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Neuroplasticity in the Phrenic Motor System

Permanent Link: http://ufdc.ufl.edu/UFE0042542/00001

Material Information

Title: Neuroplasticity in the Phrenic Motor System Intermittent Hypoxia, Spinal Cord Injury and Stem Cell Transplantation
Physical Description: 1 online resource (159 p.)
Language: english
Creator: Sandhu, Milapjit
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: cell, cord, injury, neural, phrenicotomy, plasticity, precursor, respiration, spinal, stem, transplantation
Rehabilitation Science -- Dissertations, Academic -- UF
Genre: Rehabilitation Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Neuroplasticity can be defined as a persistent change in the neural control system based on prior experience. Over the last 20 years, neuroplasticity in the respiratory control system has been extensively studied. It is hoped that by understanding how the respiratory neural control system can change with experience and/or injury, we may learn how to better promote plasticity. This doctoral dissertation presents the results of three studies of respiratory neuroplasticity using an adult rat experimental model. Perhaps the most studied model of respiratory neuroplasticity is long-term facilitation (LTF) of phrenic motor output following intermittent hypoxia. This response is usually studied in anesthetized animals with extracellular recordings of phrenic output being made from the cut central end of phrenic nerves. However, the phrenicotomy procedure removes afferent inputs to the spinal cord and also axotomizes phrenic motoneurons? both can potentially affect motoneuron excitability. Accordingly, in Aim 1 we tested the hypothesis that intermittent hypoxia induced LTF of phrenic output is greater in rats with cut than intact phrenic nerves. Our primary finding was that phrenicotomy is associated with a substantial increase in the magnitude of both the acute hypoxic response and LTF. Spinal cord injury (SCI) at the cervical level disrupts descending neural inputs to respiratory motoneurons and can paralyze respiratory muscles. Respiratory-related problems are a leading cause of morbidity and mortality in patients with cervical SCI. Therefore, an important goal of SCI research is to improve respiratory motor function by harnessing neuroplasticity within the neural system controlling breathing. In Aim 2, we investigated the neural circuitry underlying spontaneous phrenic recovery following C2 spinal cord hemisection (C2HS) injury. Our hypothesis was that synchronization between ipsilateral (IL) and contralateral (CL) phrenic motoneurons (PhrMNs) discharges, studied using cross-correlation analyses, would be affected after chronic C2HS. Our primary finding was that the conduction time to IL (vs. CL) PhrMNs is prolonged after C2HS suggesting the possibility of polysynaptic inputs to IL PhrMNs after injury. In the final aim, our goal was to investigate a potential ?neural repair? strategy for promoting motor recovery after SCI. These experiments examined the feasibility of neural precursor cells (NPC), derived from post-natal rat pups, as potential candidates for transplantation after C2HS injury. We hypothesized that post-natal NPCs will survive, migrate and improve respiratory outcome after transplantation into the injured cervical spinal cord. Our data demonstrate these cells can survive, migrate, differentiate and improve phrenic output especially during respiratory challenges. These results serve as a proof of principle that NPC transplantation is a potentially viable option for therapeutic intervention after SCI.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Milapjit Sandhu.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Fuller, David D.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042542:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042542/00001

Material Information

Title: Neuroplasticity in the Phrenic Motor System Intermittent Hypoxia, Spinal Cord Injury and Stem Cell Transplantation
Physical Description: 1 online resource (159 p.)
Language: english
Creator: Sandhu, Milapjit
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: cell, cord, injury, neural, phrenicotomy, plasticity, precursor, respiration, spinal, stem, transplantation
Rehabilitation Science -- Dissertations, Academic -- UF
Genre: Rehabilitation Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Neuroplasticity can be defined as a persistent change in the neural control system based on prior experience. Over the last 20 years, neuroplasticity in the respiratory control system has been extensively studied. It is hoped that by understanding how the respiratory neural control system can change with experience and/or injury, we may learn how to better promote plasticity. This doctoral dissertation presents the results of three studies of respiratory neuroplasticity using an adult rat experimental model. Perhaps the most studied model of respiratory neuroplasticity is long-term facilitation (LTF) of phrenic motor output following intermittent hypoxia. This response is usually studied in anesthetized animals with extracellular recordings of phrenic output being made from the cut central end of phrenic nerves. However, the phrenicotomy procedure removes afferent inputs to the spinal cord and also axotomizes phrenic motoneurons? both can potentially affect motoneuron excitability. Accordingly, in Aim 1 we tested the hypothesis that intermittent hypoxia induced LTF of phrenic output is greater in rats with cut than intact phrenic nerves. Our primary finding was that phrenicotomy is associated with a substantial increase in the magnitude of both the acute hypoxic response and LTF. Spinal cord injury (SCI) at the cervical level disrupts descending neural inputs to respiratory motoneurons and can paralyze respiratory muscles. Respiratory-related problems are a leading cause of morbidity and mortality in patients with cervical SCI. Therefore, an important goal of SCI research is to improve respiratory motor function by harnessing neuroplasticity within the neural system controlling breathing. In Aim 2, we investigated the neural circuitry underlying spontaneous phrenic recovery following C2 spinal cord hemisection (C2HS) injury. Our hypothesis was that synchronization between ipsilateral (IL) and contralateral (CL) phrenic motoneurons (PhrMNs) discharges, studied using cross-correlation analyses, would be affected after chronic C2HS. Our primary finding was that the conduction time to IL (vs. CL) PhrMNs is prolonged after C2HS suggesting the possibility of polysynaptic inputs to IL PhrMNs after injury. In the final aim, our goal was to investigate a potential ?neural repair? strategy for promoting motor recovery after SCI. These experiments examined the feasibility of neural precursor cells (NPC), derived from post-natal rat pups, as potential candidates for transplantation after C2HS injury. We hypothesized that post-natal NPCs will survive, migrate and improve respiratory outcome after transplantation into the injured cervical spinal cord. Our data demonstrate these cells can survive, migrate, differentiate and improve phrenic output especially during respiratory challenges. These results serve as a proof of principle that NPC transplantation is a potentially viable option for therapeutic intervention after SCI.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Milapjit Sandhu.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Fuller, David D.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042542:00001


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1 NEURO PLASTICITY IN THE PHRENIC MOTOR SYSTEM: INTERMITTENT HYPOXIA, SPINAL CORD INJURY AND STEM CELL TRANSPLANTATION By MILAPJIT SINGH SANDHU 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 2010

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2 2010 Milapjit Singh Sandhu

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3 To my pare nts who ta ught me the value of perseverance ; and t o my wife for her unconditional love and sup port in this endeavor.

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4 ACKNOWLEDGMENTS The writing of this dissertation has been an exciting and yet sometimes challenging experience for me. It has been an undertaking that I could not have completed without the help and support of a number of people in my life. M y deepest gratitude goes to my advisor Dr. David Fuller Every doctoral student should be so lucky to work with a mentor like Dave. (The converse is not necessarily true). I am forever grateful to him for devot ing a huge amount of effort to help me grow as a scientist and always expressing a genuine concern for my professional and personal well being I also owe him special thanks for his willingness to allow me to work on several interesting projects. I am thankful to Dr. Paul Neuromei Reier, who has been like a second advisor to me. H is leadership is exemplary as is his amazing sense of humor. remember coming out of a meeting with him not feeling completely motivated I want to express my gratitude to Dr. Donald Bolser fo r his genius and excellent suggestions especially with the cross correlation study ; and Dr. Danny Martin for helping me refine my experiments across various studies Many thanks are expressed to Dr. Heather Ross for teaching me the fundamentals of stem cel l biology and for being a great friend I t h as been a real pleasure work ing with her. I would like to thank all the present and past members of the Fuller lab for helping me with various experiments, course corrections proof reading (Elisa) and for makin g it a great place to work: Dr. Kun Ze Lee, Brendan Dougherty, Dr. Kai Qiu, Luther Gill, Elisa Gonzalez Rothi, Sandy Morrison and Rachel Mattio. A s pecial thanks to Nick Doperalski for his friendship and support. I also want to thank all the members of the Reier lab, especially Dr Michael Lane, Barbara and Dr. Todd

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5 White for their enormous help during the last 5 years I owe special thanks to Dr. Peter Kirkwood for his invaluable suggestions and for educating me through several long emails from London I w ould like to allude to the tremendous support and advice I have gotten from my parents Kuldip and Neena, without whom none of this would have been possible. I extend my heartfelt gratitude to my uncles, Kirpal and Narinder for hel ping me when I needed the most ; and all my cousins and friends for their friendship and support. I w ould like to say a big thank you to m y wife, Aman, whose support and sacrifices have been critically important to me Thanks for being my best friend and fo r bearing with me. Finally, I want to thank the A lmighty for all the incredible blessings I have been given in my life.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURES ................................ ................................ ................................ ........ 10 ABSTRACT ................................ ................................ ................................ ................... 12 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 14 Neuroplasticity ................................ ................................ ................................ ........ 1 4 Neural Control of Respiration ................................ ................................ .................. 15 Brainstem Respiratory Centers ................................ ................................ ........ 15 Medullary respiratory nuclei ................................ ................................ ....... 15 Pontine respiratory nuclei ................................ ................................ ........... 18 Cerebral Control of Respiration ................................ ................................ ........ 18 Spinal Cord ................................ ................................ ................................ ....... 19 Descending respiratory tracts. ................................ ................................ ... 19 Respiratory motoneurons ................................ ................................ ........... 20 Propriospinal interneurons and control of breathing ................................ ... 21 Control of Phrenic Motoneurons ................................ ................................ ............. 23 Eupnea ................................ ................................ ................................ ............. 23 Hyperoxia ................................ ................................ ................................ ......... 23 Hypoxia ................................ ................................ ................................ ............ 23 Acute response ................................ ................................ .......................... 25 Short term potentiation (STP) ................................ ................................ .... 25 Progressive augmentation ................................ ................................ ......... 26 Long term facili tation ................................ ................................ .................. 26 Hypercapnia ................................ ................................ ................................ ..... 30 Diaphragm Afferents ................................ ................................ ........................ 30 Lung Afferents ................................ ................................ ................................ .. 32 Spinal Cord Injury ................................ ................................ ................................ ... 34 Effect of cervical spinal cord injury on respiratory function in humans ............. 34 Cervical Hemisection Injury Model ................................ ................................ ... 35 Crossed Phrenic Phenomenon ................................ ................................ ......... 36 Therapeutic Strategies for Treatment of SCI ................................ .......................... 40 Targeted Respiratory Rehabilitation after SCI ................................ .................. 41 Clinical respiratory rehabilitation ................................ ................................ 41 Experimental respiratory rehabilitation ................................ ....................... 42 Cell Transplantation Strategies ................................ ................................ ........ 43 Supportive structures ................................ ................................ ................. 43

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7 Neuronal cell type transplantation ................................ .............................. 46 Neural Precursor Cells ................................ ................................ ..................... 46 Neural Precursor Cell Transp lantation after SCI ................................ .............. 47 2 OUTLINE OF EXPERIMENTS ................................ ................................ ................ 50 Overall Objectives ................................ ................................ ................................ ... 50 Experiment One: Determine the impact of acute phrenicotomy on the expression of hypoxia induced plasticity. ................................ ............................. 51 Experiment Two: Examine changes in synchrony between IL and CL PhrMN discharge after chronic C2HS using cross correlation analyses .......................... 52 Experiment Three: Explore the feasibility of postnatal brain derived NPC transplantation to affect respiratory recovery after chronic C2HS in rat s. ............ 53 3 PHRENICOTOMY ALTERS PHRENIC LONG TERM FACILITATION (LTF) FOLLOWING INTERMITTENT HYPOXIA IN ANESTHETIZED RATS ................... 55 Methods ................................ ................................ ................................ .................. 57 Animals ................................ ................................ ................................ ............. 57 Experimental Preparation ................................ ................................ ................. 57 Experimental Protocols ................................ ................................ ..................... 59 Data Analyses ................................ ................................ ................................ .. 61 Results ................................ ................................ ................................ .................... 61 Baseline Phrenic Activity ................................ ................................ .................. 62 Intermittent Hypoxia ................................ ................................ ......................... 63 Phrenic Long Term Facilitation ................................ ................................ ......... 63 ................................ ............................. 65 Discussion ................................ ................................ ................................ .............. 66 Implications for the Study of Respiratory LTF ................................ ................... 67 Axotomy of Phrenic Motoneurons and Afferent Neurons: Potential Impact on LTF ................................ ................................ ................................ ........... 70 Summary ................................ ................................ ................................ ................ 73 4 CHANGE IN PHRENIC MOTONEURON SYNCHRONY AFTER CHRONIC HIGH CERVICAL HEMISECTION ................................ ................................ .......... 86 Methods ................................ ................................ ................................ .................. 87 Animals ................................ ................................ ................................ ............. 87 Spinal Cord Injury ................................ ................................ ............................. 87 Neurophysiology ................................ ................................ ............................... 88 Spinal Cord Histology ................................ ................................ ....................... 90 Data Analyses ................................ ................................ ................................ .. 90 Overview of the Cross Correlation Technique ................................ .................. 90 Results ................................ ................................ ................................ .................... 91 Discussion ................................ ................................ ................................ .............. 92 Summary ................................ ................................ ................................ ................ 96

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8 5 TRANSPLANTATION OF POST NATAL DERIVED NEURAL PRECURSOR CELLS AFTER HIGH CERVICAL HEMISECTIO N ................................ ................. 99 Materials and Methods ................................ ................................ .......................... 102 Animals ................................ ................................ ................................ ........... 102 Cell culture ................................ ................................ ................................ ..... 102 In vitro Immunocytochemistry ................................ ................................ ......... 102 Spinal Cord Injury ................................ ................................ ........................... 104 Cell Transplantation ................................ ................................ ....................... 104 Plethysmography ................................ ................................ ............................ 105 Phrenic Nerve Recordings ................................ ................................ .............. 105 Spinal Cord Histology ................................ ................................ ..................... 108 Data Analyses ................................ ................................ ................................ 109 Results ................................ ................................ ................................ .................. 110 In vitro Characterization of NPCs ................................ ................................ ... 110 Survival, Migration and Differentiation of NPCs after Transplantation ............ 110 Ventilation in Transplant vs. Control Rats ................................ ...................... 111 Phrenic Motor Output in Transplant vs. Control Rats ................................ ..... 112 Discussion ................................ ................................ ................................ ............ 113 Migration of Transplant ed NPC within the Spinal Cord ................................ .. 113 In vitro and In vivo differentiation of NPCs ................................ ...................... 114 Potential Neural Mechanisms for Respiratory Recover y ................................ 115 Summary ................................ ................................ ................................ .............. 118 6 CONCLUSION ................................ ................................ ................................ ...... 131 Overview ................................ ................................ ................................ ............... 131 Summary of Conclusions ................................ ................................ ...................... 131 Effect of Phrenicotomy on Hypoxia induced Phrenic Long Term Facilitation 131 Effect of C2HS on Phrenic Motoneuron Synchrony ................................ ........ 132 Neural Precursor Cells as a Potential Candidate for Treatment of SCI .......... 133 LIST OF REFERENCES ................................ ................................ ............................. 134 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 159

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9 LIST OF TABLES Table page 3 1 Mean arterial bloo d pressure, partial pressure of arterial carbon dioxide, oxygen, and arterial pH during long term facilitation ................................ .......... 75 3 2 Inspiratory and expiratory duration before and 1, 30 and 60 min post phrenic otomy ................................ ................................ ................................ ...... 75 3 3 Inspiratory and expiratory duration during selected times of the LTF or sham LTF protocols ................................ ................................ ................................ ...... 76 4 1 Apneic threshold and me an arterial blood pressure in c ontrol, 2 week post injury an d 12 week post injury group. ................................ ................................ 96 5 1 Me an arterial blood pressure partial press ure of arterial carbon dioxide, oxygen and arterial pH during baseline, hypoxia, a nd 6 min post hypoxia ...... 119 5 2 P values obtained using two way repeated measures Analysis of Variance for respiratory parameters assessed by plethysmography. .............................. 120 5 3 P values obtained using two way repeated measures Analysis of Variance for phrenic output parameters assessed by phrenic neurophysiology. ............. 121

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10 LIST OF FIGURES Figu re page 1 1 Depiction of n eural regulation of respiration ................................ ....................... 48 1 2 Depiction of ventral and dorsal medullary respiratory neuro nal projections. ....... 49 1 3 Depiction of descending respiratory pathways in the spinal cord ....................... 49 3 1 Representative examples of phrenic neur ograms ................................ .............. 77 3 2 Phre nic inspiratory burst frequency and phrenic burst amplitude during normoxic baseline in rats with cut or intact phrenic nerves ................................ 78 3 3 Representative examples of phrenic activity and arterial blood pressure during intermittent hypoxia in ra ts with phrenic nerves cut or intact .................... 79 3 4 Mean phrenic burst frequency an d peak amplitude during intermittent hypoxia. ................................ ................................ ................................ .............. 80 3 5 Representative examples of phrenic bursting and arterial blood p ressure during baseline and both 25 and 55 min following intermittent hypox ia .............. 81 3 6 Mean phrenic burst frequency and peak am plitude at 25 and 50 min following intermittent hypoxia ................................ ................................ ............................ 82 3 7 Example of phrenic ac tivity be fore, during and following phrenicotomy .............. 83 3 8 Representative phrenic neurograms du ring and following acute phrenicotomy .. 84 3 9 Mean data showing the impact of phrenicotomy on phrenic burst frequency and amplitude ................................ ................................ ................................ ..... 85 4 1 Representative histological sections of the cervical sp inal cord following hemisection injury ................................ ................................ ............................... 97 4 2 Examples of phrenic neurograms and correlograms ................................ .......... 98 5 1 In vitro differentiation prolif eration and differentiation of neural precurs or cell s grown as free floating neurospheres ................................ ................................ 121 5 2 N eural precursor cell injection site at 8 weeks post transplant. ........................ 122 5 3 Serial D AB stained transverse sections through the spinal cord injury site demonstrate rostrocaudal migr ation after cell transplantation .......................... 123 5 4 Transverse section immediately caudal to transplant site s howing robust migration of DAB st ained cells with ipsilateral white matter .............................. 124

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11 5 5 Representative airflow traces during quiet breathing and res piratory challenge with hypoxia and hypercapnia in a transplant rat. ............................ 125 5 6 Change in baseline frequency, tidal vo lume and minute ventilation from 4 to 8 wks in tran splant and control rats ................................ ................................ ..... 126 5 7 Change in hypoxic frequency, tidal volume and minute ventilation from 4 to 8 wks in trans plant and control rats ................................ ................................ ..... 127 5 8 Change in hypercapnic frequency and tidal volume from 4 to 8 wk s in transplant and control rats ................................ ................................ ................ 128 5 9 Representative phrenic neuro grams recorded ipsilateral and contralateral to high cervical hemisection during baseline and isocapnic hypoxia. ................... 129 5 10 Impact of transpla nt on phrenic output after high cervical hemisection ............ 130

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12 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy NEURO PLASTICITY IN THE PHRENIC MOTOR SYSTEM: INTERMITTENT HYPOXIA, SPINAL CORD INJURY AND STEM CELL TRANSPLANTATION By Milapjit Singh Sandhu December 2010 Chair: David Fulle r Major: Rehabilitation Science Neuroplasticity can be defined as a persistent change in the neural control system based on prior experience Over the last 20 years, neuroplasticity in the respiratory control system has been extensively studied. It is h oped that by understanding how the respiratory neural control system can change with experience and/or injury, we may learn how to better promote plasticity. This doctoral dissertation presents the results of three studies of respiratory neuroplasticity u sing an adult rat experimental model. Perhaps the most studied model of respiratory neuro plasticity is long term facilit ation ( LTF) of phrenic motor output following intermittent hypoxia. This response is usually studied in anesthetized animals with extrac ellular recordings of phrenic output being made from the cut central en d of phrenic nerves However, the phrenicotomy procedure removes afferent inputs to the spinal cord and also axotomizes phrenic motoneurons both can potentially affect motoneuron excit ability. Accordingly, in Aim 1 we tested the hypothesis that intermittent hypoxia induced LTF of phrenic output is greater in rats with cut than intact phrenic nerves. Our primary finding was that

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13 phrenicotomy is associated with a substantial increase in the magnitude of both the acute hypoxic response and LTF. Spinal cord injury (SCI) at the cervical level disrupts descending neural inputs to respiratory motoneurons and can paralyze respiratory muscles. Respiratory related problems are a leading cause o f morbidity and mortality in patients with cervical SCI. Therefore, an important goal of SCI research is to improve respiratory motor function by harnessing neuroplasticity within the neural system controlling breathing. In Aim 2 we investigated the neura l circuitry underlying spontaneous phrenic recovery following C2 spinal cord hemisection (C2HS) injury. Our hypothesis was that synchronization between ipsilateral (IL) and contralateral (CL) phrenic motoneurons (PhrMN s ) discharges studied using cross cor relation analyses, w ould be affected after chronic C2HS Our primary finding was that the conductio n time to IL (vs. CL) PhrMNs is prolonged after C2HS suggesting the possibility of polysynaptic inputs to IL PhrMNs after injury I n the final aim our goal was to investigate promoting motor recovery after SCI. These experiments examined the feasibility of neural precursor cells (NPC) derived from post natal rat pup s as potential candidate s for transplantation after C2HS injury We hypothesized tha t post natal NPC s will survive, migrate and improve respiratory outcome after transplantation into the injured cervical spinal cord. Our data demonstrate these cells can survive, migrate, differentiate and improve phrenic o utput especially during respiratory challenges. These results serve as a proof of principle that NPC transplantation is a potentially viable option for therapeutic intervention after SCI.

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14 CHAPTER 1 INTROD UCTION Neuroplasticity William James once wrote th at plasticity, in the broadest sense of the word, means possession of a structure weak enough to yield to an influence, but strong enough not to yield all at once. In the 1890s, he was the first person to introdu ce the concept of plasticity and observed th (James, 1890) However, this concept was largely neglected for the next 50 years. Such was the conventional dogma against plasticity of the nervous system that Ramon y Caja l, widely considered as the founder of modern neuroscience, said in 1913 that the adult human brain was "fixed, ended, 1914) Evidence against this dogma started slowly emerging in subsequent decades with conclusive data laboratories across several species (Bach y Rita, 1967; Hebb, 1949; Paul et al., 1972) Although neuroplasticity is now viewed as a fundamental property of the nervous system at all levels, it is not well defined and the term neuroplasticity has different meanings to resear chers in different subfields. Therefore, for the purpose s of this document, we will use the definition that was originally set forth by Mitchell and Johnson (2003) in the neural control system (morphology and/or function) Similar to other regions of the nervous system, the respiratory control system also exhibits extensive neuroplasticity. Movement s of the respiratory musculature result i n airflow into and out of lungs. Control of th ese movement s is critical for metabolic and

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15 acid base equilibrium (Forster and Dempsey, 1981) Several perturbations in everyday life place variable metabolic demands on the respiratory syste m. Such perturbations may be short term (e.g. locomotion, coughing, swallowing, vocalization micturition, defecation, respiratory infections, and altitude changes ) or long term (e.g. pr egnancy, changes in body weight). Th e refore, the neural mechanisms con trolling the respiratory system must be extre mely plastic and reliable and adaptable to changing conditions by neuroanatomical and/or functional plasticity Neural Control of Respiration In the last 50 years, great advances have been made in the delineatio n of the neural networks controlling respiration. Premotor r espiratory centers, which are respon sible for the generation and maintena nce of respiratory rhythm, are primarily locat ed within well defined nuclei of the brainstem. Descending phasic drive is co nducted from these centers to the spinal and cranial motoneurons which control the respiratory related muscles in mammals. N eural control of respiration is summarized in Fig. 1 1. Brainstem Respiratory Centers Premotor r espiratory neurons within the brain stem are responsible for generation and transmission of respiratory rhythm to the cranial and spinal motoneurons. These p remotor neur ons also are the primary site for feedback control of respiratory motor output mediated by afferent pathways. These neurons are aggregated in two areas within the brainstem : medulla and p ons Medullary respiratory nuclei The medulla contains three groups of respiratory neuron: the p re B tzinger complex the ventral respiratory column and th e dorsal respiratory column

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16 Pre B tzinger complex ( pre Bt C) The Pre Bt C is located caudal to the facial nucleus in the rostral ventrolateral medulla. The neurons of the p re Bt C are primarily propriobulbar interneurons and display intrinsic bursting activities that occur in phase w ith the respiratory cycle (Feldman, 1986) These neurons are connected to cranial motor neurons and paraambigual premotor neurons which have descending projections to upper airway respiratory muscles and spinal motor neurons, respectively (Kalia, 1977; Merrill and Fedorko, 1984) The precise role of pre Bt C neurons is not clear, however, they are considered essential for the generation of rhythmic respiratory drive (Lipski and Merrill, 1980) Pre Bt C is sometimes considered a part of r ostral ventral respiratory column (see bel ow) since it is a functional rather than an anatomical entity. Ventral respiratory c olumn (VRC) VRC is a functionally defined population of respiratory neurons located in the ventrolateral medulla of the brainstem originally described by Archard and Buche r (1954) The rostral aspect of VRC (rVRC) extends from the level of the obex to the facial motor nucleus (Bianchi, 1971) These neurons lie in close proximity to the nucleus ambiguus (nA) o f the brainstem so occasionally the term nucleus Para Ambiguus (nPA) is a lso used to describe them (Kalia, 1981) It should be noted however that the function of rostral VRC is not related to th e functions of the nA, which is a predominantly a vagal and glossopharyngeal motor nucleus and is not a part of the central respiratory network (Merrill, 1972) The caudal pa rt of VRC (cVRC) e xtends from the level of the obex to the border between the s pinal cord and medulla (Bianchi, 1971) The neurons in cVRC lie i n close relation to the nucleus retroambiguus (Merrill, 1975)

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17 VRC includes laryngeal and pharyngeal motoneurons which drive t he laryngeal and pharangeal muscles respectively as well as propriobulbar neurons which coordinate the activity of pump and upper airway muscles (Ellenberger and Feldman, 1990) The r VRC co ntain s primarily inspiratory neurons (Bianchi, 1974) which are fur ther subdivided into two types: early and late, on the basi s of their firing pattern and projections (Mitchell and Herbert, 1974) The early inspiratory (early burst) neurons are rapidly depolarized and have peak firin g rate s early during inspiration They actively inhibit e xpiratory neurons in the cVR C during rhythmic firing. T he late inspiratory cells have ramp like depolarization and achieve peak firing rate s late in the inspiratory phase. The se neurons cross the midline rostral to the obex and project through the spinal cord to both phrenic and inte rcostal motor nuclei (Merrill, 1974) The expiratory neurons, segregated in the cVRC show an increased firing frequency as expiration progresses (Baumgarten et al., 1957; Merrill, 1970) The y cross the midline caudal to the obex and pr oject to the expiratory intercostal and abdominal motoneurons in the contralateral spinal cord (Merrill, 1974; Nakayama and Baumgarten, 1964) The project ions of ventral respiratory neurons in medulla ar e summarized in Figure 1 2. Dorsal respiratory c olumn (DRC) DRC refers to the ventrolateral division of th e Nucleus Tractus Solitarius located dorsally in the medulla near the exit of the glossopharyngeal nerve (Batsel and Lines, 1973; Baumgarten and Kanzow, 1958) Their efferent projections terminate at various locati ons within medulla the nucleus ambiguus, hypoglossal nucleus, dorsal motor nucleus of vagus nerve, as well as the contralateral phrenic motoneuron ( PhrMNs ) in the spinal cord via the lateral funiculus

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18 (Berger et a l., 1984; Otake et al., 1989) DRC neurons receive afferent projections from the pulmonary stretch receptors, carotid and aortic chemoreceptors, and the C fiber endings in the lung via the vagus nerve (Bianchi, 1971) Results from studies utilizing electrical potentials in the medulla and neuronal labeling show that DRC neurons also receive input from contralateral cVRC, pre Bt C and ipsilatera l PRG (Bystrzycka, 1980; Kalia et al., 1979) The majority of DRC neurons discharge during the inspiratory phase of respiration (Batsel, 1964) and play a role in the mediation of visceral and somatic reflexes while also transmitting input from the nucleus of the solitary tract to other respiratory groups within t he brainstem (Otake et al., 1989) Pontine r espiratory nuclei Another brainstem respiratory group of importance is located within the dorsolateral pons, namely in the nucleus parabrachialis media lis and Klliker Fuse nucleus (Baxter and Olszewski, 1955; St John and Wang, 1977) In addition to other areas in brainstem, pontine respiratory nuclei receive bilateral projections from nucleus ambiguus, nucleus paraambiguus and pre Bt C (Kalia, 1981) Efferent projections from pontine respiratory nuclei travel in both rostral and caudal direction. Most PRC neuron s fire tonically reaching peak firing frequencies at the transitions between res piratory phases. Such a firing pattern suggests a role of these neurons in the transition between respiratory phases and/or in stabilization of respiratory pattern (Ballantyne et al., 1988; Douse and Duffin, 1993) Cerebral Control of Respiration In addition to involuntary control of respiration by the brainstem respiratory centers, it can also be affected voluntarily in humans. T he neur ophysiological basis for this control appear s to reside in the cerebral cortex. Voluntary inspiratory efforts result in

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19 concomitant activation of bilateral dorsal primary motor cortex just lateral to the vertex, the supplementary motor area, the right late ral premotor cortex, and left ventrolateral thalamus. Similarly voluntary expiratory efforts also cause activation of the same areas along with bilateral ventrolateral thalamus and ventrolateral motor cortices and cerebellum (Ramsay et al., 1993) The h ypothalamus also has paraventricular projections to the medullary respiratory sites, and stimulation of both the dorsal and ventral hippocampus has been shown to elicit marked effects on the phrenic output (Ruit and Neafsey, 1988) It has been suggested that cortex affects respiration either through projections to the brainstem respiratory areas (Ramsay et al., 1993) or directly to the spinal motoneurons (Corfield et al., 1998) Spinal Cord Descending respiratory tracts. Both involuntary (medullary) and voluntary (cortical) drive is relayed to the spinal cord and is presumably integrated at the spinal level. In humans as well as experimental animals, the involuntary (automatic) rhythmic respiratory drive is transmitted via axons that originate in the medullary nuclei decussate at the level of the brainstem and project to the ventrolateral column of the contralateral spin al cord (Davis and Plum, 1972; Gad and Marinesco, 1892) Within this column, the expiratory axons lie medial to the inspiratory axons. At the level of the PhrMNs 25% of descending axons arborize while the remainder descend toward intercostals and ab dominal motoneurons (Merrill, 1974) The involuntary non rhythmic (tonic) drive travels from the medial reticular formation to the spinal segments in the ventral and ventrolateral columns of ipsilateral spinal cord (Pitts, 1940) Finally, descending spinal pathways associated with voluntary respiratory drive the corticospinal or corticorubrospinal tracts are located with in the dorsolateral

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20 column of the spi nal cord in humans and the dorsomedial column in rats (A minoff and Sears, 1971) Descending respiratory fibers are depicted in Figure 1 3. Respiratory m otoneurons Phrenic motoneurons (PhrMNs). PhrMNs constitute a narrow longitudinal column of motoneurons in the medial ventral horn of the cervical spinal cord. This column extends from C3 C5 in humans and rats, and C4 C6 in cats (Kuzuhara and Chou, 1980; Webber et al., 1979) and contains about ~250 motoneurons per side (Furicchia and Goshgarian, 1987; Mantilla et al., 2009) PhrMNs receive excitatory synaptic inputs duri ng inspiration f rom the inspiratory bulbospinal pre moto r neurons in the VRC and DRC (Berger, 1979) The pathways between medullary respir atory neurons and PhrMNs are either monosynaptic and/or polysynaptic through spinal respirato r y interneurons (Davies et al., 1985; Fedorko et al., 1981) PhrMNs rhythmically activate diaphragm via the phrenic nerve s populations relative to their onset of activity during inspiration (Lee et al., 2009b; St John and Bartlett, 1979) Intercostal and Abdominal motoneurons Intercostal motoneurons involved in respiratory function are located laterally in the gray horn and extend the entire length of the thoracic spinal cord (Larnicol et al., 1982a; 1982b) Their descending input is similar to Phr MNs and they innervate the internal and external intercostals muscles which assist in expiration and inspiration, respectively. The abdominal motoneuron s occupy the lower thoracic and upper lumbar spinal cord segments, but the details of their morphology, size and organization are un known. They receive synaptic input from pre motor neurons in the caudal regions of VRC (Miller et al., 1985) and are responsible for

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21 cont rolling abdominal muscles during expiration (Fedorko and Merrill, 1984; Long and Duffin, 1986) Propriospinal inter neurons and control of breathing The central respiratory drive to phrenic and intercostal motoneuro ns is provided by inspiratory bulbospinal neurons in the medullary respiratory column (as described earlier ). However, a controversy exists as to whether the drive reaches spinal motoneurons through monosynaptic or polysynaptic pathways. On one hand, earli er electrophysiological studies provide evidence for monosynaptic pathways (Cohen et al., 1974; Hilaire and Monteau, 1976) O n the other hand, anatomical evidence for the existence of spinal respiratory interneuron s suggest s an oligosynaptic transmission. C orrelation of brainstem pre moto neurons and PhrMNs activity suggest s that monosynaptic connections exist but they cannot account for the total depolarization of the PhrMNs (Davies et al., 1985) Therefore, the majority of inputs to PhrMNs must be polysynaptic and relayed via spinal interneurons. T he electrophysiological and neuroanatomical evidence for the role of interneurons in the control of respiration is disc ussed below Electrophysiological Evidence The first study to provide a direct evidence of respiratory interneurons in the cervical spinal cord was done by Palisses et al. (1989) They recorded from various spontaneously firing cervical interneurons bursting in phase with the respiratory drive. Based on the results, they classified cervical interneurons into the following categories: 1. Inspirat ory interneurons: which were active during phrenic bursting but silent during expiratory phase. 2. Tonic interneurons: which fire throughout the respiratory phase with peak discharge frequencies during either inspiration or expiration.

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22 3. Expiratory interneuron s: which fire at a low and regular during expiration. Although direct monosynaptic connections between inspiratory interneurons and PhrMNs has not been demonstrated, one study showed a common excitation pattern during inspiration (Duffin and Iscoe, 1996) By cross correlating the activity of the inspiratory int erneurons with ipsi and contra PhrMNs they found that inhibition of PhrMNs results from inputs from inspiratory interneurons activated by descending respiratory drive. Conversely, the expiratory interneurons have been demonstrated to make monosynaptic in hibitory connections with the phrenic nucleus. These interneurons may therefore aid in modulating the drive to the PhrMNs during expiration (Douse and Duffin, 1993) Neuroanatomical Evidence Dobbins and Feldman (1994) were the first to report labeling of the cervical interneurons by tran synaptic transport of pseudorabies virus (PRV) from the phrenic nerves. A recent study from our lab oratory further inve stigated the distribution and synaptic relations of interneurons using anterograde and transneuronal synaptic tracing using PRV (Lane et al., 2008b) We found labeling of a diverse population of interneurons in the lamina VII and X, and the dorsal horn of the cervical spinal cord with PRV. We also saw ventral respiratory column projections to pre phrenic interneurons suggesting the role of interneurons as relays between medullary and PhrMN s. Dual labeling studies sh owed that the interneurons project bilaterally to PhrMN pools suggesting that they may also play a role in bilateral integration of the two phrenic nuclei amongst each other or with other respiratory motor circuits. Based on the current evidence it can be speculated that the inspiratory interneurons are driven by bulbospinal motoneurons in the ventral and dorsal

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23 respiratory columns in medulla and act as relays of inspiratory drive to the PhrMNs Similarly, expiratory interneurons receive inputs from the pr e Bt C or the expiratory neurons in caudal VRC (Fedorko and Merrill, 1984) and are likely involved in the inhibition of PhrMNs during expiration. Control o f Phrenic Motoneurons Eupnea Eupnea is defined as nor mal, unlabored breathing in an unanesthetized state As discussed earlier, expression of eupnic pattern of breathing re flects the output of a pont o medullary neuronal circuit S t. John and Paton (2003 ) reported that phrenic activity during eupnea shows a ch aracteristic ramp like rise at the onset of bursting and increments until termination. Hyperoxia Hyperoxia (i.e PaO 2 > 21%) does not occur naturally in mammals and is only experienced in clinical or experimental conditions (Fuller et al., 2005) A brief bout of hyperoxia (10 min, 100% O 2 ) enhances glutamate release in the nucleus of the solitary tract, activates NMDA receptors and induces nitric oxide formation. These processes enhance hypoxic ventilator response assessed shortly after exposure to hyperoxia (Honda et al., 1996a; b) However, s ustained hyperoxia ( 100 O 2 >12 hrs) causes type I cell necrosis in the carotid bodies and reduces their sensitivity (Mokashi and Lahiri, 1991) The reduced carotid body activity results in blunted hypoxic responses in rats and cats (Bavis et al., 2002; Bavis et al., 2003) Hypoxia Hypoxia or hypoxemia (reduction in PaO 2 ) can occur due to a decrease in the percentage of oxygen in inspired air (FIO 2 ) or an increase in metabolic rate (eg. during

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24 exerci se) Reduced PaO 2 is sensed by peripheral chemorecepto rs located in the arterial circulation and transmitted to the medulla. This leads to an increase in discharge from the respiratory pre moto r neurons, resulting in an increased phrenic output. There are two types of peripheral chemoreceptors: carotid and aor tic bodies. Carotid bodies first reported by Heymans et Al. (1930) are located bilaterally at the bifurc ation of the common carotid arteries into its internal and external branches (O'Regan and Majcherczyk, 1982) The carotid bodies have very high rates of perfusion allowing these organs to detect and respond to changes in PaO 2 rapidly. The afferent fibers synapse with the type I cells and travel through the glossopharyngeal (IX cranial) nerve to make synaptic connections with the dorsomedial subnuclei of the solitary tract, and the inspiratory neurons in the dorsal respiratory column (Donoghue et al., 1984) (McDonald, 1981) Aortic bodies are located along the ascending arch of the aorta and its branches. Afferents from aortic bodies form the aortic nerve and travel through the vagus (X cranial) nerve to the medullary respi ratory centers (McDonald, 1981) Aortic bodies are the secondary site for detection of PaO 2 after the carotid bodies (Feldman, 1986) As previously stated, c hemoreceptor activity is affected by a reduction in PaO 2 The lower the PaO 2 the higher will be chemoreceptor activity, and therefore higher the phrenic bursting. T he increased phrenic nerve output helps to improve the matching of ventilation ( E ) to blood flow in lungs (Berger et al., 1977a; 1977b; 1977c) Phrenic response to hypoxia depends on the pattern and intensity of hypoxia exposure. Following a brief exposure to hypoxia (2 5 minutes), two distinct physiological mechanisms affecting the amplit ude of phrenic output are seen :

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25 Acute r esponse Acute response to hypoxia is an immediate increase in phrenic output (and E ) as a function of decreasing PaO 2 (Bisgard and Neubauer, 1995) Such a response occurs secondary to an increase in the chemoreceptor afferent input into the solitary tract (M cCrimmon DR, 1995) and the resulting reflex excitation of the respiratory neurons in brainstem (Eldridge and Millhorn, 1986; Fregosi and Mitchell, 1994; Smith et al., 1993) Short term potentiation (STP) STP imme diately follows the acute response and lasts from a few seconds up to a minute. During this phase, there is a further increase in the phrenic output determined by the prevailing level of arterial PaO 2 (Eldridge and Millhorn, 1986) Although the exact mechanism s underlying STP are not known, it is suggested that STP may be the result of either enhanced neurotransmitter release a t the pre synaptic terminals (Wagner and Eldridge, 1991) or modulatory neuropeptide release at key locations in the respiratory neural control system (McCrimmon et al., 1997) Accordingly, there is evidence that both brainstem and spinal cord mechanisms contribute to STP (Lee et al., 2009b) Electrical stimulation of the carotid sinus nerve potentiates evoked responses in the NTS nuclei within brainstem (Mifflin, 1997) On the other hand, s pinal cord stimulation can evoke STP like changes in respiratory motor output in spinalized rats (McCrimmon et al., 1997) Alternatively, mechanisms under ly ing STP may also include changes in PhrMNs or spinal circuits including respiratory interneurons (Fuller et al., 2005; Lane et al., 2008b) Following an intermittent exposure to hypoxia, two distinct mechanisms affecting the ph renic nerve amplitude are seen:

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26 Progressive a ugmentation P rogressive augmentation is an increase in phrenic output observed during successive episodes of identical hypoxia stimuli. Although the exact mechanism s of PA are not known, it is believed to occur independent of the long term facilitation (see below) (Powell et al., 1998) Long term facilitation After a single, continuous exposure to hypoxia, respiratory motor output or E returns to pre hypoxia levels within a few minutes. However, brie f periods of intermittent hypoxia lead to a persistent enhancement of respiratory motor activity known as long term facilitation (LTF) (Feldman et al., 2003; Powell et al., 1998; Sandhu et al., 2010) LTF is often expressed as an increased phrenic motor output (pLTF) in anesthetized animals (Fuller et al., 2005; Mitchell et al., 2001a) and as an increase d tidal volume (VT) or breathing frequency in awake animals (McGuire and Ling, 2005; Olson et al., 2001; Turner and Mitchell, 1997) In addition to repeated hypoxia, LTF can also be inducted by chemical stimulation of carotid chemoreceptors, electrical stimulation of carotid sinus nerve or brainstem midline (Bach and Mitchell, 1996; Hayashi et al., 1993; Millh orn, 1986; Millhorn et al., 1980a; Morris et al., 2003) LTF was first demonstrated in anesthetized cats after episodic electrical stimulation of the carotid sinus nerve (Millhorn et al., 1980a; b) It has since been demonstrated in both awake (Harris et al., 2006; Lee et al., 2009a) and sleeping humans (Pierchala et al., 2008) and a wide range of animal species (Cao et al., 1992; Fuller et a l., 2000; Mitchell et al., 2001b; Wagner and Eldridge, 1991) There are several factors that can affect the magnitude of LTF expression such as species, age, gender, previous hypoxic exposures etc. In addition, the experimental preparations can also enhan ce or reduce

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27 LTF expression. LTF tends to be reduced and more difficult to elicit in unanesthetized and spontaneously breathing animals (Olson et al., 2001) as compared to anest hetized and ventilated animals (Baker Herman and Mitchell, 2002; Fuller et al., 2001b) In fact, LTF of respiratory output in spontaneously breathing animals is more often expressed as a persistent increase in brea thing frequency rather than inspiratory volume or diaphragm EMG burst amplitude (Baker Herman and Mitchell, 2008; Olson et al., 2001) Consistent with these observations, studies in humans show ventilatory LTF that is substantially lower than in typically seen in anesthetized rats (Lee et al., 2009a) The difference in LTF expression between spontaneously breat hing vs. anesthetized preparations does not represent the impact of anesthesia alone. For example, LTF of diaphragm EMG is not evident in anesthetized and spontaneously breathing r ats (Janssen et al., 2000) and c ats (Mateika and Fregosi, 1997) despite using a st imulation regimen which evokes robust LTF of phrenic output (Bach and Mitchell, 1996; Fuller et al., 2001b) An additional confound in these studies is that the PaCO 2 values differ widely in spontaneously breathing vs. anesthetized animals. In spontaneously breathing animals, the PaCO 2 levels are well above apneic threshold in anesthetized animals (Janssen et al., 2000) and so the phrenic motor output is also relatively during baseline conditions (Kong and Berger, 1986; St John and Bartlett, 1979) There fore, there may be a reduced capacity for increased phrenic motoneuron recruitment during the post hypoxic period resulting in reduced LTF. Consistent with this idea, pLTF is difficult to evoke in phrenic neurograms recorded contralateral to cervical spina l cord hemisection injury, a condition which results in robust compensatory increase in contralateral phrenic

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28 output (Doperalski and Fuller, 2006) Therefore, PaCO 2 levels also appear to an important determinant of respiratory LTF express ion pLTF is often studied in anesthetized animals after phrenicotomy (PhrX) with subsequently recordings being made from the proximal stump of the transected phrenic nerve. Our lab recently demonstrated that PhrX procedure is associated with a substantia l increase in p LTF (Sandhu et al., 2010) The detailed mechanisms underlying this effect o f PhrX on LTF are un known We speculate, however, that PhrX may result in increased PhrMN excitability due to axotomy of efferent or afferent axons in the phrenic nerve. In addition to PhrX r emoval of lun g volume inhibitory feedback via vagotomy also can affect the magnitude of LTF. Golder and Martinez (2008) demonstrated that under otherwise similar conditions vagotomized rats have substantially diminished pLTF compared to vagal intact rats. Pharmacological studies indicate that intermittent hypoxia activates medullary raphe neurons and triggers serotonin (5 HT) release near PhrMNs in the cervical spinal cord (Erick son and Millhorn, 1994) Serotonin receptor (5 HT2) activation initiates intracellular signaling pathways necessary for expression of LTF (Fuller et al., 2001b) These downstream signaling pathways include new protein synthesis, particularly brain derived neurotrophic factor (BDNF) and activation of the high affinity BDNF receptor, TrkB. BDNF seems to be necessary and sufficient for phrenic LTF because pharmacological bl ockade of TrkB prevents LTF and spinal application of BDNF mimics LTF (Baker Herman et al., 2004) T he balance of kinase/phosphatase activation is also a critical regulator of pLTF following intermittent hypoxia. TrkB activation result s in : a) phosphorylation of

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29 extracellular signal regulated kinase, b) mitogen activated protein kinases and c) protein kinase B (Mahamed and Mitchell, 2007; Wilkerson et al., 2008) These activated kinases are hypothesized to improve post synaptic glutamate receptor function which presumably increases synaptic strength between bra instem respiratory pre motor n eurons and PhrMNs resulting in pLTF (Wilkerson et al., 2008) Increased glutamatergic synaptic transmission during pLTF is consistent with observations that NMDA recepto rs are necessary to maintain intermittent hypoxia induced pLTF in anesthetized rats (McGuire et al., 2005) Serine/threonine phosphatases also have been suggested to have a prominent role in expression of pLTF (Wilkerson et al., 2008) Inhibition of okadaic acid sensitive serin e/threonine phosphatases in the cervical spinal cord revealed pLTF after sustained hypoxia (25 min duration), a stimulus not able to elicit pLTF (Wilkerson et al., 2008) Reactive oxygen species such as superoxide anion, H 2 O 2 and hydroxyl radicals are mediators of oxidative stress and play a critical role in normal cell ular processes that are critical to life. Intermittent hypoxia induced pLTF requires ROS since pre treatment with antioxidants abolishes pLTF (MacFarlane and Mitchell, 2008) Since ROS regulate the balance of kinase/phosphatase activities, they may shift the balance in favor of kinase ac tivation and net phosphorylation of key proteins involved in pLTF Furthermore, it is speculated that ROS may be formed via NADPH oxidase activity since inhibition of NADPH activity also blocks pLTF (Ma cFarlane and Mitchell, 2009) Reactive nitrogen species (RNS), such as nitric oxide, are also important in expression of LTF. Pharmacological blockade of nitric oxide or nitric oxide synthase 1 enzyme knockout mice do not show LTF after intermittent hypo xia. It has been

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30 suggested that nitric oxide may permit LTF by modulating serotonin release from raphe neurons or by prolonging the actions of 5 HT on respiratory neurons (Kline et al., 2002) Although we do not have a full understanding of the role of ROS and RNS in respiratory plasticity, it appears that despite their potential to elicit cell damage, low levels of these molecul es are required for normal cell signaling and expression of pLTF (Macfarlane et al., 2008; Valko et al., 2007) Hypercapnia Exposure to increased amount s of inspired CO 2 leads to an increase in the arterial PCO 2 level resulting in hypercapnia. As CO 2 is extremely soluble in tissue and blood, it has a high diffusion capacity. So the elevation of PCO 2 is sensed rapidly by the central chemoreceptors located in brainstem and in part by the peripheral chemoreceptors (Nattie, 1999) An increase in PaCO 2 above the normal level causes strong stimulation of respiration, and similarly a decrease in i ts level attenuates phrenic output leading ultimately to cessation of phrenic bursting during extreme drop of PaCO 2 in blood. Therefore an optimal balance of CO 2 is needed to maintain normal respiratory drive (Nattie, 1999) Diaphragm Afferents The first study suggest ing role of diaphragmatic afferents in respiratory control (control of PhrMNs ) was published in 1960 (Nathan and Sears, 1960) The authors reported the occurrence of diaphragmatic weakness in persons who had undergone therapeutic sectioning of the c ervical dorsal roots. Since that seminal study the influence of diaphragmatic sensory receptors on the phrenic nerve output has been extensively explored In the diaphragm, four types of sensory receptors can be categorized based on their afferent nerve fibers.

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31 Free nerve endings. Free nerve endings are usually associated with group III or IV non myelinated nerve axons whose diameter are between 0.5 (Stacey, 1969) Free nerve endings ar e not specific to any one type of tissue but respond to local pressure (Iggo, 1959) chemical, temperature an d pain stimuli (Hinsey and Phillips, 1940) Electrical stimulation of the small diameter afferent fibers (group III) results in bilateral reduction of phrenic out put (Jammes et al., 1986) A dditionally stimulation of these fibers simultaneously mediates an excitatory effect on the dorsal respira tory group (but not the ventral respiratory group). Despite this excitation the net effect of stimulation/activation of small diameter fibers is an overall inhibition of the PhrMN activity. Electrical stimulation as well as chemical stimulation of the sm all diameter group IV fibers by capsaicin produces an excitatory effect on phrenic output (Jammes et al., 1986) Pacinian and Pacinifo rm Corpuscles These receptors are usually associated with group III or group II myelinated nerve axons whose diameter are between 2 12 (Corda et al., 1965) The role of these receptors in diap hragm control is not clear Golgi Tendon Organ s These receptors are usually associated with group Ib myelinated nerve axons whose diameter are between 12 (Hudson, 1966) Golgi t endon organs are located close to the tendinous portion of the diaphragm muscle and respond to a change in the muscle tension (Road, 1990) The diaphragm contains mo re Golgi tendon organs than muscle spindles (see below) therefore majority of proprioceptive afferents (slowly adapting receptors) arise from the tendon organs (Corda et al., 1965)

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32 Muscle spindle s There are two types of muscle spindles in the diaphragm primary and secondary. Primary muscle spindles are usually connected with group Ia myelinated afferent nerve fibers whose diameters are between 12 secondary muscle spindles are usu ally connected with group II afferent nerve fibers whose diameters are between 6 (Hudson, 1966) Muscle spindles respond to changes in the muscle l ength. Electrical stimulation of the large afferent fibers (group Ia, Ib, II) causes a bilateral of phrenic output (Gill and Kuno, 1963; Jammes et al., 1986; Speck and Revelette, 1987a) There is a controversy over whether this effect is mediated at spinal or supraspinal level. Gill and Kuno (1963) demo nstrated contralateral inhibition of phrenic output by electrical stimulation of phrenic afferents, which was not abolished by spinal section at the atlanto occipital membrane. On the other hand, Duron et. al. (Duron et al., 1976) showed i psilateral inhibition of phrenic activity, which was abolished by removal of higher structures. Lung Afferents Stimuli within the lungs can cause sensations of pain, ache, and irritation resulting in an urge to cough all of which can affect respiration. These sensations are mediated by receptors within the respiratory airways and travel to the brainstem respiratory centers via the vagus nerve (Widdicombe, 1964) The most commonly studied receptors are : 1) slowly adapting pulmonary stret ch receptors (SAR s ), 2) rapidly adapting receptors (RARs), and 3) pulmonary and bronchial C fibers (Schelegle et al., 2000) Despite progress in elucidating their functional roles, questions remain concerning the contribution of these lung receptors to the re spiratory control. In general,

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33 these receptors for m the airway defense which responds to lung irritants (such as dust, ammonia, histamine) by initiating protective behaviors such as cough, bronchoconstriction, mucus secretions, gasps etc (Widdicombe, 2009) R AR s are located in the upper airways between the nasopharynx to the bronchi (Knowlton and Larrabee, 1946) These receptors respond with a rapidly adapting discharge to lung inflation and deflation, and to mechanical stim ulation. RARs in the trachea are stimulated by inflation and deflation of the airway, and some are active, with a respiratory phase in eupnea RARs have also been suggested to cause bronchoconstriction since, under several conditions, their activation is accompanied by airway constriction (Sellick and Widdicombe, 1969) SARs are another category of afferent endings within the airways that inner vate the tracheobronchial tree. Their role w as first documented by Heri ng and Breuer (1868) who showed an early termination of inspiration when the lungs are inflated during inspiration, and a prolongation of the expiratory pause when a prolonged inflation is applied at the end of inspiration In addition, these receptors play a role in the regulation of airway s mooth muscle tone, system ic vascular tone and heart rate (Schelegle, 2003) Pulmonary and bronchial C fibers are unmyelinated vagal afferents that innervate the lungs and airways. C fibers are weakly mechanosensitive and can be stimulated by external irritants (e.g. smoke and dust) and internal chemicals (e.g. histamine, prostaglandin and bradykinin) (Janczewski and Feldman, 2006; Lee et al., 2008; Lee et al., 1989) Laboratory pharmacological chemicals such as capsaicin can also activate these receptors (Lu et al., 2006) T he two types of C fibers i.e. pulmonary and bronchial have different sensitivity to various drugs but their reflex action is identical Activation of

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34 C fibers can evoke both respiratory and central nervous reflexes. R espiratory re flexes include reduced res piratory related upper airway nerve and muscle activity reduced phrenic bursting (at lower doses), laryngeal closure, apne a, and/or rapid shallow breathing (Coleridge and Coleridge, 1984; Lee et al., 2007; Lu et al ., 2006) The central nervous reflexes of C fiber activation include bronchoconstriction, mucus secretion, hypotension, bradycardia, laryngoconstriction and airway mucosal vasodilatation (Lee and Pisarri, 2001) Spinal Cord Injury The mammalian spinal cord is a complex and highly specialized neural network comprised of neurons, astrocytes, oligodendrocytes and microglia. Injury to this system unleashes a cascade of damaging events that result in permanent motor and /or sensory deficiency below the level of injury The initial injury consists of mechanical disruption of tissue and hemorrhage of small blood vessels within the spinal cord Damage to the spinal cord is not isolated to the initial insult, and does not stop immediately after the primary impact but can continue for hours an d even days post injury The secondary phase of injury features edema, necrotic cell death, inflammation, demyelination, formation of free radicals and programmed cell death. The late secondary phase of SCI is characterized by Wallerian dege ne ration of inj ured axons, astroglial scar formation and formation of cysts or cavities (Norenberg et al., 2004) Effect of cervical spinal cord injury on respiratory function in humans Spinal cord injury (SCI) affects thousands of people worldwide with massive healthcare and other associated socioeconomic cos ts (Horner and Gage, 2000) It is estimated that in the US alone, 12,000 SCIs occur each year and m ore than quarter of a million people are living with disabilities related to SCI More than half of these

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35 patients have inj uries at the level of the cervical spinal cord High cervical SCI (rostral to C4 segment) interrupts the rhythmic drive to the spinal respiratory motoneurons and results in paralysis of the respiratory muscles Therefore, persons with such injuries usually require long term mechanical ventilator y support. In persons with incomplete SCI, spontaneous respiration may be spared; however, they often exhibit a rapid and shallow pattern of breathing ( i.e. their respiratory rate increases and VT decreases ) Additio nally, individuals with c ervical SCI also may have an ineffective cough and difficulty clearing secretions; such features predisposes these individuals to mucus retention, atelactasis, pulmonary infections and ultimately to significant morbidity and mortal ity (DeVivo et al., 1993; Fishburn et al., 1990; Schilero et al., 2009) Cervical Hemisection Injury Model There are several rodent and nonhuman primate models of SCI that are used to simulate SCI pathology and e xplore potential therapeutic strategies. Experiment al contusion type models use devices that displace or impact exposed spinal cord to create a lesion (Stokes and Jakeman, 2002) Compression injuries may be s imulated by clip compression or subdural insertion and inflation of a balloon. Laceration injuries such as lateral hemisection can be performed by surgical incision. A relevant model to investigate post injury respiratory plasticity and therapeutic interventions is unilat eral high cervical spinal cord hemi section. Spi nal cord hemisection at the level of the second cervical segment (C2HS) will sever the continuity of the bulbospinal respiratory pathways and interrupt pre motor drive to PhrMNs on one side The primary advant age of the cervical hemisection model is that it is reproducible, quantifiable and has a low mortality rate in rats (Fuller et al., 2005) In addition, this model allows for the study of existing, but typically inactive spinal pathways that cross the spinal midline caudal to

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36 the injury and project to the PhrMN s (cross phrenic pathways; reviewed below) (Fuller et al., 2005) Crossed Phrenic Phenomenon The phrenic ne rve and consequently ipsilateral diaphragm are silent in the days to weeks followin g C2HS However, augmentation of the central respiratory drive by interruption of the contralateral phrenic nerve produces ipsilateral phrenic nerve activity below the hemisection. Restoration of respiratory activity in the ipsilateral phrenic nerve result s from activation of latent bulbospinal pathways that cross the spinal midline from the contralateral side and synapse onto ipsilateral Ph r MNs (Goshgarian, 2003) This effect is termed as the Crossed Phrenic P henomenon (CPP). The C PP is an example of functi onal plasticity after SCI and provides an opportunity to study underlying mechanisms related to plasticity and recruitment of la tent respiratory pathways. CPP was first demonstrated by Porter (1895) and has subsequently been confirmed in several species including dogs (Deason and Robb, 1911; Lewis and Brookhart, 1951; Rosenblueth and Ortiz, 1936) cats (Rosenbaum and Renshaw, 1949) rabbits (Chatfield and Mead, 1948) rats (Goshgarian, 1981; Sandhu et al., 2010) mice (Minor et al., 2006) guinea pigs (Guth, 1976) and woodchucks (Rosenblueth and Ortiz, 1936) Functional recovery of ipsilateral hemidiaphragm paralyzed by C2HS can also occur spontaneously without any additional intervention if sufficient time is allowed to elapse aft er injury (Nantwi et al., 1999b) This was first reported by Nantwi et al. (1999) who showed that IL phrenic activity is absent at 4 wks post C2HS but becomes relatively robust by 10 16 wks. Multiple groups have subsequently confirmed this finding, however, the duration of time which precedes the appearance of IL PhrMN bursting varies markedly between studies Spontaneous CPP has been observed as

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37 early as 2 weeks post injury in paralyzed, mechanically ventilated, and vagotomized rat pr eparations. On the other hand, anesthetized and spontaneously breathing vagally intact rats show no IL Phr nerve or hemi diaphragm activity at 2 wks post injury. In either case, the result is that IL phrenic bursting, which initially is abs ent after C2HS, r eturns during baseline quiet breathing (Fuller et al., 2005) The underlying mechanism associated with spontaneous CPP and recovery of IL phrenic nerve output and hemidiaphragm function is not yet completely understood although several laboratories have explore d the morphological and molecular changes resulting from C2HS Goshgarian and colleagues have described synaptic remo deling in and around IL PhrMNs occurring w ithin hours post injury which may enhance the effectiveness of pre existing synapses resulting in an increase d inspiratory input to Ph r MNs (Sperry and Goshgarian, 1993) Mantilla et al. showed that size of PhrMNs decreases at 2 weeks post C 2HS which may affect postsynaptic excitability and phrenic output Recently, Lane et al (Lane et al., 2008b) provided evidence that cervical pre phrenic interneurons may act as relays between descending pathways a nd PhrMNs and therefore can modulate the onset and degree of CPP after cervical SCI In addition, several molecules have been shown to contribute to phrenic output recovery after C2HS such as serotonin (Ling et al. 1994; Zhou et al., 2001) cAMP (Kajana and Goshgarian, 2008) adenosine (Nantwi and Goshga rian, 1998) and neurotrophins including BDNF (Fuller et al., 2002; Johnson et al., 2000) and glutamate (AMPA and NMDA ) receptor complexes (Huang and Goshgarian, 2009) Although it is well known that that CPP be comes spontaneously active over weeks to months post SCI, the extent to which this increased muscle activity translates

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38 into a functional increase in respiratory capacity is not clear. While E can describe the recovery of breathing af ter SCI, s uch measures reveal little about the mechanism of recovery (e.g. plasticity and compensation). Nevertheless, s everal studies have examined the impact of C2HS on E (Fuller et al., 2008 ; Fuller et al., 2006; Golder et al., 2003; Golder et al., 2001; Goshgarian et al., 1986; Nantwi et al., 1999a) The initial report was from Goshgarian and colleagues (1986) who demonstrated that fema le rats breathe with an elevated frequency at approximately 24 hr post C2HS. Arterial blood gases were consistent with hyperventilation as reflected by increased p a O 2 and a tendency for decreased p a CO 2 (Goshgarian et al. 1986). It was subsequently reported that r ats transiently hypoventilate for a few hours after C2HS (Fuller et al., 2005) but by 2 wks post injury their arter ial blood gases are not different than uninjured control rats (Miyata et al., 1995) Golder et al. (2001) used pneumotachography to study the pattern of breathing in anesthetized, tracheotomized C2HS female rats breathing room air. Relative t o uninjured control animals, C2HS rats had increased breathing frequency (fB) and reduced VT at both 1 and 2 months post injury. Golder et al. (2001b) also showed that sighs or augmented breaths occur more frequently after C2HS. The first investigation of E in unanesthetized, unrestrained rats after chronic C2HS was conducted by Fuller et al. (2006) using barometr ic plethysmography (Mortola and Frappell, 1998) In that study, E was examined in male rats during a baseline period (21% O 2 ) and a hypercapnic challenge (21%O 2 7% CO 2 ) at 2 5 wks post injur y. The respiratory challenge is particularly important because chemical stimulation of breathing (i.e. hypoxia, hypercapnia) can activate IL phrenic pathways after C2HS Rats maintained E with a rapid, shallow breathing pattern (red uced VT, increased

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39 breathing frequency ) that persisted through the duration of the study. Deficits in E were revealed during the hypercapnic challenge and reflected reduced VT. There was no evidence for recovery of VT or E over the 5 wk post injury period. However, in that study VT or E were compared to pre injury measurements in the same rats (i.e. repeated measures). This approach may not adequately control for differences in body mass or age between the injured and spinal intact conditions. Further, Nantwi et al. (1999a) showed progressive improvements in diaphragm EMG activity over intervals > 5 wks post C2HS suggesting that E recovery might be more robust at la ter time points. Accordingly, our lab examined E up to 3 months post C2HS in male rats and compared the data to age, weight and sex matched controls (Fuller et al., 2008) Similar to prior r eports (Fuller et al., 2005; Fuller et al., 2006) a t 2 wks post C2HS E was maintained during baseline conditions (21% O 2 ) but was substantially blunted during hypercapnic challenge (68% of E in uninjured, weight matched rats). However, by 3 mo nths the injured rat s achieved a hypercapnic E that was 85% of control. Thus, the ability to increase E during respiratory challenge showed a modest b ut significant recovery by 3 months post C2HS. C2HS rats also exhibited augmented breaths with reduced volume and greater frequency than controls (Fuller et al. 2008). Augmented breath volu me tended to be greater at 3 months (vs. 2 wks) post injury but remained well below values observed in control rats. Thus, some degree of E recovery occurs after C2HS, but endogenous neuromuscular plasticity and/or compensation appears to be insufficient to promote full respiratory recovery.

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40 Recovery of E following SCI may be different between males and fe males (Doperalski et al., 2008) Indeed, there are numerous reports of improved functional and/or histological outcomes in female vs. male rodents following central nervous system inj ury (Roof and Hall, 2000) and exogenous es trogen therapy can improve motor recovery after SCI (Chaovipoch et al., 2006) Sex hormone s can act as respiratory stimulants and also modulate the expression of plasticity in respiratory motoneurons (Behan and Wenninger, 2008) Accordingly, our lab recently compared respiratory recovery between male and female rats following C2HS (Doperalski et al., 2008) Significant differences in the pattern of breathing were seen between sexes following C2HS although assessments were only completed at 2 weeks post injury In particular, post injury reductions in VT o bserved during hypercapnic challenge were significantly more pronounced in males vs. females. This gender difference was reduced considerably when females were ovariectomized prior to C2HS suggesting that it may have been mediated by ovarian sex hormones. Therapeutic Strategies for T reatment of SCI Although ipsilateral Ph r MN function returns spon taneously following C2HS the amplitude of phrenic output is often well below normal even after long pos t injury period s (Fuller et al., 2008) The primary causes for the suboptimal recovery of phrenic function are the lack of intrinsic growth capacity of adult neurons and the nonpermissive environmental impediments present in the injured CNS, which prevent neuronal regeneration after SCI There is an impressive number of promising approaches which are being investigated for facilitation of regeneration as well as limiting secondary neuronal damage after injury Such approaches include promoting the intrinsic neuron

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41 capacity for regenerat ion (Neumann et al., 2002; Qiu et al., 2002) blocking axonal growth inhibitory molecules (GrandPre et al., 2002; Li and Strittmatter, 2003) delivering trophic factors to decrease cell death and stimulate axonal growth (Himes et al., 2001) modulating the immune resp onse (Rapalino et al., 1998) and cellular transplantation as growth supporting structures (Li et al., 2003; Takami et al., 2002) or cell replacement (Bregman and Reier, 1986; Reier et al., 2002) The end goal of such treatments is to help restore function that was lost due to injury. Targeted Respiratory Rehabilitation after SCI Due to the development of respiratory fa ilure after acute spinal cord injury, most patients with higher cervical injuries are initially supported by mechanical ventilation. Although mechanical ventilation is the most efficient mode of restoring adequate ventilation and sustaining life, it is ass ociated with significant discomfort, limitation in mobility and complications such as pneumonia, atelactasis, barotrauma and diaphragm atrophy/weakness (Claxton et al., 1998; Fishburn et al., 1990) Therefore, alte rnative noninvasive techniques are often considered, whenever possible, to restore inspiratory muscle function after respiratory failure secondary to cervical spinal cord injury. Clinical respiratory r ehabilitation Phrenic Nerve Pacing. Phrenic nerve paci ng or diaphragm pacing is the rhythmic application of electrical impulses to the phrenic nerve, resulting in respiration for patients dependent on a mechanical ventilator. It has been a clinically accepted modality in the management of patients with cervic al spinal cord injury (Glenn and Phelps, 1985; Glenn et al., 1986) The stimulating electrodes are placed surgically around the phrenic nerve, either in the neck or in the chest. As phrenic nerve pacing involves co

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42 over mechanical ventilation including a subjective sensation of a more normal breathing, increased mobility and speech, and reduced overall costs (DiMarco, 2009) Respiratory muscle training Paralysis of inspiratory muscles leads to decrease in inspiratory force, resulting in a reduction of vital capacity. In addition, the mechanical properties of chest wall are altered due to diaphragm paralysis causing stiffening of the rib cage and a reduction in lung compliance. Both these factors can lead to an increase in the work of breat hing, which predisposes SCI patients to development of fatigue resulting in acute respiratory failure (Grassino et al., 1979) Inspiratory muscle training increases both strength and endurance of respiratory muscles, a nd protects against fatigue (Gross et al., 1980) Furthermore, r espiratory muscle training regimen also improve s expiratory muscle stre ngth, vital capacity and residual volume after SCI. Animal studies suggest that improve ment of respiratory motor function by exercise occurs due to neural plasticity through serotonin, BDNF or glutamate receptor signaling within the spinal cord (Zimmer et al., 2007) Experimental respiratory r ehabilitation Intermittent Hypoxia Intermittent hypoxia administered acutely has been shown to induce spinal respirator y plasticity (e.g. LTF) in both intact and spinally injured animals (Doperalski and Fuller, 2006; Fuller et al., 2000) Therefore, daily acute intermittent hypoxia or chronic intermittent hypoxia can augment endoge nous plasticity in spared respiratory pathways and promote function al recovery after cervical SCI (Fuller et al., 2003) CIH pre treatment enhances AIH induced pLTF in anesthetized (Ling et al., 2001; Zabka et al., 2003) and awake rats (McGuire et al., 2003) However, CIH has limited therapeutic application as it several deleterious effects such as hypertension, hippocampal cell death and learning deficits (Fletcher et al., 1992; Gozal

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43 et al., 2001; Row et al., 2002) Daily acute intermittent h ypoxia exposes rats to fewer hypoxic episodes and is one alternative to CIH. dAIH increases expression of key proteins (e.g. BDNF) near PhrMNs and elicits the beneficial effects of CIH but without the negative side effects (McGuire et al., 2002; Wilkerson and Mitchell, 2009) Data from the effects of AIH on pLTF in SCI animals suggests that there is considerable heterogeneity in the time course of these effects and the rat strains studied (Golder and Mitchell, 2005; Vinit et al., 2009) Thus, the effectiveness of IH treatment on restoring respiratory function may vary in patients with chronic vs. acute SCI as well as among individuals of different races. Cell Transplantatio n Strategies Many reports have suggested that the adult CNS harbors robust regenerative capacity after injury if provided with an environment conducive for promoting plasticity Therefore, cellular transplantation is a promising therapeutic strategy for r epairing the injured spinal cord not only in the traditional sense of cell repl acement but also by providing tr ophic support and protection. Accordingly, both supportive structures (see below) and neuronal cell type transplantation have been explored for i mprove ment of neurological function after SCI in animal models. Supportive s tructures Supportive structures do not possess the ability to directly replace the mature neuronal cell types; instead, their main advantage lies in their ability to serve as subst rates for deliver ing trophic factors, modulating host immune response and providing growth permissive interactions with the regenerating axons. Primary s upport ive structure transplants include activated m acrophages p eripheral nerve grafts, Schwann c ells olfactory ensheathing c ells and adult bone marrow derived stromal cells

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44 Activated Macrophages. Macrophages play an important role in the maintenance, restoration and defense of the damaged tissue. Their primary role is to remove necrotic cellular debris f rom the site of injury, and supply the trophic factors (such as cytokines, growth factors) required for the healing process to occur. The recruitment and activation of macrophages i s both restricted and delayed after CNS injury, also called the restricted post injury inflammatory response (Hirschberg and Schwartz, 1995; Perry et al., 1987) T he lack of regeneration after CNS injury has been attributed, in part, to spread of damage from directly in jured neurons to sp ared neurons Therefore, implantation of pre activated macrophages leads to efficient clearance of myelin debris and acquisition of growth supportive properties (Lazarov Spieg ler et al., 1998) Work by Michael Schwartz and colleagues (Rapalino et al., 1998 ) has demonstrated that transplantation of activated macrophages promotes regeneration and behavioral recovery in completely transected thoracic SCI models. Peripher al nerve grafts and Schwann cells Peripheral nerve grafts refer to transplants of the Sc hwann cell containing segments of the peripheral nerve. The rationale for using peripheral nerves is that they provide an environment known to support and stimulate axonal regeneration. However, this support is one way and the regenerating axons grow into and through the graft, but fail to cross the host to graft boundary required for re establishment of complete function (Levi et al., 2002) Since S chwann cells are the growth promoting cellular element s of peripheral ner ve graft s, transplantation of isolated S chwann cells is an alternative to using peripheral nerve tissue. Schwann cells express a variety of molecules conducive to axonal regeneration such as growth factors, cell adhesion molecules and extracellular matrix components,

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45 which support re growth and remyelination of CNS axons (Stichel and Muller, 1998) An advantage of using Schwann cells over peripheral nerves is that they can be easily cultured and expanded from donor, allowing for autolog ous transplantation. Schwann cells can also be modified during their culture period to express various kinds of neurotrophic factors. Olfactory Ensheathing Cells Olfactory Ensheathing Cells are a group of glial cells that share both Schwann cell and astr ocytic like characteristics (Gong et al., 1994; Williams et al., 2004) They are located in the primary olfactory system, a part of the mammalian nervous system that has preserved the capacity to continuously regen erate during adulthood (Cuschieri and Bannister, 1975) Their primary function is to provide e n sheathment for the unmyelinated olfactory axons within both the CNS and PNS portions of the olf actory nerve (Doucette, 1984) Olfactory Ensheathing Cells pr esent an attractive a pproach due to their capacity to fully integrate with the CNS environment and migrate through connective tissue, thus making them a more favorable candidate than s chwann cells or peripheral nerves. Adult Bone Marrow Derived Stromal Cells (MSCs) MSCs are a dult stem cells located in bone marrow that can differentiate into cells of the mesenchymal lineage and create embryonic like niche for hematopeoitic stem cells in the marrow Rat and human MSCs can differentiate into a variety of cell types, including neu ral cells (Woodbury et al., 2000) These cells also appear to induce remyelination in animal models of SCI MSCs can be easily obtained from bone marrow and transplanted back into the original donor, minimizing the r isk of immune rejection post transplant Consequently MSCs are considered an attractive source of transplantation from a clinical perspective. Recent

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46 studies have suggested that MSCs are capable of secreting ameliorative trophic factors such as cytokines, growth factors and brain natriuretic peptides which can contribute to functional improvement after transplantation (Lu et al., 2003; Neuhuber et al., 2005; Song et al., 2004) Neuronal cell type t ransplantation F etal Spinal Cord Tissue F etal spinal cord tissue transplant is a source of several classes of potentially beneficial cell types, including mature neurons, neural stem cells and non n euronal supportive cells. Accordingly, t ransplantation of these cells can deliver trophic factors, reduce local toxicity at the injury site, decrease glial scar formation, reverse changes in neuronal membrane properties and provide a substrate for growth of injured neurons in the host spinal cord. Fetal transplants have been ex tensively studied by Reier and colleagues (Bregman and Reier, 1986; Reier et al., 1988; Reier et al., 1992) who report that these grafts demonstrate robust survival after transplantation into injured CNS and beca use of their solid nature, are ideally set up to develop into structures that resemble normal spinal cord. Despite their potential benefits, fetal transplants alone are not very effective in eliciting regeneration in adult hosts. Although intrinsic CNS pro jections span the length of the graft, these axons do not traverse the grafts to enter the host spinal cord (Jakeman and Reier, 1991) Neural Precursor Cells Neural precursor cell s (NP Cs) refer to the multipotent stem cells found within the mammalian CNS. The y have the ability to s elf renew for long periods of time and differentiate into specialized cells within the CNS i.e. neurons, a strocytes, and oligodendrocytes Thus, in principle, NPCs can be used to replace lost neuronal cells after SCI. Interestingly, f etal spinal cord transplants also consist primarily of NPCs

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47 (Kalyani and Rao, 1998) Reynolds and Weiss (1992) were the first to isolate NPC s from the subventricular zone of the adult mice brain s NPCs have subsequently been isolated from various areas of the adult brain and spinal cord in various species. In the adult CNS, there are two primary locations of NPCs i.e. the subventricular zone along lateral ventricles and the subgranular zone of the dentate gyrus in the hippocampus (Alvarez Buylla et al., 2002) In addition, NPCs with varying capacity for self renewal and differentiation have also been isola ted from other regions of the CNS, including from human white matter (Nunes et al., 2003; Palmer et al., 1995; Weiss et al., 1996) In rodents the new cells formed in the subventricular zone migrate in the rostral migratory stream from the ventricular walls to the olfactory bulb where they replace granule cells and periglomerular cells, whereas NPCs in the subgranular zone mainly differentiate to granular cells (Doetsch and Alvarez Buylla, 1996; Gage, 2000) N eural P recursor C ell T ransplantation after SCI NPCs present a number of advantageous properties which lead many to consider ideal candidates for cell therapy after SCI They are expandable in culture without any change in their properties, can be genetically manipulated and stored for long periods of time without loss of functional properties NPCs are also ethically appealing as they can be derived, in theory, from the brain s of the transplant recipient ( i.e. autograf t) using stereotactic approaches, from the brains of human organ donors or from temporal lobectomy specimens derived during neurosurgical correction of refractory epilepsy ( i.e. allograft) (Karimi Abdolrezaee et al. 2006c) These cells are also capable of survival in a toxic environment, migrate to the lesion area, differentiate into appropriate cell types, integrate with the host circuitry and contribute to functional recovery.

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48 Fig ure 1 1 D epiction of neural regulation of respiration Descending drive from brainstem respir atory neurons activates cranial and spinal (phrenic and intercostal) motoneurons, which causes contraction of respiratory muscles resulting in ventilation. Ventilation maintains the blood lev els of O 2 CO 2 and pH which are sensed by peripheral and central chemoreceptors. Afferents from chemoreceptors synapse at brainstem respiratory neurons and affect the phrenic motor output. The respiratory neurons also receive afferent feedback from the mec hanoreceptors in airways and respiratory muscles. Adapted from Feldman (1986)

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49 Fig ure 1 2 Depiction of ventral and dorsal medullary respiratory neuronal projections Fig ure 1 3 D epiction of descending respiratory pathways in th e spinal cord. voluntary (corticospinal) axons in rats.

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50 CHAPTER 2 OUTLINE OF EXPERIMEN TS Spontaneous respiratory plasticity occurs at the spinal level in adult rats after exposure to intermittent hypoxia or following chronic high cervical spinal cord injury. Although respiratory plasticity after intermittent hypoxia is extensively studied, the effect of phrenicotomy, which is performed to maintain stable extracellular recordings from the phrenic nerve, is un known. Accordingly, A im 1 of this dissertation was designed to test the hypothesis that intermittent hypoxia induced LTF of phrenic inspiratory burst amplitude is greater in rats with cut rather than intact phrenic nerves. In A im 2 we reasoned that if time dependent recovery of IL PhrMN activity following chronic C2HS involves activation of propriospinal cervical interneurons, then meas urements of synchrony between IL and CL PhrMN discharges might reveal some features of the circuitry involved. Thus, using cross correlation analyses we tested the hypothesis that synchronization between IL and CL PhrMN bursting would improve in parallel with IL phrenic recovery after chronic C2HS Finally, for Aim 3 we investigated the the potential of NPC s as candidate s for spinal cord repair and, ultimately, for promoting respiratory recovery after C2HS Accordingly, A im 3 tested the hypothesis tha t post natal SVZ derived NPC s are capable o f survival and differentiation after transplantation into the injured cervical spinal cord. In summary, these aims investigate neural circuit remodeling in response to respiratory challenges, cervical spinal cord injury and stem cell transplantation. Ove rall Objective s 1. To determine the impact of acute phrenicotomy on the expression of hypoxia induced plasticity.

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51 2. To examine spinal circuitry change s involved in the recovery of PhrMN activity after chronic spinal cord injury, using cross correlation analyse s 3. To explore the feasibility of postnatal brain derived stem cell transplantation as a potential strategy for respiratory recovery after cervical spinal cord injury Experiment One: Determine the impact of acute phrenicotomy on the expression of hypoxia induced plasticity. Rationale: Intermittent hypoxia i nduces LTF characterized by a persistent increase in neural drive to the respiratory muscles LTF of phrenic inspiratory activity is often studied in anesthetized animals after phrenicotomy (PhrX) with subsequent recordings made from the proximal stump of the phrenic nerve. However, severing both efferent and afferent axons in the phrenic nerve has the potential to alter the excitability of PhrMNs, w hich is an important determinant of phrenic output and pLTF. Hypothesis I : Intermittent hypoxia induced LTF of phrenic inspiratory burst amplitude is greater in rats with cut than intact phrenic nerves. Hypothesis II : Acute phrenicotomy influences efferent phrenic bursting during baseline conditions. Experimen tal Design: Adult male Sprague Dawley rats were anesthetized, mechanically ventilated, bilaterally vagotomized and paralyzed. Phrenic neurograms were recorded with intact phrenic nerves (n = 9 ) or bilateral phrenicotomy (n = 8 ). Data were obtained before ( i.e. baseline) during and after three 5 min bouts of isocapnic hypoxia (inspired O 2 fraction i.e. FIO 2 = 0.14 0.16) Blood samples were obtained during baseline, the first episode of hypoxia and 25 and 55 min after hypoxia. Sham animals with intact (n=8) or cut phrenic nerves (n= 7 ) received same FIO 2 as baseline throughout the protocol. In separate experiments, acute phrenicotomy was performed while

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52 electrical activity was being recorded from the phrenic nerves (n = 6). Peak integrated phrenic amplitude and burst frequency, averaged over 1 min for each data point, were expressed in millivolts and bursts per min, respectively, and were also normalized to values recorded during baseline. Experiment Two : Examine changes in synchrony between IL and CL PhrMN discharge after chronic C2HS using cross correlation analyses Rationale: Following C2HS, inspiratory burst amplitude in the IL phrenic nerve increases over weeks months. This time dependent recovery of IL PhrMN activity following chronic C2HS has been attr ibuted to the activation of a monosynaptic, bulbospinal pathway which crosses the spinal midline caudal to the injury However, recent neuroanatomical data from our lab suggests that the descending control of PhrMNs in the rat may also involve propriospina l cervical interneurons We reasoned that if recovery of IL PhrMN activity following chronic C2HS involves activation of propriospinal cervical interneurons, then measurements of synchrony between IL and CL PhrMN discharges might reveal some features of th e circuitry involved. Hypothesis: Synchronization between IL and CL PhrMN bursting would improve in parallel with IL phrenic recovery after chronic C2HS. Experimental Design: Bil ateral phrenic neurograms were recorded in three groups of anesthetized adul t male Sprague Dawley rats: uninjured (n=5) and either 2 (n=5) or 12 (n=12) wks post C2HS. The raw inspiratory phrenic signal was recorded for ~3 hours and then converted into events C ross correlation was done between IL and CL events trains and the resu lting peaks were analyzed to examine synchronous activity as well as common synaptic input. After the completion of neurophysiological

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53 recordings, a ll C2HS injured rats were perfused and lesions were histologically confirmed to extend to the spinal midline Experiment Three : Ex plore the feasibility of postnatal brain d erived NPC transplantation to affect respiratory recovery after chronic C2HS in rats Rationale: Endogenous subventricular zone (SVZ) derived neural precursor cells (NPCs) have shown an extens ive capacity for self renewal and multipotency in vitro. These cells are regarded as a viable therapeutic alternative to embryonic stem cells as they obviate the potential ethical issues involved with the use of embryonic stem cells T he ability of NPCs, d erived from the SVZ of post natal rats, to survive, differentiate and affect respiratory outcomes following C2HS h as not been explored Hypothesis I : Post natal SVZ derived NPCs are capable of survival and differentiation afte r transplantation into the inj ured cervical spinal cord. Hypothesis II : NPCs do not negative ly impact the respiratory recovery after C2HS Experimental Design: NPCs were isolated from the SVZ of green fluorescent protein (GFP) expressing rat pups at post natal day 4 and maintained in c ulture. Resulting neurospheres were mechanically dissociated and then injected (~500,000 cells suspended in NeuroCult NS A basal medium, 5 l total volume) immediately caudal to an acute C2HS injury in adult female, Sprague Dawley rats. In a subset of rat s, daily cyclosporine A immunosuppression as used to enhance the survival of transplanted NPCs. Ventilation was assessed in unanesthetized rats at both 4 and 8 weeks post transplantation using whole body plethysmography. Phrenic motor output was also recor ded at 8 wk post transplant ation in anesthetized, paralyzed, vagotomized and ventilated rats. Rats were perfused at the end of each experiment and cervical

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54 spinal cords were dissected for immunocytochemical characterization of the transplanted cells.

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55 CH APTER 3 PHRENICOTOMY ALTERS PHRENIC LONG TERM FA CILITATION (LTF) FOLLOWING INTERMITTE NT HYPOXIA IN ANESTHETIZED RATS Exposure to intermittent hypoxia over short periods ( e.g. min to hours) can evoke a persistent increase in respiratory motor output termed long term facilitation or LTF (Lee et al., 2009a; McKay et al., 2004; Mitchell and Johnson, 2003) LTF has been observed in both awake (Harris et al., 2006; Lee et al., 2009 a) and sleeping humans (Pierchala et al., 2008) and a wide range of animal species (Fuller et al., 2001b; Mateika and Fregosi, 1997; McKay et al., 2004) The mechanisms underlying LTF have been studied extensively ov er the last 10 15 years, primarily in anesthetized animals (reviewed in MacFarlane et al., 2009; Mitchell et al., 2001a; Mitchell and Johnson, 2003) In anesthetized animals, LTF is typically manifest as an increa se in the inspiratory burst amplitude of phrenic (Fuller et al., 2000) and/or hypoglossal (XII) extracellular nerve r ecordings (Fuller et al., 2001a) Recor dings of in vivo (Fuller et al., 2000) and in vitro (Bocchiaro and Feldman, 2004) respiratory motor LTF are typically made from cut respiratory muscle nerves. More specifically, the phrenic and/or XII nerves are cut and subsequent extracellular recordings are made from the central end of the nerve. This procedure can provide stability to the neurophysiological recording procedures, particularly when a dorsal surgical approach is used, but also has the potential to alter respiratory motor output and the subsequent expression of plasticity. For example, although LTF of phrenic burst amplitude is typically robust in anesthetized rats with cut phrenic nerves ( i.e. phrenicotomy, PhrX) (Baker Herman and Mitchell, 2002; Fuller et al., 2001b) LTF of diaphragm electromyogram (EMG) activity is absent in anesthetized, spontaneousl y breathing rats with intact phrenic nerves (Janssen et al., 2000) Th is difference probably does not reflect vagally mediated inhibition of LTF

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56 during spontaneous breathing because Golder and Martinez (2008) demonstra ted that under otherwise similar conditions vagotomized rats have substantially diminished phrenic LTF compared to vagal intact rats. Similarly, LTF of inspiratory volume (which correlates with phrenic burst amplitude, Eldridge, 1971) ) is reduced in unanesthetized, spontaneously breathing animals (Olson et al., 2001) as compared to phrenic LTF in anesthetized PhrX rats (Baker Herman and Mitchell, 2002; Fuller et al., 2001b) Consistent with these observations, studies in humans s how ventilatory LTF that is substantially less than is typically seen in PhrX, anesthetized rats (Lee et al., 2009a) Collectively, these observation s are consistent with the notion that PhrX may create preconditions which enhance the subsequent expression of LTF of phrenic burst amplitude following intermittent hypoxia. There are at least two potential mechanisms that can be put forth to explain thi s fact First, axotomy can alter neuronal excitability. Indeed a substantial body of evidence shows that chronic axotomy increases neuronal excitability as indicated by a decrease in the amount of electrical current needed to evoke an action potential (i .e. decreased rheobase current) (reviewed in Titmus and Faber, 1990) Studies of acute axotomy reveal relatively rapid changes in the morphology of mammalian neurons (Chuckowree and Vickers, 2003) and increases in intracellular calcium concentration in both invertebrate (Sattler et al., 1996) and vertebrate axons (Mandolesi et al., 2004) Accordingly, phrenic motoneuron excitability may be incr eased after PhrX. Another possibility is that removal of afferent signals which normally travel in the phrenic nerve could alter the neural control of PhrMNs (Frazier and Revelette, 1991; Road, 1990) Indeed afferent fibers make up 40 45% of the phrenic nerve (Landau et al., 1962;

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57 Langford and Schmidt, 1983) and these afferents project to both spinal (Goshgarian and Roubal, 1986; Song et al., 1999) and supraspinal structures (Chou and Davenport, 2005; Speck, 1987) Although a direct role of phrenic a fferents in modulating the excitability of PhrMNs has not been definitively shown, there is indirect evidence suggesting that afferents can reflex ive ly affect the phrenic motor drive (Duron et al., 1976; Gill and Ku no, 1963; Jammes et al., 1986; Rijlant, 1942; Speck and Revelette, 1987a) Our purpose was to test the hypotheses that : 1) intermittent hypoxia induced LTF of phrenic inspiratory burst amplitude is greater in rats with cut vs. intact phrenic nerves, and 2) conditions. Preliminary results have been presented in abstract form (Sandhu et al., 2009) Methods Animals All procedures were approved by the University of Florida Institutional Ani mal Care and Use Committee. Adult male Sprague Dawley rats (Harlan Inc., Indianapolis, IN, USA) were divided into four groups 1) bilateral PhrX with intermittent hypoxia (PhrX LTF; n=8), 2) phrenic nerves intact with intermittent hypoxia (PhrI LTF; n=9), 3) Sham; n=7) and 4) phrenic nerves intact with sham hypoxia (PhrI Sham; n=8). Six additional rats were used to investigate the effect of acute PhrX on phrenic output. Experimental Preparatio n The general procedures have been described recently (Doperalski and Fuller, 2006; Lee et al., 2009c) Rats were anesthetized with isoflurane (5% in 100% O 2 ) in a

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5 8 closed chamber and then transferred to a nose cone (2 3% isoflurane in 50% O 2 balance N 2 ). The trachea was cannulated with PE 240 tubing and rats were mechanically ventilated for the remainder of the experiment. The lungs were briefly hyperinflated (2 3 seconds) approximately once per hour to minimize at electasis. The tracheal pressure was monitored with a pressure transducer (Statham P 10EZ pressure transducer, CP122 AC/DC strain gage amplifier, Grass Instruments, West Warwick, RI, USA) connected to the tracheal cannula. A catheter (PE 50) was inserted i nto the femoral vein, and rats were converted from isoflurane to urethane anesthesia (1.6 g/kg, i.v.; 0.12 g/ml distilled water). The adequacy of anesthesia was monitored during this period by observing limb withdrawal response to toe pinch and supplementa l urethane was given if indicated (0.3 g/kg, i.v.). A femoral arterial catheter (PE 50) was inserted to measure blood pressure (Statham P 10EZ pressure transducer, CP122 AC/DC strain gage amplifier, Grass Instruments, West Warwick, RI, USA) and to periodic ally withdraw blood samples (see protocol below ). Rats were bilaterally vagotomized to prevent entrainment of phrenic motor output with the ventilator and paralyzed with pancuronium bromide (2.5 mg/kg, i.v.) to eliminate spontaneous breathing efforts. Fol lowing paralysis, the adequacy of anesthesia was monitored by observing blood pressure and phrenic nerve response to toe pinch. A slow infusion of lactated Ringer's solution and sodium bicarbonate (3:1, 1.5ml/h) was maintained to promote acid base balance (Baker Herman et al., 2009; MacFarlane and Mitchell, 2009) Arterial partial pressures of O 2 (PaO 2 ) and CO 2 (PaCO 2 ) as well as pH were determined from 0.2 ml arterial blood samples using an i Stat blood gas analyze r (Heska, Fort Collins, CO, USA). Blood gas and pH values were corrected to the rectal

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59 temperature measured at the time of the blood sample. The end tidal CO 2 partial pressure (PET CO2 ) was measured throughout the protocol using a rapidly responding mainstr eam CO 2 analyzer positioned a few cm from the tracheostomy tube on the expired line of the ventilator circuit (Capnogard CO 2 monitor, Novametrix Medical Systems, Wallingford, CT, USA). Rectal temperature was maintained within 371 C (see results) using a rectal thermistor connected to a servo controlled heating pad (model TC 1000, CWE Inc., Ardmore, PA, USA). Both phrenic nerves were isolated within the caudal neck region medial to the brachial plexus, using a ventral surgical approach. Electrical activit y was 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 f ull wave rectified and moving averaged (time constant 100 ms; model MA 1000; CWE Inc., Ardmore, PA, USA). Data were digitized using a CED Power 1401 data acquisition interface and recorded on a PC using Spike2 software (Cambridge Electronic Design Limited, Cambridge, England). Experimental Protocols Long Term Facilitation. The LTF protocol was similar to prior studies (Bach and Mitchell, 1996; Fuller et al., 2001b; Kinkead et al., 1998) After an adequate plane of anesthesia was established, the PET CO 2 was maintained at 402 mmHg for 5 10 min; the inspired O 2 fraction (FI O 2 ) was held at 0.5. The end tidal CO 2 apneic threshold for inspiratory phrenic activity was then determined by gradually increasing the ventilator pump rate until inspiratory bursting ceased in both phrenic nerves. Apnea was maintained for one min, and the ventilator rate was then gradually decreased until

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60 inspiratory bursting resumed in the phrenic nerves. The PET CO2 associated with the onset of in spiratory bursting was noted, and the ventilator rate was adjusted to maintain PET CO2 at 2 mmHg above this value throughout the experiment. The PET CO2 measurements, however, were merely a guide to help maintain isocapnia, and conclusions regarding isocapni a were determined exclusively by arterial blood analyses. After a 10 20 min baseline period, an arterial blood sample was drawn. Rats were then exposed to either three, 5 min episodes of hypoxia (FI O2 = 0.14 0.16), separated by 5 min of hyperoxic recovery or sham hypoxia (FI O2 same as baseline). Blood samples were obtained during the first episode of hypoxia, and 25 and 55 min post hypoxia. At the conclusion of the protocol, another 5 min episode of hypoxia was given in an effort to confirm the integrity of the nerve electrode contact. If phrenic burst amplitude was diminished by more than 10% relative to the initial hypoxic response this was taken as an indicator that the nerve electrode contract was unstable. Based on this criterion, data from a single r at was excluded from the final analyses. Influence of PhrX on phrenic bursting. The intact phrenic nerves were placed on electrodes and the nerve electrode contact area was covered with a silicone elastomer (Kwik Sil, World Precision Instruments, Saras ota, FL, USA). This procedure ensured that the contact was preserved during the subsequent PhrX. Once a stable phrenic recording was obtained, PhrX was performed while electrical activity was being recorded from the phrenic nerves. In 4 of 6 rats, arter ial blood samples were taken before and after the PhrX procedure. These blood samples were drawn at the end of the baseline period and PET CO 2 was maintained at pre PhrX level for 60 min. Another

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61 blood sample was drawn at the end of the recording to confir m isocapnia relative to pre PhrX. Data Analyses The analyses of phrenic neurograms was described in our recent paper (Lee et al., 2009c) Peak integrated phrenic amplitude ( Phr) and burst frequency, averaged over 1 min for each recorded data point, were expressed in mV (i.e. arbitrary units, a.u.) and bursts/min, respectively, and were also normalized to values recorded during baseline. Statistical tests were done using SigmaStat v. 2.03 software. Differences in phrenic LTF and hypoxic responses, and arterial blood gases between groups (e.g. PhrX LTF vs. PhrI LTF; PhrX LTF vs. PhrX sham, etc.) were determined using a two way repeated measures analysis of variance (RM ANOVA). The Student Neuman Keuls test was used for post hoc analyses. The effects of acute PhrX on phrenic output were examined using a one way RM ANOVA. Body weight w as compared between groups using one way ANOVA. Differences were considered statistically significant when the P Results Body weight (g) was similar between the five experimental groups: PhrX LTF=4109, Phr I LTF=4197, PhrX Sham=41510, PhrI Sham=41113 and acute PhrX=40711 ( P = 0.91). PaO 2 decreased during hypoxia in the LTF groups as expected ( P < 0.001). Rectal temperature did not change by more than 0.6 C over the course of the experimental protocol i n any animal, and mean temperatures were similar between groups (baseline values: PhrX LTF=37.40.1, PhrI LTF=37.50.1, PhrX Sham=37.30.1, PhrI Sham=37.30.2 and acute PhrX=37.40.1; P = 0.68). The PET CO 2 recruitment threshold for phrenic bursting was sim ilar between PhrI (411

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62 mmHg) and PhrX rats (421 mmHg, p=0.54). Baseline and post hypoxia PaO 2 values did not differ between groups (Table 3 1). PaCO 2 also was similar between groupsand was maintained within 2 mmHg of baseline values throughout the inte rmittent hypoxia or sham protocols (Table 3 1). The mean arterial blood pressure (MAP) tended to be lower in PhrX LTF rats compared to the other groups. However, this effect was only statistically significant compared to the sham group (Table 3 1). MAP d ropped transiently during hypoxic episodes ( P < 0.01) as previously reported in anesthetized rats (Bavis and Mitchell, 2003; Fuller, 2005) The relative decrease in MAP during hypoxia was similar in PhrX LTF ( 20 3% baseline) and PhrI LTF rats ( 286% baseline, P = 0.25). Similar to prior phrenic LTF studies (Bavis and Mitchell, 2003; Doperalski and Fuller, 2006; MacFarlane and Mitchell, 2008) MAP also tended to decrease slightly over the course of the experimental protocol, however statistical significance was reached in only PhrI LTF rats ( P = 0.035; Table 3 1). Baseline Phrenic A ctivity Figure 3 1 shows representative neurograms recorded during baseline conditions. An afferent burst that was distinct from the inspiratory phrenic burst was clearly distinguishable in 15 of 17 (88%) PhrI rats. The afferent burst occurred in phase with lung deflation in all cases (Fig. 3 1), and was immediately abolished by acute PhrX (see below). We observed anecdotally that recordings from the distal stump of the phrenic nerve after PhrX (i.e. removing all efferent bursting) showed a clear rhythmic burst during lung deflation (Fig. 3 1C). Phrenic inspiratory burst frequency (bursts/min) was similar between groups during baseline (PhrX=472; PhrI=492 ; Fig. 3 2 ). The vs. PhrI rats, but the difference did not reach statistical significance (t test, P = 0.076 ; Fig. 3 2 ).

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63 Intermittent Hypoxia Example neurograms depicting a typical response to intermitten t hypoxia are provided in Fig. 3 3 A and B. The initial hypoxic episode evoked a significant increase in assessed during the initial 30 sec (onset) of the first hypoxic ep isode was significantly greater in PhrX LTF (17411 % baseline) vs. PhrI LTF rats (1459 % baseline) (Fi g. 3 4 A P = 0.006). The hypoxia onset phrenic burst frequency showed a decrease over sub sequent hypoxic episodes (Fig. 3 4 A ). In addition, difference s in the hypoxia onset burst frequency between PhrX LTF and PhrI LTF groups were not observed during hypoxic episodes 2 and 3 (Fig. 3 4 A ). Burst frequency assessed over the final minute of hypoxia was similar across all three hypoxic episodes in both Phr X LTF and PhrI LTF rats (Fig. 3 4 B). LTF vs. PhrI LTF when the data were expressed as a.u. ( P = 0.07, Fig. 3 4C ). However, the as significantly greater in PhrX LTF compared to PhrI LTF rats ( P < 0.01, Fig 3 4D ). Both PhrX LTF and PhrI LTF rats tended to show a slight progressive augmentation (Powell et al., 1998) amplitude during succe ssive hypoxic episodes (Figs. 3 4D ), but this was statistically significant only in the PhrX rats ( P < (data not shown). Phrenic Long Te rm F acilitation Representative examples of phrenic neurograms recorded during baseline and the post hypoxia period are shown in Fig. 3 5 Phrenic burst frequency (bursts/ min, Fig.

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64 3 6 A; % baseline, Fig. 3 6 B ) was stable during the post intermittent hypoxia period with no evidence for an increase relative to baseline values. Although there was no that changes in the timing of the phrenic bursts may have occurred, particul arly in the PhrX LTF group. Compared to baseline, inspiratory (Ti) and expiratory duration (Te) tended to be lower after intermittent hypoxia in both PhrX LTF and PhrI LTF rats but statistical significance was observed only in Ti at 55 min post hypoxia i n PhrX LTF animals (P < 0.05, Table 3 2A). As expected, v alues of Ti also weresignificantly lower during hypoxia in both groups compared to the corresponding sham data points (P < 0.001, Table 3 3 ). Analyses of p ost significant interaction between treatment ( i.e. PhrX LTF or PhrI LTF) and time ( i.e. 25 or 55 min) ( P < 0.001; Fig. 3 6 a.u. and % baseline) between PhrX LTF and Phr I LTF rats were more pronounced at 55 t han 25 min post hypoxia (Fig. 3 6 C and D P < 0.05) in PhrX LTF r ats at both time points (Fig. 3 6 D PhrX LTF and PhrI LTF rats was significantly greater than the value recorded in the corresponding sham groups at 55 min post intermittent hypoxia (PhrX Sham:1065 % baseline; PhrI Sham:1053 % baseline; both P < 0.05 vs. corresponding LTF group). as induced in both PhrX LTF and PhrI LTF rats, but the relative magnitude was much greater in the PhrX LTF group. the values obtained in the corresponding sham group. However, c omparison of the post i.e. 100% in all rats) also

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65 resulted in the same conclusions. Specifically, relative to baseline, LTF was significant at 55 min in both PhrX LTF and PhrI LTF groups, but was n ot present in either sham group. We also quantified changes in the amplitude of the afferent burst in the PhrI LTF rats following intermittent hypoxia. This analysis indicated that the afferent burst was ent hypoxia. Specifically, the amplitude of the burst was 974 and 898 %baseline at 25 and 55 min post hypoxia, respectively (both p>0.05). Impact of A Additional studies were performed in which we recorded phrenic activity before and for 60 min following PhrX in the same animals. Great care was taken to prevent any change in the nerve electrode contact during the PhrX procedure (see methods). Blood samples taken during baseline (pre PhrX) and at 60 min after PhrX (post) showed that the values of PaO 2 (pre: 13216, post: 13420 mmHg), PaCO 2 (pre: 323, post: 323 mmHg) and pH (pre: 7.400.01, post: 7.400.02) were similar before and after PhrX. We were unable to collect blood samples in two animals, however, the phrenic nerve activity in these rats was not different in any way from the others, and therefore they were included in the overall group average. Blood pressure did not significantly change after PhrX ( P = 0.58). E xample s of the impact of PhrX on phreni c bursting are provided in Fig s 3 7 and 3 8 Acute PhrX abolished afferent bursting and led to an increase in ipsilateral efferent several cases (4/6), the PhrX procedure caused an immediate increase in the amplitude of the inspiratory phrenic burst that was sustained for a pproximately 60 min (e.g. Fig. 3 7 3 8 ). However, in the remaining instances (2/6),

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66 there was a more gradual inc rease in phrenic burst amplitude after PhrX. On average, (i.e. pre PhrX) only at 30 min post PhrX ( P < 0.05, Fig. 3 8 amplitude at 1 (p= 0.07) and 5 min (p=0.08) post PhrX did not reach statistical significance. The PhrX also lead to an immediate increase in non rhythmic tonic bursting in the ef ferent phrenic recording (Fig. 3 7 3 8 ). This response occurred in all PhrX experiments, and mo st likely reflects high frequency spike activity in axons and/or motoneurons secondary to axotomy. Indeed, the response shown after PhrX is quite similar to in vitro recordings of efferent bursting following acute axotomy (Mandolesi et al., 2004; see discussion) The tonic phrenic bursting decreased gradu ally over time post PhrX (Fig. 3 7 B C 3 8 B C ). Phrenic inspiratory burst frequency was n ot influenced by PhrX (Figs. 3 9 A). Analyses of the respiratory cycle before a nd after PhrX revealed no significant changes in Ti and Te (Table 3 2 P > 0.05). rats do not show evidence of facilitation of inspiratory burst amplitude. During time c ontrol experiments there was a delay of at least 30 min after PhrX and before beginning the baseline period. In addition, the PET CO 2 apneic and recruitment thresholds were established after the PhrX procedure in the time control rats. Discussion This is the first comprehensive analyses of the impact of acute PhrX on the expression of hypoxia induced respiratory plasticity. Our primary finding was that PhrX is associated with a substantial increase in the magnitude of both the acute hypoxic response and I H induced LTF. Efferent phrenic motor output and the capacity for respiratory plasticity are thus influenced by axotomy of afferent and/or efferent axons in

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67 the phrenic nerve, and the integrity of the phrenic nerve should be taken into account when interpr eting mechanisms of phrenic motor plasticity in anesthetized animals. We speculate that PhrX could change PhrMN excitability by removing inhibitory afferent input, by injury related mechanisms that lead to motoneuron depolarization, or a change in intrinsi c membrane properties. Implications for the Study of R espiratory LTF Many published studies of respiratory LTF have used in vivo (Fuller et al., 2000; Mahamed and Mitchell, 2007) in situ (Tadjalli et al., 2007) or in vitro (Bocchiaro and Feldman, 2004) preparations with recordings of efferent motor output from the distal end of the cut phrenic and/or hypoglossal (XII) nerves. These studies demonstrate predictable variability across laboratories, preparations and species, as well as g enetic variations between rat strains and sub strains (Fuller et al., 2001a) However, a review of the literature indicates that LTF of efferent phrenic burst amplitude is considerably more robust in the aforementioned preparations as compared to LTF assessed in spontaneously breathing animals (diaphragm EMG data) and humans (tidal volume data) with intact respiratory nerves (reviewed i n Fuller et al., 2005) Indeed, LTF of respiratory output in spontaneously breathing animals is more often expressed as a persistent increase in breathing frequency rather than inspiratory volume or EMG burst amplitude (Baker Herman and Mitchell, 2008; Olson et al., 2001) The difference in LTF expression between spontaneously breathing vs. ventilated preparations probably does not represent the impact of anesthesia on plasticity (Janssen et al., 2000; Mateika and Fregosi, 1997) For example, Janssen and Fregosi (2000) could not induce LTF of diaphragm EMG b urst amplitude in urethane anesthetized and spontaneously breathing rats despite using an anesthetic and IH regimen which evokes robust LTF in ventilated

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68 rats with PhrX (Bach and Mitchell, 1996; Fuller et al., 2001b ) Similarly, LTF of diaphragm EMG activity is not evident in anesthetized and spontaneously breathing cats following intermittent stimulation of the carotid sinus nerve (Mateika and Fregosi 1997) Interestingly, LTF of upper airway muscle activity ( e.g. genioglossus) can be evoked in spontaneously breathing animals in the absence of diaphragm LTF (Mateika and Fregosi, 1997; Tadjalli et al., 2008) A similar result was inferred from measuremen ts of decreased pulmonary airflow resistance following IH in sleeping humans (Aboubakr et al., 2001; Shkoukani et al., 2002) Therefore, the mechanisms which restrain phrenic/diaphragm LTF during spontaneous breat hing (30) may not exert a parallel influence on hypoglossal or other upper airway respiratory motor outputs. Janssen et al. (2000) hypothesized that the (comparative) lack of phrenic/diaphragm LTF expression during spontaneous breathing reflects the relatively higher PaCO 2 values in spontaneously breathing vs. ventilated animals. Since even small increases in PaCO 2 will increase the overall output of PhrMNs (Kong and Berger, 1986; St John and Bartlett, 1979) elevated PaCO 2 may impair or reduce subsequent LTF expression via a (pre IH) conditions there may be a reduced capacity for increased motoneuron recruitment or rate coding during the post hypoxic period (Doperalski and Fuller, 2006) Consistent with this idea, phrenic LTF is difficult to evoke in phrenic neurograms recorde d contralateral to cervical spinal cord hemisection injury (Doperalski and Fuller, 2006) a condition which results in robust compensatory increases in contralateral phrenic output (Miyata et al., 1995) In contrast, Lee and colleagues (2009a) recently demonstrated that raising ETCO 2 by approximately 4 mmHg above eupneic values is a

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69 prerequisite for induction of ventilatory LTF (including both increased frequency and tidal volume) in spontaneously breathing humans. Thus, in some circum stances PaCO 2 elevations appear to be necessary for respiratory LTF, and factors other than PaCO 2 levels may be responsible for differences in LTF between spontaneously breathing and ventilated preparations. Based on our data, we suggest that the conditio n of the phrenic nerve ( i.e. cut vs. intact) contributes to the observed LTF differences. We therefore put forward the working hypothesis that PhrX creates preconditions which enhance the subsequent induction of phrenic LTF. To be clear, we are not sugge sting that PhrX is a necessary precondition for LTF, but rather that PhrX primes the phrenic motor system, possibly by increasing phrenic motoneuron excitability, and enables more robust increases in phrenic burst amplitude following IH or other stimuli (e.g. intermittent apnea, Mahamed and Mitchell, 2008; Tadjalli et al., 2008) Many studies of phrenic LTF in rats have used unilateral (vs. bilateral) PhrX (Baker Herman et al., 2009; Baker Herman et al., 2004; Bavis and Mitchell, 2003; MacFarlane and Mitchell, 2008; 2009; MacFarlane et al., 2009; Mahamed and Mitchell, 2008; Tadjalli et al., 2007) In the current study, we used a bilateral PhrX in order to remove all phreni c afferent inputs to the spinal cord. It will be interesting in future work to examine the impact of unilateral PhrX on bilateral phrenic LTF (i.e. one phrenic nerve cut and the other intact). Based on the immediate impact of cutting the phrenic nerve (i .e. increased phasic and toni c bursting in that nerve, Fig. 3 7 ), we speculate that such an experiment would result in enhanced LTF only in the cut phrenic nerve. In any case, our data underscore the importance of considering the effect(s) of PhrX when i nterpreting and comparing LTF data from different experimental setups.

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70 Below we briefly discuss two potential candidate mechanisms to explain the impact of PhrX on phrenic motor output and LTF. Axotomy of Phrenic Motoneurons and A fferent Neurons: Potentia l I mpact on LTF The PhrX procedure used in the current and prior LTF studies will sever all the axons in the main trunk of the phrenic nerve. To interpret the effects of PhrX, the impact of axotomy on neuronal discharge and membrane properties should be c onsidered. Long term changes in neuronal membrane properties after chronic axotomy have been extensively studied (reviewed in Titmus and Faber, 1990) Over time frames ranging from days to weeks, axotomy results in changes in neuronal properties that favor increased discharge (e.g. increased resistance, decreased rheobase). Relatively fewer studies have investigated the potential for acute changes in neuronal properties immediately following axotomy. Mandolesi et al. (2004) showed that transecting the axons of cultured rat neurons initiates a rapid depolarization at the injury site followed by a burst of action potentials. This effect appears to be initiated at the site of axotomy, and then the depolarization travels back from the lesion site to the soma where it triggers vigorous spiking activity and sustained depolarization lasting up to 10 min (Mandolesi et al., 2004) Similarly, peripheral nerve crush in Aplysia californica results in a transient (~60 sec) bursting of action potentials in motoneurons that were silent prior to the injury (Lin et al., 2003) The acute, axotomy induced alterations in neuronal activity are as sociated with disruption of ionic regulation across the axon and/or soma membrane. For example, depolarization of the cell membrane after axotomy activates voltage sensitive calcium (Ca 2+ ) channels triggering a rise of intracellular Ca 2+ (Mattson et al., 1990; Sanchez Vives et al., 1994; Sattler et al., 1996; Strautman et al., 1990; Ziv and Spira, 1993)

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71 There is also a gradual increase of intracellular sodium (Na + ) after axotomy of in vitro mammalian neurons (Mandol esi et al., 2004) Increased Ca 2+ has been linked to activation of several molecular mechanisms that regulate various functions in the cell, including modulation of firing patterns and neuronal excitability (Ambron et al., 1996) Furthermore, blocking Ca 2+ influx after axotomy reduces excitability and prevents firing of in vitro mice neurons (Hilaire et al., 2005) Axotomy may also trigger a change in neuromodulatory inputs to motoneurons. Chronic axotomy of cervical spinal afferents (including phrenic afferents) via dorsal rhizotomy enhances serotonergic innervation of PhrMNs an d augments serotonin dependent LTF of phrenic motor output (Kinkead et al., 1998) However, little is known about potential change s in serotonin receptor expression acutely following axotomy of mammalian neurons. Interestingly, serotonin exposure for 20 min causes molecular changes (activation of mitogen activated protein kinase) that are similar to what was seen after severing axon s (Lin et al., 2003) These data present the possibility that axotomy evoked electrical discharge or molecular changes might promote serotonin receptor expression in motoneurons or re organization of pre motor sero tonergic input (Lin et al., 2003) Interruption of axonal continuity also disrupts normal retrograde transport of trophic signals from the axon terminal which can affect the synthesis of cyclic AMP and gene express ion (Bedi et al., 1998; Lewin and Walters, 1996; Liao et al., 1999) The potential for rapid changes in neurotrophic factor expression after axotomy is of particular interest in regard to phrenic LTF. Baker Herman et al. (2004) demonstrated that spinal brain derived neurotrophic factor (BDNF) is both necessary and sufficient for phrenic LTF in PhrX rats. Accordingly, if phrenic axotomy influences

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72 BDNF expression in or around PhrMNs this could have a profound effect on the subsequent induction of LTF. The PhrX procedure will also abruptly elimin ate inputs associated with activation of phrenic afferent fibers. It is not always appreciated that a large portion ( i.e. 40 45%) of the axons in the phrenic nerve are afferent in origin (Landau et al., 1962; Langford and Schmidt, 1983) These afferent fibers carry information from the proprioceptors (muscle spindles and tendon organs), rapidly adapting mechanoreceptors ( P acinia n corpuscles) and nociceptors in the diaphragm as well as free nerve endings in the pericardium and pleural surface of diaphragm (Holt et al., 1991; Road, 1990) Many studies have reported that electrical stimulation of phrenic afferents has an inhibitory effect on phrenic motoneuron activity (Duron et al., 1976; Gill and Kuno, 1963; Jammes et al., 1986; Rijlant, 1942; Speck and Revelette, 1987a) For example, Jammes et al. (1986) showed that s timulation of both large diameter and thin afferent fibers in the phrenic nerve of anesthetized cats caused a contralateral reduction of phrenic motoneuron impulse frequency and duration of phrenic activity, respectively. Similarly, an inhibitory effect on ipsilateral phrenic activity in response to phrenic afferent stimulation has also been demonstrated (Rijlant, 1942) Additionally, Cheeseman et al. (1990) showed that a sudden change in diaphragm length causes a reflex reduction of integrated diaphragm electromyogram amplitude (diaEMG) in anesthetized cats, and this effect was not observed after interruption of afferent input via cervical dorsal rhizotomy. On the other hand, activation of phrenic afferents may stimulate breathing in some cases. Speck et al. (1987b) showed that about one fourth of DRG respiratory modulated neurons are excited by phrenic afferents. Based on conduction veloci ty measurements, they

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73 attributed this effect to activation of predominantly small, type III myelinated fibers. However, in spite of this excitation, the overall effect of phrenic afferent stimulation was inhibition of phrenic motoneuron activity. In any c ase, it is clear that sensory feedback from the diaphragm can modulate the respiratory drive. However, the impact of phrenic afferents on phrenic motor output in the anesthetized, ventilated, paralyzed rat preparation is unclear. Our data demonstrate tha t acute PhrX eliminates an afferent signal originating distal to the recording site ( e.g. Fig. 3 8 ). This phrenic afferent bursting occurred in phase with lung deflation as indicated by the trac heal pressure recordings (Fig. 3 1 ). Therefore, it is possib le that cutting the phrenic nerve removes phrenic afferent mediated modulation of phrenic output, either at the level of the spinal cord (Jammes et al., 1986) or via ascending projections to medullary respiratory centers (Speck, 1987) Finally, our data also showed that the increase in phren ic burst frequency (bursts/min) during the initial hypoxic episode was significantly greater in PhrX vs. PhrI rats. Accordingly, the PhrX procedure may have removed inhibitory inputs to the brainstem respiratory rhythm/pattern generator, which in turn cou ld potentiate the acute hypoxic response. However, the effects of PhrX were only transient in this case as differences in the hypoxic frequency response between PhrX vs. PhrI rats were not observed after the initial hypoxic episode. Summary Our data indic ate that PhrX affects phrenic output during baseline and hypoxic conditions, and the magnitude of LTF in response to intermittent hypoxia in anesthetized rats. Therefore, along with other parameters including the integrity of the vagus nerves (Golder and Martinez, 2008) anesthesia, and mechanical ventilation, the

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74 effect of PhrX should be taken into consideration when interpreting LTF data from PhrX animals. The imp act of PhrX may be especially important in studies investigating the molecular mechanisms of phrenic LTF. An investigation of the specific mechanism(s) through which PhrX affects phrenic motoneuron excitability and LTF should be the subject of future studi es.

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75 Table 3 1 Mean arterial blood pressure (MAP), partial pressure of arterial carbon dioxide (PaCO 2 ), oxygen (PaO 2 ), and arterial pH during baseline, the first hypoxic episode, 25 min and 55 min post hypoxia. *, different from baseline values, different from corresponding sham data point. Baseline Hypoxia 25min 55min MAP (mmHg) PhrX LTF 86 5 66 7 85 5 84 4 Phr I LTF 107 6 78 7 103 8 93 8 PhrX Sham 111 13 109 11 107 9 107 7 Phr I Sham 114 9 113 9 101 10 104 9 PaCO 2 (mmHg) PhrX LTF 39 3 37 3 38 3 38 3 Phr I LTF 41 2 39 2 42 2 41 2 PhrX Sham 37 4 35 5 39 3 37 4 Phr I Sham 35 2 36 3 35 3 36 3 PaO 2 (mmHg) PhrX LTF 163 13 34 1 155 9 162 9 Phr I LTF 162 8 33 1 152 10 155 11 PhrX Sham 142 11 152 11 145 14 158 11 Phr I Sham 185 8 181 9 173 11 173 11 pH PhrX LTF 7.37 0.02 7.34 0.02 7.35 0.03 7.34 0.03 Phr I LTF 7.35 0.01 7.33 0.01 7.33 0 .01 7.32 0.01 PhrX Sham 7.38 0.02 7.39 0.02 7.40 0.02 7.41 0.02 Phr I Sham 7.36 0.02 7.35 0.02 7.36 0.02 7.38 0.02 Table 3 2 Inspiratory (Ti) and expiratory (Te) duration before (pre PhrX) and 1, 30 and 60 min post PhrX. *, differ corresponding sham data point. pre Phrx 1 min 30 min 60 min Ti 0.35 0.02 0.35 0.02 0.36 0.02 0.36 0.03 Te 0.98 0.12 0.95 0.09 1 0.05 0.97 0.07

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76 Table 3 3 Inspiratory (Ti) an d expiratory (Te) duration during selected times of the LTF Group Baseline Hypoxia 25 min 55 min Ti PhrX LTF 0.38 0.03 0.25 0.01* 0.35 0.02 0.32 0.02* Phr I LTF 0.35 0.02 0.23 0.01* 0.33 0.02 0.33 0.01 PhrX Sham 0.38 0.02 0.37 0.03 0.39 0.03 0.36 0.03 Phr I Sham 0.39 0.02 0.40 0.02 0.36 0.01 0.36 0.01 Te P hrX LTF 0.99 0.07 0.88 0.04 0.87 0.04 0.89 0.04 Phr I LTF 0.94 0.08 0.93 0.05 0.92 0.05 0.93 0.05 PhrX Sham 0.88 0.07 0.91 0.1 0.90 0.09 0.81 0.07 Phr I Sham 0.86 0.07 0.88 0.06 0.90 0.06 0.91 0.05

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77 Fig ure 3 1 Rep resentative examples of phrenic neurograms. The moving averaged or signal (Phr). PInsp is the pressure recorded in the inspired line of the ventilator circuit. Panel A depicts recordings obtained from an intact phrenic nerve (PhrI). Note that the signal is composed of two distinct and phasic bursts. The larger burst is the typical inspiratory burst, whereas the smaller bursts (indicated by the arrowheads) occurred in phase wit h lung deflation as reflected by the PInsp (dashed lines). These afferent bursts were completely eliminated following PhrX (Panel B). An additional recording was made in a single rat from the distal stump of the cut phrenic nerve (Panel C). Therefore, in this example the rhythmic activity cannot reflect activity of phrenic motoneurons, and must reflect activity in afferent neurons within the phrenic nerve (see text for further description).

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78 Fig ure 3 2 Phrenic inspiratory burst frequency ( left panel) and (right panel) during normoxic baseline in rats with cut or intact phrenic nerves. Since baseline conditions were identical in all groups, the LTF and sham rats were combined for this analysis, and the PhrX group represents the combi ned PhrX LTF and PhrX Sham data, and the the PhrI group includes the combined PhrI LTF and PhrI amplitude in the PhrI group did not reach significance ( P = 0.076).

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79 Fig ure 3 3 ivity and arterial blood pressure during intermittent hypoxia in rats with phrenic nerves cut (PhrX LTF, A) or intact blood pressure trace (mmHg). The top panel depicts appr oximately 35 min of data including each of the three hypoxic episodes. The bottom panel depicts during baseline (i, iv), and both the onset (ii, v) and end (iii, vi) of the initial hy poxic episode.

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80 Fig ure 3 4 during intermittent hypoxia. Data are presented from PhrX onds) of hypoxia; panel B shows burst frequency during the final minute of hypoxia. amplitude (D). *, P < 0.02 vs. the PhrI LTF group; #, P < 0.05 vs. hypoxic episode 1. A B C D

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81 Fig ure 3 5 (Parterial) during baseline and both 25 and 55 min following intermittent phrenic nerves (PhrX LTF, top panel). A smaller but statistically significant LTF (see Fig. 3 6 ) was seen in rats with phrenic nerves intact (PhrI LTF, bottom panel). 0.05 mV 55 min P arterial P arterial PhrI LTF PhrX LTF Baseline 25 min 100 50 50 100 10 sec

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82 Fig ure 3 6 at 25 and 50 min following intermitte nt hypoxia. Data are presented from PhrX expressed as a.u. (C) and relative to baseline (D). P < 0.01. *, indicate s significantly higher than the PhrI LTF group ( P < 0.05). #, indicates significantly higher vs. 25 minutes, P < 0.05. A B C D

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83 Fig ure 3 7 Example of phrenic activity before, during and following PhrX. The top panel depicts approximately 1 hour of Phr data including the pre PhrX baseline period, the moment of PhrX (indicated by the arrow) and the post PhrX period. Note the immediate increase in the Phr signal after PhrX. The PhrX procedure always caused an increase in phrenic burst amplitude, and this respo nse was abrupt (as shown in this example) in 60% of the experiments but was more gradual in the remaining 40%. Expanded time scale traces showing several neural breaths are provided for the time points indicated by A, B, C and D in the top panel. In addi tion, the time scale is expanded even further (bottom panels) to show raw phrenic bursting at the points indicated by i vii. These traces depict the post PhrX increase in phrenic activity during the inspiratory burst (iii and v), and suggest an increase i n tonic activity during the expiratory period (iv and vi).

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84 Fig ure 3 8 Representative phrenic neurograms during and following acute PhrX. Both the raw (Phr) and integrated ( Phr) phrenic signal is shown in each trace. Panel A depicts the imme diate impact of PhrX (occurring at the upward arrow) on phrenic bursting. Note the abrupt increase in the baseline of the Phr signal indicating increased tonic phrenic discharges. This response occurred in 100% of the PhrX experiments. The smaller down ward arrows in Panel A indicate afferent bursting prior the PhrX. Panels Ai and Aii depict a single neural breath at the times indicated in the top panel. Time dependent PhrX) and C (60 min post Phr X ). Note that the tonic phrenic discharge (indicated by the horizontal dotted lines) gradually decreased over time following PhrX. Panels Biii and Biv show single neural breaths at the indicated time points.

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85 Fig ure 3 9 Mean data s amplitude. Data are shown during the pre PhrX baseline period (pre) and at 1, 5, 30 and 60 mins post PhrX. No significant changes in burst frequency were noted (panel A). While most animals showed an abrubt increase in phrenic bursting after PhrX (e.g. Fig. 5), a few showed a more gradual increase. Accordingly, the increase in Phr burst amplitude (% pre PhrX amplitude) did not reach statistical significance until 30 min post PhrX (panel B). P < 0. 05 vs. baseline (pre PhrX).

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86 CHAPTER 4 CHANGE IN PHRENIC MOTONEURON SYNCHRONY AFTER CHRONIC HIGH CERVICAL HEMI SECTION Hemisection from the midline to lateral edge of the cervical spinal cord has been used extensively to study respiratory plasticity follow ing spinal cord injury (SCI) (Fuller et al., 2005; Goshgarian, 2003; Lane et al., 2008a) The basic premise is that C2 hemisection (C2HS) interrupts descending bulbospinal pathways from t he medulla to PhrMNs locate d ipsilateral (IL) to the lesion. Thus, the IL hemidiaphragm is transiently paralyzed but contralateral (CL) diaphragm activity persists. Compensation via activation of CL PhrMNs and other respiratory pathways is sufficient to maintain minute ventilation ( E) thereby enabling the animal to survive the lesion. Subsequently, recovery processes affecting respiratory motor output can be studied. The appearance of IL PhrMN inspiratory activity following both acute (i.e. min to days post i njury) and chronic C2HS has been attributed to activation of a monosynaptic, bulbospinal pathway which crosses the spinal midline caudal to the injury (Goshgarian 2003). Goshgarian and colleagues have provided clear anatomical evidence for the existence of (Goshgarian, 2003) However, recent neuroanatomical data from our group suggests that the descending control of PhrMNs in the rat may also involve propriospinal cervical intern eurons (Lane et al. 20 08a, 2008b ). There is neurophysiological evidence that some PhrMNs may be activated by descending, polysynaptic pathways in spinal intact rats (Duffin and van Alphen, 1995; Tian and Duffin, 1996) Similarly, neuro physiological evidence of polysynaptic inputs to PhrMNs in C2HS rats was provided by Ling et al. (1995) Specifically, electrical stimulation of the ventral funiculus in the CL spinal cord evoked compound potentials in

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87 the IL phrenic nerve with both short (i.e. ~ 1.0 ms) and relatively long onset latencies (i.e. 5 7 ms). The long late ncy peaks are consistent with polysynaptic inputs in IL PhrMNs (Ling et al., 1995). While definitive evidence of a role for cervical interneurons in respiratory recovery after SCI is lacking, there is strong evidence in other motor pathways (e.g. corticos pinal) that cervical interneurons can promote functional recovery (Bareyre et al., 2004) We reasoned that if time dependent recovery of IL PhrMN activity following chronic C2HS involves activation of propriospinal cervical interneurons, then measurements of synchrony between IL and CL PhrMN discharges might reveal some features of the circuitry involved. Thus, we used cross correlation analyses (Kirkwood, 1979) to examine the synchrony of bursting between IL and CL PhrMNs after chronic C2HS. We hypothesized that C2HS will induce a persistent a lteration in respiratory control and delayed synchronization of IL and CL phrenic bursting Methods Animals All procedures were done following approval by the Institutional Animal Care and Use Committee at the University of Florida. Experiments were condu cted using a dult (250 280 g) male Sprague Dawley rats obtained from Harlan Inc. ( Indianapolis, IN, USA) Phrenic neurophysiology data were obtained from a group of uninjured rats (n=5) and rats with anatomically complete C2HS lesions at either 2wk (n=5) or 12wk (n=12) post injury. Spinal Cord Injury Animals were anesthetized with injections of xylazine ( 10 mg/kg s.q. ; Phoenix Pharmaceutical, Inc., St. Joseph, MO ) and ketamine (12 0 mg/kg i.p. ; Fort Dodge Animal

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88 Health, Fort Dodge, IA ). An incision was made e xtending from the base of the skull to the third cervical segment, and a subsequent laminectomy was made at the second cervical segment. A dural incision was made and lateral hemisection was performed on the left side of the spinal cord with a microscalpel and gentle aspiration using a pulled micropipette and suction The dura was closed with interrupted 10 0 sutures and durafilm placed over the dura. The overlying m uscle s were closed with polyglycolic acid sutures (4 0 vicryl) and the skin closed with 9 mm stainless steel wound clips. Following surgery, an analgesic (buprenorphine, 0.03 mg/kg, s.q.) was given every 10 12 h for 2 days Lactated ringer solution (12 ml/day, s.q.) was provided for 1 3 days, until adequate volition al drinking resumed. Neurophys iology Isoflurane anesthesia was induced in a closed chamber and rats were transferred to a nose cone where they breathed 2 3% isoflurane. The trachea was then cannulated with PE 240 tubing and rats were mechanically ventilated for the remainder of the exp eriment. A catheter (PE 50) was inserted into the femoral vein, and rats were converted from isoflurane to urethane anesthesia (1.6 g/kg, i.v.; 0.12 g/ml distilled water). The adequacy of anesthesia was monitored during this period by observing limb withdr awal and palpebral reflexes. A femoral arterial catheter (PE 50) was inserted to enable blood pressure measurements (Statham P 10EZ pressure transducer, CP122 AC/DC strain gage amplifier, Grass Instruments, West Warwick, RI, USA). The vagus nerves were se ctioned in the 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 press ure to the paws. A 3:1 solution of lactated

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89 Ringer's and sodium bicarbonate was continually infused via the venous catheter (1.5 ml/h) to help maintain arterial blood pressure and acid base regulation. The end tidal CO 2 partial pressure (PETCO 2 ) was measur ed throughout the protocol using a rapidly responding mainstream CO 2 analyzer positi oned a few centimeters from the tracheostomy tube on the expired line of the ventilator circuit (Capnogard neonatal CO 2 monitor, Novametrix Medical Systems, Wallingford, CT USA). Rectal temperature was maintained at 37.5 1C using a rectal thermistor connected to a servo controlled heating pad (model TC 1000, CWE Inc., Ardmore, PA, USA). A ventral approach was used to isolate both phrenic nerves within the caudal neck reg ion, medial to the brachial plexus. Electrical activity was recorded using silver wire electrodes with a monopolar configuration, amplified (1000 x ), band pass (300 10,000 Hz) and notch (60 Hz) filtered using a differential A/C amplifier (Model 1700, A M Sy stems, Carlsborg, WA, USA). The amplified signal was full wave rectified and moving averaged (time constant 100 ms) using a model MA 1000 moving averager (CWE Inc., Ardmore, PA, USA). Data were digitized using a CED Power 1401 data acquisition interface an d recorded on a PC using Spike2 software (Cambridge Electronic Design Limited, Cambridge, England). The end tidal CO 2 was maintained 10 15 mmHg above the apneic threshold during all recordings to enable a robust signal to noise ratio in the IL phrenic neur ogram. The sensitivity of the cross correlation method improves with longer recording durations (Kirkwood, 1979) Neurograms were thus recorded for approximately 3 hours (mean = 17414 min) resulting in an average of 302,57366,026 events per correlogram.

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90 Spinal Cord Histology The cervical spinal cord was examined histologically aft er the neurophysiology experiments. Urethane anesthetized rats (see above) were euthanized by systemic perfusion with heparinized saline followed by 4% paraformaldehyde. The cervical spinal cord was then removed, blocked, and sectioned (4 vibrato me Tissue sections were slide mounted, stained with luxol fast blue followed by cresyl violet, and examined with light microscopy. Some portion of bulbospinal projections to phrenic motor neurons travel in the medial portions of the ventral funiculi. Acco rdingly, hemisections were considered anatomically complete only if there was a complete absence of apparently healthy white matter in the ipsilateral spinal cord at the lesion epicenter. An example of cervical hemisection injury is shown in Fig. 4 1. Data Analyses The raw phrenic signal was converted into events by setting a threshold just above the noise level in the phrenic neurograms (see Fig. 4 2). The left (IL) event train was then cross correlated with the right (CL) reference train using 0.2 ms bin widths using Spike2 software (Cambridge Electronic Design Limited, Cambridge, England). Correlogram baseline and peaks were discerned using the method described by Davies et al. (1985) A brief overview of the cross correlation technique is provided below. Data are presented as the mean SEM Overview of the Cross Correlation T echnique Detailed critiques and descriptions of the relevant methodologies have been published (Hamm et al., 2001; Kirkwood, 1979; Peever and Duffin, 2001) Cross correlation, as used in the present analysis is employed to examine the temporal

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91 relationship between two neural signals. In the context of C2HS, the relevant sig nals are the IL and CL phrenic neurograms. Using CL output as the reference signal, spikes recorded in the IL neurogram are correlated with CL spikes. This analysis yields a cross correlation histogram (correlogram) which presents the spike occurrences i n the IL neurogram (ordinate) relative to the time of occurrence of spikes in the CL neurogram (Kirkwood, 1979) (abscissa; Fig. 4 2). The presence of a peak in the resulting correlogram indicates that the probability of IL and CL spikes occurring simultaneously or sequentially (i.e. time locked, but delayed) is greater than should occ ur by chance (Kirkwood, 1979) A correlogram peak which is centered at zero on t he ordinate (Fig. 2) indicates a high probability of simultaneous bursting (spikes) from IL and CL PhrMNs, and a relatively narrow peak suggests a common, monosynaptic input to these cell populations (Duffin and van Alphen, 1995) For instance, a single ventral respirato ry column (VRC) inspiratory neuron which anatomically branches to innervate PhrMNs on both the IL and CL side of the spinal cord could produce just such a correlogram peak (Duffin and van Alphen, 1995) In contrast, a correlogram peak that is offset (i.e. to shifted to the right of zero on the ordinate) is consistent with delayed activation of one cell population (i.e. IL vs. CL PhrMNs). In addition, Kirkwood and colleagues (Vaughan and Kirkwood, 1997) suggest that relatively broad correlogram peaks are consistent with acti ons of common di or oligosynapt ic inputs during breathing. Results All C2HS lesions were confirmed histologically to ex tend to the spinal midline (e.g. Fig. 4 1 ). All animals displayed rhythmic inspiratory bursting in both IL and CL phrenic nerves (Fig. 4 2A). T he apneic threshold and mean arterial pressure were all similar between gr oups (all p>0.05 ; Table 4 1 ). Correlog rams computed from IL and CL

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92 phrenic neurograms in uninjured rats (n=5) displayed peaks with a mean half width of 1.080.14 ms (Fig. 4 2Ci, Cii). Correlogram peaks occurred at zero in 3 rats but showed a slight positive lag in the remaining 2 rats (0.1 and 0.6 ms). No discernable peaks were observed in correlograms from 2 wk post C2HS rats (n=5) (Fig. 4 2Ciii, Civ), despite comparable baseline counts in the histograms (and hence sensitivity for detection of peaks). In contrast, correlogram peaks were obser ved in 6 of 12 rats studied at 12 weeks post C2HS. The half widths of these peaks were similar to values in uninjured rats (1.050.11 ms). However, correlogram peaks observed at 12 wks post C2HS were shifted away from zero by the following lags, 0.3, 0.9, 1.1, 1.3, 1.3, 1.7 (mean = 1.10.19; p=0.003 vs. uninjured, Mann Whitney test). Five of these were shifted to the right (e.g. Fig. 4 2Cv), but one was shifted to the left (lag 0.9, Fig. 2Cvi). Moreover the example with the smallest shift, was a peak whic h had a k value (Sears and Stagg, 1976) of 1.77, and therefore demonstrated synchrony which was at least 10 times stronger than any of the normal examples (k between 1.009 and 1.068) or the other C2HS animals (k between 1.015 and 1.060), as illustrated in Fig. 4 2C. Discussion Cross correlation of phrenic signals in spinal intact rats reveals common activation of PhrMNs. Duffin and van Alphen (1995) computed cross correlation between the left and right phrenic nerves in anesth etized, paralyzed and mechanically ventilated adult female Sprague Dawley rats and showed robust central broad peaks in the resulting correlograms (i.e. peaks centered at time zero ) (Duffin and van Alphen, 1995; Peever and Duffin, 2001) The presence of these peaks indicates that a subset of IL and CL PhrMNs receive a common synaptic input (note: IL and CL are arbitrary terms in spinal intact rats) Subsequently, Peever and Duffin (2001) showed that the common

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93 synaptic in put is present only in adult rats. N o central peaks were observed in cross correlograms between right and left phrenic nerves in the neonatal in vitro brainstem spinal cord preparation, suggestin g that a common premotor neuron population does not excite th em. Midline tran section of the medulla abolishes phrenic nerve discharge in neonates, presumably because the ipsilateral bulbospinal pathway is not active However, s imilar to earlier results the cross correlograms between left and right phrenic nerve dis charges i n adult rats displayed robust central peaks. A common input could, in theory, reflect brainstem synchrony (e.g. precise synchronization of IL and CL medullary premotor neurons) or simultaneous excitation of IL and CL PhrMNs by shared premotor neu rons. This latter possibility could reflect simultaneous IL and CL PhrMN activation via collaterals that cross the spinal midline (Moreno et al., 1992) Bilateral PhrMN synchrony probably does not reflect activation of dendrites which cross the midline because such processes are rare in adult rat s (Prakash et al., 2000) The most likely explanation for IL and CL PhrMN synchrony in spinal intact rats is activation of medullary premotor neurons with two descending projections: one which crosses the midline in the medulla and another which descends caudally without crossing (Duffin and Li, 2006) Indeed, Peevet et al. (1998) have demonstrated that a midline transection of the adult rat brainstem results i n independent rhythmic activities of left and right phrenic nerves with no evidence of bilateral respiratory synchronization Clear IL phrenic bursting was observed at 2 wks post C2HS as previously reported (Fuller et al., 2008) However, cross correlation of IL and CL phrenic neurograms did not yield correlogram peaks. One interpretation of this apparent lack of

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94 synchrony is that distinct medullary populations are responsible for activation of PhrMNs on the IL vs. CL sides of th assessed at 2 wks post C2HS, may innervate only IL PhrMNs with no collateral projection to CL cells. It must be mentioned, however, that precisely equivalent populations of motoneurons may not be sam pled at the different time points, either because of different physiological recruitment or because of the somewhat arbitrary levels chosen for event discrimination. Motoneurons of different sizes may not be equally synchronized. In any case, the absence o f a correlogram peak at 2 wks post C2HS does not exclude a potential contribution of cervical interneurons to activation of IL PhrMNs. However, if interneurons are part of the CPP circuit at this time point they are probably not innervated by medullary ce lls also projecting to CL PhrMNs. A common synaptic input to IL and CL PhrMNs was detected in 50% of rats at 12 wks post C2HS as shown by discernable correlogram peaks (Fig. 4 2). Since correlogram peaks were not observed at 2 wks post injury, this findi ng may reflect time dependent plasticity within the phrenic motor circuitry. For example, sprouting of descending CL fibers across the spinal midline could result in progressive recovery and common innervation of IL and CL PhrMNs. Correlogram peaks prese nt at 12 wks post injury which showed a lag or rightward shift from zero are consistent with delayed onset of IL PhrMNs. One or more mechanisms could contribute to these effects. First, for the peaks with a rightward shift, slower conducting axons may pla y a larger role in the transmission of excitatory drive to the IL vs. CL side. Second, IL PhrMNs may be less excitable after C2HS. However, Mantilla and Sieck (2003) reported a reduction in PhrMN soma size after C2HS, a finding which argues against a reduction in overall

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95 excitability. Another possibility, however, is that the peak lag reflects a more complex (polysynaptic) circuit (Vaughan and Kirkwood, 1997) controlling IL PhrMNs in at le ast a subset of rats with chronic C2HS. In addition, the appearance of a peak to the left of zero indicates delayed activation of PhrMNs on the right (CL) with respect to those on the left (IL), possibly reflecting an input which originates on the left. Th e input concerned would most likely be non respiratory, e.g. intrinsic spinal cord interneurons tonically firing. This would also be the most obvious hypothesis for the single example with very strong synchrony (i.e. high k value). Evidence for the release of tonic interneuron activity acting to support the respiratory phased discharges of inspiratory thoracic motoneuron s was provided by Kirkwood et al (1984). Overall the present results suggest a heterogeneous group of inputs becoming active to support th e recovered IL PhrMN activity following C2HS. Such a view is also consistent with the concept that polysynaptic circuit via the interneurons may affect spontaneous recovery in the chronically injured spinal cord. Lipski et al. (1993) reported first anatom ical evidence for inspiratory neurons in the upper cervical spinal cord of adult rat. Although majority of axons in their study projected towards intercostals motoneurons, some collaterals were seen directed towards PhrMNs. Further anatomical evidence was provided in a study by Dobbins and Feldman (1994) which used retrograde pseudorabies virus (PRV) tracing and reve al ed interneurons in the Rexed laminae VII and X at the second cervical segment and laminae VII and IX at the level of the PhrMNs. However, the se authors were not able to conclusively identify these cells are pre phrenic second order interneurons. R ecent data from our lab (Lane et al., 2008a) using u nilateral labeling of the PhrMN pool with

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96 pseudorabies virus delivered to the hemidiaphragm demonstrated a substantial number of second order, bilaterally distributed cervical interneurons predominantly in the dorsal horn and around the central canal. In addition, c ombined anterograde (biotin dextran amine (BDA) injection in the VRC) and retrograde (using PRV delivery to ipsilateral hemidiaphragm) tracing revealed BDA labeled VRC projections to the soma and dendrites of PRV labeled, pre phrenic interneurons around the central canal suggesting that some propriospinal relays exist between medullary neurons and the phrenic nucleus. A direct test of this hypothesis must await more detailed analyses of cervical interneuron behavio r, both neuroanatomical and neurophysiological, after C2HS. Summary C2HS induces a persistent alteration in respiratory control as shown by neurophysiological outcomes The spontaneous respiratory recovery after C2HS reflects time dependent plasticity within the p hrenic motor circuitry The delayed synchronization of IL and CL phrenic bursting post injury raise the possibility that recovery of IL phrenic output may involve a population of premotor cervical spinal interneurons. Further, these data suggest that the n eural circuitry underlying IL PhrMN recovery may be more complex tha n previously envisioned Table 4 1 Apneic threshold and m ean arterial blood pressure (MAP) in control, 2 week post injury (2wk) and 12 week post injury (12wk) group Control 2wk 12wk A pneic threshold 39.1 2.3 39.7 1.2 40.1 0.6 MAP (mmHg) 97.9 6.2 101.1 8.2 98 3.8

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97 Figure 4 1 Representative histological secti ons of the cervical spinal cord following hemisection Panel A presents both longitudinal and transverse sec tions of a anatomically complete C2HS injury. Panel B provides longitudinal and transverse section s of an incomplete C2H S with ventromedial tissue sparing. The arrow in panel B indicates the area of midline tissue sparing. The vibratome) are labeled with antibodies to pseudo rabies virus (PRV; as part of a separate study); the transverse vibratome) are stained with luxol fast blue and cresyl voilet. Scale bars are 1mm.

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98 Figure 4 2 Examples of phrenic neurograms and correlograms. Panel A presents phrenic neurograms recorded both ipsilateral and contralateral to C2HS lesion at 2 (middle) and12 (bottom) wks post injury. Bilateral phrenic bursting in an uninjured rat is shown in th e top traces of Panel A. Time and amplitude scaling are identical in each trace; phrenic signals were amplified 1000x. Panel B shows an expanded time scale of the phrenic burst area indicated by the gray line in panel A. In addition, the event markers g enerated via spike2 software to enable cross correlation (see methods) are presented immediately above or below the corresponding neurogram signal. Panel C shows correlograms generated via cross correlation of phrenic event markers (see methods). The num ber of counts is shown on the Y axes. In panel C, the top correlograms (i.e. Ci, Ciii and Cv) were generated from the complete record (i.e. ~ 3 hr of data) associated with the phrenic data depicted in panel A. The bottom correlograms in panel C (i.e. Cii Civ, Cvi) are provided as additional representative examples. While correlogram peaks were always observed in uninjured rats (Ci, Cii), they were never observed at 2 wks post C2HS (Ciii, Civ) and occurred in 50% of rats (6/12) at 12 wks post C2HMx (Cv, Cvi). The arrow in panel Cvi indicates the presence of a peak occurring with a negative lag (see results).

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99 CHAPTER 5 TRANSPLANTATION OF POST NATAL DERIVED NEURAL PRECURSOR CELLS AFTER HIGH CERVICAL HEMISECTION Mo st spinal cord injuries (SCI) occur at the cervical level, accounting for nearly half of all SCI cases. High cervical SCI interrupts axonal conduction between respiratory pre motoneurons in the brainstem and PhrMNs in the cervical spinal cord. This results in the loss of descending rhythmic drive a nd paralysis of the respiratory muscles usually requiring long term mechanical ventilatory support (Goshgarian, 2009; Zimmer et al., 2008) Respiratory complications are the leading cause of morbidity and death b oth in the short and long term after SCI (NSCIS, 2010) T herefore recovery of respiratory f unction is a priority among individuals with cervical spinal cord injury Injury to the spinal cord unleashes a cascade of damaging cellular events that result in permanent motor and/or sensory deficiency below the level of injury (Kakulas, 1999; Tator, 1995) Neu rological dysfunction results fro m the loss of cells within the CNS due to primary mechanical impact as well as from secondary damage which involves complex biological changes such as edema, inflammation, ischemia, reperfusion and lipid peroxidation (Dusart and Schwab, 199 4; Tator and Fehlings, 1991) Concomitant with these processes that result in cell loss gliosis and cyst formation at the lesion site may also hinder axonal reconnection and functional recovery (Reier, 2004) One potential neural repair strategy for promoting motor recovery after SCI is transplantation of stem cells These cells are particularly attractive due to their potential to form myeli n, promote and guide axonal growth, and bridge the site of injury. In addition, stem cells secrete trophic factors, which may have neuroprotective effects and/or promote plasticity in the spared spinal cord. In fact, t he adult CNS harbors a

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100 population of e ndogenous stem cells that remain mitotically active through the lifespan, generating less committed progenitor/precursor cells and their differentiated progeny. This cell population is likely responsible for normal turnover of cells (Eriksson et al., 1998; Temple and Alvarez Buylla, 1999) and is collectively referred to as neural precursor cells (NPCs) NPCs are multipotent and retain the capacity to generate neurons, oligodendroglia and astroglia. However, the pro liferative capacity of endogenous NPCs is too limited to support significant self repair after SCI. Thus exogenous NPCs have been explored as a candidate for cell transplantation therapy NPCs are predominantly found in the periventricular region ( in the s ub ventricular zone, SVZ) and in the subgranular zone (SGZ) of the dentate gyrus in the hippocampus (Kuhn et al., 1996; Reynolds and Weiss, 1992) Several reports have also identified NPCs in the white matter of th e adult brain and spinal cord of various species including humans (Alvarez Buylla and Lois, 1995; Clarke and Frisen, 2001; Reynolds and Weiss, 1992) They support neurogenesis within restricted areas throughout adu lthood, can undergo extensive in vitro expansion and, therefore, have been proposed as a renewable source of neural precursors for transplantation after SCI (Horner and Gage, 2000; Teng et al., 2002) Adult NPCs a void the potential ethical issues involving embryonic stem (ES) or fetal cells for human clinical studies. Therefore, these cells have become an important focus in pre clinical research over the past decade and are regarded a viable therapeutic alternative to ES or fetal cells. Several studies have investigated trans plantation of these cells into the spinal cord with variable differentiation, migration and functional outcome For example, Karimi Abdolrezaee et al. (2 006c) transplanted

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101 adult mouse NPC grown as neurospheres into adult female rat spinal cords after a compression SCI (T8) and showed robust migration and differentiation into oligodendrocytes after 6 wks In comparison, Cao et al. (2001 ) who also transp lanted adult NPCs into a dult female rat spinal cords after thoracic contusion injury (T8) found that these cells mainly differentiated into astrocytes with no o ligodendrocyte differentiation after 8 wks Therefore, even though the development of NPC based therapies for the treatment of SCI have made signifi cant progress in the laboratory, critical limitation s remain before they are realized as reliable and effective approaches in the clinic. The m ost pressing translational issues include whether transplant ed cells remain at the site of injection or migrate elsewhere, whether they survive, and their mode of benefi t (if any). Most studies using adult/postnatal deri ved NPCs have been done in thoracic SCI models. On the contrary, only a handful of studies have investigated the viability of adult/postnatal derived NPC/stem cell transplantation after cervical SCI (Schaal et al., 2007; Vroemen et al., 2003) Furthermore t he effect of NPC transplantation on respiratory reco very after cervical SCI has not been investigated In this study, w e investigated the ability of post natal SVZ derived NPC transplantation to survive, migrate, differentiate and improve respiratory outcomes following hemisection lesions of the spinal cord above the level of the phrenic motoneuron pool ( i.e. C2HS) in a rat model Functional reco very of breathing was assessed using plethysmography and recording of phrenic nerve activity To our knowledge, t his is the first study to report respiratory recover y after post natal NPC transplantation into cervical spinal cord

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102 Materials and Methods Animals Experiments were conducted using adult female Sprague Dawley rats ( Harlan Inc., Indianapoli s, IN, USA) and neonatal Sprague Dawley rat pups engineered to express green fluorescent protein (EGFP) ( a kind gift from Dr. Brandi Ormerod ). All procedures were approved by the University of Florida Institutional Animal Care and Use Committee. C ell c ulture NPCs were derived from EGFP expressing transgenic neonatal Sprague Dawley rats (postnatal day 4; in house breeding ). Animals were sacrificed by rapid decapitation, and b rains were immediately removed The subventricular zone containing tissue of the forebrain was dissected, incubated briefly in trypsin, and dissociated in to a single cell slurry with a series of decreasing diameter fire polished glass pipettes. The slurry was plated overnight in growth media ( Rat Neuroc ult, Ste mcell Technologies Inc., Canada) Rat Proliferation Supplement, basic fibroblast growth factor (10 ng/ml, bFGF], and epidermal grow th factor [ 10ng/ml, EGF]). To isolate neurosphere forming cells (NFCs) after 24 hrs the slurry was aspirated, pelleted by centrifugation, and incubated in trypsin for 2 minutes. Cells were gently triturated, washed, and res uspended. NFC s were then plated in nonadherent flasks at clonal density (10,000 cells/cm 2 ) in growth medium. Cells were amplified as neurospheres in growth medium for two to three rounds of passage In vitro Immunocytochemistry Fully formed NS were selecte d by a hand held pipette under a phase contrast microscop e, transferred to glass coverslips coated with poly L ornithine (Sigma #P3655,

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103 10 g/ml), and maintained in growth media without the growth factor s bFGF and EGF. NS were allowed to attach and differ entiate for 2 3 days. Coverslips were then fixed for 1 hour with 4% paraformaldehyde and processed for immuno labeling to assess the expression of a proliferation marker and also of neuronal and astrocytic lineage markers. Cells plated on poly L ornithine coated coverslips were fixed i n 4% paraformaldehyde for 30 min at room temperature (RT) a nd blocked for 30 min at RT with PBS containing 0.01% Triton X 100 and 10% FBS). Primary antibody (rabbit anti III tubulin 1:200; rabbit anti glial fibrillary acidi c protein (GFAP) 1:2000; rabbit anti Ki67, 1:1000) was applied overnight at 4 C. Coverslips were washed 2 x 10 minutes in wash buffer (PBS, 0.01% Triton X 100) and incubated with fluorescence conjugated secondary antibody (goat anti mouse, goat anti rabbi t 1:500 each ) for 3 hours at room temperature. Slips were washed 2 x 10 minutes in wash buffer, mounted on positively charged glass slides (Fisherbrand Superfrost/Plus, Fisher Scientific, Pittsburg, PA) and cover slipped in Vectashield containing DAPI co unterstain (Vector Laboratories, Burlingame, CA). Fluorescence micrographs were obtained with a Leica DM5000B epifluorescence microscope equipped with a color Spot cooled CCD digital camera. Image analysis was conducted using Leica Application Suite Ver sion 3.50 software with post analysis using Microsoft Photoshop CS2 Version 9.0 software. To quantitate cells, a predetermined five field grid was established, and the microscope stage adjusted to those fields prior to analysis. All NS within these prede termined fields were analyzed. Total cells (DAPI) and then total cells positive for Ki67, III tubulin or GFAP were counted. Data is expressed as percent positivity for the marker of interest, standard deviation.

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104 Spinal Cord Injury Adult Animals were randomly divided into two groups 1) NPC transplantation ( NPCtxpt ; n=8 ; weight = 2693 g); 2 ) Control i.e. C2HS only or C2HS +growth medium transplantation (n=8 ; weight = 2603 g ). As no differences were seen in the C2HS alone or C2HS+growth medium transpl antation rats, they were combined as one group i.e. control. Animals were anesthetized with injections of xylazine (10 mg/kg s.q. ; Phoenix Pharmaceutical, Inc., St. Joseph, MO ) and ketamine (12 0 mg/kg i.p. ; Fort Dodge Animal Health, Fort Dodge, IA ). An inc ision was made extending from the base of the skull to the third cervical segment, and a subsequent laminectomy was made at the second cervical segment. A dural incision was made and lateral hemisection was performed on the left side of the spinal cord wit h a microscalpel and gentle aspiration using a pulled micropipette. The dura was closed with interrupted 10 0 sutures and durafilm was a placed over it. The overlying muscles were closed with polyglycolic acid sutures (4 0 vicryl) and the skin closed with 9 mm stainless steel wound clips. Following surgery, an analgesic (buprenorphine, 0.03 mg/kg, s.q.) was given every 10 12 h for 2 days. Lactated ringer solution (12 ml/day, s.q.) was provided for 1 3 days, until adequate volitional drinking resumed. Cell T ransplantation Seven days post injury rats were re anestheti z ed as described above For NPC transplantation, the rats were injected with a cell suspension of postnatal NPCs. To prepare the cell suspension, neurosphere s were mechanically dissociated into s ingle cells. Cell viability was assessed by trypan blue. After gaining surgical access to the injured region, a total volume of 5l cell suspension, containing approximately 500,000 live cel ls diluted in growth medium was injected 1mm caudal to the lesion site in the

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105 dorsal spinal cord, next to the midline. T ransplants were made using a 10 l Hamilton glass syringe, connected to a 31 gauge needle and attached to a micromanipulator. Sham animals received injections of neurocult growth medium only. The animals received a daily subcutaneous injection of cyclosporine A (10 mg/kg, Sandimmune;Novartis, East Hanover, NJ) starting 2 d ays before transplantation and continuing until the end of the experiments. Plethysmography Ventilation was measured in awake unrestrai ned sham and transplanted rats using a commercially available whole body barometric plethysmograph y system (Buxco Inc., Wilmington, NC, USA) Calibration was accomplished by injecting known volumes of air using a 5 ml syringe into the recording chamber. Th e chamber pressure, temperature and humidity, rectal temperature of the rat, and atmospheric pressure were used in the equation described by Drorbaugh and Fenn (1955) to calculate respiratory volumes including tidal volume (VT, ml/breath) and minute ventil ation (V E ml/min). Overall breathing frequency ) was calculated from the airflow traces. During the experiments, gas mixtures flowed through the chamber at a rate of 2 L/min to enable control of inspired gases. Baseline recordings lasted 1 1. 5 h, and were made while the chamber was flushed with 21% O 2 (balance N 2 ) (i.e. eucapnic normoxia). Rats were then exposed to hypoxia (10% O 2 balance N 2 ) and then hypercapnia (7% CO 2 21% O 2 and balance N 2 ) for 5 mins each min with a 5 min normoxic inter val between the two exposures Phrenic Nerve Recordings Rats were anesthetized with isoflurane (5% in 100% O 2 ) in a closed chamber and then transferred to a nose cone (2 3% isoflurane in 50% O 2 balance N 2 ). The

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106 trachea was cannulated with PE 240 tubing an d rats were mechanically ventilated for the remainder of the experiment. The lungs were briefly hyperinflated (2 3 seconds) approximately once per hour to minimize atelectasis. The tracheal pressure was monitored with a pressure transducer (Statham P 10EZ pressure transducer, CP122 AC/DC strain gage amplifier, Grass Instruments, West Warwick, RI, USA) connected to the tracheal cannula. A catheter (PE 50) was inserted into the femoral vein, and rats were converted from isoflurane to urethane anesthesia (1.6 g/kg, i.v.; 0.12 g/ml distilled water). The adequacy of anesthesia was monitored during this period by observing limb withdrawal response to toe pinch and supplemental urethane was given if indicated (0.3 g/kg, i.v.). A femoral arterial catheter (PE 50) wa s inserted to measure blood pressure (Statham P 10EZ pressure transducer, CP122 AC/DC strain gage amplifier, Grass Instruments, West Warwick, RI, USA) and to periodically withdraw blood samples (see protocol). Rats were bilaterally vagotomized to prevent entrainment of phrenic motor output with the ventilator and paralyzed with pancuronium bromide (2.5 mg/kg, i.v.) to eliminate spontaneous breathing efforts. Following paralysis, the adequacy of anesthesia was monitored by observing blood pressure and phren ic nerve response to toe pinch. A slow infusion of lactated Ringer's solution and sodium bicarbonate (3:1, 1.5ml/h) was maintained to promote acid base balance (Baker Herman et al., 2009; MacFarlane and Mitchell, 20 09) Arterial partial pressures of O 2 (PaO 2 ) and CO 2 (PaCO 2 ) as well as pH were determined from 0.2 ml arterial blood samples using an i Stat blood gas analyzer (Heska, Fort Collins, CO, USA). Blood gas and pH values were corrected to the rectal temperatu re measured at the time of the blood sample. The end tidal CO 2 partial

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107 pressure (PET CO2 ) was measured throughout the protocol using a rapidly responding mainstream CO 2 analyzer positioned a few cm from the tracheostomy tube on the expired line of the venti lator circuit (Capnogard CO 2 monitor, Novametrix Medical Systems, Wallingford, CT, USA). Rectal temperature was maintained within 371 C (see results) using a rectal thermistor connected to a servo controlled heating pad (model TC 1000, CWE Inc., Ardmore, PA, USA). Both phrenic nerves were isolated within the caudal neck region medial to the brachial plexus, using a ventral surgical approach. Electrical activity was recorded using silver wire electrodes with a monopolar configuration, amplified (1000x) a nd 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 Power 1401 data acquisition interface and recorded on a PC using Spike2 software (Cambridge Electronic Design Limited, Cambridge, England). After an adequate plane of anesthesia was established, the PETCO 2 was ma intained at 402 mmHg for 10 min. The PETCO 2 threshold for inspiratory phrenic activity was then determined by gradually increasing the ventilator pump rate until inspiratory bursting ceased in both phrenic nerves. After 1 min, the ventilator rate was gr adually decreased until inspiratory bursting resumed in the contralateral phrenic nerve. The PETCO 2 was then maintained 2 mmHg above the value associated with the onset of contralateral inspiratory bursting by adjusting the ventilator rate. After a 10 min baseline period, an arterial blood sample was drawn. Rats were t hen exposed to a 5

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108 min period of hypoxia (FIO 2 = 0.12 0.14), and a blood sample was obtained d uring the final minute. After 10 min rats were exposed to a 5 min bout of hypercapnia (PETCO 2 80 mmHg) achieved by raising the inspired CO 2 concentration. Ten minutes after the hypercapnic exposure, a brief asphyxic challenge was performed by stopping the ventilator for 10 20 s. Spinal Cord Histology After the neurophysiology experime nts, adequat e anesthesia was confirmed and rats were euthanized by systemic perfusion with heparinized saline followed by 4% paraformaldehyde. The cervical spinal cord was then removed, blocked, and sectioned (4 vibratome For fluorescence immunostaining, th e following antibodies were used: chicken anti green fluorescent protein ( a nti GF P, 1:10 00; Aves Lab s OR ) for transplanted neuronal precursor cells mouse anti o ligodendrocyte O4 ( anti O4; 1:1 00; MAB345, Millipore MA) for oligodendrocytes and mouse anti n euronal nuclei ( anti NeuN ; 1:500; MAB377, Millipore MA ) for neurons Sections were blocked with 10% (v/v) norma l goat s erum in 0.1 M PBS for 60 min, incubated with primary antibody overnight at 4C, washed three times and then incubated with goat anti chicken or anti mouse secondary antibody conjugated to Alexa Fluor 488 or 568 (1:500, Invitrogen CA ) for 2 h r Tissue sections were washed three times with PBS, slide mounted and coverslipped with mounting medium containing DAPI to counterstain the nuclei Immunofluorescent images were take n using a n Olympus 1X81 DSN spinning disk confocal microscope equipped with a Hamamatsu CCD digital camera Image analysis was done by 3i slidebook software and post analysis was done in photoshop. For brightfield micros copy, spinal cord sections were rinsed with PBS and incubated overnight with chicken anti green fl uorescent protein (anti GFP, 1:40, 000; Aves Labs,

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109 OR). Section w ere washed three times with PBS and incubated with goat anti chicken DSB X biotin secondary a ntibody (1: 2 00, Invitrogen, CA) for 2hr Sections were then followed by incubation in ABC reagent (Vectastain ABC kit, Vector laboratories, Burlingame, CA, USA) for 2hr and then developed in freshly prepared diaminobenzidine (DAB) peroxidase substrate sol ution to detect GFP (conjugated with biotin, brown). Tissues were also counter stained with cresyl violet for morphology and then examined with light microscopy Some portion of bulbospinal projections to phrenic motor neurons travel in the medial portions of the ventral funiculi. Accordingly, hemisections were considered anatomically complete only if there was a complete absence of apparently healthy white matter in the ipsilateral spinal cord at the lesion epicenter. One animal did not meet this criterion and was excluded Data Analyse s Plethysmography data were analyzed using custom software (Buxco Inc.). Data were averaged over a 10 min period during baseline, and over the last 1 min of the hypoxic and hypercapnic challenge. Respiratory volume data were expressed per 100 g body mass. A two way repeated measures analyses of variance (ANOVA) followed by the Student Neuman Keuls post hoc test was used to compare ventilation data before and after C2HS: factor 1 = time (4 or 8 wk), factor 2 = treatment group ( transplant or control). The analysi s of phrenic neurograms was done with Spike 2 software. The integrated phrenic neurogram was used to calculate phrenic amplitude ( Phr) and burst frequency, averaged over 1 min fo r each recorded data point and expressed i n mV (i.e.

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110 arbitrary units, a.u.) and bursts/min, respectively, and were also normalized to values recorded during baseline and in the contralateral phrenic recordings Statistical tests were done using SigmaStat v. 2.03 software. Statistical differences were determined using a two way repeated measures analysis of variance (RM ANOVA). The Student Neuman Keuls test was used for post hoc analyses. Differences were considered EM. Results In vitro Characterization of NPCs In order to allow for progeny cells to exit the cell cycle and proceed through terminal differentiation pathways n eurospheres were plated in the absence of mitogenic growth factor for 3 4 days. The resulti ng cells exhibited a low proliferative profile, as assessed by Ki67 which is a nuclear antigen associated with cell division (2.5 2.2% positivity Figure 5 1 ). With regard to terminal differentiation, a low percent of these cells labeled positively for GFAP which is an astrocyte specific intermediate filament protein (9.65 4.5% positivity Figure 5 1 ). A larger percent of these cells were positive for III tubulin which is a neuron specific microtubule protein (32.2 13.2% Figure 5 1 ). Survival, Mi gration and Differentiation of NPCs after Transplantation Morphological analysis was performed on a ll spinal cords at 8 wk after transplantation. We did not observe any signs of tumorgenesis macroscopically or microscopically along the length of the transp lanted spinal cords. NPC s were found predominantly within the ipsilateral ( IL ) spinal cord in dorsomedial, ventromedial and ventrolateral wh ite matter (Figure 5 2 and 5 3 ). Cells are found up to 3 mm away from the lesion site in the rostral caudal directi on, confirming their migratory capacity (Figure

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111 5 3) NPC s survive d readily following transplantation into the sub acutely injured spinal cord as indicated by the widespread detection of GFP labelled cells (Figure 5 4) Detection of NPC decreases caudally an d rostrally to the lesion site. I n the contralateral (CL) spinal cord transplanted cells were usually present near the midline suggesting that their transverse migration is restricted The extent of gliosis at the transplant spinal cord interface varied among animals. Morphological analysis of tissue sections showed that t ransplanted cells differentiated primarily into glial cells. We tested for the presence of oligodendrocytes (O4) and astrocytes (GFAP) however these markers did not reveal conclusive r esults. We therefore cannot comment on the specific phenot ypic identity of the differentiated cells We observed very few G FP positive cells expressing NeuN (neural marker). Ventilation in T ransplant vs. Control R ats Representative airflow traces recorded using plethysmography are provided in Fig. 5 5 During b aseline breathing frequency decreased sign ificantly from 4 to 8 wks (P < 0.05 Figure 5 6A ) however, it was not different between the two experimental groups at either time point There was a tende ncy for higher baseline VT and V E expressed both as raw values (Figure 5 6B and D) as well as normalized for weight (Figure 5 6C and E), in the transplant group but these values did not reach statistical significance. During hypoxia, there was a strong te ndency (P = 0.051) for a blunted frequency response in the transplanted group at 8 wks. Similar to baseline, VT tended to be higher in transplanted vs. control rats (Figure 5 7B, C and D), however statistical significance was reached only in raw values at 8 wks (Figure 5 7C). V E data did not differ between the two groups during hypoxia. A trend for higher VT was also present in transplanted rats during hypercapnia challenge (Figure 5 8B, C, D) but statistical signific ance was

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112 reached only in raw values at 8 wks (Figure 5 8C) N o differences were observed in frequency (Figure 5 8A) and V E (Figure 5 8E and F) between the two groups P values obtained using 2 way RM ANOVA for respiratory parameters assessed by plethysmography are reported in Table 5 2 Phrenic Motor Output in Transplant vs. Control Rats A representative example of neurophysiological recordings of phrenic nerve activity in anesthetized rats is provided in Figure 5 9 Consistent with prior reports (Doperals ki et al., 2008; Fuller et al., 2008) hypoxia caused a reduction in MAP (Table 5 1 ) Blood gases were within physiologically normal ranges, and Pa O2 and pH were not significantly different between groups Pa CO2 was reduced during hypoxia and hypercapnia in the treatment group (P < 0.05, Table 5 1) Chemical respiratory challenge was provided by hypoxia, hyperc apnia, and asphyxia (induced by transiently stopping the mechanical ventilator). The overall phrenic burst frequency was similar between the groups ; however, it was significantly h igher in first 30 sec of hypoxic response vs. other condi tions in both gr oups (Figure 5 10 A) approaches Analyses of IL phrenic burst amplitu de (volts) revealed a significant interaction between condition (i.e. baseline, hypercapnia, hypoxia, and asphyxia) and treatment (i.e. transplant or control ) ( P < 0.00 1). Specifically, burst amplitude differences were not present during baseline but duri ng hypoxia hypercapnia and asphyxia transplant rats had substantially greater IL phrenic burst amplitude (all P < 0.02 Figure 5 10 C ). IL phrenic burst data expressed relative to CL values showed strong tendency for higher amplitude during hypoxia (P = 0.06) and hypercapnia (P = 0.067) but a significan t difference was seen only during asphyxia ( P <

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113 0.05, Figure 5 10 B). CL phrenic values were not significantly different between the two groups (P > 0.05, Figu re 5 10 D). When phrenic amplitude data was norma lized relative to baseline values, no significant difference was found between the two groups (Figure 5 10 E and F) A sphyx ia values within each group in the normalized data were significantly higher than the hy poxia and hypercapnia values. P values obtain ed using 2 way RM ANOVA for phrenic output parameters assessed by phrenic nerve neurophysiology are reported in Table 5 3 Discussion This study provides the first comprehensive description of the impact of post natal derived NPC transplantation on phrenic motor output and ventilation following chronic cervical SCI The se data demonstrate that NPCs are capable of s urvival, migration and differentiation after transplantation into the injured spinal cord and their presence improve s phrenic motor output espec ially during respiratory challenges at 8 wks post transplant Our results provide a proof of principle for feasibility and efficacy of the postnatal/ adult derived NPCs as a potential therapeutic intervention after SCI Migration of Transplanted NPC within the Spinal Cord This study provides important insights into the feasibility of adult NPC transplantation The observation that NPCs, transplanted in to the spinal cord caudal to the lesion were detected in the ipsilateral spinal cord 2 3mm rostral ly indica tes that engrafted NPCs underwent robust migration along the lesion perimeter T his is a very promising finding which suggests that these cells are not sealed off by the surrounding host spinal cord, in contrast to other cell types used for transplantation such as fibroblasts or Schwann cells (Grill et al., 1997; Weidner et al., 1999) Thus, adult NPC could have the capacity to build a continuum with the host tissue

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114 Previous studies have show n that exogenously deli vered NPCs migrate within CNS using navigational cues provided by inflammatory response a common characteristic of CNS injury (Carbajal et al., 2010) One component consid ered critical in this process is the infl ammatory chemoattractant stro mal cell derived factor 1 1 regulated after injury (Imitola et al., 2004) NP Cs express chemokine receptor 4 (CXCR4) the cognate receptor for SDF (Imitola et al., 2004) therefore exposure of NPCs to SDF crucial signaling cue responsible for cell migration seen in our study The primary source of increased SDF is the large number of reactive astrocytes within the injured regions (Patel et al., 2010) In our study, we observed relatively few transplanted cells migrated to the CL white matter therefore we speculate that reactive astrocytes were not present o n the CL spinal cord to express S DF An additional source of SDF be the blood vessels within the injured area a region where end othelial cells also up regulate SDF (Imitola et al., 2004; Miller et al., 2005) Nonetheles s, g uiding the cells to a n injury site provides promise to c ervical SCI patients since transplantation at the cervical region can be highly risky Therefore, transplantation can be done either in the safer areas caudal to the cervical region or via a less invasive route and the cells would still be expected to migrate to wards the injury sites. In vitro and In vivo differentiation of NPCs In vitro characterization of neurospheres (NS) suggest s that a low percentage of cells are still mitotic (i.e. dividin g into cells) which is consistent with plating the neurosphere progeny cells in a permissive environment for exiting the cell cycle and induce terminal differentiation. A yield of 32% neuronal cells is a favorable percent, as

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115 yields under standard cultur e conditions have been reported to range from 10 18% (Karimi Abdolrezaee et al., 2006b; Studer et al., 2000) These cells exhibited a surprisingly low yield of astrocytic cells, as others have reported an astrocyt ic yield as high as 60% (Karimi Abdolrezaee et al., 2006b) We tested for the presence of oligodendrocytes (CNPase, Olig1, O4), however these markers did not reveal conclusive results. We therefore cannot comment on the phenotypic identit y of the remaining ~48% cells. Overall, our expectation of modest neuronal and oligodendrocytic yield (20%) coupled with a high astrocytic yield (Karimi Abdolrezaee et al., 2006b) was not reflected in this data. In vivo immun o histochemistry and spinning disk confocal microscopy of tissue sections suggested that the majority of transplanted cells morphologically resemble glial lineage cells although we were not able to specify the astrocy tic or oligodendrocytic differentiation. One of the reasons for lack of labeling with the O4 oligodendrocyte marker could be due to the fact that O4 antigen is only expressed in intermediate or mature oligodendrocytes. Therefore, if the differentiated NPCs belonged to immature oligodendrocytes precursors this marker would not label them. Future analysis will seek to address this by using antibodies for markers expressed in early stage oligodendrocytes differentiation. Only a minority of transplanted cells were positive for neuronal marker, NeuN. These data are in agreement with other studies reporting adult NPCs differentiation after transplantation into the injured spinal cord (Cao et al., 2001; Karimi Abdolrezaee e t al., 2006a) Potential Neural M echanism s for Respiratory R ecovery The experimental model of cervical SCI used in this study ( i.e. high cervical hemisection from midline to lateral edge of the spinal cord) removes bulbospinal

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116 projections to the ipsilate ral phrenic motor pool and thus silences the ipsilateral hemidiaphragm. However, following chronic C2HS, IL phrenic motor output is gradually enhanced via endogenous mechanisms (Nantwi et al., 1999b) In particular inspiratory burstin g resumes in IL PhrMNs weeks to months post injury, even during conditions of quiet breathing (baseline). This phenomenon is known as the crossed phrenic phenomenon (CPP) (Goshgarian, 2003) In this study we showed that IL phrenic output was more pronounced in transplanted vs. control rats under carefully controlled conditions This could reflect an augmentation of CPP in the transplanted group, which might be responsible for an enhanced recovery of VT observed in transplant rats du ring hypoxia Determination of the exact mechanisms by which any cell type provides benefit after transplantation in to the injured spinal cord is challenging given the wide range of possible effects of such procedures T here are a variety of means by which NPC transplantation could have contributed to the respiratory recovery we observed in our experiments. One is the function of glial cells derived from transplanted cells. Oligodendrocytes derived from the NPCs could remyelinate spared fibers that had been demyelinated as a r esult of injury and restored connectivity. Following SCI, oligodendrocytes undergo apoptosis during Wallerian degeneration (Crowe et al., 1997) and result in demyelination of spared axons W e injected NPCs 7 days post injury and demonstrated presence of donor derived cell with glial morphology around the injury site. Therefore, these donor derived oligodendrocytes could have improved remyelinated and prevented the functional loss caused by de myelination.

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117 A lternatively, astrocytes derived from donor NPCs might have played an active role in neural repair. Fetal brain derived astrocytes have been shown to regulate neurite out growth (Garcia Abreu et al., 2000) and promote morphological development of neuronal cells in vitro (Blondel et al., 2000) Davies et al. (2006) demonstrated that transplantation of astrocytes derived from glial restricted precursors promote axon growth and improvement of locomotor function after acute transection injur ies of the adult rat spinal cord. Therefore, astrocytes can provide a neuroprotective environment that supports axon growth after SCI and have the ability to reduce atrophy of axotomized axons. There are other ways, in addition to local differentiation, by which the transplanted NPCs could contribute to respiratory recovery. These cell s can locally generate neu ro trophic factors that promote neuronal survival or axonal outgrowth Llado et al (2004) showed that neura l stem cells, placed adjacent to spinal cord sections, secrete glial cell line derived factor (GDNF) and nerve growth factor (NGF) C onditioned medium from NSCs cultures has also been shown to induce axonal outgrowth P re incubation of the conditioned medi um with GD NF blocking antibodies abolishes axonal outgrowth, suggesting that these cells secrete soluble factors that have trophic properties (Llado et al., 2004) Transplanted NPCs could also contribute to neurop rotection and phrenic output by participation in local immune modulation. A recent study transplanted adult NPCs in combination with T cell based v accination, a type of white blood cell that is of key importance to the immune system, after SCI in mice. The y demonstrated better tissue preservation, increased neurogenesis from endogenous precursors, and improved

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118 functional recovery in dual treated mice suggesting that the immune cells ( T cells and microglia) and the adult NPCs provide an infrastructure for ti ssue repair by modulating activity (Ziv et al., 2006) Summary These results serve as a proof of principle that neonatal SVZ derived NPC transplantation is a feasible and potentially viable option for treatment of cervical SCI due to their robust survival, migration, lack of tumor formation and improved phrenic output We have established a baseline dataset, which can be used to compare future combinatorial strategies that can have a synergistic effect for treating SCI

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119 Table 5 1 Mean arterial blood pressure (MAP), partial pressure of arterial carbon dioxide (PaCO 2 ), oxygen (PaO 2 ), and arterial pH during baseline, hypoxia and 6 min post hypoxia. *, different from baseline values, different from c orresponding control data point. Baseline Hypoxia Post hypoxia MAP (mmHg) NPCtxpt 97 8 58 8* 103 9 Control 98 4 70 7* 110 4 PaCO 2 (mmHg) NPCtxpt 44 1 42 1 45 1 Control 46 2 46 2 4 9 1 PaO 2 (mmHg) NPCtxpt 159 13 33 2* 11 2 5 Control 150 9 3 2 1 103 4 pH NPCtxpt 7.3 0.01 7.28 0.02 7.26 0.02 Control 7.3 0.0 1 7.2 9 0.0 1 7.26 0.0 1

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120 Table 5 2 P values obtained using two way repeated measures Analysis of V ariance (RM ANOVA) fo r respiratory parameters assessed by plethysmography. Treatment Time Interaction Baseline Frequency 0.741 0.004 0.702 VT 0.119 0.164 0.756 VT/100g 0.550 0.263 0.666 MV 0.092 0.706 0.828 MV/100g 0.616 0.624 0.990 Hypoxia Frequency 0.22 0.77 0.051 VT 0.03 0.109 0.583 VT/100g 0.195 0.196 0.536 VT/100g/baseline 0.238 0.906 0.735 MV 0.496 0.739 0.309 MV/100g 0.990 0.834 0.416 Hypercapnia Frequency 0.971 0.267 0.518 VT 0.022 0.708 0.822 VT/100g 0.189 0.586 0.692 VT /100g/baseline 0.429 0.165 0.824 MV 0.434 0.426 0.721 MV/100g 0.798 0.456 0.713

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121 Table 5 3 P values obtained using two way repeated measures Analysis of V ariance (RM ANOVA) for phrenic output parameters assessed by phrenic neurophysiology Trea tment Time Interaction Baseline Frequency 0.572 <0.001 0.902 CL Amp 0.565 <0.001 0.800 IL Amp 0.011 <0.001 0.001 IL Amp/CL 0.086 0.007 0.101 CL Amp/BL 0.565 <0.001 0.124 IL Amp/BL 0.691 <0.001 0.806 Figure 5 1 In vitro differentiation proliferation and differentiation of NPCs grown as free floating neurospheres (NS) NS were plated in absence of growth factors 2 3 days to promote terminal differentiation: Ki67, marker for proliferation; GFAP ( glial fibrillary acidic protein ), marker for astrocytes; and 3T ( III tubulin ), marker for neurons.

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122 Figure 5 2 NPCs injection site at 8 weeks post transplant. Top panels show p resence of DAPI and GFP labeling confirming healthy transplanted cells in the host tissue. Three dimensional rendering i n the bott om panels from image in the top right panel confirm DAPI and GFP labeling within the same cell.

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123 Figure 5 3 Serial DAB stained transverse sections through the spinal cord injury site demonstrate rostrocaudal migration after NPC transplantation. Scale bar, 500 m

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124 Figure 5 4 Transverse section immediately caudal to transplant site showing robust migration of DAB stained NPCs with IL white matter. Scale bar, 500 m

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125 Figure 5 5 Representative airflow traces recorded via plethysmography during quiet breathing (baseline) and respiratory challenge with hypoxia (10%O2, balance N2) and hypercapnia (7% CO 2 21% O 2 balance N 2 ) in a transplant rat.

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126 Figure 5 6 Change in baseline (21% O 2 balance N 2 ) frequency tidal volume (VT) and minute ventilation (MV) from 4 to 8 wks in transplant and control rats The statistical significance symbol denote s differences as follows: #, different than 4 wks

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127 Figure 5 7 Change in hypoxic ( 10 % O 2 balance N 2 ) frequency tidal volume (VT) and minute ventilation (MV) from 4 to 8 wks in tra The statistical significance symbol denote s differences as follows: *, different than control rat s.

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128 Figure 5 8 Change in hypercapnic (7% CO 2 21% O 2 balance N 2 ) frequency and tidal volume (VT) from 4 to 8 wks in tra and control rats The statistical significance symbol denote s differences as follows: *, different than control rat s.

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129 Figure 5 9 Representative phrenic neurograms recorded ipsilateral (IL) and contralateral (CL) to C2HS during baseli ne and is ocapnic hypoxia

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130 Figure 5 10 Impact of transplant on phrenic output after C2HS. Data were collected in Burst frequency is presented in panel A for the conditions of baseline, early hypoxia, lat e hypoxia and hypercapnia The raw IL and CL phrenic neurogram burst ampl itudes are presented in panels C and D respectively. IL phrenic amplitude data normalized to CL output is shown in panel B. Panels E and F represent IL and CL phrenic amplitude norma lized to baseline values. The statistical significance symbols denote differences within each condition as follows: *, different than corresponding controls corresponding hypercapnia values

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131 CHAPTER 6 CONCLUSION Overview The function of the central nervous system is to produce appropriate behavior. Behavior is largely determined by current and past experience This phenomenon is termed neuroplasticity and defined as a modification in neural control that changes the b ehavior and persis ts longer than the stimuli that evoke it The mammalian neural network which controls phrenic output and ventilation undergoes extensive neuro plasticity throughout life and has many different mechanisms. This doctoral dissertation provides insights into r espiratory neuroplasticity within the spinal cord following three separate stimuli: intermittent hypoxia, spinal cord injur y and stem cell transplantation; and considers their implications for understanding plasticity as wel l as for restoring respiratory f unctions lost due to injury Summary of Conclusions Effect of Phrenicotomy on Hypoxia i nduced Phrenic Long Term Facilitation Repeated hypoxic bouts trigger a form of respiratory plasticity that functions to strengthen the output of respiratory motoneurons This type of plasticity known as long term facilitation (LTF) serves to deepen breathing and improve lung ventilation. Hypoxia induced LTF is typically studied in ventilated, anesthetized rats and t o ensure that respiratory feedback does not interfere w ith LTF mechanisms, mechanical feedback from lung receptors is removed by cutting the vagus nerve (vagotomy) C hemical feedback is also tightly controlled by maintaining the levels of arterial O 2 and CO 2 throughout the experiment. LTF of PhrMNs is shown by changes in the phrenic nerve activity Nerve recordings are generally made from the proximal end of the cut

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132 nerves. However, cutting the phrenic nerve ( phrenicotomy PhrX) removes afferent inputs to the spinal cord, and also axotomized the PhrMNs We hyp othesized that b oth of these things can potentially change motoneuron excitability and impact LTF expression; therefore, we studied hypoxia induced LTF in anesthetized rats with and without intact phrenic nerves. We found that PhrX alone can trigger an LTF like phenomenon that lasted up to 60 min post PhrX We also found that hypoxia response and episodic hypoxia induced LTF was significantly higher in PhrX rats. We conclude therefore that although PhrX itself is not necessary for activating LTF, it sets u p certain preconditions that strengthen it s expression This study is the first to document the impact of cutting phrenic nerve on LTF. Future work is required to determine the exact mechanisms underlying this effect Effect of C2HS on Phrenic Motoneuron Synchrony Loss of respiratory function is arguably the most serious consequence of cervical SCI as respiratory failure is the leading cause for impaired quality of life and death in chronically injured patients. Therefore u nderstanding how cervical SCI alt ers the control of breathin g is important. In Aim 2, we investigated the neural circuitry underlying phrenic recovery following C2 spinal cord hemisection (C2HS) injury. Our rational was that if time dependent recovery of ipsilateral phrenic motoneuron (IL PhrMN) activity following chronic C2HS involves activation of propriospinal cervical interneurons, then measurements of synchrony between IL and contralateral (CL) PhrMN discharges would reveal features of the circuitry involved. Our hypothesis was that s ynchronization between IL and CL PhrMN bursting, studied using cross correlation analyses, would be delayed after chronic C2HS. Our primary finding was that IL PhrMN activity recovers in a time dependent manner after C2HS, and prolonged conduction time to IL (vs. CL)

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133 PhrMNs suggesting the possibility of polysynaptic inputs to IL PhrMNs after chronic C2HS. The observations made in this study reveal valuable insights into the temporal changes associated with incomplete cervical SCI Such fundamental informati on is a prerequisite for investigations involving novel treatments designed to restore function or facilitate self repair. Neural Precursor Cells as a Potential Candidate for Treatment of SCI In the final study our goal was to examine the feasibility of n eural precursor cells (NPC) derived from the post natal rat pups as a potential candidate for transplantation after C2HS injury. We hypothesized that post natal NPCs will survive, migrate and improve respiratory outcome after transplantation into the injur ed cervical spinal cord. Our results demonstrated that these cells can survive chronically post injury, robustly migrate in a rostro caudal manner, differentiate primarily along a glial lineage and improve phrenic output especially during respiratory chall enges. Th is study provides an important proof of principle demonstration that neonatal derived NPC s have the therapeutic promise for intervention after cervical SCI. We are optimistic that this work could be eventually applied to human patients, however se veral major hurdles must be addressed first. The se include a detailed understanding of the mechanisms by which NPCs improve function safety issues related to the risk of tumorigenesis by transplanted cells long term survival and phenotypic stability of s tem cell derived neurons or glial cells in the spinal cord. In addition, practical issues such as determining the best source for generating human NPCs as well as the optimal methods and sites for transplantation of cells need to be resolved Thus, the dev elopment of neural precursor stem cell based therapies for SCI is still in a very early phase and it is crucial that scientists and clinicians progress with great care.

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159 BIOGRAPHICAL SKETCH Milap jit Sandhu was born in Jalandhar India. He attended St. Francis School, Amritsar and Khalsa College, Amritsar for his high school and senior secondary education, respectively. degree in physical therapy from the Guru Nanak Dev University, Amritsar in 2003 Subsequently, he worked as a physical therapist at the Apollo Physiotherapy and Rehabilitation Center in Amritsar until 2004 He joined the doctoral program in rehabilitation science at the University of Florida in the fa ll of 2004. He graduated in December 2010 with a Ph.D. in rehabil itation science