C2HS) transiently paralyzes the hemidiaphragm (Goshgarian, 2003; Lane et al. 2008a; Goshgarian, 2009; Vinit & Kastner, 2009) However, a partial return of ipsilateral PMN inspiratory bursting occurs over a period of weeks to months following C2HS (Nantwi et al. 1999; Golder et al. 2003; Fuller et al. 2006; Fuller et al. 2008) This response h as been termed the spontaneous crossed phrenic phenomenon (sCPP) (Goshgarian, 2009; Lane et al. 2009; Sandhu et al. 2009a) The sCPP provides an important experimental model of neuroplasticity and associated funct ional recovery (i.e. phrenic bursting) after spinal cord injury (SCI) (Goshgarian, 2009; Lane et al. 2009; Sandhu et al. 2009a) However, the functional contribution of the sCPP to ventilation ( E ) has not been definitively established. In other words, it is unclear if the relatively small amount of electrical activity that has been measured in the ipsilateral phrenic nerve after chronic C2HS is sufficient to alter inspiratory tidal volume (V T ). Thus, it is unknown if C2HS induced, spontaneous neuroplastic changes associated with the sCPP (Goshgarian, 2003, 2009) have a meaningful impact on the respiratory system (Golder et al. 2003) The functional impact of the sCPP has been examined to a limited extent (Golder et al. 2003; Fuller et al. 2006; Fuller et al. 2008) Correlations between phrenic nerve activit y recorded under anesthesia and E measured in unanesthetized rats suggest that the sCPP makes a small contribution to V T (Golder et al. 2003; Fuller et al. 2006; Fuller et al. 2008) In the most comprehensive study to date (Golder et al. 2003)

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55 Golder et al (Golder et al. 2003) evaluated V T from causing diaphragm contraction). Spontaneously breathing poikilocapnic rats w ith the dual injury had similar V T levels as those with C2HS alone. However, t he volume of augmented breaths (Golder et al. 2005) was reduced after the dual injury. These results lead to the hypothesis that the sCPP makes relatively little contribution to eupneic breathing, but becomes funct ionally relevant when respiratory drive is increased (Golder et al. 2003; Sandhu et al. 2009a) However, a potential confound to this interpretation is that eliminating the sCPP may enhance the compensatory plast icity (Lane et al. 2009; Sandhu et al. 2009a) that occurs in other respiratory motor pools (e.g. contralateral phrenic motoneurons; intercostal motoneurons, etc.) following C2HS. Indeed, even when both phrenic n erves are intact, C2HS injury causes a robust enhancement of contralateral phrenic output (Miyata et al. 1995; Rowley et al. 2005; Doperalski & Fuller, 2006; Fuller et al. 2006) We reasoned, therefore, that me asuring inspiratory V T immediately before and after an acute ipsilateral phrenicotomy procedure could enable a more definitive assessment of the functional significance of the sCPP. In other words, the immediate T resulting f rom disruption of the sCPP should provide a quantitative estimate of its importance. We used this approach to test the hypothesis that the functional significance of the sCPP increases in parallel with respiratory drive. Further, we hypothesized that the functional significance of the sCPP increases in a time dependent manner over weeks to months post C2HS. Our final purpose was to establish an experimental method that will enable more direct testing of the functional

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56 efficacy of therapeutic interventions aimed at enhancing the sCPP (Alilain et al. 2008; Alilain & Silver, 2009; Dougherty BJ 2010; White et al. 2010) Portions of this work have been presented in abstract form ( Dougherty BJ 2010) In addition, this cha pter was recently published in the Journal of Applied Physiology (Dougherty et al. 2011) Materials and Methods All experimental procedures were approved by the Institutional Animal Care and Use Committee at the U niversity of Florida. Animals A total of 37 adult, male Sprague Dawley rats were obtained from Harlan Laboratories Inc. (Indianapolis, IN, USA). Rats receiving C2HS injury were grouped by the following post 3 days), 2 weeks ( 2wk) or 8 weeks (8wk). Uninjured control rats were age matched to the 2wk injury group (N=4) or the 8 wk group (N=5). A summary of the experimental groups is presented in Table 3 1 Spinal Cord I njury Our anesthesia and injury methods have been previously described (Fuller et al. 2008; Fuller et al. 2009) Rats were anesthetized by injection of xylazine (10 mg/kg, s.q.) and ketamine (140 mg/kg, i.p., Fort Dodge Animal Health, IA, USA). The spinal cord was exposed at the C2 level via a dorsal approach, and a left C2HS lesion was induced using a microscalpel followed by aspiration. The dura and overlying muscles were sutured and the skin closed with stainless steel wound clips (Stoelting, IL, USA). Rats were given an injection of yohimbine (1.2 mg/kg, s.q., Lloyd, IA, USA) to reverse the effect of xylazine. Following surgery, animals received an analgesic (buprenorphine, 0.03 mg/kg, s.q., Hospira, IL, USA) and sterile lactated Ringers solution (5 ml s.q.). Post surgic al care included administration of buprenorphine (0.03 mg/kg, s.q.) during

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57 the initial 48 hours post injury and delivery of lactated Ringers solution (5 ml/day, s.q) and oral Nutri cal supplements (1 3 ml, Webster Veterinary, MA, USA) until adequate voliti onal drinking and eating resumed. Experimental P reparation These procedures were adapted from our prior publications (Doperalski et al. 2008; Fuller et al. 2008; Fuller et al. 2009; Lane et al. 2009; Sandhu et al 2009a) Isoflurane anesthesia (3 4% in O 2 ) was induced in a closed chamber followed by i.p. injection of urethane (1.6g/kg, Sigma, St. Louis, MO, USA). The adequacy of urethane anesthesia was confirmed by testing limb withdrawal and palpebral reflexes Rats were maintained in a supine position throughout the protocol. The trachea was cannulated in the mid cervical region and connected in series to a custom designed, small animal pneumotachograph and volumetric pressure transducer (Grass Instruments, Qu incy, MA, USA) for measurement of respiratory air flow. Partial pressure of arterial oxygen (P a O 2 ) was maintained above 150 mmHg by delivering a hyperoxic gas mixture (F I O 2 =0.50, balance N 2 de sign (Fuller et al. 1998) The femoral vein was catheterized (PE 50) to enable supplemental urethane anesthesia (0.3 g/kg, i.v., Sigma, St. Louis, MO, USA) if indicated. Another PE 50 catheter was placed in the femoral artery and connected to a pr essure transducer (Statham P 10EZ pressure transducer; amplifier CP122 AC/DC strain gauge amplifier, Grass Instruments, West Warwick, RI, USA) for arterial pressure and blood gas measurements. The ipsilateral phrenic nerve was isolated in the cervical reg ion via a ventral approach (Sandhu et al. 2009a; Lee et al. 2010; Sandhu et al. 2010) The exposed nerve was covered in mineral oil but not manipulated at this time. Arterial blood samples (0.2 ml) were drawn d uring the baseline period (see

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58 Experimental Protocols) and analyzed for P a O 2 carbon dioxide partial pressure (P a CO 2 ) and pH (i STAT, Waukesha, WI). Blood gas measures were corrected to rectal temperature which was monitored by rectal thermistor and mainta ined at 37.5 1 C by a servo controlled heating pad (model TC 1000, CWE Inc., Ardmore, PA, USA). Experimental Protocols The ipsilateral phrenic nerve was cut during expiration during either the baseline period (N=20) or during a hypercapnic respiratory c hallenge (N=17) ( Table 3 1 ). Baseline breathing was established over a 20 minute period during which rats breathed the hyperoxic gas mixture described above. Baseline was followed by a five minute hypercapnic respiratory challenge (7% CO 2 50% O 2 balanc e N 2 ; e.g Fig. 3 2). Rats were then returned to baseline conditions, and once breathing had returned to the pre hypercapnic values, the ipsilateral phrenic nerve was cut (i.e., baseline phrenicotomy group). After 5 minutes had elapsed, the hypercapnic ch allenge was repeated. In a separate group, the ipsilateral phrenic nerve was cut during the third minute of the second hypercapnic challenge (i.e., hypercapnic phrenicotomy group). This time corresponded to a stable period of hypercapnic V T that was simil ar to the initial hypercapnic response. All rats were returned to baseline conditions following the second hypercapnic challenge. A small sample of rats with C2HS (N=4) were studied to confirm our assumption that reflexive increases in contralateral respir atory muscle activity would not occur during the initial breath following ipsilateral phrenic nerve section. Thus contralateral external intercostal electromyogram (EMG) activity (first intercostal space; N=2) or contralateral hemidiaphragm EMG activity (medial costal region; N=2) was assessed during spontaneous breathing in urethane anesthetized rats following C2 hemisection injury. EMG activity was measured using intramuscular fine

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59 wire electrodes as previously described (Fuller et al. 1998) Baseline conditions were established as described above. After a stable period of poikilocapnic baseline breathing, the ipsilateral (left) phrenic nerve was sectioned and we examined the immediate impact on the EMG activity of the contralateral respirator y muscles. Spinal Cord H istology All C2HS lesions were confirmed to extend to the spinal midline as previously described (Fuller et al. 2008; Fuller et al. 2009; Sandhu et al. 2009a) At the conclusion of the phr enicotomy experiment, rats were euthanized by systemic perfusion with saline followed by 4% paraformaldehyde (Sigma, St. Louis, MO, USA). The cervica l spinal cord was removed, and 4 0 m sections were made in the transverse plane using a vibrotome. Tissue sections were mounted on glass slides (Fisher Scientific, Pittsburgh, PA, USA), stained with Cresyl violet and evaluated by light microscopy. A histological example o f a C2HS lesion is shown in Figure 3 1 Consistent with our previous publications (Fuller et al. 2008; Lane et al. 2008b; Fuller et al. 2009; Sandhu et al. 2009a) the apparent absence of healthy white matter in the ipsilateral C2 spinal cord was taken as confirmation of an anatomically complete C2 HS (Fuller et al. 2009) Data Analysis Calibration of the pneumotachograph was accomplished using a series of constant volume injections with varying airflow rates. Respiratory airflow signals were amplified (x100K ; CP122 AC/DC strain gage amplifier, Grass Instruments) and recorded on a PC using Spike2 software (Cambridge Electronic Design Limited). The inspiratory phase of the airfl (Cambridge Electronic Design Limited) and V T was then calculated off line. Inspiratory and expiratory duration (T I and T E respectively) were calculated (Lee et al. 2009) based

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60 on the integrated airflow traces as indicated in Figure 3 3 Respiratory frequency (f, breaths*min 1) was ca lculated as 60/(TI + TE). Rats occasionally showed augmented breaths (ABs; Figure 3 2). These were identified by characteristic two phase airflow patterns as previously described (Cherniack et al. 1981; Golder et al 2005; Fuller et al. 2008) The first phase of the AB was indistinguishable from the preceding tidal breath. However, at the peak of inspiration there was a further, distinct increase in the rate of rise of inspiratory flow followed by a prolonged T E One way analysis of variance (ANOVA) was used to compare body weight, blood gases, and mean arterial blood pressure (MAP) across groups. Pre phrenicotomy data including initial baseline and hypercapnic V T and relative increases in V T during hypercapnic challenge were also assessed using one way ANOVA. Respiratory parameters (e.g., T I T E f and V T ) were averaged over the 10 breaths that immediately preceded the phrenicotomy. These values were compared to values measured at the 1st, 3rd and 5th breaths following phrenicotomy as well as the breath 1 minute following phrenicotomy. These data were compared using 2 way repeated measures (RM) ANOVA and the Student Newman Keuls post hoc test. For this ANOVA, factor 1 was acute phrenicotomy). Changes in V T T ) following phrenicotomy were normalized to the pre phrenicotomy values as follows: %V T decline = [1 (Post phrenicotomy V T / Pre phrenicotomy V T )] x 100 %. The final 30 seconds of the pre and post phrenicotomy hypercapnic challenges were averaged fo r comparison using 2 way RM ANOVA [factor 1: treatment (lesion group), factor 2: condition (pre or post phrenicotomy)]. Changes in baseline AB f and

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61 volume following phrenicotomy were assessed in a subset of C2HS rats (see results) using a one way ANOVA. Further, since hypercapnia induced ABs in both control and C2HS rats, we analyzed changes in hypercapnic AB f and volume using 2 way RM ANOVA [factor 1: treatment (lesion group), factor 2: condition (pre or post phrenicotomy)]. All data are presented as t he mean standard error. A P value of < 0.05 was considered statistically significant. Results A time dependent change in body mass occurred following C2HS as previously reported (Fuller et al. 2006; Doperalski et al. 2008; Fuller et al. 2008; Fuller et al. 2009) Thus, both the 1 3 day and 2wk post C2HS groups weighed less than control and 8wk post injury rats ( Table 3 1 ). P a O 2 was similar between groups during baseline breathing ( Table 3 2 ). However, the acu tely injured rats (1 3 days post injury) showed evidence for hypoventilation and arterial acidosis as reflected by increased P a CO 2 and decreased pH (P<0.05 vs. other groups; Table 3 2 ). Consistent with prior reports (Doperalski & Fuller, 2006; Fuller et al. 2008) no group differences in MAP were observed ( Table 3 2 ). Effect of C2HS on V entilation Representative examples of airflow signals in control and C2HS rats are provided in Figures 3 2/3 3. Before describing t he impact of phrenicotomy, we first provide an overview of how C2HS influenced breathing during the initial (i.e. pre phrenicotomy) measurements. C2HS did not significantly influence f, T I or T E during the initial baseline period ( Table 3 3 ). A tendency for increased f was noted during baseline breathing in the 8 wk C2HS groups, but this did not reach statistical significance (P = 0.16, Table 3 3 ). Some between group differences in f were noted during hypercapnia

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62 ( Table 3 3 ). Control (1012% baseline) a nd 1 3 day post C2HS rats (995% baseline) did not alter their breathing f during hypercapnic challenge. In contrast, both 2 wk (913% baseline) and 8 wk rats (904% baseline) showed a decline in f during hypercapnia reflecting an elongation of T I (P < 0. 001, Table 3 3). The significance of an elongated T I during hypercapnia is not clear, but may represent an adaptive strategy to elongate the period of gas exchange during hypercapnic challenge in chronic C2HS. The C2HS injury caused a substantial reductio n in V T (ml/breath) during both Fig ure 3 4 ). Injured rats in the 1 3 days group produced the smallest baseline V T (P<0.05 vs. all other groups). A progressive increase in base line V T was measured over the 8wk period following C2HS. In particular, 2wk C2HS rats had increased V T compared to the 1 3 day post injury group, and 8 wk rats had greater V T than both 2wk and 1 3 day groups ( Fig ure 3 4A ). However, baseline V T remained be low control values in the 8wk C2HS rats (P<0.05; Figure 3 4A ). All C2HS rats also had lower V T during hypercapnia co mpared to controls (P<0.05; Figure 3 4B ). As with the baseline condition, the smallest V T values were observed in the 1 3 days group. Howeve r, since P a CO 2 in these rats was already elevated ( Table 3 2) a reduced hypercapnic V T response was anticipated. Partial recovery of hypercapnic V T was seen in 2 and 8wk C2HS rats (Fig ure 3 4B ). The relative increase in hypercapnic V T (% baseline) was simi lar between control (26623%) and chronic C2HS rats (2 wk: 22217%; 8 wk: 21820%; data not shown), but this response was substantially blunted in the 1 3 days post C2HS group (16614%; P < 0.05).

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63 Immediate Impact of Ipsilateral P hrenicotomy Representative examples of airflow and V T before and after phrenicotomy in control and C2HS rats are provided in Figure 3 3. Reductions in V T following acute phrenicotomy in the two control groups (i.e., age matched to the 2 and 8 wk C2HS groups, respectively) were sim ilar during baseline (P = 0.27) and hypercapnia (P = 0.95). These data were therefore combined to form a single control group ( Table 3 1 ) for each condition. Likewise, similar reductions in V T following ipsilateral phrenicotomy occurred at 1 day and 3 days post C2HS during baseline (P = 0.64) and hypercapnia (P condition. In control rats, baseline phrenicotomy caused an immediate reduction in f that reflected an elongation of T E ( Table 3 4 ). In contrast, baseline phrenicotomy did not significantly affect breathing pattern in any of the C2HS groups ( Table 3 4 ). Phrenicotomy during the hypercapnic challenge did not influence f in control or C2HS rats (Table 3 5 ). The immed iate impact of phrenicotomy on V T was assessed in both absolute Figure 3 4) and relative to the preceding breaths (i.e. % decline Figure 3 5 ). Our primary outcome measure was the % decline in V T immediately (next breath) following phrenicotomy. Baseline phreni cotomy in control rats caused an immediate reduction in V T by 492 % (Figure 3 3 thru 3 5). Control rats were able to compensate for the loss of hemidiaphragm activity as evidenced by the progressive increase in V T over the subsequent breaths (Figure 3 4A ) However, V T did not return to pre phrenicotomy values within 60 sec post phrenicotomy (Figure 3 4A ). The decline in V T following phrenicotomy in C2HS rats was considerably less than in

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64 controls. Indeed, phrenicotomy caused no measurable changes in basel ine V T at 1 3 days post C2HS ( VT = 13%; Figures 3 4 and 3 5 ). However, by 2 wks post C2HS ipsilateral phrenicotomy caused an immediate 162% reduction in V T ( Figures 3 4 and 3 5 ). Phrenicotomy triggered a similar decline of V T in the 8wks post C2HS gro up (164%; Figure 3 5). Rats with chronic C2HS were able to rapidly compensate for the loss of ipsilateral diaphragm activity. In both groups, V T had returned to baseline (pre phrenicotomy) values within three breaths ( Figure 3 4 ). Phrenicotomy during t he hypercapnic challenge produced qualitatively similar responses to what was observed during baseline (Figures 3 3 thru 3 5). Control rats showed an immediate decline in V T of 471% (Figure 3 5). As with the baseline response, C2HS rats showed a comparati vely smaller change in V T after hypercapnic phrenicotomy. Surprisingly, the relative drop in V T during hypercapnia was similar at each post injury time point (1 3 days: 75, 2 wk: 134, 8 wk: 92%; Figure 3 5). In other words, a time dependent and progres sive increase in sCPP contribution to hypercapnic V T was not apparent. The relative magnitude of the change in V T following phrenicotomy and hypercapnia in 1 3 days injured rats merits a further comment. Because the 1 3 day C2HS rats had much lower absol ute V T compared to the other groups, the ~7% reduction in V T following phrenicotomy ( Figure 3 5 ) corresponded to a change of just 0.10.1 ml/breath. As such, the contribution of the sCPP to hypercapnic V T at 1 3 days post injury should be interpreted cauti ously. Finally, the compensatory responses to phrenicotomy were similar to what was observed during the baseline condition. Specifically, hypercapnic V T returned to pre phrenicotomy levels within 3

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65 breaths in all injured groups, while in control rats the reduced V T persisted for 60 seconds following phrenicotomy ( Fig ure 3 4B ). Effect of Phrenicotomy on Subsequent Hypercapnic Ventilatory R esponses Hypercapnic challenge was performed both before and after phrenicotomy in all control and C2HS rats in the base line phrenicotomy group. Phrenicotomy in control rats caused a blunting of the subsequent hypercapnic ventilatory response that reflected reductions in both f and V T (Figure 3 6). These blunted ventilatory responses, however, remained significantly greate r than the response o f phrenic intact C2HS rats ( Figure 3 6 ). All C2HS rats maintained V T f and E at pre phrenicotomy values when assessed during the post phrenico tomy hypercapnic challenge (Figure 3 6). Thus, in contrast to the cont rol rats, C2HS animals were able to fully compensate for the acute loss of ipsilateral hemidiaphragm activity. The normalized hypercapnic ventilation data (i.e., % pre phrenicotomy) were qualitatively similar to the absolute values (not shown). Effect of Phrenicotomy on Spontaneous A ugmente d B reaths Representative examples of AB behavior during spontan eous breathing are shown in Figure 3 2. they were observed in a subset of C2HS rats. Of those rats exhibiting ABs, no apparent differences could be detected across the three C2HS groups. Specifically, AB f (P=0.38) and volume (P=0.34) were similar across C2HS groups and thus these data were pooled. Overall, 40% of C2HS rats showed ABs during baseline conditions at a rate of 309*hr 1. Phrenicotomy significantly reduced the rate of AB occurrence to just 43*hr 1 (P = 0.02). However, the phrenicotomy procedure did not diminish baseline AB volume in C2HS rats (11792%). Hypercapnic challe nge induced the appearance of ABs in control rats, and increased the f of ABs in C2HS groups (e.g., Figure 3 2 ). The

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66 average f of ABs was similar in control (8817*hr 1) and C2HS rats (10022*hr 1) during hypercapnia (P=0.66). Phrenicotomy did not influen ce hypercapnic AB f either within or between groups (not shown). As expected, phrenicotomy diminished hypercapnic AB volume in contro l rats (6221%, P = 0.004) ( Figure 3 7). However, similar to the baseline response hypercapnic AB volume was not altered f ollowing phrenicotomy in C2 HS rats (1023%, P = 0.63) ( Figure 3 7). Effect of Phrenicotomy on Contralateral Respiratory Muscle EMG A ctivity Anecdotally, we noted in supplemental experiments that acute phrenicotomy caused no discernable immediate (next brea th) change in the contralateral EMG activity of the hemidaphragm or external intercostal muscles in C2HS rats ( Figures 3 8 and 3 9 ). Thus, changes in V T following ipsilateral phrenicotomy result from primarily, if not exclusively, the immediate paralysis of the ipsilateral hemidiaphragm. Compensatory EMG responses in accessory muscles and the contralateral diaphragm are not rapid Discussion There are two particularly nov el aspects of our results. First, contraction of the ipsilateral hemidiaphragm following C2HS makes a meaningful contribution to V T during quiet breathing (i.e. baseline conditions) as early as two weeks following C2HS. However, the impact of the sCPP on ventilation was similar during both baseline and respiratory challenge. Therefore, the functional significance of the sCPP does not appear to increase relative to respiratory demand. This observation contrasts with previous assertions that the sCPP is re levant primarily during periods of increased respiratory drive (Golder et al. 2003) Second, progressive increases in the functional impact of the sCPP occurred only during the initial two weeks following C2HS. In other

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67 words, after chronic C2HS injury, ther e was not an increased (or preferential) reliance on the sCPP to generate tidal volume. This observation does not necessarily preclude a time dependent increase in the output of the sCPP (Nantwi et al. 1999; Fuller et al. 2006; Fuller et al. 2008) rather, it indicates that any increases in ipsilateral phrenic motor output are occurring in parallel with compensatory increases in other motor outputs (e.g. intercostal muscles). Commentary on Methods and Diaphragm B i omechanics Here we provide a brief discussion regarding the phrenicotomy method and the interpretation of subsequent changes in V T First, the phrenicotomy procedure necessitated that the rats be deeply anesthetized. Anesthesia has the potential to supp ress sCPP activity, and accordingly could lead to an underestimation of the potential contribution of ipsilateral phrenic activity to V T in unanesthetized animals. Second, we recognize that the immediate decrease in V T following ipsilateral phrenicotomy d oes not necessarily represent decreases in transdiaphragmatic pressure (P di ) that directly resulted from diaphragm paralysis. Rather, alterations in the biomechanical relationships between the diaphragm, rib cage, abdominal cavity and accessory respirator y muscles may alter P di secondary to diaphragm paralysis. It should be emphasized that the biomechanical relationship between diaphragm contraction and generation of P di is complex (De Troyer, 1986) Contraction of the increases in abdominal pressure) (Urmey et al. 1988) The net action of the diaphragm (and resultant P di ) will depend on the balance of these two forces. In our experiments,

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68 we did not attempt to characterize how C2HS or the ph renicotomy induced cessation of diaphragm activity altered diaphragm and chest wall biomechanics. Regardless of the underlying biomechanical changes, however, the immediate change in V T following phrenicotomy provides a quantitative evaluation of the impo rtance of hemidiaphragm contraction. In this regard, it is striking that V T decreased dramatically immediately following phrenicotomy in spinal intact rats, as might be predicted if diaphragm contraction is a primary contributor to P di under these conditi ons. A final comment is warranted regarding postural impacts on diaphragm function. Similar to prior reports (Fuller et al. 2006; Doperalski et al. 2008; Fuller et al. 2008; Fuller et al. 2009; Sandhu et al. 2 010) rats were studied in the supine position to provide suitable access to the trachea and phrenic nerves. Studies in humans have clearly demonstrated that posture can impact diaphragm function. For example, the diaphragm expands the rib cage less when contraction takes place in the supine as compared to the upright position (De Troyer, 1986) However, the impact of supine vs. prone breathing on pulmonary biomechanics in quadrupedal rodents is l ess clear. The C ontribution of the sCPP to Tidal Volume F ollowing C2HS Even months after C2HS the ipsilateral phrenic nerve and/or hemidiaphragm (Nantwi et al. 1999; Fuller et al. 2003; Fuller et al. 2006; Fuller et al. 2008; Dow et al. 2009; Fuller et al. 2009) It is difficult to say precisely how much inspiratory activity is present since the actual number of active ipsilateral phreni c motor units during spontaneous breathing after C2HS is unknown. Recruitment of even a relatively low number of motor units as has been suggested by prior studies (Nantwi et al. 1999; Fuller et al. 2003; Fuller et al. 2006; Fuller et al. 2008; Fuller et al. 2009) could make a meaningful contribution

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69 to V T Our data are consistent with this possibility since ipsilateral phrenicotomy during baseline conditions significantly reduced V T as early as 2 wks post C2HS. Thus, the current results indicate a functional role for the sCPP in the recovery of quiet breathing during the initial weeks following chronic spinal cord injury (Dow et a l. 2009) The relative importance of the sCPP to baseline V T was similar across 2 8 wks post injury. There are several potential explanations for this plateau in the functional hemidiaphragm and thereby minimize paradoxical movements and enhance the effectiveness of contralateral hemidiaphragm contractions. This action might require only a relatively small number of active ipsilateral phrenic motor units as wou ld be expected to occur by 2 wks post injury (Fuller et al. 2006; Fuller et al. 2008) Secondly, in the spontaneously breathing rat, the output of the sCPP may not increase significantly after the first few weeks post C2HS injury. In this scenario the spontaneously occurring spinal cord plasticity associated with increased ipsilateral phrenic output (see (Goshgarian, 2003; Zimmer et al. 2008; Goshgarian, 2009) ) may be esse ntially complete within 2 wks. In addition vagal inhibition of ipsilateral phrenic activity is much more pronounced at eight vs. two wks following C2HS (Lee et al. 2010) Therefore, potential increases in sCPP output in spontan eously breathing, vagally intact animals could be constrained by activation of vagal afferent neurons. It follows that acute phrenicotomy in vagotomized rats may have revealed a time dependent increase in the contribution of the sCPP to V T We emphasize t hat the current data do not necessarily preclude a progressive increase in ipsilateral phrenic motor output after chronic C2HS as previously reported

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70 (Nantwi et al. 1999; Fuller et al. 2006) Nantwi et al (Nantwi et al. 1999) were the first to rep ort that spontaneous inspiratory activity recorded from the ipsilateral phrenic nerve increases over weeks to months post (Nantwi et al. 1999) anesthetized and spontaneously breathing rats were studied at intervals following C2HS. Spontaneous CPP activity could n ot be detected at 4 wks post C2HS but was present in ~50% of rats at 6 8 wks post C2HS, and occurred in 100% of rats at 12 16 wks post C2HS. In the current experiments ipsilateral phrenic nerve activity was not measured, and accordingly we draw no conclusi sCPP rather we used this preparation to examine the relative contribution of the ipsilateral phrenic nerve in C2HS rats to V T The apparent discrepancy between ks post injury) and the current data (i.e., a functional contribution of the ipsilateral phrenic nerve at 2 wks post C2HS) may indicate that tonic bursting in the ipsilateral phrenic nerve is functionally important in the first few weeks following C2HS. I n any case, if neural activity associated with the sCPP becomes more robust over time post injury (Nantwi et al. 1999; Fuller et al. 2006) then our data indicate that this is occurring in parallel with compensator y increases in other respiratory motor outputs (e.g. intercostal muscles). In this scenario, the relative contribution of the sCPP would remain static as shown by the current data. Numerous studies have shown that ipsilateral phrenic bursting after chron ic C2HS increases substantially during chemical respiratory challenge (Fuller et al. 2003; Fuller et al. 2006; Fuller et al. 2008; Fuller et al. 2009) Similarly, progressive improvements in hypercapnic E (whole body plethysmography) occur over weeks to months following C2HS (Fuller et al. 2008 ) These observations are consistent with the hypothesis that

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71 the primary role of the sCPP is to enable respiratory behaviors requiring large V T (Golder et al. 2003) Why then did ipsilateral phrenic activity make only modest contributions to V T when respirat ory drive was substantially increased with hypercapnia? We hypothesize that parallel increases in the contribution of the contralateral hemidiaphragm and/or intercostal muscles during respiratory stimulation ensures that the relative contribution of the s CPP to V T remains stable. In this regard, we again emphasize that the number of active motor units in the ipsilateral hemi diaphragm after chronic C2HS is unknown. For example, if the number of active phrenic motor units is relatively low under baseline conditions, the robust increase in ipsilateral phrenic nerve activity seen during chemical challenge in C2HS rats (Fuller et al. 2003; Fuller et al. 2006; Fuller et al. 2009) could reflect a very modest degree of motor unit recruitment. In this scenario, the apparently large increase in relative phrenic activity (% baseline), may actually make a small contribution to pulmonary biomechanics (and V T ), particularly during ongoing recruitment of accessory respiratory muscles. Compensation Following P hrenicotomy Changes in V T following diaphragm paralysis or paresis are mitigated by compensatory increases in the activity of other respiratory muscles (Sherrey & Megirian, 1990; Br ichant & De Troyer, 1997; Winslow & Rozovsky, 2003; Lane et al. 2009) For example, both rostral (1st space) and caudal (6 10th space) intercostal muscle EMG activity is substantially increased following bilateral phrenicotomy in rats (Sherrey & Megirian, 1990) Indeed, respiratory compensatory responses are so rob ust that rats maintain E after bilateral phrenicotomy with virtually no disturbance to their sleep

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72 wake cycles (Sherrey & Megirian, 1990) Spontaneously breathing, awake dogs also have only minor reductions (Katagiri et al. 1994) or even no change in V T (Stradling et al. 1987) following bilateral diaphragm paralysis. Unilateral diaphragm paralysis also evokes compensatory increases in accessory respiratory muscle activity incl uding the contralateral parasternal muscles (Teitelbaum et al. 1993; Katagiri et al. 1994) and transverse abdominus (Katagiri et al. 1994) These compensatory responses remain robust after vagotomy suggesting that vagally mediated lung afferents are not essential to this proces s (Teitelbaum et al. 1993) Rather, the increased output of other respiratory mu scles after diaphragm paralysis appears to reflect a diminished influence of inhibitory phrenic afferent neurons with additional contribution from arterial hypercapnia (Teitelbaum et al. 1993; Brichant & De Troyer, 1997) It is difficult, however, to compare the time course (e.g., onset time) of the compensatory responses across studies since prior work has induced diaphragm paralysis with selective anesthesias (e.g. lidocaine, bupivacaine, vecuronium) (Teitelbaum et al. 1993; Katagiri et al. 1994; Brichant & De Troyer, 1997) Accordingly, the onset of diaphragm paralysis will have a slower time course as compared to phrenicotomy, and to our knowledge prior studies have no t attempted to examine immediate (i.e., next breath) changes in V T Nevertheless, these previously described compensatory responses almost certainly contributed to the time dependent recovery of V T that occurred over the initial breaths following phrenicot omy (e.g. Fig ure 3 4). Axotomy and Phrenic Afferent N eurons Axotomy can trigger neuronal depolarization (Mandolesi et al. 2004) and increases in phrenic nerve activity (Sandhu et al. 2010) However, axotomy effects should be limited to ipsilateral phrenic motoneurons with l imited, if any impact on

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73 contralateral motor pools. In addition to axotomy induced changes in membrane potential, the acute removal of ipsilateral phrenic afferent activity could have a rapid impact on contralateral phrenic output. Approximately 45% of ph renic nerve axons are afferent fibers (Langford & Schmidt, 1983) and a few studies have shown that phrenic afferents can inhi bit phrenic motor activity on the contralateral side of the spinal cord (Goshgarian, 1981; Jammes et al. 1986) Removal of inhibitory phrenic afferents could therefore disinhibit contralateral phrenic motoneurons ( and possibly intercostal motoneurons (De Troyer, 1998) ) and thus facilitate the short term recovery of V T seen in C2HS rats. However, in a few supplemental experiments, we could not detect any rapid (i.e. within 1 3 breaths) changes in the EMG activity of the contralateral hemidiphragm or external intercostal muscles following phrenicotomy ( Figures 3 8 and 3 9 ). Phrenic afferents are also capab le of exerting excitatory effects on supraspinal respiratory neurons (Speck & Revelette, 1987b) Consistent with th is notion, we noted that phrenicotomy induced immediate changes in respiratory frequency in uninjured rats. However, phrenicotomy did not alter respiratory frequency in C2HS rats a finding that is consistent with plasticity in supraspinal respiratory neu rons following C2HS (Golder et al. 2001) Our data also suggest that phrenic afferent s are involved in triggering ABs following C2HS. Two prior studies indicate that ABs occur with m uch greater frequency following C2HS, but with substantially reduced volumes as compared to uninjured controls (Golder et al. 2003; Fuller et al. 2008) In the current study, we noted a sharp decline in AB frequen cy observed following phrenicotomy in C2HS animals. Accordingly, afferent information from the ipsilateral hemidiaphragm may be responsible

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74 for the increase in AB frequency that occurs after chronic C2HS injury (Go lder et al. 2003; Fuller et al. 2008) Regarding the contribution of the sCPP to the volume of ABs, a prior study which utilized a dual injury method (simultaneous C2HS and ipsilateral phrenicotomy) indicated that the sCPP made a significant contributio n (Gol der et al. 2003) However, in our study AB volumes in C2HS rats were similar both before and after the acute ipsilateral phrenicotomy. We suggest that determination of the direct contribution of the sCPP to AB volume would require phrenicotomy during an AB cycle, a technical challenge to say the least. Summary The sCPP makes a significant contribution to post injury breathing following C2HS during eupneic conditions. However, taken as a whole, our results indicate that increased output of the contralat eral diaphragm and other accessory respiratory muscles (i.e., compensation) is probably more important than the sCPP in promoting respiratory recovery after C2HS. Specifically, a time dependent increase in V T occurred over a two month period post injury d espite a plateau in the contribution of the sCPP (Figure 3 5 ). Accordingly, we hypothesize that spontaneous improvements in V T beyond the initial weeks post C2HS primarily reflect plasticity taking place in inspiratory motor circuits other than the sCPP. Plasticity in the neurons and/or networks controlling the accessory muscles has received considerably less attention than the phrenic circuitry (Mitchell et al. 2001; Mitchell & Johnson, 2003) However, at least o ne prior report indicates that intercostal motoneurons have a greater capacity for plasticity than do phrenic motoneurons (Fregosi & Mitchell, 1994) Functionally, the sCPP may serve as compensation in other motor pathways. This concept is addressed in Chapter 4.

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75 Our result s also validate the C2HS model for use in rehabilitation studies. The limited contribution of the sCPP to recovery (current data, (Fuller et al. 2008) ) provides a template for testing strategies intended to increase spinal synaptic efficacy after spinal cord injury (Fuller et al. 2003; Alilain et al. 2008; Alilain & S ilver, 2009; Vinit & Kastner, 2009; Dale Nagle et al. 2010) Moreover, the methods described herein may prove useful to evaluate the impact of treatments intended to strengthen the sCPP such as intermittent hypoxia (Fuller et al. 2003; Vinit & Kastner, 2009) activation of spinal neurons via channel rhodopsin (Alilain et al. 2008; Alilain & Silver, 2009) and neuronal replacement strategies (Polentes et al. 2004; Dougherty BJ 2010; White et al. 2010)

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76 Figure 3 1 A representative histological section illustrating a C2HS lesion. This 40 m transverse section was taken from the second cervical segment (C2) at 8 wks post injury and stained with Cresyl violet (image shown in grey scale) The absence of white and grey matter in the ipsilateral (IL) spinal cord suggests an anatomically complete C2HS. CL: contralateral; CC: central canal; VH: ventral horn; DH: dorsal hor n. Scale bar: 200 m

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77 Figure 3 2. Representative examples of V T during spontaneous breathing at baseline and hypercapnic respiratory challenge. The images show the integra ted 3 days, 2 wks and 8 wks following C2HS injury. The shaded area represents the hypercapnic challenge (7% inspired CO 2 ). The spikes in the record are augmented breaths (see te xt); the augmented breaths were harder to detect at 1 3 days post C2HS and are indicated by arrows. C2HS resulted in decreased V T during both baseline and hypercapnic challenge at all post injury time points. In addition, a reduction in the volume of augm ented breaths was observed following C2HS. Scaling is identical in all panels.

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78 Figure 3 3. Representative airflow traces showing the impact of phrenicotomy on V T Examples are provided from a control (uninjured) rat, and from separate rats studi ed 1 3 days or 8 wks following C2HS injury. The phrenicotomy procedure was done to the left phrenic nerve of all rats (C2HS injury was to the left side of the spinal cord). Phrenicotomy occurred between breaths and is denoted by the dashed lines. The i mmediate (i.e. next breath) change in V T T ) following phrenicotomy was used to estimate the contribution of signals traveling in the ipsilateral phrenic nerve to inspiratory V T This example shows that phrenicotomy caused an approximately 50% reduction in V T in the control rat. In contrast, phrenicotomy did not change V T at 1 3 days post C2HS and caused a transient reduction of approximately 20% at 8 wks. Scaling is identical in each panel. T I : inspiratory duration. T E : expiratory duration

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79 Figure 3 4. The effect of phrenicotom y on V T (ml/breath) during baseline breathing (A) and hypercapnic respiratory challenge (B). Phrenicotomy caused an immediate (next breath) and robust decline in V T in uninjured rats. In contrast, phrenicotomy caused modest declines in V T in C2HS rats. *: P < 0.05, ***: P < 0.001 compared to pre phrenicotomy values; #: P < 0.05, ##: P 3 days post C2HS.

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80 Figure 3 5. The immediate (next breath) decline in V T following phrenicotomy. The change in V T was expressed relative to the pre phrenicotomy baseline ( V T % decline). Note that performing the phrenicotomy procedure during the hypercapnic challenge caused declines in V T similar to what occurred during the baseline condition. ***: P < 0.001 compared to all C2HS; ##: P < 0.01 compared to 1 3 days post C2HS; ###: P < 0.001 from 1 3 days post C2HS.

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81 Figure 3 6. The impact of phrenicotomy on the hype rcapnic ventilatory response. Tidal volume (V T ), respiratory frequency (f) and ventilation ( E) were measured during hypercapnic respiratory challenge before and after (> 60 seconds) phrenicotomy in control uninjured rats and rats wit h C2HS injury. The phrenicotomy procedure blunted the hypercapnic ventilatory response in uninjured rats, but did not impact the response in C2HS rats. A progressive recovery in the hypercapnic ventilatory response was observed whereby E was highest in rats at 8 wks following injury. ***: P<0.001, **: P<0.01, *: <0.05 compared to uninjured; #: P<0.05 compared to 1 3 days post C2HS

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82 Figure 3 7. The impact of phrenicotomy on the volume of spontaneous augmented breaths (AB). The data presented here were collected during a hypercapnic respiratory challenge. Two way ANOVA was not possible with the poikilocapnic baseline data since ABs were not observed in control uninjured rats at baseline. Only the uninjured animals sh owed a reduction in AB volume following phrenicotomy. ***: P < 0.001, ** P< 0.01, *: P< 0.05 compared to uninjured.

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83 Figure 3 8. E xample of contralateral intercostal EMG (first intercostal space) recorded at 8wks post C2HS. P hrenicotomy of the left nerve (i.e., ipsilateral to C2HS) was performed at the dashed red line (top) These results demonstrate no following the phrenicotomy. The procedure was done in N =2 rats A mean response is shown in the bottom panel.

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84 4 Days 2 Weeks Figure 3 9. Examples of contralateral diaphragm EMG (4 days [ A ] and 2 weeks [ B ] ) following C2HS. In both examples, a phrenicotomy of the left nerve (i.e., ipsilate ral to C2HS) was performed at the red arrow. We were able to detect

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85 Table 3 1. Age and weight values for all experimental groups. C2HS rats were grouped at 1 3 days, 2wks, and 8wks post injury. Baseline Phrenicotomy Hypercapnic Phrenicotomy N Age (days) Weight (g) N Age (days) Weight (g) Uninjured 5 131 13 382 17 4 131 13 421 35 1 3 days 6 91 1 302 6*** 4 97 1 326 18* 2wk 5 109 1 333 5** 4 111 1 359 9 8wk 4 152 1 413 8 5 153 0 397 16 Values are mean SE ***: P < 0.001; **: P < 0.01; *: P < 0.05 from uninjured. Statistics represent o ne way ANOVA. Table 3 2. Blood gas a nd mean arterial blood pressure (MAP) values taken during baseline ventilation. P a O 2 P a CO 2 pH MAP (mmHg) Uninjured 190 14 45 2 7.30 0.01 90 5 1 3 days C2HS 193 7 66 6*** 7.16 0.03*** 80 7 2wk post C2HS 199 6 45 2 7.29 0.02 81 5 8wk post C2HS 172 8 47 2 7.28 0.02 91 6 Values are mean s SE. ***: P < 0.001 from all other groups; one way ANOVA.

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86 Table 3 3. Respiratory frequency (f), inspiratory (T I ) duration and expiratory (T E ) duration during pre ph renicotomy baseline (BL) and hypercapnic respiratory challenge (CO 2 ). Values are mean SE using 2 way RM ANOVA.*: P < 0.05 ***P < 0.001 from baseline. f (breaths/min) T I (sec) T E (sec) BL CO 2 BL CO 2 BL CO 2 Uninjured 97 3 98 5 0.24 0.01 0.23 0.01* 0.38 0.03 0.38 0.04 1 3 days C2HS 77 10 75 8 0.34 0.05 0.35 0.05*** 0.47 0.12 0.46 0.10 2 wk C2HS 97 9 89 9* 0.28 0.04 0.30 0.04*** 0.36 0.09 0.41 0.11 8 wk C2HS 110 11 100 15* 0.26 0.03 0.28 0.03*** 0.29 0.08 0.35 0.11

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87 Table 3 4. Respiratory frequency (f), inspiratory duration (T I ) and expiratory duration (T E ) pre and post ipsilateral phrenicotomy (PhrX) during baseline ventilation. f (breaths/min) Pre PhrX Breath 1 Bre ath 3 Breath 5 60 sec Uninjured 983 90 2 *** 88 2 *** 87 2 ** 9 3 2 1 3 days C2HS 84 8 84 8 82 8 83 7 85 8 2wk C2HS 94 6 96 5 94 5 9 3 6 92 6 8wk C2HS 10 9 10 11 2 9 11 0 10 11 2 10 111 9 T I (sec) Uninjured 0.23 0.01 0.20 0.01 0.2 2 0.01 0.2 4 0.0 0 0.2 8 0 .0 4 1 3 days C2HS 0. 22 0.0 1 0.21 0.0 1 0. 21 0.0 1 0. 22 0.0 1 0. 27 0.0 4 ** 2wk C2HS 0. 29 0.0 4 0. 28 0.01 0. 29 0.0 4 0. 29 0.0 4 0. 31 0.0 5 8wk C2HS 0. 22 0.0 1 0. 22 0.0 1 0. 22 0.0 1 0. 23 0.0 1 0. 24 0.0 1 T E (sec) Uninjured 0. 38 0.0 2 0.47 0.01 ** 0.46 0.0 2 ** 0. 4 5 0 .0 2 ** 0. 37 0.0 5 1 3 days C2HS 0. 53 0.0 6 0.53 0.0 6 0.54 0.0 6 0.53 0.06 0.4 7 0.0 6 ** 2wk C2HS 0. 36 0.0 7 0. 36 0.0 7 0. 36 0.0 7 0.36 0.0 8 0. 3 5 0.0 8 8wk C2HS 0.3 4 0.0 4 0.3 3 0.0 3 0.3 4 0.0 4 0.3 2 0. 04 0.31 0.0 3 Values are mean SE using 2 way RM ANOVA.*: P < 0.0 5; **: P < 0.01; ***: P < 0.001 from pre phrenicotomy.

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88 Table 3 5. Respiratory frequency (f), inspiratory duration (T I ) and expiratory duration (T E ) pre and post ipsilateral phrenicotomy (PhrX) during hypercapnic respiratory challenge. f (breaths/min) Pre PhrX Breath 1 Breath 3 Breath 5 60 sec Uninjured 908 86 9 86 8 83 8 83 7 1 3 days C2HS 70 4 71 4 69 4 71 6 70 5 2wk C2HS 102 8 105 9 103 10 103 8 105 9 8wk C2HS 91 6 92 6 90 6 82 7 *** 88 4 T I (sec) Uninjured 0.25 0.01 0.22 0.01 *** 0.24 0.01 0.25 0.01 0.27 0.01 ** 1 3 days C2HS 0.41 0.02 ### 0.40 0.02 0.40 0.02 0.40 0.02 0.44 0.01 *** 2wk C2HS 0.45 0.01 ### 0.45 0.01 0.44 0.01 0.45 0.01 0.46 0.01 8wk C2HS 0.36 0.05 # 0.36 0.05 0.37 0.05 0.37 0.04 0.37 0.05 T E (sec) Uninjured 0.44 0.07 0.51 0 .09 0.48 0.07 0.50 0.08 0.48 0.08 1 3 days C2HS 0.46 0.04 0.46 0.04 0.47 0.04 0.46 0.06 0.42 0.05 2wk C2HS 0.15 0.03 # 0.14 0.03 0.15 0.04 0.14 0.03 0.13 0.04 8wk C2HS 0.30 0.08 0.30 0.08 0.31 0.08 0.38 0.10 ** 0.31 0.08 Values are mean SE using 2 way RM ANOVA.*: P < 0.05; **: P < 0.01; ***: P < 0.001 from pre phrenicotomy values; #: P < 0.05; ##: P < 0.01; ###: P < 0.001 from Uninjured

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89 C HAPTER 4 SPONTANEOUS RECOVERY OF INSPIRATORY INTER COSTALS FOLLOWING HIGH CERVICAL HEMISE CTION IN RATS Spontaneous and progressive improvements in inspiratory tidal volume (V T ) are observed in rats following complete hemisection of the C2 spinal cord (C2HS) (see Chapter 3 (Fuller et al. 2008) ). However, the fundamental pattern of neuromuscular recruitment facilitating V T recovery has not been demonstrated. Plasticity in phrenic motor pathways restores partial functionality to the ipsilateral hemidiaphragm (i.e. the (Lane et al. 2008b; Goshgarian, 2009) Yet, significance of this restored function as it relates to generation of V T appears to plateau by 2 weeks post injury in spontaneously breathing rats (see Chapter 3). Thus, complementary muscle groups, aside from the ipsilateral diaphragm, are likely the primary facilitators of spontaneous long term V T recovery (see Chapter 3 (Golder et al. 2003) ). In the C2HS model of cervical SCI, bulbospinal respiratory drive to phrenic motoneurons opposite (i.e. contralateral) to the lesion remains intact. Thus, progressive recovery of V T may conceivably develop from on going plasticity in contralateral phrenic neu ral and neuromuscular elements. Previous studies report increased contralateral diaphragm activity following hemisection injury or unilateral diaphragm paralysis (Teitelbaum et al. 1993; Katagiri et al. 1994; Miyat a et al. 1995; Golder et al. 2001; Rowley et al. 2005) Therefore, it is probable that increased contralateral diaphragm activity plays an important role in V T maintenance, especially in the acute phase following C2HS. However, it is unclear whether pl asticity in the contralateral phrenic motor nucleus or at the level of the neuromuscular junction is capable of the adaptations required for the progressive recovery of V T observed between 2 and 8 weeks post

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90 C2HS (see Chapter 3). Indeed, increased demand p laced o n contralateral phrenic motoneurons diminish the ir ability to express (Fuller et al. 2005; Doperalski & Fuller, 2006; Fuller et al. 2006) Should this be the case, recruitment of additional inspiratory muscles, like the external intercostals (IC), may be required to facilitate long term V T recovery. Plasticity in IC muscle groups in response to respiratory relat ed stimulation (e.g. repeated carotid sinus nerve stimulation) is robust, perhaps more so than in the phrenic motor system (Fregosi & Mitchell, 1994) Yet studies examining progressive changes in IC activation following C2HS are limited (Zimmer et al. 2007) Here we examined IC muscle activation using electromyography (EMG) in the weeks following C2HS in anes thetized, spontaneously breathing adult rats. We hypothesized that IC muscles in particular those ipsilateral to the C2HS, would display progressive augmentation in neural output following C2HS during both poikilocapnic baseline conditions and during hype rcapnic respiratory challenge. Since inspiratory IC muscles are generally activated along a rostral to caudal activation gradient (Gandevia et al. 2006; Butler & Gandevia, 2008) we focused our studies on activity within rostral inspiratory IC s of the first rib space. Further, since descending bulbospinal innervation to inspiratory IC motoneurons appears primarily polysynaptic through thoracic interneurons (Merrill & Lipski, 1987; Tian & Duffin, 1996; Saywell et al. 2011) we hypothesized that any progressive augmentation of IC activity may be associated with changes in thoracic interneuron circuitry (Lane et al. 2008b)

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91 Materials an d Methods All experimental procedures were approved by the Institutional Animal Care and Use Committee at the University of Florida. Animals A total of 27 adult, male Sprague Dawley rats were obtained from Harlan Laboratories Inc. (Indianapolis, IN, USA). Rats receiving C2HS injury were grouped by the following post 3 days), 2 weeks (2wk) or 8 weeks (8wk). Uninjured control rats were age matched to the 2wk injury group (N= 3 ) or the 8 wk group (N= 4 ). A summary of the experimen tal groups is presented in Table 4 1 Spinal Cord Injury Our anesthesia and injury methods have been previously described (Fuller et al. 2008; Fuller et al. 2009) Briefly, r ats were anesthetized by injection of x ylazine (10 mg/kg, s.q.) and ketamine (140 mg/kg, i.p., Fort Dodge Animal Health, IA, USA). The spinal cord was exposed at the C2 level via a dorsal approach, and a left C2HS lesion was induced using a microscalpel followed by aspiration. The dura and over lying muscles were sutured and the skin closed with stainless steel wound clips (Stoelting, IL, USA). Rats were given an injection of yohimbine (1.2 mg/kg, s.q., Lloyd, IA, USA) to reverse the effect of xylazine. Following surgery, animals received an anal gesic (buprenorphine, 0.03 mg/kg, s.q., Hospira, IL, USA) and sterile lactated Ringers solution (5 ml s.q.). Post surgical care included administration of buprenorphine (0.03 mg/kg, s.q.) during the initial 48 hours post injury and delivery of lactated Rin gers solution (5 ml/day, s.q) and oral Nutri cal supplements (1 3 ml, Webster Veterinary, MA, USA) until adequate volitional drinking and eating resumed.

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92 Experimental P reparation These procedures were adapted from our prior publications (Doperalski et al. 2008; Fuller et al. 2008; Fuller et al. 2009; Lane et al. 2009; Sandhu et al. 2009a) Isoflurane anesthesia (3 4% in O 2 ) was induced in a closed chamber followed by i.p. injection of urethane (1.6g/kg, Sigma, St. Louis, MO, USA). The adequacy of urethane anesthesia was confirmed by testing limb withdrawal and palpebral reflexes. Rats were maintained in a supine position throughout the protocol. The trachea was cannulated in the mid cervical region and connected in series to a custom designed, small animal pneumotachograph and volumetric pressure transducer (Grass Instruments, Quincy, MA, USA) for measurement of respiratory air flow. Partial pressure of arterial oxygen (P a O 2 ) was maintained above 150 mmHg by deli vering a hyperoxic gas mixture (F I O 2 =0.50, balance N 2 design (Fuller et al. 1998) The femoral vein was catheterized (PE 50) to enable supplemental urethane anesthesia (0.3 g/kg, i.v. Sigma, St. Louis, MO, USA) if indicated. Another PE 50 catheter was placed in the femoral artery and connected to a pressure transducer (Statham P 10EZ pressure transducer; amplifier CP122 AC/DC strain gauge amplifier, Grass Instruments, West Warwick, RI USA) for arterial pressure and blood gas measurements. Rostral and caudal intercostal muscles were exposed ventrolaterally following reflection of overlying pectoral musculature from sternal midline. The ipsilateral phrenic nerve was isolated in the cer vical region via a ventral approach (Sandhu et al. 2009a; Lee et al. 2010; Sandhu et al. 2010) The exposed nerve was covered in mineral oil but not manipulated at this time. Electromyography (EMG) electrodes w ere custom fabricated from Teflon coated tungsten wire ( A M Systems, Sequim, WA ). Wire was threaded through a 25 gauge hypodermic needle (Tyco

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93 Healthcare, Mansfield, MA) and the ends (~2 3mm) stripped of their Teflon coating. EMG electrodes were inserted bilaterally into external intercostal muscles of either the first (i.e. rostral) or the sixth intercostal space (i.e. caudal) to record inspiratory muscle activation. EMG b urst signals were matched to the inspiratory phase of the airflow trace to confirm a ccurate placement in the external intercostals Arterial blood samples (0.2 ml) were drawn during the baseline period (see Experimental Protocols) and analyzed for P a O 2 carbon dioxide partial pressure (P a CO 2 ) and pH (i STAT, Waukesha, WI). Blood gas measu res were corrected to rectal temperature which was monitored by rectal thermistor and maintained at 37.5 1 C by a servo controlled heating pad (model TC 1000, CWE Inc., Ardmore, PA, USA). Experimental Protocols Our primary goal was to determine the spo ntaneous recovery of intercostal EMG activity during baseline and respiratory stress following C2HS. Therefore, b aseline EMG activity was recorded over a 20 minute period during which rats breathed the hyperoxic gas mixture described above. This b aseline period was followed by a five minute hypercapnic respiratory challenge (7% CO 2 50% O 2 balance N 2 ) Rats were then returned to baseline conditions O nce breathing had returned to the pre hypercapnic values (i.e. baseline) a second hypercapnic challenge wa s initiated in a subset of rats (N=20, Table 4 1 ). The previously exposed ipsilateral phrenic nerve was cut during the third minute of the second hypercapnic challenge to assess changes in compensatory intercostal activity following the complete removal o f ipsilateral diaphragm contributions ( see Chapter 3 ) This time point corresponded to a stable period of hypercapnic V T that was similar to the initial hypercapnic response. All rats were returned to baseline conditions following the second hypercapnic ch allenge.

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94 Anatomical Tracing Protocols Recombinants of the Bartha strain of pseudorabies virus (PRV) or cholera toxin subunit (CT ) 0.1% in distilled water were used as anatomical tracers to examine neural circuitry associated with the rostral inspiratory intercostal s in a small cohort of uninjured rats (N=2) CT is a monosynaptic tracer and will label only those cells projecting to the site of tracer application (i.e. intercostal motoneurons) (Yates et al. 1999) whereas PRV is transynaptically transported and will infect t he entire intercostal circuit over time. 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). Propagation and culture methods for the PRV have been extensively detailed (Banfield et al. 2003; Lane et al. 2008b) Motoneuron projections to the rostral external ICs were retrogradely labeled using PRV (10 l per injection) in combination with CT (1:1, 10 l per injection). A small incision was made laterally under the ipsilateral axilla (i.e. left) in spinal intact control rats, and the skin and muscles retracted to expose the external ICs. Tracers were injected into external ICs within the firs t three rib spaces (10 l per IC, 30 l total volume per rat) via Hamilton syringe (Hamilton Company, Reno, NV). Animals were left to survive for 96 hours following tracer injection. This time course was based on pilot data showing primary IC motoneuron co l abeling and transneuronal labeling of thoracic interneurons. Spinal Cord H istology and Immunohistochemistry At the conclusion of the phrenicotomy experiment s rats were euthanized by systemic perfusion with saline followed by 4% paraformaldehyde (Sigma, S t. Louis, MO, USA). The cervica l spinal cord was removed, and 4 0 m sections were made in the transverse plane using a vibrotome. For histological analysis, t issue sections were

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95 mounted on glass slides (Fisher Scientific, Pittsburgh, PA, USA), stained wit h Cresyl violet and evaluated by ligh t microscopy. An example o f a C2HS lesion is shown in Figure 4 1 Consistent with our previous publications (Fuller et al. 2008; Lane et al. 2008b; Fuller et al. 2009; Sandhu et al. 2009a) the apparent absence of healthy white matter in the ipsilateral C2 spinal cord was taken as confirmation of an anatomically complete C2HS (Fuller et al. 2009) For immunohistochemical analysis of PRV and CT labeling, transverse vibratome sections were washed in PBS (0.1 M, 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 n onspecific protein labeling (10% serum in 0.1 M PBS with 0.03% Triton X). Sections were then incubated at 4C overnight with primary antibodies against PRV (rabbit anti PRV; Rb133/Rb134, raised against whole, purified PRV particles that were acetone inacti vated), 1:10,000 (generously provided by Dr. Lynn Enquist) and CT (polyclonal goat antiserum to purified CT B (choleragenoid isolated from Vibrio cholerae type Inaba 569B; List Biological Laboratories, Campbell, CA; product No. 703, lot No. 7032A5, 1:10,000). On the following day, tissue was washed in PBS (0.1 M, 3 X 5 minutes), incubated for 2 hours at room temperature in either a biotinylated secondary antibody (donkey anti rabbit; Jackson Immunocytochemicals, West Grove, PA; 1:200) or fluorescently conjugated secondary antibody (donkey anti rabbit FITC, 1:100, or don key antigoat Texas red, 1:100; Jackson Immunocytochemicals) and rewashed in PBS (3 X 5 minutes). Tissue processed for light microscopy was further incubated for 2 hours in an avidin biotin complex (ABC Elite Vectastain Kit; Vector Laboratories, Burlingame, CA),

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96 given a third series of washes in PBS, and processed for antigen visualization with diaminobenzidine (DAB;Sigma, St. Louis, MO). Tissue processed for fluorescence miscroscopy was washed in PBS following secondary antibody incubation and slide mounted Tissue sections from negative control animals (i.e. not used for tracing) showed no labeling with either antibody (not shown). Data Analysis Calibration of the pneumotachograph was accomplished using a series of constant volume injections with varying a irflow rates as previously reported (see Chapter 3). Respiratory airflow signals were amplified (x100K; CP122 AC/DC strain gage amplifier, Grass Instruments) and recorded on a PC using Spike2 software (Cambridge Electronic Design Limited). The inspiratory phase of the airflow signals was a customized Spike2 software script (Cambridge Electronic Design Limited) as an indicator of V T Inspiratory and expiratory duration (T I and T E respectively) were calculated (Lee et al. 2009) based on the integrated airflow traces as previously reported (see Chapter 3). Respiratory frequency (f, breaths*min 1 ) was calculated as 60/( T I + T E ). One way analysis of variance (ANOVA) was used to compare body weight, blood gases, and mean arterial blood pressure (MAP) across groups. Respiratory parameters including T I T E f and V T during baseline and hypercapnic respiratory challenge were compared using two way repeated measures (RM) ANOVA and Student Newman Keuls post hoc test. For this ANOVA, factor 1 was treatment (i.e. control or lesion group), factor 2: condition (baseline or respiratory challenge). Additionally, in rats undergoing p hrenicotomy, all respiratory parameters were averaged over the 10 breaths that immediately preceded ipsilateral phrenic nerve sectioning. These values were

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97 compared to values measured at the first breath following cut and at 30 and 60 seconds following ph renicotomy. Data were compared using 2 way RM ANOVA and the Student Newman For statistical an alysis of bilateral integrated IC EMG activity, a 30 second period of stable bursting during baseline and within the last minute of hypercapnic respiratory challenge were analyzed using two way RM ANOVA with factors of time post C2HS and condition (i.e. ba seline or hypercapnia). Ipsilateral EMG signals were compared relative to contralateral signals during both baseline and hypercapnic challenge The data for this comparison were not normally distributed, therefore, a one way ANOVA on ranks ethod for pairwise multiple comparisons was used All data are presented as the mean standard error. A P value of < 0.05 was considered statistically significant. Results A time dependent change in body mass occurred following C2HS as previously reported (Fuller et al. 2006; Doperalski et al. 2008; Fuller et al. 2008) Thus, both the 1 3 day and 2wk post C2HS groups weighed less than uninjured and 8wk post injury rats overall ( Table 4 1 ) P a O 2 was similar betwe en groups during baseline breathing ( Table 4 2 ) likely as a result of oxygen supplementation during our experiments However, the acutely injured rats (1 3 days post injury) were hypoventilating (Table 4 3 ) as reflected by increased P a CO 2 (P < 0.001 vs. o ther groups; Table 4 2) and decreased pH (P < 0.05 vs. other groups; Table 4 2 ). Consistent with prior reports (Doperalski & Fuller, 2006; Fuller et al. 2008) no group differences in MAP were observed (Table 4 2).

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98 Effect of C2HS on V entilation Before describing the recovery of intercostal activity following C2HS, we first report the effects of injury on breathing. Table 4 3 illustrates changes in V T f, T I and T E in the days and weeks following C2HS. As anticipate d, rats 1 3 days following injury produced the smallest V T during both baseline (P < 0.05 vs. other groups) and hypercapnic respiratory challenge (P < 0.001 vs. other groups; Table 4 3 ). By 2 wks post C2HS, rats were able to produce equivalent V T when comp ared to controls during both baseline and hypercapnic challenge. This result differs from our previous study (see Chapter 3) where 2 wk C2HS rats showed significantly blunted V T compared with uninjured control rats. Variables likely contributing to this di fference include lower baseline P a CO 2 values (~39 mmHG compared to ~45 mmHG in Chapter 3) in uninjured control rats within this study compared to Chapter 3 and higher mean weights (~348g compared to ~333g) in 2 wk C2HS rats within this study compared to C h apter 3 Prior studies have demonstrated a compensatory increase in respiratory f to counterbalance declines in V T following C2HS for maintenance of E (Fuller et al. 2003; Fuller et al. 2006; Fuller et al. 2008; Fuller et al. 2009) Here, we noted significant declines in f during both baseline and respiratory challenge (P < 0.01, Table 4 3 ) in the 1 3 day C2HS group associated with lengthening of T E (P < 0.01, Table 4 3 ). This change in f m ay have resulted from experimental conditions employed (e.g. oxygen supplementation or anesthesia) or may be related to declines in MAP seen in this group of rats. The significance of these findings as they relate to recovery of IC activity is unclear. Als o, the effects of C2HS on f were opposite in 2wk C2HS rats. These animals displayed

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99 increased f compared to all other groups (P < 0.001, Table 4 3 ) resulting from declines in T E (P < 0.05, Table 4 3 ). Progressive Changes in Rostral Intercostal EMG Activity Following C2HS Representative examples of bilateral rostral IC EMG activity during baseline and hypercapnic respiratory challenge are shown in Figure 4 2 C2HS eliminated activation of ipsilateral ICs during baseline breathing over the first three days po st injury. Modest EMG activity was observed in the ipsilateral ICs during respiratory challenge, but appeared reduced compared to controls ( Figure 4 2 ). As anticipated following C2HS, reductions in ipsilateral IC EMG activity were mirrored by enhanced EMG activity in the contralateral inspiratory ICs. This was observed under both baseline and challenge conditions in the first three days post injury ( Figure 4 2 ). Remarkably, by 2 wks post C2HS, robust ipsilateral EMG activity was observed during both respira tory conditions and contralateral activity appeared to diminish (Figure 4 2). This trend persisted 8 wks following C2HS, with further augmentation of the ipsilateral IC EMG burst amplitudes and reduction of contralateral output during both baseline and res piratory challenge ( Figure 4 2 ). Quantification of EMG activity during baseline and hypercapnic respiratory challenge are shown in Figures 4 3 and 4 4 Uninjured control rats showed increased ipsilateral EMG activity during respiratory challenge (0.20 0.0 4 a.u., P < 0.001) compared with baseline (0.120.04 a.u., Figure 4 3). Acutely (1 3 days) following C2HS, rats displayed reduced ipsilateral IC activity during both baseline (0.010.01 a.u.) and respiratory challenge (0.030.01 a.u., P < 0.01) and showed an inability to significantly increase ipsilateral IC output during respiratory challenge (Figure 4 3 ). However, b y 2 and 8 wks post C2HS, baseline ipsilateral IC activity appeared to

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100 normalize Specifically, baseline EMG amplitudes returned to 0.1 0.02 a.u. by 2 wks post C2HS and 0.14 0.03 a.u. by 8 wks post C2HS. In addition, ipsilateral EMG activity during hypercapnic challenge appeared to fully recover. Rats 2 wks post C2HS produced ipsilateral EMG amplitudes of 0.16 0.03 a.u. and by 8 wks this had in creased to 0.20 0.04 a.u., both similar to uninjured control rats (Figure 4 3). Comparatively little change in contralateral IC EMG activity was observed following C2HS (Figure 4 3 ) and rats retained their ability to augment contralateral IC muscle activit y in response to respiratory challenge (all groups P < 0.05 compared to baseline, Figure 4 3 ). Ipsilateral IC EMG activity was standardized as a percentage of contralateral IC activity to determine if rats preferentially activate ipsilateral ICs following C2HS (Figure 4 4). In the first 3 days following C2HS, minimal activation of the ipsilateral IC muscles was observed during either baseline (12 6%) or respiratory challenge (16 5%) when compared to contralateral ICs (Figure 4 4 ). However, by 2 wks post in jury a significant shift was observed such that rats preferentially utilized ipsilateral ICs during both baseline (332 105%, P < 0.05 compared to 1 3 day group) and hypercapnic challenge (269 63%, P < 0.05 compared to 1 3 day group; Figure 4 4 ). This trend continued 8 wks post C2HS as ipsilateral EMG activity continued to increase relative to contralateral activity during baseline (710 542%, P < 0.05 compared to 1 3 day group) and hypercapnic challenge (370 212%, P < 0.05 compared to 1 3 day group; Figure 4 4 ). I mpact of I psilateral P hrenicotomy on Ventilation Acute section of the ipsilateral phrenic nerve (i.e. left) during hypercapnic respiratory challenge caused immediate declines in V T similar to our previous report (see Chapter 3 and (Dougherty et al. 2011) ). Specifically, significant (P < 0.01 compared to pre phrenicotomy, Table 4 4 ) reductions in V T were observed in the first

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101 breath following phrenicotomy in uninjured control rats and in rats 2 and 8 wks post C2HS. As V T during respiratory challenge was already greatly reduced in rats 1 3 days post injury (see above), no significant declines were expected following phrenicotomy (see Chapter 3). Also in agreement with prior reports (see Chapter 3), rats in the C 2HS groups reestablished V T following phrenicotomy within 30 seconds, whereas control rats required >60 seconds to bring V T back to pre phrenicotomy levels (P < 0.001, Table 4 4 ). In addition, phrenicotomy caused reduced frequency in control rats (P < 0.05 Table 4 4 ), but not in C2HS groups. We have previously hypothesized that this reduced frequency in uninjured control rats may result from acute disruption of ipsilateral diaphragm afferent signals following phrenicotomy (see Chapter 3). Immediate Impact of Ipsilateral P hrenicotomy on Intercostal EMG Activity We previously reported that acute removal of IL diaphragm activity during hypercapnic respiratory challenge caused modest reductions in V T that were quickly compensated for by other inspiratory muscle groups following C2HS (see Chapter 3). To determine if the rostral inspiratory ICs were involved in this short term compensatory response, we explored changes in IC EMG activity following acute ipsilateral phrenicotomy. The immediate impact of phrenicotom y on rostral IC EMG activity was assessed in both absolute values (i.e., arbitrary units; a.u., Figure 4 5) and relative to the preceding breaths (i.e. % of pre phrenicotomy values, Figure 4 6). In uninjured control rats, phrenicotomy during respiratory ch allenge caused immediate (i.e. next breath) recruitment of ipsilateral IC musculature. Specifically, ipsilateral EMG amplitudes increased from 0.18 0.03 a.u. to 0.21 0.03 a.u. (P < 0.5, Figure 4 5 ) in the first breath following phrenicotomy, and remained e levated (~0.22 0.02 a.u.) to 60 seconds post phrenicotomy (P < 0.05, Figure 4 5 ). Conversely, no changes in ipsilateral

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102 IC EMG activity were observed following phrenicotomy in any of the C2HS groups (Figure 4 5 ). As expected, ipsilateral IC EMG activity wa s sharply reduced in the 1 3 day C2HS group (P < 0.05, Figure 4 5) consistent with previous results (see above and Chapter 3). In addition, no significant changes in contralateral IC EMG activity were observed following ipsilateral phrenicotomy in any grou p (Figure 4 5). The relative change in IC EMG amplitude following phrenicotomy is highlighted in F igure 4 6 Although an apparent trend for gradually enhanced ipsilateral IC activation following phrenicotomy was observed in uninjured control rats (Figure 4 6 ), this did not reach statistical significance (P = 0.19, 2 way RM ANOVA). However, 60 seconds post phrenicotomy, uninjured control rats displayed a 12812% increase in ipsilateral IC activation (compared to pre phrenicotomy) (Figure 4 6 ). This was sign ificantly elevated (P < 0.05) compared to each of the C2HS groups. As suggested by the absolute EMG data (Figure 4 5 ), no progressive changes in contralateral IC activity were observed following ipsilateral phrenicotomy (Figure 4 6 ). Neural Circuitry of th e Rostral Inspiratory Intercostals Figure 4 7 illustrates initial results with retrograde anatomical tracing of the rostral inspiratory intercostals using both transynaptic (PRV) and monosynaptic (CT ) tracers. Co injection of these tracers into the rostr al three rib spaces (left side only) of uninjured rats produced labeling of primary IC motoneurons ipsilateral to the injection (Figure 4 7 A). Co labeling of CT and PRV appeared in a portion of labeled motoneurons (Figure 4 7A). However, a majority of ce lls appeared to stain predominately with either CT or PRV (i.e. no co labeling, Figure 4 7A), highlighting the need for improved injection or immunodetection methods for future quantification studies. The 96 hour time point was sufficient to allow propa gation of the PRV to a set of pre motor thoracic interneurons

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103 near the s pinal midline (Figure 4 7B). Thus, our data provide preliminary proof of principle evidence to support the use of PRV injected into the inspiratory intercostals for future quantificati on of IN circuitry changes associated with C2HS. These studies are currently on going. Caudal Intercostal EMG Activity Following Ipsilateral Phrenicotomy The preceding results illustrate d changes in IC EMG activity recorded in rostral (i.e. first) rib spac es following C2HS and in response to ipsilateral phrenicotomy. In a subset of rats, EMG recordings were made from caudal (i.e. sixth rib space, see Methods) external IC muscles to asses s differential compensatory activation. Ultimately, this recording meth od was proven unreliable for accurate quantification of IC EMG contamination below). However, recordings of caudal EMG activity in uninjured control rats (n=10 total) during ipsilateral phrenicotomy led to a series of experiments exploring a possible as additional exploratory studies are described here. Most individual study components were completed u sing 3 rats or less, insufficient for proper statistical analysis. Therefore, qualitative data are presented with supporting figures. Figure 4 8 depicts an uninjured control rat that underwent ipsilateral phrenicotomy with simultaneous recording of caudal IC EMG activity. In this rat, as with several other (n=6) uninjured controls, showed a marked reduction in ipsilateral caudal IC EMG activity was observed immediately (i.e. next EMG burst) following ipsilateral phrenicotomy. This was matched by an immedia te, though less robust, increase in the contralateral caudal IC EMG signal (Figure 4 8 ). It is important to note that similar responses were never observed in the caudal IC EMG of rats following C2HS (not

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104 shown). Additionally, when the ipsilateral phrenico tomy was followed some time later (~15 20 minutes) with a contralateral phrenicotomy, similar immediate declines in the contralateral caudal IC EMG signal were seen (Figure 4 9 ) prior to the progressive recruitment of inspiratory ICs for survival. These ob servations led to the hypothesis that an excitatory, phrenic afferent mediated reflex pathway connected the ipsilateral hemidiaphragm with caudal inspiratory IC muscles. de cline in caudal IC EMG by painting a local anesthetic (lidocaine, 1% in 100 l saline) onto the intact phrenic nerve of an uninjured control rat (Figure 4 10 ). As predicted, introduction of the anesthetic produced a decline in ipsilateral IC EMG activity an d parallel enhancement of contralateral IC EMG activity consistent with the phrenicotomy results, albeit with a slower time course (Figure 4 10 ). These results supported our reflex hypothesis, but did not prove the existence of the proposed pathway. There fore, we attempted to directly activate the exci tatory reflex using electrical stimulation. The proximal end of a cut phrenic nerve in an uninjured (i.e. spinal intact) control rat was stimulated across various stimulation intensities and configurations to isolate parameters activating our proposed reflex (Figure 4 11 and 4 12). Initially, 100 millisecond trains of 100 A stimulation (pulse duration of 0.5 milliseconds) produced transient changes in caudal IC EMG activity (Figure 4 11 ) suggesting a direct re flex connection. However, caudal IC EMG activity reduced with electrical stimulation; opposite to the excitatory response expected based on phrenicotomy results. In addition, repeated follow up studies using identical experimental conditions failed to rep roduce the original stimulation outcomes (Figure 4

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105 12 fibers), unable to consistently be activated u sing the electrical stimulation parameters chosen. Therefore, in a separate uninjured control rat with phrenic nerves intact we attempted to directly activate c fiber endings in the diaphragm using capsaicin, the active ingredient in chili peppers, painted onto the ipsilateral diaphragm (Lee et al. 2007; Lee et al. 2008) Similar capsaicin activation of phrenic afferents has been utilized in dogs (Revelette et al. 1988; Huss ain et al. 1990) but d espite repeated attempts using a range of dosages (10 1000ppm), no obvious changes in caudal IC EMG activity could be elicited (Figure 4 13 ). However, an important caveat to this methodology must be considered ; it is unknown whethe r the TRPV 1 receptor necessary for capsaicin binding is present within the diaphragm of rat s Therefore, it is possible that this technique did not facilitate activation of the phrenic afferent fibers of interest. Following multiple unsuccessful attempts to elicit a motor response via our phrenicotomy induced decline in caudal IC EMG activity was, in actuality mediated by phrenic afferent fibers. It was plausible, based o n our collective results that observed caudal IC changes with phrenicotomy may represent a phrenic efferent phenomenon as opposed to an afferent reflex pathway. Therefore, in two uninjured control rats, we repeated the acute ipsilateral phrenicotomy experi ment following ipsilateral dorsal rhizotomy of C2 through C6 (Figure 4 14 ). Despite removal of phrenic afferent input to the spinal cord, phrenicotomy still resulted in a robust decline in ipsilateral caudal EMG

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106 activity (Figure 4 14 ). This result, in comb ination with the stimulation and capsaicin results, strongly opposed the existence of a direct Consequently we investigated other mechanisms that may explain the robust decline in caudal IC EMG activity observed following a cute phrenicotomy For example w e proposed that the effect may be mediated by a segmental excitatory IC reflex dependant on contraction of the diaphragm. In this scenario, as the diaphragm contracted and displaced the ribs of the lower thorax, stretch rec eptors in the caudal ICs would be activated, triggering a reflex loop to the thoracic cord and enhancing activation of the ICs. This would provide stability to the lower thorax and allow the diaphragm to contract against a fixed ribcage, enhancing its effi cacy. Conversely, we speculated that the caudal IC EMG electrodes may simply be picking up electrical activity from the nearby ipsilateral diaphragm contaminating our signal This hypothesis seemed logical based on the insertion of the diaphragm muscle o n surrounding ribs of the lower thorax (Netter, 1989) but also unlikely due to the location and size of our electrodes Either way, w e attempted to dis cern if these proposed mechanisms (segmental mechanical reflex or electrical contamination) may be the source of our observations. I n two uninjured control rats, the ipsilateral phrenic nerve was isolated and cut per previous experiments. Thereafter, we st imulated the distal cut end of the phrenic nerve (i.e. innervating the diaphragm) and observed evoked electrical potentials in EMG recordings of the ipsilateral diaphragm, ipsilateral caudal ICs and ipsilateral rostral ICs (first IC space) consistent with our previously described experiments (see above). Figure 4 15 illustrates an evoked potential recorded in the ipsilateral diaphragm and caudal inspiratory ICs with relatively modest phrenic stimulation (10 A pulse, 0.5

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107 millisecond duration). The amplitude of the IC evoked potential approximated the amplitude of the integrated EMG signals recorded in our spontaneously breathing rats (not shown). We therefore concluded that a large percentage of IC EMG activity recorded from the external IC of the sixth rib s pace may in fact, represent electrical artifact from the diaphragm. Consequently, no reliable conclusions could be drawn from EMG data recorded using the se methods. W e did not observe the presence of evoked potentials in the rostral IC EMG signal with phr enic stimulation (Figure 4 15 ), affirming the validity of this measure as an indicator of compensation following C2HS and ipsilateral phrenicotomy. It should be noted, however that even though electrical contamination appeared to be the likely source of o ur observations, the presence of evoked potentials in the caudal IC EMG signal did not necessarily rule out the existence of the hypothesized excitatory IC segmental reflex pathway. This is further discussed below. Discussion This study provides the first comprehensive assessment of IC motor recovery in spontaneously breathing rats following C2HS. These data demonstrate that plasticity within spinal IC circuits facilitate near complete functional recovery of inspiratory associated EMG activity in rostral i nspiratory ICs by 2 wks post C2HS. Further, this plasticity may involve recruitment of thoracic INs (Kirkwood et al. 1988; Lane et al. 2008b; Saywell et al. 2011) and is associated with a n apparent shift in activ ation towards ipsilateral inspiratory ICs over contralateral ICs during conditions of spontaneous; poikilocapnic baseline and hypercapnic challenge by 8 wks post C2HS. Finally, these results support our previous hypothesis that rats rely predominately on r ecruitment of supplementary muscles of inspiration (i.e. inspiratory ICs) for long term

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108 improvement in spontaneous V T following C2HS (see Chapter 3 and (Golder et al. 2003; Fuller et al. 2006) ). Commentary on Met hods and Intercostal Biomechanics As with our prior studies (see Chapter 3), phrenicotomy experiments and IC EMG recordings necessitated that the rats be deeply anesthetized Anesthesia has the potential to suppress central respiratory activity and accord ingly could lead to an underestimation of the progressive recovery of IC function over time. Also, the depth of anesthesia during individual aspects of our preparation likely varies slightly from animal to animal; though identical tests of anesthetic depth were employed across all groups (i.e. absence of reflexive responses, see Materials and Methods). Variation in anesthetic depth may have impacted our ventilatory measures, specifically frequency in the 1 3 day and 2 wk C2HS rats (Table 4 3). Consistent w ith previous reports of respiratory recovery following C2HS, rats were maintained in supine throughout the neurophysiological experiments to allow suitable access to the trachea and phrenic nerves (see Chapter 3 and (Fuller et al. 2006; Doperalski et al. 2008; Fuller et al. 2008; Fuller et al. 2009; Sandhu et al. 2010) ). The impact of supine versus prone positioning on breathing has not been extensively studied in quadrupedal animals. However, the amplitude of I C EMG can be modulated by trunk positioning (Rimmer et al. 1995; Hudson et al. 2010) For example, EMG studies in humans suggest that rotational movements of the thorax activate muscle spindle afferents of ICs sit uated in the lateral chest wall (Rimmer et al. 1995) This, in turn, enhances the gain of gamma loop reflexes onto IC motoneurons (Critchlow & Von, 1963; Eklund et al. 1964; Sears, 1964) and amplifies inspiratory related IC EMG activity (Rimmer et al. 1995) Further, rats and humans display a physiological gradient

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109 of IC muscle activation such that inspiratory IC muscles rostral in the rib cage are activated pr ior to those situated more caudally (Gandevia et al. 2006) This was a principal rationale for recording external IC EMG activity from the first IC space in the current study. However, the activation gradient places additional priority on activation of dorsal versus ventral ICs and medial versus lateral ICs (Butler & Gandevia, 2008) Together, these results imply that supine positioning will likely effect IC motor recruitment and subsequent generation of V T On the other hand, as rats were maintained in a position of neutral trunk rotation and the positioning in supine restricts dorsal expansion of the rib cage, it is reasonable to speculate that while situated in supine, IC activation would shift ventrolaterally for rib cage expansion during inspiratory activity. Thus, recording IC EMG activity from a ventrolateral approach would seem appropriate in the current context. Finally, commentary on the complex and multifunctional nature of IC bio mechanics is warranted. It is now well established that external IC muscles function primarily during inspiration and internal ICs during expiration (De Troyer et al. 2005) Therefore, contraction of external ICs, especially those in the rostral rib spaces, act to elevate and expand the rib cage to facilitate lung expansion and inspiration (De Troyer et al. 2005) In addition, IC musculature plays an important role in axial rotation and postural control (see above and (Rimmer et al. 1995; Hudson et al. 2010) ). Diaphragm par alysis resulting from C2HS likely causes fundamental alterations in the biomechanical relationships between the diaphragm, rib cage, abdominal cavity and IC muscles Thus, comparison of IC EMG activity between uninjured rats and rats following C2HS begins from a dissimilar biomechanical platform. Yet, high cervical SCI in humans affecting

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110 phrenic motoneurons or bulbospinal input to phrenic motoneurons may produce similar alterations to respiratory muscle biomechanical relationships. For example, Silver et a l. (1969) noted increased abdominal expansion, and inward pulling of the ribs during inspiratory actions in tetraplegic patients compared with uninjured control subjects (Silver & Moulton, 1969) The authors surmised that the alterations in thoracic biomechanics resulted from a breakdown in highly coordinated inspiratory muscle activation patterns following injury (Silver & Moulton, 1969) Therefore, studies related to compensatory muscle recruitment for maintenance of breathing in these conditions are warranted. Progressive Recovery of Ipsil ateral Rostral Intercostals Following C2HS Modest, spontaneous recovery of ventilation and ipsilateral phrenic motor output has been reported following C2HS in rats (Fuller et al. 2006; Fuller et al. 2008) Resear ch emphasis related to this spontaneous recovery has focused primarily on the contribution of ipsilateral phrenic motor output via bulbospinal and/or propriospinal projections crossing below C2 (i.e. the CPP (Goshgar ian, 2009; Lane et al. 2009; Sandhu et al. 2009a) ). However, direct contribution of CPP associated neural activity to V T following C2HS in spontaneously breathing rats was shown to plateau by 2 wks post injury (see Chapter 3), while V T continued to impr ove (see Chapter 3 and (Fuller et al. 2008) ). We speculated that the combination of progressive inhibitory constraints to ipsilateral phrenic output (i.e. vagal mediated inhibition (Lee et al. 2010) ) and parallel neuroplastic changes in other inspiratory motor outputs (e.g. inspiratory ICs) triggered this occurrence (see Chapter 3). Our current data support this hypothesis, as rostral ipsilateral IC activity was restored by 2 wks post C2HS and appeared amplified by 8 wks post injury (Figures 4 2 and 4 3). It is interesting to note that this dramatic return of

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111 ipsilateral IC activity f ollowed a 1 3 days post C2HS period of quiescence (Figures 4 2 and 4 3). C2HS, therefore, appears to have eliminated a preponderance of primary bulbospinal inputs to rostral IC motoneurons. Multiple reports suggest, however, that most descending respirator y inputs to IC motoneurons are polysynaptic through thoracic INs versus monosynaptic connections to the motoneurons directly (Lipski & Duffin, 1986; Kirkwood et al. 1988; Lipski et al. 1994) Thus, it is interesti ng to speculate that the smaller quantity of direct ipsilateral connections to IC motoneurons interrupted by C2HS injury may be the key drivers of most inspiratory related IC activity observed in spontaneously breathing, poikilocapnic rats (Figure 4 2). Th is idea would only be applicable if polysynaptic connections to IC motoneurons crossed the spinal midline, which is suggested by our initial anatomical tracing studies (Figure 4 7) In any case the return of ipsilateral IC activity following initial quies cence points to plasticity within a Though progressive recovery of inspiratory IC activity following C2HS supports our primary hypothesis, the apparent shift towards activation of ipsilateral versus contrala teral rostral IC was surprising (Figure 4 4) We g a ve careful consideration to the interpretation of this result, however, given the variability in the 8 wk C2HS group. Activation of inspiratory ICs likely serves two fundamental functions: active expansion of the lungs to facilitate inspiration (De Troyer, 1986; De Troyer et al. 2005; Butler, 2007) and stabilization of the rib cage to provide a solid base for efficient diaphragm contraction (Guttmann & Silver, 1965; Silver & Moulton, 1969; Feldman 1986; De Troyer, 2005) As C2HS likely results in biomechanical changes (see above) associated with significant V T reduction (see chapter 3 and Table 4 3), plasticity facilitating b oth

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112 functions would appear vital to long term recovery. Thus, the shift in IC activation patterns might make sense. In this scenario, initial restoration of ipsilateral IC activity may be more important for lung expansion, as V T significantly increases bet ween 3 days and 2 wks post C2HS (see Chapter 3 and Table 4 2). Thereafter, progressively increasing ipsilateral IC activity may represent continued lung expansion and thoracic stability, underlying the progressive gains in V T observed between 2 and 8 wks p ost C2HS (see Chapter 3). Few studies have explored plasticity of inspiratory activity following cervical SCI (Zimmer et al. 2007) but the capacity of inspiratory ICs to express motor plasticity is robust. Fregosi and Mitchell (1994) used repeated carotid sinus nerve stimulation to test whether inspiratory ICs would express long term facilitation (LTF), a serotonin dependent form of spinal motor plasticity (Fuller et al. 2001; Baker Herman & Mitchell, 2002; Dale Nagle et al. 2010) The results suggested that inspiratory ICs posses the cha racteristics necessary for LTF. In addition, facilitated motor activity from inspiratory ICs exceeded that of phrenic activity (Fregosi & Mitchell, 1994) Further, since little variation in respiratory frequency was noted, the authors determined that LTF was likely the result of spina l mechanisms (Fregosi & Mitchell, 1994) .The progressive recovery of spontaneous ipsilateral IC EMG a ctivity observed here may have similar underlying mechanisms allowing for gradually enhanced output following C2HS, though these mechanisms were not specifically explored (see Summary ). The Effect of Acute Ipsilateral Phrenicotomy on Intercostal Motor Act ivity As previously reported, c hanges in V T following diaphragm paralysis or paresis in spinal intact animals are mitigated by compensatory increases in the activity of other respiratory muscles (Sherrey & Megirian, 1990; Brichant & De Troyer, 1997; Winslow &

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113 Rozovsky, 2003; Lane et al. 2009) We anticipated that progressive plasticity, leading to increased reliance on IC motor output for V T production following C2HS would reduce the effects of ipsilateral diaphragm paralysis following acute phrenicotomy (see Chapter 3). Indeed, the lack of significant changes to IC activity following phrenicotomy in rats receiving C2HS seem to support this idea (Figures 4 5 and 4 6). Further, the progressive increase in IC activatio n in uninjured rats following phrenicotomy supports a slowly adapting compensatory process likely resulting from a diminished influence of inhibitory phrenic afferent neurons with additional contribution from arterial hypercapnia (Teitelbaum et al. 1993; Brichant & De Troyer, 1997) A I t is generally accepted that a majority of bulbospinal inputs to IC motoneurons are polysynaptic relays through thor acic INs (Lipski & Duffin, 1986; Merrill & Lipski, 1987; Kirkwood et al. 1988; Lipski et al. 1994) This makes sense given the dual roles of IC musculature in respiration and postural control (De Troyer, 1986; Rimmer et al. 1995) For example, a polysynaptic relay could facilitate layering of respiratory activation over a more tonic, monosynaptic postural input to IC motoneurons (Rimmer et al. 1995 ) Regardless, the presence of a population of pre motor IC inputs (Lane et al. 2008b) especially those already receiving bulbospinal innervation (Lipski & Duffin, 1986; Mer rill & Lipski, 1987; Kirkwood et al. 1988; Lipski et al. 1994) may provide the anatomical substrate underlying the robust recovery of IC function seen here. Should this be the case and remodeling of thoracic pre sed we might expect to observe more thoracic INs retrogradely labeled with PRV injected into the rostral inspiratory ICs. Future studies

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114 using the combined CT and PRV injections into inspiratory IC muscles following C2HS noted above should provide important evidence related to this idea. Commentary on Observed Effects of Phrenicotomy on Caudal Intercostal Activity The dramatic reduction in caudal IC EMG activi ty following ipsilateral phrenicotomy (e.g. Figure 4 8) warrants further comment. The evoked potential observed in the ipsilateral caudal IC EMG signal following stimulation of the phrenic nerve (Figure 4 contamination jacent diaphragm. As, such, we cannot reliably use the EMG recording methods employed to make definitive conclusions about the activity of caudal inspiratory ICs following C2HS. However, the presence of an evoked potential may not completely discount the d ata either. A previous report validates caudal IC recording procedures using fine wire electrodes into the inspiratory ICs, similar to our current methods, in humans following cervical SCI (Silver & Lehr, 1981) In this study, electrical activity originating from the diaphragm was clearly dif ferentiated from IC EMG activity from the third through the eighth IC spaces (Silver & Lehr, 1981) Therefore, our EMG results from the sixth IC space likely capture significant inspiratory activity from the external ICs. We proposed that observed declines in the caudal IC EMG signal follow ing phrenicotomy may suggest a disruption of segmental reflex pathways in the thoracic cord. Specifically, if IC stretch receptors were activated as a result of diaphragm contraction and ensuing pull on the lower rib cage, they could increase the excitabil ity of IC motoneurons via afferent connections. Guttman and Silver (1965), studying reflex pathways in the IC muscles of patients following high cervical SCI made the following observations:

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115 ely the lower ribs on account of its anatomical attachment, thus causing a stretch effect on the intercostal muscles which acts as afferent stimulus of a stretch reflex. This stretch reflex increases in intensity commensurate with the subsiding of the spin al shock and the return of the reflex activity in the spinal cord. It is, therefore, not surprising that in a case of unilateral impaired diaphragmatic activity in cervical cord lesions; the reflex activity of the corresponding intercostal muscles is great The intercostal muscles regain their tone once the spinal shock has subsided and, in due course help to restore (by their reflex contractions especially during inspiration) the tension and rigidity of the intercostal spaces essential for a more powerful function of the diaphragm; they thus contribute to a better ventilation of the lungs which enables the tetraplegic in later stages to lead a more active life (Guttmann & Silver, 1965) In other words, the authors speculate on the apparent existence of an excitatory IC reflex similar to the one proposed here based on the caudal IC EMG results. This excitatory reflex loop activated by stretch receptors in inspiratory ICs was later confirmed electrophysiologically in animals (Kirkw ood & Sears, 1982) Therefore, it is likely that the observed decline in caudal IC EMG activity following acute ipsilateral phrenicotomy results, as least in part, from elimination of afferent reflex modulation with ipsilateral diaphragm paralysis (Silver & Moulton, 1969; Silver & Lehr, 1981; Kirkwood & Sears, 1982) Summary Spontaneous recovery of V T in rats following C2HS is associated with robust plasticity of inspiratory IC output. Our results suggest that time dependant recovery of IC activity represents a functional shift towards activation of rostral inspiratory ICs for long term V T recovery, in preference to the spontaneous CPP (see Chapter 3). Further, functional plasticity in ipsilateral inspiratory ICs ap pears more robust than that obser ved in contralateral ICs and may be associated with recruitment of thoracic INs. Although these results did not specifically address neural mechanisms underlying robust

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116 functional IC plasticity, we hypothesize that serotoni n mediated processes are likely involved. LTF of IC output observed by Fregosi and Mitchell (1994) was eliminated following pre treatment with methysergide, a serotonin receptor antagonist (Fregosi & Mitchell, 1994) Further, t here appears to be a greater density of serotonergic terminals near retrogradely labeled inspiratory intercostal motoneurons (Jiang & Shen, 1985) relative to phrenic motoneurons (Holtman et al. 1984; Zhan et al. 1989) suggesting the possibility that serotonergic influences on IC m otoneurons is relatively greater (Zhan et al. 2000) Regardless, preferential activation of inspiratory IC muscles, particularly ipsilateral to the lesion, to facilitate V T following C2HS represents a novel finding that may have implications for the study of res piratory recovery following high cervical SCI. Specifically, we suggest that neuroplasticity in inspiratory IC output may be sufficiently robust to mitigate some respiratory compromise associate with SCI. As such, inspiratory ICs may be an appropriate targ et for future treatments aimed at enhancing functional respiratory recovery in the cervical SCI population (Brown et al. 2006; DiMarco et al. 2006; DiMarco & Kowalski, 2010)

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117 Figure 4 1 A repres entative histological section illustrating a C2HS lesion. This 40 m transverse section was taken from the s econd cervical segment (C2) at 2 wks post injury and stained with Cresyl violet. The absence of white and grey matter in the ipsilateral (IL) spinal cord suggests an anatomically complete C2HS. CL: contralateral; CC: central canal; VH: ventral horn; DH: dorsal horn. Scale bar: 200 m

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118 Figure 4 2 Representative examples of rostral inspiratory IC EMG during poikilocapnic baseline and hypercapnic respiratory challenge. The images show the raw (EMG) and integrated EMG EMG ) signals from a control (uninjured) rat, and rats studied 1 3 days, 2 wks and 8 wks following C2HS injury. C2HS resulted in decreased ipsilateral (IL) EMG activity 1 3 days post C2HS during both baseline and hypercapnic challenge. However, robust return of IL EMG was observed at 2 wks and 8 wks post C2HS, while contralateral (CL) IC EMG signals were attenuated. Scaling is identical in all panels. Scale bar: 1 sec.

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119 Figure 4 3 The effect of C2HS on rostral IC EMG amplitude (arbitrary u nits, a.u.) during baseline breathing and hypercapnic respiratory challenge Ipsilateral (IL, left) IC EMG amplitudes were reduced in rats 1 3 days post C2HS in each condition. IL EMG amplitudes recovered in 2 wk and 8 wk C2HS rats in each condition. In co ntrast, C2HS caused little change in contralateral (CL) IC EMG amplitudes (right) compared with uninjured controls. : P < 0.0 01 from baseline; : P < 0.05 from baseline; : P < 0.01 from all other groups within condition.

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120 Figure 4 4 The relative activation of rostral ipsilateral (IL) versus contralateral (CL) inspiratory intercostals following C2HS EMG amplitude during baseline breathing and hypercapnic respiratory challenge was standardized as a ratio of ipsilateral to contralateral act ivity. Activation of ipsilateral ICs during baseline and respiratory challenge matched uninjured control rats by 2 wk post C2HS. In addition, upward shifts towards activation of ipsilateral inspiratory ICs during both conditions is suggested by 8 wks post C2HS. : P < 0.0 5 from 1 3 days C2HS group.

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121 Figure 4 5 The effect of phrenicotomy on rostral IC EMG. Phrenicotomy (PhrX) caused an immediate (next breath) increase in ipsilateral IC EMG activity (arbitrary units, a.u.) in uninjured rats that persisted for > 60 seconds In contrast, phrenicotomy caused little change in IC EMG activity in C2HS rats. : P < 0.05, **: P < 0.0 1 compared to pre PhrX values; #: P < 0.05, compared to all other groups within condition.

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122 Figure 4 6 Rela tive change in IC EMG amplitude following phrenicotomy. By 60 seconds post phrenicotomy (PhrX), the relative activation of ipsilateral (IL) ICs in uninjured rats was significantly higher than rats receiving C2HS. In contrast, little relative change in IC EMG activity was observed in the contralateral (CL) IC EMG signal following PhrX. #: P < 0.05, ## : P < 0.01 compared to uninjured controls.

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123 Figure 4 7. Retrograde anatomical tracing of inspiratory intercostals. A polysynaptic retrograde tracer (pseudo rabies virus, PRV green) and a monosynaptic retrograde tracer (cholera toxin CT red) were injected into the left external intercostals of the first three rib spaces (10l per space 30l total volume per rat) of spinal intact rats for visualization of intercostal neural circuitry Animals were left to survive for 96 hours fol lowing tracer injection. Primary intercostal motoneurons are co labeled with both tracers (yellow arrows in A) A portion of motoneurons appeared to label with only CT (red arrows in A) or PRV (green arrows in A). Additionally, pre intercostal interneuron s on either side of the spinal midline were labeled with PRV at this time point (B). Magnifications: 10 x (A) and 20x (B). Scale bars: 100m (A) and 200m (B). IL: ipsilateral, CL: contralateral.

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124 Figure 4 8. Example of caudal IC EM G decline with acute phrenicotomy. The arrow indicates ipsilateral (i.e. left) phrenicotomy during poikilocapnic baseline in an uninjured rat. Phrenicotomy resulted in an immediate (i.e. next breath) reduction of caudal IC EMG amplitude with parallel incre ases in contralateral (i.e. right) caudal IC EMG amplitude. BP: blood pressure. Scale Bar: 10 sec.

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125 Figure 4 9. Example of IC EMG activity following contralateral phrenicotomy. Following acute ipsilateral (i.e. left) phrenicotomy, an uninju red rat (same as in Figure 4 8) maintained poikilocapnic, baseline breathing for ~15 20 minutes. This was followed by contralateral (i.e. right) phrenicotomy as indicated with arrow. This second phrenicotomy resulted in immediate declines in caudal IC EMG activity on the right and parallel increases on the left. The effect was similar (but opposite) to Figure 4 8. These initial short term responses were followed by rather large increases in caudal IC EMG activity bilaterally to compensate for complete loss of diaphragm activity. BP: blood pressure. Scale Bar: 10 sec.

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126 Figure 4 10. The effects of lidocaine application to the ipsilateral phrenic nerve on IC EMG activity. The local anesthetic lidocaine was painted on the intact (i.e. uncut) ipsilateral (i.e. left, L) phrenic nerve (PhrN) of an uninjured rat. Immediate declines in ipsilateral (IL) caudal EMG activity and parallel increased activity in contralateral (CL) caudal IC EMG activity was observed. This result supported o pathway. Vt: integrated inspiratory tidal volume trace. Scale Bar: 10 sec.

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127 Figure 4 11. Example of caudal intercostal EMG response s to phrenic nerve stimulation. To test the hypothesis of a reflex pathway connecting the phrenic nerve a nd the caudal inspiratory ICs, we stimulated the proximal end of a cut phrenic nerve in an uninjured rat. This initial experiment demonstrated reductions in ipsilateral (IL) caudal IC EMG activity with stimulation, consistent with a possible reflex connect ion. CL: contralateral, Flow: spontaneous respiratory airflow trace. Scale Bar: 1 sec.

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128 Figure 4 12. Example of caudal IC EMG response to phrenic stimulation in follow up studies. Repeated attempts to validate the stimulation resul t shown in Figure 4 11 were unsuccessful. Identical stimulation parameters failed to suggest a reflex connection between the phrenic and caudal inspiratory ICs. IL: ipsilateral, CL: contralateral, Flow: spontaneous respiratory airflow trace. Scale Bar: 1 s ec.

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129 Figure 4 13. Effects of capsaicin on caudal intercostal EMG activity. To determine if our phrenic afferent fibers, we painted capsaicin, the active ingr edient in chili peppers, onto the diaphragm of an uninjured rat in order to directly activate c fiber endings. Despite repeated attempts using a range of dosages (10 1000ppm), no obvious changes in caudal IC EMG activity could be elicited. IL: ipsilateral, CL: contralateral, Vt: integrated inspiratory tidal volume trace. Scale Bar: 2 sec.

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130 Figure 4 14. Effects of i psilateral dorsal rhizotomy on phrenicotomy induced intercostal reflex wa s mediated by phrenic afferent fibers, we cut the ipsilateral dorsal roots (i.e. rhizotomy) at spinal levels C2 to C6, disrupting all phrenic afferent pathways. We followed with acute phrenicotomy of the ipsilateral (IL) phrenic nerve in an uninjured rat d uring poikilocapnic baseline. Phrenicotomy resulted in a sharp decline of caudal ipsilateral IC EMG activity as previously shown (Figure 4 8). This result, combined with results outlined above (Figures 4 12 and 4 13) ran counter to our reflex hypothesis. S cale Bar: 5 sec.

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131 Figure 4 15. Evoked potentials measured in the caudal ipsilateral intercostal EMG signal. Electrical stimulation to the distal end of a severed phrenic nerve in an uninjured control rat resulted in short latency evoked poten tials in both ipsilateral diaphragm (i.e. left Dia) and ipsilateral caudal (i.e. left Cdl) IC EMG signals. The amplitude of the caudal IC evoked potential approximated the amplitude of IC EMG signal s recorded in an anesthetized, spontaneously breathing rat Thus, no firm conclusions can be made based on caudal IC EMG observations alone following phrenicotomy, as our EMG signal likely includes electrical carry over from the diaphragm. Of note, no evoked potential was noted in the ipsilateral rostral (i.e. le ft Ros) IC EMG signal.

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132 Table 4 1 Age and weight values for spinal intact control rats and rats 1 3 days, 2 w ks, and 8 w ks post C2HS. Phrenicotomy of the ipsilateral phrenic nerve during hypercapnic respiratory challenge was completed in a subset o f rats. Total Phrenicotomy N Age (days) Weight (g) N Age (days) Weight (g) Uninjured 7 133 29 371 24 3 105 4 347 15 1 3 days 4 97 1 326 18 & 3 97 1 ** 317 22 2 w ee k s 9 111 1 348 6 & 7 111 1 349 10 8 w ee k s 7 15 5 1 399 11 7 155 1*** 399 11* Values are mean SE using 1 way ANOVA. &: P < 0.05 from 8 week group within condition. ***: P < 0.001, **: P < 0.01, *: P < 0.05 from all other groups within condition. Table 4 2 Blood gas and mean arterial blood pressure (MAP) values taken during baseline ventilation. P a O 2 P a CO 2 pH MAP (mmHg) Uninjured 190 18 39 2 7.31 0.02 95 6 1 3 days 207 7 65 7*** 7.19 0.04* 75 7 2 weeks 205 9 45 2 7.32 0.02 83 4 8 weeks 164 10 43 2 7. 28 0.02 89 5 Values are mean SE using 1 way ANOVA. ***: P < 0.001, *: P < 0.05 from other groups within condition.

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133 Table 4 3 Tidal volume (V T ), r espiratory frequency (f), inspiratory (T I ) duration and expiratory (T E ) duration during pre phrenico tomy (phrX) baseline (BL) and hypercapnic respiratory challenge (CO 2 ). V T (ml/breath) f (breaths/min) T I (sec) T E (sec) BL CO 2 BL CO 2 BL CO 2 BL CO 2 Uninjured 1.72 0.1 3.57 0.3*** 96 3 94 6 0.29 0.02 0.28 0.02 0.36 0.02 0.39 0.03 1 3 days C2HS 0.79 0. 1 # 1.16 0.1 ### 73 4 ### 71 3 ### 0.28 0.03 0.28 0.03 0.55 0.06 ## 0.57 0.67 ## 2wk C2HS 1.84 0.1 3.37 0.3*** 121 3 ### 116 4* ### 0.25 0.01 0.24 0.01 0.24 0.02 # 0.28 0.02* # 8wk C2HS 1.78 0.2 3.53 0.4*** 98 3 93 5 0.26 0.02 0.27 0.02 0.36 0.04 0.38 0.04 Value s are mean SE using 2 way RM ANOVA.*: P < 0.05; **: ***P < 0.001 from BL; #: P < 0.05, ##: P < 0.01; ###: P < 0.001 from other groups within condition.

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134 Table 4 4. Tidal volume (V T ), respiratory frequency (f), inspiratory duration (T I ) and expiratory d uration (T E ) pre and post ipsilateral phrenicotomy (p hrX) during hypercapnic respiratory challenge. V T (breaths/min) pre phrX post phrX 30 secs 60 secs Uninjured 4.1 0.1 1.9 0.1*** 3.4 0.1*** 3.5 0.2*** 1 3 days C2HS 1.0 0.1 ### 0.9 0.1 0.8 0.1 1.1 0.1 2wk C2HS 3.5 0.3 3.1 0.3*** 3.4 0.3 3.5 0.3 8wk C2HS 3.4 0.3 3.1 0.3** 3.4 0.3 3.4 0.3 f (breaths/min) Uninjured 88 13 81 12* 76 10** 76 10*** 1 3 days C2HS 72 5 72 6 71 6 69 7 2wk C2HS 110 7 & # 115 7* # 112 7 105 7 8wk C2HS 91 4 91 3 88 4 90 4 T I ( sec) Uninjured 0.27 0.03 0.23 0.02*** 0.28 0.02 0.28 0.04* 1 3 days C2HS 0.29 0.05 0.29 0.05 0.29 0.05 0.29 0.05 2wk C2HS 0.25 0.01 0.24 0.01 0.25 0.01 0.25 0.01 8wk C2HS 0.27 0.02 0.27 0.02 0.27 0.02 0.27 0.02 T E (sec) Uninjured 0.42 0.07 0.53 0.0 9*** 0.52 0.08*** 0.52 0.07*** 1 3 days C2HS 0.55 0.10 0.56 0.11 0.56 0.11 0.59 0.12 2wk C2HS 0.31 0.03 0.29 0.23 0.30 0.25 0.32 0.03 8wk C2HS 0.39 0.04 0.40 0.04 0.42 0.04 0.40 0.04 Values are mean SE using 2 way RM ANOVA.*: P < 0.05; **: P < 0.01; ***: P < 0.001 from pre phrenicotomy ; &: P < 0.01 from 1 3 days; #: P < 0.05, ###: P < 0.001 from other groups within condition.

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135 C HAPTER 5 TRANSPLANTATION OF E MBRYONIC MEDULLARY R APH CELLS ENHANCES TIDAL VOLUME AND PHR ENIC BURSTING FOLLOW ING C2 HEMISECTI ON IN RATS Serotonin (5 HT) is a potent modulator of neuronal function throughout the central nervous system. In the phrenic motor system, 5 HT modulates respiratory motoneuron excitability and is associated with neural plasticity (McCrimmon, 1995; Golder et al. 2001; Goshgarian, 2003; Mitchell & Johnson, 2003; Teng et al. 2003) Following C2 hemisection (C2HS) normal serotonergic innervation to ipsilateral phrenic motor neurons is disrupted, feasibly diminishing their excitability and slowing recovery (Golder & Mitchell, 2005) Partial restoration of 5 HT innervation to ipsilateral phrenic motoneurons occurs spontaneously over time (Ful ler, Doperalski unpublished results and (Golder & Mitchell, 2005) ), likely via collateral branching from contralateral raphe spinal tracts (Saruhashi et al. 1996) This modest recovery of 5 HT innervation has been s uggested to underlie activation of phrenic motoneurons via previously latent crossed (Rosenblueth & Ortiz, 1936; Zhou & Goshgarian, 1999; Goshgarian, 20 09) In fact, as 5 HT fiber density increase s to ipsilateral phrenic motonucleus, the amplitude of ipsilateral phrenic motor output is enhanced with strong correlation (Golder & Mitchell, 2005) Nevertheless, phrenic motor recovery does not recover fully even 3 months following C2HS (Fuller et al. 2008) Pharmacological manipulations of the CPP support the significance of 5 HT for ipsilateral phrenic motor recovery. In acutely injured C2HS rats, the CPP is revealed with introduction of the 5 HT precursor 5 Hydroxytryptophan (Ling et al. 1994; Zhou & Goshgarian, 1999, 2000; Fuller et al. 2003) and this functional improvement is

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136 eliminated with the broad spectrum 5 HT receptor antagonist methysergide (Z hou & Goshgarian, 1999, 2000) The mechanism of 5 HT involvement in activation of the CPP is still under investigation (Hadley et al. 1999b; Golder et al. 2001) However, strong evidence points to the excitatory 5 HT 2A receptor on phrenic motoneurons as a primary target for these effects. Indeed, pharmacological activation of 5 HT 2A receptors elicits crossed phrenic activity (Zhou et al. 2001a) Interestingly, following SC I there is an upregulation of 5 HT 2A receptors on motoneurons below the injury (Ung et al. 2008; Kong et al. 2010) This includes phrenic motoneurons below a C2HS (Fuller et al. 2005) Given the important role of 5 HT for modulation of motoneuron output (Heckman et al. 2009; Murray et al. 2011) and the profound decline in 5 HT following SCI, cells expressing 5 HT have been used as transplants to augment functional recovery in models of SCI. For example, embryonic cells from the brainstem raphe nuclei (RN) of developing rats (gestational day 14; E14) have been injected into the dorsal columns of the spinal cord below complete thoracic transection lesions (Ribotta et al. 2000; Majczynski et al. 2005; Eaton et al. 2008) These studies demonstrate that embryonic serotonergic cells can reinnervate target pools of neurons be low an injury (Dumoulin et al. 2000; Ribotta et al. 2000) Specifically, immunohistochemical analysis showed transplanted cells forming dense axonal bundles within areas of the cord normally receiving raphe spinal input (e.g. ventral horn motoneurons, dorsal horn laminae I, (Dumoulin et al. 2000; Ribotta et al. 2000) ). Further, the return of 5 HT coincided with modest recovery of locomotor function (Feraboli Lohnherr et al. 1997; Feraboli Lohnherr et al. 1999; Dumoulin et al. 2000; Ribotta et al. 2000) In follow up studies,

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137 Majczynski et al. (2005) demonstrated that locomotor improvements were depend e nt on increased reflexive re sponses to sensory afferent feedback resulting from enhanced excitability of the CPG from new serotonergic input (Majczynski et al. 2005) Although these studies could not relate the functional improvements to spec ific reinnervation of motoneurons with new serotonergic synapses, ultra structural studies have shown a high likelihood of new synaptic formation between transplanted 5 HT cells and surviving motoneurons (Privat et a l. 1989; Rajaofetra et al. 1992) For example, examination of the ventral horns below 5 HT transplants in the lumbar cord detected numerous axodendritic synapses of 5 HT fibers onto larger motoneuron dendrites, similar to what is seen in uninjured rats (Privat et al. 1989) The noted effects, however, may have resulted from paracrine release of serotonin from axons approximating surviving motoneurons (Privat et al. 1989) Paracrine release (or volume transmission; (McCrimmon, 1995; Hentall et al. 2006) ) is an inherent property of medullary RN neurons. Regardless, these studies suggest that 5 HT cell transplants can enhance motor recovery following SCI. Based on the role of 5 HT in phrenic control and plasticity (Fuller et al. 2000; Fuller et al. 2001; Mitchell et al. 2001; Mitchell & Johnson, 2003) the encouraging literature in locomotor systems related to serotonergic cell transpla ntation (Privat et al. 1989; Feraboli Lohnherr et al. 1997; Dumoulin et al. 2000; Ribotta et al. 2000; Hains et al. 2002) and our own observations we proposed to examine the impact of fetal RN cell transplants below C2HS injuries on recovery of phrenic motor output. We hypothesized that transplanted RN cells would augment endogenous neuroplasticity seen in the phrenic motor system following C2HS. Specifically, increased

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138 5 HT innervation would be associated wit h increased amplitudes of ipsilateral phrenic motor output (Golder & Mitchell, 2005) and enhanced inspiratory tidal volume in the weeks following C2HS. Our results highlight the importance of 5 HT neuromodulator supplementation for maximizing motor recovery following SCI. Materials and Methods All experimental procedures were approved by the Institutional Animal Care and Use Committee at the University of Florida. Animals A tota l of 20 adult, male Sprague Dawley rats were obtained from Harlan Laboratories Inc. (Indianapolis, IN, USA). Rats were divided into the following groups: sham transplants ( cell culture medium, sham, N= 8 ), graft controls (i.e. fetal spinal cord cells, FSC, N= 5 ) and embryonic raph neuron (RN) transplants (N= 7 ). A summary of the experimental groups is presented in Table 5 1 Spinal Cord Injury Our anesthesia and injury methods have been previously described (Fuller et al. 2008; Fuller et al. 2009) Rats were anesthetized by injection of xylazine (10 mg/kg, s.q.) and ketamine (140 mg/kg, i.p., Fort Dodge Animal Health, IA, USA). The spinal cord was exposed at the C2 level via a dorsal approach, and a left C2HS lesion was induced using a microscalpel followed by aspiration. The dura and overlying muscles were sutured and the skin closed with stainless steel wound clips (Stoelting, IL, USA). Post hoc histological analysis (see below) confirmed the extent of hemisection and animals with spared ipsilateral tissue were excluded from the study (Fuller et al. 2009) Rats were given an injection of yohimbine (1.2 mg/kg, s.q., Lloyd, IA, USA) to reverse the effect of xylazine. Following surgery, animals received an analgesic (buprenorphine,

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139 0.03 mg/kg, s.q., Hospira, IL, USA) and sterile lactated Ringers solution (5 ml s.q.). Post surgical care included administration of buprenorphine (0.03 mg/kg, s.q.) during the initial 48 hours post i njury and delivery of lactated Ringers solution (5 ml/day, s.q) and oral Nutri cal supplements (1 3 ml, Webster Veterinary, MA, USA) until adequate volitional drinking and eating resumed. Cell S uspensions Embryonic donor tissue was taken from the same str ain (i.e. Sprague Dawley) as the host animals. Embryos were taken after laparotomy from pregnant rats under xylazine (10 mg/kg, s.q.) and ketamine (140 mg/kg, i.p.) anesthesia at embryonic day 14 (E14). The day after mating was considered day 0. The microd issection of the tissue has been previously described in detail (Konig, 1989; Jakeman & Reier, 1991) Briefly, the caudal rhombencephalon, extending from pontine flexure to the rostral cervical spinal cord and conta ining the medullary raph nuclei (Figure 5 1) was dissected out in chilled Hanks Balanced Salt Solution (HBSS, Invitrogen, Grand Island, NY). For graft control experiments using FSC transplants, meninges/dorsal root ganglion free spinal cords were isolate d in HBSS and subsequently processed in the same manner After initial mincing of the tissue with a sterilized scalpel blade, mechanical dissociation by gentle pipetting in HBSS was completed. Tissue was mixed with trypsin EDTA (0.25% trypsin, 1.0M EDTA, A tlanta Biologicals, Lawrenceville, GA) and incubated for 7 minutes in a 37C water bath. Quenching of trypsin activity with fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) was followed by manual trituration of dissociated tissue through progres sively smaller bore Pasteur pipettes until maximum tissue dissociation was observed. The suspension was centrifuged at 80 X g for 10 min utes resuspended

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140 in minimal essential culture medium (Invitrogen Corporation, Grand Island, NY), and adjusted to a fina l concentration of 50,000 75,000 cells/l. Cell Transplantation Animals were transplanted 1 week following C2HS using procedures described earlier (Rajaofetra et al. 1992) because this period was found optimal for graft su rvival and development (Ribotta et al. 2000) Briefly, the cervical spinal cord was re exposed and 4 5l of dissociated cell suspension (RN or FSC) or minimal essential medium (sham) was injected into the spinal cord (0.5 1.0 mm below the pial surface) with a 31 guage needle (45 degree beveled tip) connected to a Hamilton microsyringe (Hamilton Company, Reno, NV). The injection site was ~1.0mm from the caudal edge of the C2HS lesion in the spinal midline near the C2 C3 border. The needle was withdrawn 2 minutes following the end of the injection to avoid suspension reflux. The musculature was sutured, and the animals were treated as described above. Barometric Plethysmography Whole body plethysmography (Buxco Inc. Wilmington, NC, USA) (Fuller et al. 2008) was used to obtain measures of breathing in unanesthetized rat s six weeks following cell transplantation. The plethysmograph was calibrated by injecting known volumes of air into a Plexiglas recording chamber using a 5 ml syringe. The chamber pressure, temperature and humidity, and rectal temperature of the rat were used in the Drorbaugh and Fenn equation (Drorbaugh & Fenn, 1955) to calculate respiratory parameters including breathing frequency (f; breaths/minute), tidal volume (V T ; ml/breath) and minute ventilation ( E ml/min ute ). Baseline recordings were made for 60 minutes while the chamber was flushed with 21% O 2 (balance N 2 2 l/min ute ). Subsequently, rats wer e given a 5 minute hypercapnic respiratory challenge (7% CO 2

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141 21% O 2 balance N 2 2 l/min ute ). Mean values for analyses were obtained from a 10 minute period during baseline and during the last 2 minutes of the hypercapnic challenge. Neurophysiology Prepa ration These procedures were adapted from our prior publications (Doperalski et al. 2008; Fuller et al. 2008; Fuller et al. 2009; Lane et al. 2009; Sandhu et al. 2010) and were carried out 6 weeks post cell tra nsplantation I soflurane anesthesia (3 4% in O 2 ) was induced in a closed chamber followed by i.p. injection of urethane (1.6g/kg, Sigma, St. Louis, MO, USA). The adequacy of urethane anesthesia was confirmed by testing limb withdrawal and palpebral reflexe s. Rats were maintained in a supine position throughout the protocol. V T measurement in spontaneously breathing rats The V T response to acute ipsilateral phrenicotomy (see Chapters 3 and 4) was measured in a subset of transplanted rats. Thus, the trachea was cannulated in the mid cervical region and connected in series to a custom designed, small animal pneumotachograph and volumetric pressure transducer (Grass Instruments, Quincy, MA, USA) for measurement of respiratory air flow. Partial pressure of arter ial oxygen (P a O 2 ) was maintained above 150 mmHg by delivering a hyperoxic gas mixture (F I O 2 =0.50, balance N 2 design (Fuller et al. 1998) The femoral vein was catheterized (PE 50) to enable supplemental urethane anesthesia (0.3 g/kg, i.v., Sigma, St. Louis, MO, USA) if indicated. Another PE 50 catheter was placed in the femoral artery and connected to a pressure transducer (Statham P 10EZ pressure transducer; amplifier CP122 AC/DC stra in gauge amplifier, Grass Instruments, West Warwick, RI, USA) for arterial pressure

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142 and blood gas measurements. The bilateral phrenic nerves were isolated in the cervical region via a ventral approach (Sandhu et al. 2009a; Lee et al. 2010; Sandhu et al. 2010) The exposed nerves were covered in mineral oil but not manipulated at this time. Arterial blood samples (0.2 ml) were drawn during the baseline period (see below ) and analyzed for PaO 2 carbon dioxide par tial pressure (P a CO 2 ) and pH (i STAT, Waukesha, WI). Blood gas measures were corrected to rectal temperature which was monitored by rectal thermistor and maintained at 37.5 1 C by a servo controlled heating pad (model TC 1000, CWE Inc., Ardmore, PA, USA ). To determine if RN cell transplantation may affect activation of the spontaneous CPP, the ipsilateral phrenic nerve was cut during hypercapnic respiratory challenge in a subset of anesthetized spontaneously breathing rats as previously described (see C hapter s 3 and 4). Briefly, baseline V T was recorded during a 20 minute period during which rats breathed the hyperoxic gas mixture described above and baseline blood samples were drawn This baseline period was followed by a five minute hypercapnic respir atory challenge (7% CO 2 50% O 2 balance N 2 ) in accordance with our prior studies Rats were then returned to baseline conditions. Once breathing had returned to the pre hypercapnic values (i.e. baseline) a second hypercapnic challenge was initiated. The previously exposed ipsilateral phrenic nerve was cut during the third minute of the second hypercapnic challenge to assess immediate changes in V T following the complete removal of ipsilateral phrenic nerve contributions (see Chapter s 3 and 4). This time p oint during hypercapnic challenge corresponded to a stable period of hypercapnic V T that was similar to the initial hypercapnic response. All rats were returned to baseline conditions following the second hypercapnic challenge.

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143 Phrenic nerve recording in m echanically ventilated rats Following completion of the acute phrenicotomy preparation, or once adequate anesthetic depth was attained; rats were maintained on mechanical ventilation for the remainder of the experiment. The vagus nerves were sectioned in t he mid cervical region and the rats were paralyzed with pancuronium bromide (2.5 mg/kg, i.v.). Following paralysis, the adequacy of anesthesia was monitored by observing blood pressure and respiratory responses during application of deep pressure to the pa ws. The carbon dioxide partial pressure (P ET CO 2 ) was measured throughout the protocol using a rapidly responding mainstream CO 2 analyzer positioned a few centimeters from the tracheostomy tube on the expired line of the ventilator circuit (Capnogard neonat al CO 2 monitor, Novametrix Medical Systems, Wallingford, CT, USA). Efferent phrenic nerve compound action potentials were recorded using silver wire electrodes with a monopolar configuration, amplified (1000X) and filtered (band pass=300 10,000 Hz, notch =60 Hz) using a differential A/C amplifier (Model 1700, A M Systems, Carlsborg, WA, USA). The amplified signal was full wave rectified and moving averaged (time constant 100 ms; model MA 1000; CWE Inc., Ardmore, PA, USA). Data were digitized using a CED Po wer 1401 data acquisition interface and recorded on a PC using Spike2 software (Cambridge Electronic Design Limited, Cambridge, England). While maintaining an adequate plane of anesthesia, rats were ventilated for 30 45 minutes with P ET CO 2 at 402 mmHg. T he end tidal CO 2 apneic threshold for inspiratory activity was then determined by gradually increasing the ventilator pump rate until inspiratory bursting ceased in both phrenic nerves. The ensuing apnea was maintained for 2 3 minutes, and the ventilator r ate was gradually decreased until inspiratory activity reappeared. The P ET CO 2 associated with onset of inspiratory bursting was noted, and

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144 the ventilator rate was adjusted to maintain P ET CO 2 2 mmHg above this value ET CO 2 measurements, however, were merely a guide to help maintain isocapnia, and CO 2 levels were determined exclusively by arterial blood analyses. An arterial blood sample was drawn a few minutes prior to hypoxic challenge as a baseline measureme nt. Rats were subsequently exposed to a five minute bout of hypoxia (F I O 2 ~0.13) and returned to baseline. Prior to terminating the experiment, a subset of rats was given injections of Ketanserin (1mg/kg, i.v.), a pharmacological antagonist with high affin ity to the 5 HT 2A receptor, to assess the potential role of this receptor in graft associated physiological improvements. At the conclusion of the experiment, rats were euthanized via systemic perfusion (see below). Spinal Cord Histology and Immunohistoche mistry All C2HS lesions were confirmed to extend to the spinal midline as previously described (Fuller et al. 2008; Fuller et al. 2009; Sandhu et al. 2009a) At the conclusion of the neurophysiology experiments, rats were euthanized by systemic perfusion with saline followed by 4% paraformaldehyde (Sigma, St. Louis, MO, USA). The cervical spinal cord was removed, and 40 m sections were made in the transverse plane using a vibrotome. All sections were processed fo r 5 HT immunodetection. Prior to incubation with the primary antibody, sections were washed in PBS (0.1 M, 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 20 minutes), rewashed in PBS, treated with sodium borohydride (1% in dH 2 O, incubated for 30 minutes) given a third and final wash with PBS and blocked against nonspecific protein labeling (3% goat serum in 0.1M PBS with 0.03% Triton X). Sections were then incubated wi th a rabbit polyclonal antibody against 5 HT (1:20,000; Immunostar, Hudson, WI) with 1% nonspecific goat

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145 serum and 0.03% Triton X overnight at 4C. On the following day, tissue was washed in PBS with 0.3% Triton X (0.1 M, 3 X 5 minutes), blocked against no nspecific protein labeling (3% goat serum in 0.1M PBS with 0.03% Triton X, 30 minutes) and incubated for 30 minutes at room temperature in a biotinylated secondary antibody (goat anti rabbit; Vector Laboratories, Burlingame, CA; 1:100). This was followed b y a second round of washes in PBS with 0.3% Triton X (3 X 5 minutes), a second round of blocking against nonspecific protein labeling (3% goat serum in 0.1M PBS with 0.03% Triton X, 30 minutes) and incubation in rabbit peroxidase anti peroxidase (Sigma, St Louis, MO; 1:400, 30 minutes). Sections were then given a third series of washes in PBS with 0.3% Triton X, and antigen was visualized with nickel enhanced diaminobenzidine (DAB with 1% nickel ammonium sulfate; Sigma, St. Louis, MO) in the presence of H 2 O 2 A subset of tissue sections were additionally counterstained with Cresyl violet for visualization of neuronal cell bodies. A representative histological example of a C2HS is provided in Figure 5 2 c onsistent with our previous publications (Fuller et al. 2008; Lane et al. 2008b; Fuller et al. 2009; Sandhu et al. 2009a) Data Analysis Plethysm ography data were analyzed in 10 second bins per our previously published reports (Fuller et al. 2006; Fuller et al. 2008) For the baseline condition, data represent the average of consecutive bins over a stable 10 minute period just prior to hypercapnia. For hypercapnia, we report the average of the last 2 min utes of exposure. Ca libration of the pneumotachograph was accomplished using a series of constant volume injections with varying airflow rates as previously reported (see Chapter 3). Respiratory airflow signals were amplified (x100K; CP122 AC/DC strain gage amplifier,

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146 Grass I nstruments) and recorded on a PC using Spike2 software (Cambridge Electronic using a customized Spike2 software script (Cambridge Electronic Design Limited) as an indicat or of V T Data corresponding to V T reduction following acute phrenicotomy during hypercapnic respiratory challenge in anesthetized, spontaneously breathing rats represents the mean amplitudes of integrated airflow signals over 10 breaths prior to phrenicot omy (i.e. pre PrX) and the first breath following phrenicotomy (i.e. post PhrX) Phrenic neurograms were quantified in terms of 1) absolute voltage (i.e., arbitrary u nits a.u.), 2) relative to the ampli tude in the contralateral phrenic nerve (% contralat eral) and 3) relative to the amplitude during baseline (% baseline). Spontaneous inspiratory phrenic nerve activity was averaged over a stable 10 second period just prior to respiratory challenge. During baseline conditions, the peak amplitudes of moving t ime averaged, spontaneous inspiratory phrenic bursts were quantified as an absolute voltage (i.e., arbitrary units) and as a percentage of the amplitude in the contralateral nerve (i.e. % contralateral).During the hypoxic period, the increase in phrenic bu rst amplitude was quantified as an absolute voltage, as a percent increase from baseline burst amplitude (i.e. % baseline), and as a percentage of the amplitude in the contralateral nerve. In addition, for rats receiving injections of ketanserin, phrenic n eurograms were quantified as absolute voltages (i.e. arbitrary units) over stable 10 second periods preceding injection and 2 minutes following injection and as a percentage decrease (i.e. % decline) in amplitude between these time periods.

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147 Statistical ana lyses were performed using commercially available software (Sigma Stat, SPSS, Chicago, IL). In cases where variables had comparable units for both RN transplanted rats and control rats (e.g. V T a.u.) or ipsilateral and contralateral nerve activity (e.g. a.u.), data were compared using two way analysis of variance (2 w ay ANOVA) and the Student Neuman Keuls post hoc test. Variables for which data could not be directly compared (e.g., ipsilateral phrenic amplitude expressed as a percentage of the contralater al amplitude) were analyzed using a n unpaired t test Comparisons of arterial blood gases, mean arterial blood pressure (MAP), ages and weights were also made using unpaired t test Data comparing changes in phrenic nerve amplitudes during hypoxic challeng e as a percentage of baselines (i.e. % baseline) were not normally distributed. Therefore, a 1 way w ANOVA on pairwise multiple comparisons was used for analysis All data are presented as the mean standard error. A P value of < 0.05 was considered statistically significant. Results Anatomical Characterization of Cell Transplants Spinal cord sections from rats receiving sham, RN or FSC transplants were visualized with light microscopy once processed for 5 HT immunodetection RN and FSC transplants survived to 6 weeks post transplantation as clusters of cells within the dorsal white matter ( Figure 5. 3 ). T ransplants appeared to survive and integrate more con sistently when approximating gra y matter, consistent with previous obs ervations (Nornes et al. 1983) FSC transplants never stained positively for 5 HT (Figure 5. 3 ). RN transplants, on the other hand, displayed 5 HT positive cell bodies and neuronal fibers within the graft emana ting towards the ipsilateral gra y matter (Figure 5. 3 ).

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148 RN transplants were associated with enhanced 5 HT immunodetection about the central canal and in the ipsilateral ve ntral horn at C3 (Figure 5 3 ). Control rats (i.e. rats receiving sham or FSC transplants) displayed reduced 5 HT staining within the se same regions (Figure 5 3 ). Qualitatively similar 5 HT staining was observed in the contralateral ventral horns of control and RN rats, though all appeared somewhat reduced compared with uninjured controls ( N=2, Figure 5 3 ). Since our hypothese s centered on augmentation of 5 HT to the vicinity of phrenic motoneurons with RN transplants, qualitative comparisons of 5 HT immunod etection was further assessed at C4 (Figure 5 4 ). Once more RN transplants were associated with enhanced 5 HT staining around the central canal and in the ipsilateral ventral horns near presumed phrenic motoneurons (Figure 5 4 ). Though the 5 HT staining a ppeared more robust than in sham or FSC transplant ed rats, the immunodetection in RN rats remained reduced compared with uninjured rat s (Figure 5 4 ). Consistent with observations at C3, qualitatively similar 5 HT staining was observed in the contralateral ventral horn of cell transplant ed and sham rats. However, this 5 HT staining appeared reduced compared with an uninjured rat. Additional e xamples of individual RN transplants are provided in Figures 5 11 through 5 14 Effect s of Cell Transplantation on Ven tilation (Unanesthetized Rats) To determine if the enhanced 5 HT immunodetection in the ipsilateral ventral horn (and around the central canal) of RN transplanted rats was associated with improved inspiratory V T barometric plethysmography was performed si x weeks post cell transplantation (or sham injection). All ventilation parameters (i.e. f, V T and E ) were similar in sham transplanted rats and rats receiving FSC transplants (P>0.15 for each)

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149 Therefore, these groups were combined i nto a single room air breathing (i.e. baseline 21% O 2 balanced N 2 ), no differences in f, V T or E were observed between control rats and rats receiving RN transplants ( Figure 5. 5 ). Control rats te nded to breathe faster during the baseline condition (9 5 4 breaths/min) compared to RN transplanted rats ( 85 3 breaths/min), however, this did not reach sig nificance (P=0.18 Figure 5 5 ). As anticipated f, V T and E all increased in r esponse to hypercapnic respiratory challenge ( 7% CO 2 21% O 2 balanced N 2 P<0.001) in control and RN rats (Figure 5. 5 ). However, RN rats produced larger V T (4.0 0.1 ml/breath) during hypercapnia compared with controls (3.5 0.1 ml/breath P<0.01 ) (Figure 5. 5) This led to enhanced E for RN rats (657 16 ml/minute vs. 582 28 ml/min for controls P<0.05) (Figure 5.5) during hypercapnic respiratory challenge. Effect s of Cell Transplantation on Tidal Volume (Anesthetized Rats) W e also exami ned the effects of RN cell transplantation on activation of the spontaneous CPP during respiratory challenge in a group of spontaneously breathing, anesthetized rats consistent with our prior studies (see Chapter 3 and 4). A subset of RN transplanted rats (N=5) and control rats (i.e. sham or FSC transplanted rats, N=9) received an acute phrenicotomy during hypercapnic respiratory challenge. Arterial blood gases during baseline spontaneous breathing were similar between groups as outlined in Table 5 2 Consi stent with our plethysmography data, rats receiving RN transplants produced larger V T during hypercapnic challenge (5.7 0.9 ml/breath) than control rats (4.40.5 ml/breath), though this did not reach statistical significance (P=0.16) (Figure 5 6) Acute ph renicotomy of the ipsilateral phrenic nerve resulted in immediate reductions of V T in both RN rats (P<0.05 from pre phrenicotomy) and control rats (P<0.01 from pre

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150 phrenicotomy) (Figure 5 6). When this reduction in V T was presented as a relative decline in V T following phrenicotomy (i.e. % decline), no significant differences emerged between RN rats (92% decline in V T ) and controls (113% decline in V T ) ( t test, Figure 5 6 ). Effect s of Cell Transplantation on Phrenic Motor Output To test our hypothesis tha t RN transplants would be associated with increased ipsilateral phrenic motor output, phrenic nerve recording s were made in a subset of rats. As with plethysmography, no differences in phrenic output were observed between sham transplanted rats and FSC tra nsplanted rats. Therefore, these groups were Arterial blood gas es were sampled during baseline conditions and are summarized in Table 5. 3 RN rats (N=6) displayed higher average P a O 2 (220 12 Torr) compa red to controls (N=8 195 6 Torr, P<0.05). However, this may have resulted from increased F I O 2 supplementation in two RN transplanted rats. No differences in P a CO 2 pH, or MAP were observed between groups. Examples of inspiratory phrenic nerve bursting ar e shown in Figure 5. 7 During baseline conditions, inspiratory phrenic burst amplitudes did not differ between RN rats and controls when expressed in arbitrary units (a.u. Figure 5. 8 ) However, consistent with the example in Figure 5.7 RN rats tended to s how enhanced ipsilateral phrenic burst amplitudes during baseline when compared relative to contralateral phrenic burst amplitude s ( i.e. % contralateral; P=0.14, Figure 5. 8 ). D uring respiratory challenge (hypoxia), RN rats produced larger ipsilateral phren ic burst amplitudes (0.085 0.02 a.u. ) compared with controls (0.036 0.01 a.u. ; P<0.01) ( Figure 5. 8 ) This also represented an increase from baseline phrenic amplitudes (P<0.01 Figure 5. 8 ) However, when ipsilateral phrenic burst amplitudes during respirato ry challenge were represented

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151 relative to baseline amplitudes (i.e. % baseline) significance was not reached (P=0.13, Figure 5. 8 ). No differences in contralateral phrenic nerve burst amplitudes were observed between groups during either baseline or respira tory chal lenge (Figure 5. 8 ) regardless of how data were quantified (i.e. a.u. or % change) The Effect s of Ketanserin on Phrenic Activity Following Cell Transplantation Plasticity in phrenic motor output has been linked specifically to activation of the 5 HT 2A receptor on phrenic motoneurons (Fuller et al. 2000; Mitchell et al. 2001; Baker Herman & Mitchell, 2002) Therefore, we initiated pharmacological studies to determine whether the 5HT 2A receptor may play a ro le in the enhanced ipsilateral phrenic motor recovery seen following RN transplantation. Ketanserin, a selective 5HT 2A antagonist, was injected intravenously (1mg/kg) (Fuller et al. 2001) at th e end of the bilateral phrenic nerve recording experiments in a subset of rats (N=3 RN rats and N=8 control rats). The effects of ketanserin on bilateral phrenic nerve amplitudes are illustrated in Figure 5. 9 Prior to injection, RN rats displayed larger i psilateral phrenic burst amplitudes (0.04 0.01 a.u.) compared with control rats (0.02 0.01 a.u.), but this did not re ach significance (P=0.09 ) ( Figure 5. 10 ) Following ketanserin injection, both groups displayed a reduction in ipsilateral phrenic burst amp litude, though the reduction measured in RN rats (0.03 0.01 a.u. P<0.001) was greater than that observed in control rats (0.01 0.00 a.u. P<0.05) (Figure 5 1 0 ). When the post ketanserin ipsilateral phrenic burst amplitudes were expressed relative to pre ket anserin values (i.e. % decline) RN rats showed a tendency for larger reduction s in phrenic activity (74 5% reduction) in response to ketanserin compared to controls (55 6% reduction) but this did not reach significance (P=0.13) (Figure 5 1 0 ) Contralatera l phrenic burst amplitudes remained relatively stable in both groups following ketanserin injection, though control rats did

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152 show a significant reduction in contralateral burst amplitude when expressed in arbitrary units (P<0.05, Figure 5. 10 ) Discussion T his study provides the first evidence to support the feasibility of chronic 5 HT replacement therapy using embryonic cell transplants to enhance respiratory r ecovery following C2HS Specifically, cell transplants derived from the developing (E14) brainstem RN survived intraspinal transplantation into the cervical spinal cord immediately caudal to a C2HS lesion and increased 5 HT immunological staining within the ipsilateral ventral horn and central grey matter of cervical spinal segments caudal to the lesio n Consistent with our hypotheses, the e nhanced 5 HT corresponded to increased inspiratory V T and larger ipsilateral phrenic burst amplitudes 6 weeks post transplantation Finally, our data support a role for the 5 HT 2A receptor in mediating t his functiona l recovery (Zhou et al. 2001a; Goshgarian, 2003; Mitchell & Johnson, 2003; Fuller et al. 2005) RN Transplants Enhance 5 HT Innervation to Target Spinal Regions E mbryonic cells from the RN of developing rats (E14) have been injected into the dorsal columns of the spinal cord to affect locomotor recovery following complete thoracic transection lesions (Ribotta et al. 2000; Majczynski et al. 2005; Eaton et al. 2008) These studies demonstrate d that RN cells c ould reinnervate target pools of neurons below an injury when implanted with this method. Specifically, immunohistochemical analysis showed transplanted RN cells forming dense axonal bundles within areas of the cord norm ally receiving raphe spinal input (e.g. ventral horn motoneurons, dorsal horn laminae I, central grey matter ) (Privat et al. 1988; Rajaofetra et al. 1989; Ribotta et al. 2000) Although these studies could not re late functional

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153 improvements to the specific reinnervation of motoneurons with new serotonergic synapses, ultra structural studies have shown a high likelihood of new synaptic formation between transplanted 5 HT cells and surviving motoneurons (Privat et al. 1989; Rajaofetra et al. 1992) The noted functional effects however, may have been the result of paracrine release of 5 HT from axons approximating surviving motoneurons (Privat et al. 1989; Hentall et al. 2006) Paracrine release (or volume transmission; (McCrimmon, 1995) is an inherent property of medullary RN neurons. Therefore, for RN transplants to exert effects on phrenic motor activity, new 5 HT fibe rs may need only to approximate phrenic motoneurons or pre phrenic interneurons (Hentall et al. 2006) Detection of serotonergic fibers following RN transplantation in this study demonstrate enhanced 5 HT immunoreactivity around the centr al canal and in the ipsilateral ventral horns caudal to graft placement (Figures 5 3 and 5 4). These regions of increased 5 HT staining correspond to the specific cervical regions supplied with 5 HT in spinal intact rats (Figures 5 3 and 5 4 (Steinbusch, 1981; Tork, 1990) ). Thus, it would appear that similar post transplantation growth patterns of serotonergic processes occurred with RN transplants following hemisection as seen previously with complete spinal transec tions (Privat et al. 1986; Privat et al. 1988; Privat et al. 1989; Ribotta et al. 2000) It should be noted, however, that c omplete spinal transection eliminates all 5 HT inputs of supra spinal origin. Therefor e, 5 HT reinnervation of segments caudal to the injury following RN transplantation in transection models can specifically be attributed to growth of new fibers originating from the graft (Privat et al. 1986; Privat et al. 1988; Privat et al. 1989; Rajaofetra et al. 1992) Since spinal hemisection only eliminates the descending innervation of 5 HT to half of the cord, w e cannot discount the possibility that embryonic

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154 RN cells may somehow facilitate enhanced spro uting of endogenous serotonergic fibers (Saruhashi et al. 1996; Golder et al. 2005) leading to the enhance 5 HT immunoreactivity observed herein. However, results from rats receiving embryonic graft control transp lants (i.e. FSC) support our hypothesis that RN transplants are directly responsible for the enhance d 5 HT present near phrenic motoneurons (and INs). Rats receiving FSC transplants derived from the same developmental time point (Reier et al. 1988; Reier et al. 1992) did not demonstrate increased serotonergic innervation of target neuronal regions (Figures 5 3 and 5 4), nor were they associated with improvements in ventilation or phrenic motor output (see below). Y et, more precise visualization or quantification of 5 HT spinal reinnervation following RN cell transplantation in a hemisection model is warranted for future studies. Alternative cell transplant sources for 5 HT supplementation may enhance the potential q uantification of fiber growth. Indeed, p revious studies of locomotor recovery after SCI have used transplant s of hNT2.19 cells, an immortalized human neuronal cell line which actively secretes 5 HT to enhance 5 HT levels near lumbar motor pools (Reier P .J., 2003; Eaton et al. 2008) Such grafts were associated with improved locomotion when placed in the dorsal lumbar spinal cord (Eaton et al. 2008) Transplantation of hNT2.19 cells in our C2HS model may allow for immun odetection of both 5 HT and human specific neuronal markers (e.g. human neurofilament) providing a means to visualize and quantify graft specific innervation (versus host sprouting). To that end, we initiated the clonal expansion and differentiation of hNT 2.19 cells with the goal of using them as intraspinal transplants below a C2HS. Figure 5 15 provides a

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155 demonstration of recent in vitro immunohistochemistry of cultured hNT2.19 cells to be used in future studies. Enhanced Inspiratory Tidal Volume Followin g RN Transplantation Following C2HS respiratory deficits appear more pronounced under conditions of respiratory stress (i.e. hypoxia or hypercapnia) (Fuller et al. 2006; Fuller et al. 2008) Our data suggest that enhanced 5 HT immunodetection in the spinal cords of rats receiving RN transplants is associated with increased inspiratory V T during hypercapnia when assessed with barometric plethysmography (Figure 5 5). C onditions of increased respiratory demand may al so activate the CPP (Golder et al. 2003; Goshgarian, 2003) providing evidence to support an increased role for the CPP in V T during respiratory challenge (Golder et al. 2001 ; Fuller et al. 2006; Fuller et al. 2008) in rats receiving RN transplants. However, acute phrenicotomy of the ipsilateral phrenic nerve (see Chapter 3) during hypercapnic challenge in anesthetized RN transplanted rats (described below) does not directl y support this hypothesis. Regardless, our results clearly demonstrate that rats receiving RN transplants more effectively respond to respiratory challenge 6 weeks post transplantation compared to controls (i.e. rats receiving either sham or FSC transplant s). Consistent with these results, p rior studies utilizing FSC transplantation following C2HS have shown minimal beneficial effects on E (White et al. 2010) Thus, these data highlight the imp ortance of 5 HT neuromodulator supplementation for maximizing functional respiratory recovery following C2HS. RN Transplants Augment Ipsilateral Phrenic Nerve Activity We hypothesized that enhanced 5 HT innervation of ipsilateral phrenic motoneurons follo wing RN transplantation would correspond with increased phrenic

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156 burst amplitudes under conditions of anesthesia, mechanical ventilation and vagotomy (Golder et al. 2001; Golder & Mitchell, 2005; Fuller et al. 2006; Fuller et al. 2008) These experimental conditions allow for controlled assessment of ipsilateral phrenic motor recovery and strengthening of crossed phrenic signaling following C2HS O ur results demonstrate that rats receiving RN transplants produce la rger ipsilateral phrenic burst amplitudes than control rats during periods of high respiratory demand (i.e. hypoxia, Figure 5 8) T he robust increase of ipsilateral phrenic output during challenge may suggest that rats receiving RN transplants are more pro ficient than controls in recruit ing ipsilateral phrenic motoneurons via the CPP in response to rising demand (Figures 5 8 ) ; although an improved 5 HT associated rate coding mechanism in ipsilateral phrenic motoneurons cannot be discounted Further, t hese d ata are consistent with p revious studies showing the impact of 5 HT and serotonergic agents o n expression of the CPP. For example, in acutely injured C2HS rats, the CPP can be revealed with introduction of the 5 HT precursor 5 Hydroxytryptophan (Ling et al. 1994; Zhou & Goshgarian, 1999, 2000; Fuller et al. 2003) and this functional improvement is eliminated with the broad spectrum 5 HT receptor antagonist methysergide (Zhou & Goshgarian, 1999, 2000) In addition, as 5 HT levels increase to the ipsilateral phrenic motonucleus following C2HS the amplitude of ipsilateral phrenic motor output has been shown to increase with near linear correlation (Golder & Mitchell, 2005) This suggests a role for 5 HT in not only the initial unmasking of the CPP, but also in the progressive amplification of ipsilateral phrenic activity following C2HS. M echanisms un derlying the possible 5 HT mediated strengthening of crossed phrenic inspiratory synapses may be similar to those observed with phrenic long term

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157 facilitation (LTF) (Fuller et al. 2000; Mitchell et al. 2001; Baker Herman & Mitchell, 2002) In this model, activation of the 5 HT 2A receptor on phrenic motoneurons is linked to amplification of phrenic burst amplitudes for 60 minutes or more following intermittent exposure to hypoxia (Fuller et al. 2000; Mitchell et al. 2001; Baker Herman & Mitchell, 2002) Specifically, 5 HT 2A receptor activation leads to a series of intracellular events involving increased respiratory signal sensitivity, strengthened glutamatergic synapses and neurotrophic factor (BDNF) translation and release (Mitchell et al. 2001; Baker Herman & Mitchell, 2002) Therefore, o nce the hypoxic stimulus is removed, prolonged phrenic motor output is believed to be a spinal level enhancement of neural signal sensitivity catalyzed by 5 HT Interestingly, this model of spinal neuroplasticity is not expressed in the contralateral phrenic nerve following C2HS in rats (Doperalski & Fuller, 2006) It was postulated that the increased motor demand place on contralateral phrenic m (Fuller et al. 2005; Doperalski & Fuller, 2006) ). Though we did not measure LTF in our study, our data support similar mechanisms of 5 HT 2 A mediated synaptic enhancement in crossed phrenic circuitry underlying augmented ipsilateral phrenic output in RN transplanted rats. Specifically, injection of the 5 HT 2A receptor antagonist ketanserin during bilateral phrenic nerve recording produced a m ore substantial reduction in ipsilateral phrenic burst amplitudes in rats receiving RN transplants than in the ipsilateral phrenic bursts of control rats or contralateral phrenic nerve outputs in either group (Figures 5 10) This comparatively robust effec t on ipsilateral phrenic output in rats receiving RN transplants suggests an enhanced role of the 5 HT 2A receptor in facilitating recovery of ipsilateral phrenic bursting. Indeed, it is important to note that 5 HT 2A receptors on ipsilateral

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158 phrenic motoneu rons are upregulated following C2HS (Fuller et al. 2005) Thus, it is feasible to conclude that increased 5 HT 2A receptor expression, combined with chronic restoration of 5 HT to the vicinity o f phrenic motoneurons following RN transplantation may combine to activate intracellular pathways leading to strengthened crossed phrenic synaptic connectivity and enhanced phrenic motor output during respiratory challenge. Effect of Acute Phrenicotomy on V T Following RN Transplantation If increased 5 HT innervation of phrenic motoneurons following RN transplantation indeed strengthen s existing glutamatergic synapses associated with the CPP via the 5 HT 2 A receptor as we suggest (Fuller et al. 2000; Mitchell et al. 2001; Baker Herman & Mitchell, 2002; Dale Nagle et al. 2010) neural activity associated with the CPP may have greater influence on inspiratory V T following C2HS than has previously been reported (see Cha pter 3 (Golder et al. 2003; Fuller et al. 2006; Fuller et al. 2008) ). We tested this idea using the acute phrenicotomy methods established in Chapter 3 in a subset of transplanted rats. Surprisingly, thoug h rats receiving RN transplants continued to demonstrate larger V T during respiratory challenge under anesthesia compared to control rats (Figure 5 6), the modest reduction in V T with removal of ipsilateral phrenic contributions was similar to controls (Figure 5 6) and consistent with our prior reports (Chapter 3). It should be emphasized that direct comparisons between these studies is difficult due to methodological variations (e.g. multiple surgical procedures, transplanted cells), however, the consistency in r esults may highlight the influence of RN transplants on motor outputs of other inspiratory networks ( e.g. inspiratory intercostal muscles, see Chapter 4) or for the increased excitability of multiple inspiratory neuron groups simultaneously For example, a cting through the 5 HT 2 A receptors, 5 HT has been implicated in the augmentation of persistent inward Ca 2+ currents (PIC) on motoneurons

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159 (Heckman et al. 2008; Heckman et al. 2009) These currents effectively depol ariz e the membrane potential of neurons closer to their firing potential, making them more readily activated with smaller excitatory synaptic inputs (Heckman et al. 2008; Heckman et al. 2009; Sukiasyan et al. 2009 ) This may represent another mechanism underlying progressive crossed spinal phrenic motor recovery following C2HS injuries ; though PICs in adult rat phrenic motoneurons have not been demonstrated In addition, 5 HT associated increases in motoneuron exc itability may occur through other putative receptors other than the 5 HT 2A receptor Indeed, 5 HT 2B (MacFarlane et al. 2011) 5 HT 2C (Zhou et al. 2001a) and 5 HT 7 (Baker Herman et al. 2010; Hoffman & Mitchell, 2011) receptors are found on phrenic motoneurons and play roles in phrenic neuroplasticity. Thus, it is possible for RN transplants to alter the membrane excitability of inspir atory neurons via multiple mechanisms. Since pre phrenic INs may play some functional role in the CPP (Lane et al. 2008b; Lane et al. 2009; Sandhu et al. 2009a; Lane, 2011) we must not overlook the possibility that RN transplants may have some effect on these cells as well Indeed, the abundance of 5 HT present around the central canal in normal and C2HS rats receiving RN transplants (Figures 5 3 and 5 4, and (Steinbusch, 1981; Tork, 1990) ) may predict the presence of putative 5 HT receptors on pre phrenic INs, though detailed characterization of these cells has yet to be completed. Pre phrenic INs also link phrenic and intercostal motor circuits (Lane et al. 2008b) As such, i t is interesting to speculate that enhanced 5 HT innervation, in particular to the regions around the central canal and in the vicinity of INs (Lane et al. 2008b) foll owing RN transplantation may lead to enhanced ou tput of inspiratory intercostal muscles If this were the case, it is

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160 logical to speculate that associated plasticity in inspiratory intercostal output following RN transplantation would mirror that of ipsila teral phrenic output (see Chapter 3), potentially explaining the consistent contribution s of ipsilateral phrenic activity to V T seen here. Of course, this idea is predicated on the idea that 5 HT directly influences cervical IN activity, which has not been conclusively shown Summary Transplantation of embryonic RN cells below a C2HS lesion led to e nhanced innervat ion of 5 HT to the ipsilateral ventral horn near phrenic motoneurons and to the area of cervical INs around the central canal This e nhanced 5 H T innervation was associated with enhanced ventilation in awake, unanesthetized rats and increased ipsilateral phrenic burst amplitudes in anesthetized rats indicative of strengthened crossed phrenic inspiratory signaling (Goshgarian, 2003; Golder & Mitchell, 2005; Fuller et al. 2006; Fuller et al. 2008; Goshgarian, 2009) Injection of ketanserin, a 5 HT 2A receptor antagonist, to rats receiving RN transplants produced a significant reduction in the ipsilateral phre nic output compared to controls Since 5 HT 2A receptors are upregulated on ipsilateral phrenic motoneurons following C2HS (Fuller et al. 2005) we conclude that the combined increase in 5 HT a vailability and receptor density following RN transplantation and C2HS likely underlie the observed functional improvements. Th ese results provide support for the possible use of chronic 5 HT supplementation as a therapeutic tool to enhance motor output fo llowing experimental SCI. Future studies will be needed to discern precise mechanism s and location s of action for RN transplants as we could not definitively link improved CPP activation with V T in spontaneously breathing rats. Nevertheless, our results a ssociate 5 HT with functional respiratory improvements following cervical SCI and provide initial support for 5 HT

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161 replacement as a possible standalone therapeutic or in combination with other cell transplant paradigms (e.g. stem cells) for respiratory rec overy following SCI

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162 Figure 5 1 Dissection of the e mbryonic raph nucleus. The medullary raph nucleus was removed from embryonic Sprague Dawley rats at day 14 of development (E14) according to previously published methods (A) (Konig, 1989) To confirm the location of midline 5 HT positive neurons within the medullary raph at E14, 40 m l ongitudinal section s were taken from the dissected raph tissue and stained with antibodies against 5 HT (with FITC secondary antibody for fluorescent visualization) Robust fluorescent staining of serotonergic cells and processes (green) was clearly visib le within the area used for transplants (B). pr: pontine raph mr: medullary raph lc: locus coeruleus. V, VII, and VIII: cranial nerves 5, 7, and 8 Scale bar: 1 00 m

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163 Figure 5 2 A representative histological section il lustrating a C2HS lesion. This 40 m transverse section was taken from the s econd cervical segment (C2) at 6 wks post injury Immunodetection of 5 HT was completed and tissue processed for light microscopy (nickel enhanced DAB staining, appears black). Tis sue was then counter stained with Cresyl violet (purple) The absence of white and grey matter in the ipsilateral (IL) spinal cord suggests an anatomically complete C2HS. CL: contralateral; GM : grey matter ; WM : white matter Scale bar: 200 m

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164 Figure 5 3 R epresentative section s illustrating cell transplant s at C3 These 40 m transverse section s were taken from the third cervical segment (C3 ) at 6 wks post transplantation for visualization with light microscopy Immunodetectio n of 5 HT was completed (nickel enhanced DAB staining, appears black) for qualitative comparison between groups Rats receiving embryonic raph cell transplants to enhance 5 HT (5 HT Tx) showed enhanced immunostaining of 5 HT around the central canal, and in the ipsilateral C3 ventral horn compared to rats receiving sham transplants (Sham Tx, culture media) or transplants of fetal spinal cord cells (FSC Tx). FSC transplant s never stained positively for 5 HT and were counter stained with Cresyl violet (purple ) for visualization of transplanted cells. CL: contralateral. Scale bar s: 1 00 m.

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165 Figure 5 4 R epresentative section s illustrating 5 HT immunodetection at C4. These 40 m transverse section s were taken from the fourth cervi cal segment (C4) of the same rats seen in figure 5 3 at 6 wks post transplantation for visualization with light microscopy. Immunodetection of 5 HT was completed (nickel enhanced DAB staining, appears black) for qualitative comparison between groups. Rats receiving embryonic raph cell transplants to enhance 5 HT (5 HT Tx) showed enhanced immunostaining of 5 HT around the central canal, and in the ipsilateral (IL) C4 ventral horn s approximating phrenic motoneurons (outlined in red; magnified in both IL and CL ventral horn columns) compared to rats receiving sham transplants (Sham Tx, culture media) or transplants of fetal spinal cord cells (FSC Tx ). CL: contralateral. Scale bars: 100 m.

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166 Figure 5 5 Effects of cell transplantation on ventil ation in unanesthetized rats. Inspiratory frequency, tidal volume and minute ventilation were recorded during quiet breathing (baseline) and respiratory challenge (hypercapnia) in rats 6 weeks post cell transplantation. All parameters were similar in rats receiving sham transplants and fetal spinal cord (FSC) transplants, so they were combined into one control group for comparison. Rats receiving raph cell transplants (5 HT transplants) demonstrated larger tidal volume production during hypercapnia compare d to controls, leading to enhanced minute ventilation. ###: P<0.001 from baseline ; : P<0.05 ** : P<0.01 from controls

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167 Figure 5 6 Effect of acute phrenicotomy on tidal volume in anesthetized rats. Phrenicotomy (PhrX) during hypercapnic respiratory challenge in anesthetized rats caused an immediate (next breath) decline in tidal volume (left). The relative decline in tidal volume (i.e. % decline) following phrenicotomy was similar between rats receiving raph cell transplants (5 HT trans plants) and rats receiving control transplants (sham or fetal spina l cord transplants; right). ** : P<0.01, : P<0.05 from pre PhrX.

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168 Figure 5 7 E xamples of phrenic motor output recorded in anesthetized rats. The images depict raw ( Phr ) and Phr ) phrenic signals from a rat receiving a sham transplant and a rat receiving an embryonic raph transplant (5 HT transplant). Note that ipsilateral phrenic signals from the sham transplant rat change little with hypoxic challenge compared t o the rat receiving the 5 HT transplant Scaling is identical in all panels. Scale Bar: 1 sec.

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169 Figure 5 8 T he effect of hypoxia on bilateral phrenic motor output. The top panels reflect raw inspiratory phrenic burst amplitudes (reported in arbitrary units, a.u.) in ipsilateral (IL) and co ntralateral (CL) phrenic nerves of rats receiving embryonic raph transplants (5 HT Tx) or rats receiving control transplants (sham or fetal spinal cord transplants). Note that only t he raw IL phrenic burst amplitudes of 5 HT Tx rats increased substantially in response to hypoxia. However, when this hypoxic response was standardized relative to baseline bursting (% baseline, BL), or when the ipsilateral phrenic burst amplitudes were co mpared relative to contralateral bursting (% contralateral) differences between groups did not reach significance. ##: P<0.01 from baseline ; ** : P<0.01 from control

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170 Figure 5 9 The effect of ketanserin on phrenic output The images depict raw (Phr) and Phr ) phrenic signals from a rat receiving a sham transplant and a rat receiving an embryonic raph transplant (5 HT transplant) before and 2 minutes after injection of ketanserin (denoted by dashed black line) Note that ipsilateral phrenic signals from the sham transplant rat reduced following injection of ketanserin, but ipsilateral phrenic bursting was nearly eliminated in the rat receiving a 5 HT transplant. Scaling is identical in all panels. Scale Bar: 1 sec.

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171 Figure 5 10 The effect of ketanserin on phrenic burst amplitudes. The reduction in raw phrenic burst amplitudes (arbitrary units, a.u.) and the relative declines in amplitude (% decline) following ketanserin injection are s hown for ipsilateral (IL) and contralateral (CL) phrenic nerves. Note that ipsilateral phrenic signals from both control (sham and fetal spinal cord cells) and transplant rats reduced following injection of ketanserin. However, the differences in relative decline of ipsilateral phrenic burst amplitudes between groups did not reach significance. Also, a small, but significant decline in the contralateral phrenic burst amplitudes was seen in control rats following ketanserin injection. *** : P<0.001, : P<0.05 from pre ket anserin injection (pre ket).

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172 Figure 5 11 Example of Immunohistochemical staining for 5 HT (Rat F11) These 40 m transverse section s were taken from C3 and C4 6 wks post embryonic raph cell transpla ntation for visualization with light microscopy. Immunodetection of 5 HT was completed using a nickel enhanced DAB staining (appears black). Higher magnification images from the ventral horns represent areas outlined in red. IL: ipsilateral. CL: contralater al Scale bars: 100 m.

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173 Figure 5 12 Example of Immunohistochemical staining for 5 HT (Rat E) These 40 m transverse section s were taken from C3 and C4 6 wks post embryonic raph cell transplantation for visualization with light micro scopy. Immunodetection of 5 HT was completed using a nickel enhanced DAB staining (appears black).Higher magnification images from the ventral horns represent areas outlined in red. IL: ipsilateral. CL: contralateral Scale bars: 100 m

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174 Figure 5 13 Example of Immunohistochemical staining for 5 HT (Rat G ) These 40 m transverse sections were taken from C3 and C4 6 wks post embryonic raph cell transplantation for visualization with light microscopy. Immunodetection of 5 HT was complete d using a nickel enhanced DAB staining (appears black).Higher magnification images from the ventral horns represent areas outlined in red. IL: ipsilateral. CL: contralateral Scale bars: 100m

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175 Figure 5 14 Example of Immunohistochemi cal staining for 5 HT (Rat 6.3) These 40m transverse sections were taken from C3 and C4 6 wks post embryonic raph cell transplantation for visualization with light microscopy. Immunodetection of 5 HT was completed using standard DAB staining (appears bro wn ) co stained with Cresyl violet .Higher magnification images from the ventral horns represent areas outlined in red. IL: ipsilateral. CL: contralateral Scale bars: 100m

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176 Figure 5 15. Example of NT2.19 cells. As an alternativ e 5 HT cell transplant source (i.e. in place of the embryonic raph cell s) culturing of the human NT2.19 cell line is ongoing according to established protocols (Eaton et al. 2008) This cell line is non t umorigenic and prod uces neurons (A light microscopy ) that endogenously produce and release 5 HT (B, stained for 5 HT with fluorescent secondary antibody) In preliminary studies (C), cultured hNT2.19 cells stained positively for the neuronal marker III Tubulin (green) and for 5 HT (red).Dapi: nuclear stain (blue).

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177 Table. 5 1. Age and weight values for rats receiving delayed (7 days) transplants of cell culture medium ( Sham Tx) fetal spinal cord cells (FSC Tx ) or embryonic serotonergic cells (5 HT Tx) Values are repres entative of six weeks post transplantation at which time all outcome measures were completed. N Age (days) Weight (g) Sham Tx 8 144 1 384 9 FSC Tx 5 145 1 372 12 5 HT Tx 7 142 1 385 10 Table. 5 2 Blood gas and mean arterial blood pre ssure (MAP) values taken during baseline ventilation in spontaneously breathing, anesthetized rats Rats receiving sham (i.e. cell culture medium) and fetal spinal cord cell transplants were combined into a single control group (Control Tx) for comparison with rats receiving embryonic serotonergic cell transplants (5 HT Tx). P a O 2 (mmHg) P a CO 2 (mmHg) pH MAP (mmHg) Control Tx 195 9 44 3 7.302 0.02 89 5 5 HT Tx 209 10 39 2 7.330 0.01 95 10

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178 Table. 5 3. Blood gas and mean arterial blood pressure (MAP) va lues taken during baseline ventilation in anesthetized mechanically ventilated rats. Rats receiving sham (i.e. cell culture medium) and fetal spinal cord cell transplants were combined into a single control group (Control Tx) for comparison with rats rece iving embryonic serotonergic cell transplants (5 HT Tx). P a O 2 (mmHg) P a CO 2 (mmHg) pH MAP (mmHg) Control Tx 195 6 31 2 7.37 0.03 97 8 5 HT Tx 220 12* 35 7 7.40 0.02 101 17 *:P<0.05, unpaired T test

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179 CHAPTER 6 CONCLUSIONS AND FUTURE DIRECTIONS Respiratory neural networks in the spinal cord demonstrate extensive plasticity in response to stimuli or in jury. This dissertation presented novel insights on spontaneous neuroplasticity within spinal respiratory motor systems following high cervical spinal cord injury in rats and a specific cell based treatment to enhance this plasticity. Chapters 3 and 4 demonstrated the recovery of respiratory neuro musc ular activity in spontaneously breathing rats following C2HS fundamental to progres sive improvements in inspiratory tidal volume. Subsequently, in Chapter 5, we demonstrated that restoration of serotonergic innervation to ipsilateral phrenic motoneurons using cell transplants of embryonic raph cells could augment phrenic motor output an d recovery of tidal volume following C2HS. These results provided important proof of principle support for chronic serotonin supplementation as a future therapeutic intervention following SCI. The Contribution of the Spontaneous Crossed Phrenic Phenomenon to Inspiratory Tidal Volume in Spontaneously Breathing Rats Following C2HS in rats, transient paralysis of the ipsilateral hemidiaphragm results from disruption of primary bulbospinal motor drive to ipsilateral phrenic motoneurons. Previous studies have de monstrated spontaneous neuroplasticity in the remaining phrenic circuit allowing for modest recovery of ipsilateral phrenic motor output (i.e. the sCPP). Yet, the minimal inspiratory bursting associated with the sCPP has never been directly linked to recov ery of tidal volume. Our study established a novel method of measuring the impact of phrenic neural output to tidal volume in spontaneously breathing rats with acute phrenicotomy. The results of this study suggested that neural activity in the ipsilateral phrenic nerve (via the sCPP) made a meaningful contribution to

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180 tidal volume by 2 weeks post C2HS. However, sCPP associated neural activity showed no progressive increase in relative importance following this time point. In other words, though strengthening of sCPP neural output may have continued, it was mirrored by parallel augmentation of other respiratory neural outputs. In addition, our results suggested that the relative activation of the sCPP was not dependent on level of respiratory drive, a finding at odds with multiple prior studies. We believe that this result further supports the idea of parallel plasticity in other respiratory motor circuits facilitating long term recovery of tidal volume after C2HS. However, future studies will be required to va lidate this hypothesis and define mechanisms underlying spontaneous plasticity in other respiratory circuits following C2HS. Finally, the results of this study support the use of acute phrenicotomy in spontaneously breathing rats as a method to evaluate th e functional contribution of phrenic neural output to tidal volume. This method may prove useful to evaluate the impact of treatments intended to strengthen the sCPP. In addition, the limited contributions of the sCPP to spontaneous tidal volume recovery f ollowing C2HS observed herein will serve as a template on which to test therapeutic strategies intended to increase spinal synaptic efficacy after spinal cord injury. Spontaneous Recovery of Inspiratory Intercostals Following High Cervical Hemisection in Rats forth in Chapter 3 by describing spontaneous recovery of inspiratory intercostal muscles following C2HS in rats. We hypothesized that neural plasticity associated wi th activation of ipsilateral external (i.e. inspiratory) intercostals (rostral > caudal) would lead to recovery of EMG activity by 2 weeks post C2HS to support on going spontaneous

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181 recovery of tidal volume. Further, we proposed that this plasticity may be associated with changes in the recruitment of thoracic interneurons known to facilitate intercostal activity. Our results demonstrated near complete recovery of inspiratory related EMG activity in rostral inspiratory intercostals during baseline and hyperc apnic challenge by 2 weeks post C2HS. Further, the results suggested a tendency for enhanced recruitment of ipsilateral versus contralateral intercostals for tidal volume maintenance by 8 weeks post C2HS. Though exciting, t his result was treated cautiously as variability in EMG recordings within the 8 week C2HS group was high. Regardless, the restoration of ipsilateral intercostal EMG activity by 2 weeks, following an initial period of inactivity, not only suggested robust spontaneous neuroplasticity in in tercostal motor output following recovery. M ore detailed anatomical tracing studies combined with phenotypic identification of thoracic interneuron s will be required t o define the role of thoracic the demonstration of robust intercostal plasticity associated with spontaneous tidal volume improvement following C2HS is in line with our hypothesis that rats exhibit paral lel plasticity in respiratory motor outputs, other than the sCPP, to facilitate on going spontaneous recovery of tidal volume. Future studies are warranted in rodent and human subjects to identify the extent of intercostal recruitment during spontaneous re spiration following SCI. The demonstration of enhanced reliance on intercostal activation for inspiratory motor recovery may shift the focus of rehabilitation towards maximizing intercostal function versus restoration of diaphragm activity.

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182 Transplantatio n of Embryonic Medullary Raph Cells Enhances Tidal Volume and Phrenic Bursting Following C2 Hemisection in Rats We transitioned our focus from patterns of spontaneous tidal volume recovery following C2HS to a cell based treatment approach to augment respi ratory recovery in Chapter 5. Specifically, we used embryonic raph cells transplanted below a C2HS lesion to enhance the serotonergic innervation of ipsilateral phrenic motoneurons. Similar cell transplants have previously been utilized in thoracic transe ction models of SCI and demonstrated the exquisite capacity to reinnervate target cell populations caudal to the lesion with serotonin (5 HT), leading to increased functional recovery. Chapter 5, however, represented the first study to use embryonic raph cell transplants in a cervical hemisection model with the goal of enhancing respiratory outcomes. Our results demonstrated that E14 raph cells transplanted intraspinally below a C2HS injury remained viable 6 weeks following transplantation. Further, rats receiving raph cell transplants presented with enhanced serotonergic immunohistochemical staining in the ipsilateral ventral horns approximating phrenic motoneurons and in the region of the central canal approximating pre phrenic interneurons compared wit h controls. This enhanced 5 HT supply was associated with increased amplitudes of ipsilateral phrenic nerve bursting in anesthetized rats and increased tidal volume during respiratory challenge in unanesthetized rats. Accordingly, we concluded that chronic restoration of 5 HT to phrenic motoneurons was associated with enhanced recovery of respiratory function following cervical SCI and may be an appropriate target for future therapeutic intervention. Though the results of this study were exciting, many que stions remained related to the underlying mechanisms of how the fetal raph cell transplants conveyed their

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183 effects. For example, although studies have previously demonstrated raph cell transplants contain the capacity to specifically reinnervate motoneur on pools previously receiving 5 HT innervation, our study could only qualitatively demonstrate enhanced 5 HT present in the ventral horn following transplant. In other words, we could not show growth of serotonergic axons from the graft to the ipsilateral phrenic pool. Follow up studies will utilize transplanted cells that can be differentiated from host tissue using immunhistochemistry (e.g. raph cells from eGFP rat colonies or human 5 HT cell lines). In addition, we hypothesized, based on a subset of rat s receiving injections of a 5 HT 2A receptor antagonist ( ketanserin ) that the mechanism underlying raph transplant associated increased phrenic motor output was based on activation of 5 HT 2 receptors. A rich literature exists to support this hypothesis, i ncluding the demonstration of increased 5 HT 2A receptor expression on phrenic motoneurons following C2HS. But, additional studies will be required to confirm this mechanism as the primary mode of raph cell transplant effects. It was also interesting to no te that relative tidal volume changes following acute ipsilateral phrenicotomy in rats receiving raph cell transplants did not support amplification of sCPP output as the major component of functional respiratory recovery in this model. This result sugges ts that embryonic raph cell transplants may exert additional effects on contralateral phrenic motor output and intercostal motor output. Follow up studies exploring the motor output of these respiratory groups with raph cell transplants will be important In summary, rats express a modicum of spontaneous respiratory recovery following C2HS. This spontaneous recovery involves utilization of crossed spinal inspiratory phrenic and intercostal motor pathways to sustain progressive tidal volume

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184 improvements. It may be possible to enhance this spontaneous recovery by restoring 5 HT supply to ipsilateral phrenic motor pools (and respiratory associate d interneurons) void of this key neuromodulatory input following injury. Use of embryonic raph cells to resupply 5 HT to phrenic motoneurons below a C2HS represented an important proof of principle study supporting long term serotonergic replacement as a therapeutic intervention for augmenting respiratory function after cervical SCI. However, future studies exploring the mechanisms underlying embryonic raph cell transplant effects may result in less invasive and more specific pharmacological interventions for functional respiratory recovery.

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209 BIOGRAPHICAL SKETCH Brendan Joseph Dougherty was born i n Syracuse, New York in 1978. He graduated from George Walton Comprehensive High School in 1996 and proceeded to Maryville University of St. Louis where he completed his Bachelor of Science degree in physical therapy in 2000. Subsequently, Brendan worked f ull time as a physical therapist in multiple clinical settings for 6 years before returning to full time graduate interdisciplinary p rogram (IDP) in biomedical sciences at the University of Florida in 2006 and gr aduated December 2011 with his Ph.D. in neuroscience.