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T Cell-Mediated Neuroprotection in the Injured Central Nervous System

Permanent Link: http://ufdc.ufl.edu/UFE0023719/00001

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Title: T Cell-Mediated Neuroprotection in the Injured Central Nervous System Gaining Insight with the Facial Nerve Axotomy Model
Physical Description: 1 online resource (86 p.)
Language: english
Creator: Ha, Grace
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

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

Notes

Abstract: Although T cells aid in maintaining homeostasis of the normal central nervous system (CNS), the significant trafficking of T cells to the CNS under conditions of pathology, infection, or injury presents a dichotomy. The presence of T cells in the CNS under certain contexts is considered detrimental, where T cells have been shown to cause or exacerbate neuropathology. Under certain experimentally-induced conditions, however, T cells have been shown to confer neuroprotection. In the interest of developing immune-based strategies to treat neurodegenerative disease or CNS injury, it is important to understand the conditions that drive T cells to act in a bi-directional manner in the CNS. To begin to address this issue, we used the well-characterized facial nerve axotomy model to study the interactions between T cells, microglia, and injured neurons. We demonstrated that the level of neuronal death can influence the magnitude and rate of T cell accumulation to the injured CNS. Moreover, T cells that are exposed to neuronal injury in early adulthood possess the ability to exhibit memory and increase their responsiveness to the same form of injury induced later in life. Although we found that the presence of T cell memory in the injured FMN was associated with modest effects on functional recovery, there was no obvious impact on measures of neuronal survival. The lack of effect on neuronal survival may have been due to the inability to detect a population of neurons that have been shown in previous studies to undergo severe atrophy and shrinkage following axotomy. It was also shown in those studies that the atrophic neurons possess the capacity to regenerate (i.e., reverse atrophy as demonstrated by the increase in neuronal cell number and size) following nerve re-injury. Using this nerve re-injury model, we found that immunodeficiency impaired the regenerative response of injured facial motor neurons but that T cells were not associated with the reversal of atrophy in wild-type mice. We propose that T cells respond to the neuronal death induced by injury and aid in promoting the long-term survival of the surrounding neurons by maintaining them in an atrophied state where they can be prompted to regenerate. Our findings provide intriguing information regarding the impact of T cells on the status of injured neurons, which may have important implications for the future development of treatment strategies that could aid in the prolonged survival of the neuron following injury.
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 Grace Ha.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Petitto, John M.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0023719:00001

Permanent Link: http://ufdc.ufl.edu/UFE0023719/00001

Material Information

Title: T Cell-Mediated Neuroprotection in the Injured Central Nervous System Gaining Insight with the Facial Nerve Axotomy Model
Physical Description: 1 online resource (86 p.)
Language: english
Creator: Ha, Grace
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

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

Notes

Abstract: Although T cells aid in maintaining homeostasis of the normal central nervous system (CNS), the significant trafficking of T cells to the CNS under conditions of pathology, infection, or injury presents a dichotomy. The presence of T cells in the CNS under certain contexts is considered detrimental, where T cells have been shown to cause or exacerbate neuropathology. Under certain experimentally-induced conditions, however, T cells have been shown to confer neuroprotection. In the interest of developing immune-based strategies to treat neurodegenerative disease or CNS injury, it is important to understand the conditions that drive T cells to act in a bi-directional manner in the CNS. To begin to address this issue, we used the well-characterized facial nerve axotomy model to study the interactions between T cells, microglia, and injured neurons. We demonstrated that the level of neuronal death can influence the magnitude and rate of T cell accumulation to the injured CNS. Moreover, T cells that are exposed to neuronal injury in early adulthood possess the ability to exhibit memory and increase their responsiveness to the same form of injury induced later in life. Although we found that the presence of T cell memory in the injured FMN was associated with modest effects on functional recovery, there was no obvious impact on measures of neuronal survival. The lack of effect on neuronal survival may have been due to the inability to detect a population of neurons that have been shown in previous studies to undergo severe atrophy and shrinkage following axotomy. It was also shown in those studies that the atrophic neurons possess the capacity to regenerate (i.e., reverse atrophy as demonstrated by the increase in neuronal cell number and size) following nerve re-injury. Using this nerve re-injury model, we found that immunodeficiency impaired the regenerative response of injured facial motor neurons but that T cells were not associated with the reversal of atrophy in wild-type mice. We propose that T cells respond to the neuronal death induced by injury and aid in promoting the long-term survival of the surrounding neurons by maintaining them in an atrophied state where they can be prompted to regenerate. Our findings provide intriguing information regarding the impact of T cells on the status of injured neurons, which may have important implications for the future development of treatment strategies that could aid in the prolonged survival of the neuron following injury.
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 Grace Ha.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Petitto, John M.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0023719:00001


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T CELL-MEDIATED NEUROPROTECTION IN THE INJURED CENTRAL NERVOUS SYSTEM: GAINING INSIGHT WITH TH E FACIAL NERVE AXOTOMY MODEL By GRACE KIM HA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008 1

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2008 Grace Kim Ha 2

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To my dad, whose fleeting presence in my life imparted everlasting moments of laughter and comfort. And to my mom, who courageously fulfilled the unexpected role of dad. 3

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ACKNOWLEDGMENTS My successes in life can be attributed to th e hard work and dedication of my mom, to whom I am eternally indebted. My sister, A nn, deserves much love for being a friend and confidante during the best and wo rst of times. And the journey up to this point would not be as adventurous had it not been sh ared with my husband and best friend, Abe, whose insatiable hunger for good food and conversation is always welcomed at the end of a long day. My mentor, Dr. John Petitto, fondly known as JP, deserves many words of gratitude for his open door and open ears. His kind manne r of mentoring has helped to influence my development as a thoughtful scie ntist and his words of wisdom regarding matters of life and science will always be remembered. I am thankful to my committee members, who have been helpful and supportive throughout the process-Dr. Mark Atkinson for his encouragement, Dr. Mark Lewis for always having an answer to my s ticky statistics questions, and Dr. Jake Streit for his knowledge on all things microglial. The lab ha s become a second home thanks to Huang Zhi, who on numerous occasions has shared advice on ma tters in and out of lab. His musical cells will be missed. Clive Wasserfall has been an instrumental advisor in the design of the immunological aspects of my experiments. I am extrememly grateful to Dr. Mike King, who on numerous occasions has shared his histological expertise and knowledge in great detail. My improvement in writing can largely be attribut ed to Dr. Sue Semple-Rowland, who made sure her own contagious enthusiasm for written disc ourse was shared with others. Moreover, her constant support of the students is greatly ap preciated. Many thanks go to the number of high school and undergraduate students who have passed in and out of the lab throughout the years. Their technical and intellectual contributions have been indispensable. And finally, my time in Gainesville would not be notewor thy had it not been marked by the many moments, formal and 4

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5 informal, potlucks and dinner gath erings, excursions down rivers, through town and out-of-town, shared with my dear friends.

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4LIST OF TABLES ...........................................................................................................................8LIST OF FIGURES .........................................................................................................................9LIST OF ABBREVIATIONS ........................................................................................................1 1ABSTRACT ...................................................................................................................... .............12 CHAPTER 1 INTRODUCTION TO THE IMMUNE SURVEILLANCE IN THE CNS ...........................14Immune Privilege in the CNS .................................................................................................14Context-Dependent Effects of T Cells in the Compromised CNS .........................................15Facial Nerve Axotomy Model ................................................................................................16Neuroimmune Response in the Injured FMN .................................................................17Role of T Cells Following Facial Nerve Axotomy .........................................................192 MATERIALS AND METHODS ...........................................................................................22Animals ....................................................................................................................... ............22Animal Surgery .......................................................................................................................22Light Immunohistochemistry ..................................................................................................23Nissl stain ................................................................................................................................24Assessment of Functional Recovery .......................................................................................25Image Analysis ................................................................................................................ .......25Quantification and Statistical Analysis ...................................................................................253 ENDOGENOUS T CELL AND MICROGLIAL RESPONSE TO FACIAL NERVE AXOTOMY: EFFECT OF GENETIC BA CKGROUND AND THE RAG-2 KO GENE ...27Introduction .................................................................................................................. ...........27Results .....................................................................................................................................29Strain-dependent Differences in the T Cell Response to Injured Facial Motor Neurons ....................................................................................................................... .29Effect of Strain Differences on the Microglial and Neuronal Death Response to Injured Facial Motor Neurons ......................................................................................29Effect of RAG-2 Gene Deletion on the Mi croglial and Neuronal Death Response to Axotom y ......................................................................................................................3 0Discussion .................................................................................................................... ...........31 6

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4 T CELL MEMORY IN THE FACIAL MOTOR NUCLEUS: EFFECT ON NEURONAL SURVIVAL AND FUNCTIONAL RECOVERY FOLLOWING INJURY ..................................................................................................................................38Introduction .................................................................................................................. ...........38Results .....................................................................................................................................39Effect of Prior Exposure to Neuronal In jury on the T Cell an d Microglial Response to Repeated Injury ........................................................................................................39Effect of T Cell Memory in the Injured FMN on the Neuronal Response to Facial Nerve Transection ........................................................................................................40Effect of T Cell Memory in the Inju red FMN on Functional Recovery Following Facial Nerve Crush ......................................................................................................41Discussion .................................................................................................................... ...........425 EFFECT OF INJURY SEVERITY ON THE RATE AND MAGNITUDE OF THE T CELL AND NEURONAL DEATH RESPONSE FOLLOWING FACIAL NERVE AXOTOMY ....................................................................................................................... .....51Introduction .................................................................................................................. ...........51Results .....................................................................................................................................52A Comparison of the Rate and Magnitude of the T Cell and Neuronal Death Response to Facial Nerv e Crush and Resection ..........................................................52Neuronal Cell Loss Following Facial Nerve Crush and Resection .................................53Discussion .................................................................................................................... ...........536 IMMUNODEFICIENCY AND REVERSAL OF NEURONAL ATROPHY: RELATION TO T CELLS AND MICROGLIA ....................................................................60Introduction .................................................................................................................. ...........60Results .....................................................................................................................................62Effect of Immunodeficiency on the Reversal of Neuronal Atrophy ...............................62Effect of Nerve Re-Injury on the T Cell Response in the FMN ......................................63Effect of Nerve Re-Injury on the Microglial Response in the FMN ...............................64Discussion .................................................................................................................... ...........657 PERSPECTIVES ................................................................................................................ ....73Future Directions ....................................................................................................................73Concluding Remarks ............................................................................................................ ..74LIST OF REFERENCES ...............................................................................................................78BIOGRAPHICAL SKETCH .........................................................................................................86 7

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LIST OF TABLES Table page 2-1. Summary of primary antibodies .............................................................................................22 8

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LIST OF FIGURES Figure page 1-1. Location of the facial nerv e in rodents (A) and schematic of the facial nerve axotomy paradigm (B). ................................................................................................................. ....21 3-1. Comparison of the number of CD3+ T ce lls in the injured FMN between B6, 129, and B6x129 (F1) mice.. ............................................................................................................34 3-2. Comparison of MHC II+ microglia in the axotomized FMN between subject groups. .......35 3-3. Representative photomicrographs of CD3+ T cells and MHC II+ microglia in the FMN of B6, B6/RAG-2 KO, 129, and 129/RAG-2 KO mice 14 days after facial nerve transection. ............................................................................................................... .......36 3-4. Comparison of perineuronal CD11b+ microg lial phagocytic clusters in the axotomized FMN between subject groups. ........................................................................................37 4-1. Quantification of CD3+ T cells (A) and MHC+ microglia (B) in the injured FMN of nave and sensitized mice at 14 days post-axotomy. .......................................................47 4-2. Immunohistochemistry for CD3+ T cells in the injured FMN of nave (A) and sensitized (B) mice. ..........................................................................................................47 4-3. Quantification of CD11b+ microglial phagocytic clusters in nave and sensitized mice at 14 days post-axotomy. ................................................................................................48 4-4. Quantitative cell counts (A) and average neuronal cell area (B) in the injured FMN of nave and sensitized mice at 49 days post-axotomy. .......................................................48 4-5. Comparison of functional recovery in naive and sensitized mice. ......................................49 4-6. Quantification of CD3+ T cells and CD 11b+ microglial phagocytic clusters in the injured FMN of nave and sensitized mice at 14 days post-crush. ..................................50 5-1. Temporal relationship betw een infiltrating CD3+ T cells (s olid lines) and the number of CD11b+ microglial phagocytic clusters (dashed lines) in the FMN following facial nerve resection ( ) and crush ( ). ....................................................................................58 5-3. Spearmans rank correlation analysis between the number of CD3+ T cells and CD11b+ microglial phagocytic clusters fo r both treatment groups combined (A), crush only (B), and resection only (C). ..............................................................................59 5-4. Photomicrographs of the uninjured and injured FMN following facial nerve crush (AB) or resection (C-D) at 49 days post-injury. ..................................................................59 9

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6-1. Quantification of mean cell counts (A) an d mean cell size (B) in chronically resected WT and RAG-2 KO mice that received ne rve re-injury or sham re-injury. ......................69 6-2. Facial motor neurons binned according to cell size following sham re-injury and reinjury. ....................................................................................................................... ..........70 6-3. Photomicrographs of Nissl stained facial motor neurons in WT and RAG-2 KO mice. ....71 6-4. Quantification of CD3+, CD4+, and CD8+ T cells in the FMN of WT mice. ....................72 6-5. Quantification of MHC2+ microgl ia in the FMN of WT mice. ..........................................72 10

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11 LIST OF ABBREVIATIONS CNS Central nervous system BBB Blood brain barrier FMN Facial motor nucleus IL-15 Interleukin-15 MHC Major histocompatibility complex NGS Normal goat serum NO Nitric oxide PBS Phosphate buffered saline PF Paraformaldehyde RAG-2 KO Recombinase act ivating gene-2 knockout scid Severe combined immunodeficiency

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Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy T CELL-MEDIATED NEUROPROTECTION IN THE INJURED CENTRAL NERVOUS SYSTEM: GAINING INSIGHT WITH TH E FACIAL NERVE AXOTOMY MODEL By Grace Kim Ha December 2008 Chair: John Petitto Major: Medical Sciences--Neuroscience Although T cells aid in maintaining homeosta sis of the normal central nervous system (CNS), the significant trafficking of T cells to the CNS under conditions of pathology, infection, or injury presents a dichotomy. The presence of T cells in the CNS unde r certain contexts is considered detrimental, where T cells have been shown to cause or ex acerbate neuropathology. Under certain experimentally-indu ced conditions, however, T cells have been shown to confer neuroprotection. In the interest of deve loping immune-based strategies to treat neurodegenerative disease or CNS injury, it is important to under stand the conditions that drive T cells to act in a bi-directional manner in the CNS. To begin to address this issue, we used the well-characterized facial nerv e axotomy model to study the in teractions between T cells, microglia, and injured neurons. We demonstrated that the level of neuronal death can influence the magnitude and rate of T cell accumulation to the injured CNS. Moreover, T cells that are exposed to neuronal injury in early adulthood posse ss the ability to exhibit memory and increase their responsiveness to the same form of injury induced later in life. Although we found that the presence of T cell memory in the injured FMN wa s associated with modest effects on functional recovery, there was no obvious impact on measures of neuronal survival. The lack of effect on neuronal survival may have been due to the inabili ty to detect a population of neurons that have 12

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13 been shown in previous studies to undergo se vere atrophy and shrinkage following axotomy. It was also shown in those studies that the atrophic neurons possess the capacity to regenerate (i.e., reverse atrophy as demonstrated by the increas e in neuronal cell number and size) following nerve re-injury. Using this nerve re-injury mode l, we found that immunodeficiency impaired the regenerative response of injured facial motor neur ons but that T cells were not associated with the reversal of atrophy in wild-t ype mice. We propose that T cells respond to the neuronal death induced by injury and aid in promoting the l ong-term survival of the surrounding neurons by maintaining them in an atrophied state where they can be prompted to regenerate. Our findings provide intriguing information rega rding the impact of T cells on the status of injured neurons, which may have important implications for the fu ture development of treatment strategies that could aid in the prolonged survival of the neuron following injury.

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CHAPTER 1 INTRODUCTION TO THE IMMUNE SURVEILLANCE IN THE CNS Immune Privilege in the CNS The concept of immune privilege in the centr al nervous system (CNS) once referred to the failure of the CNS to initia te adaptive immune responses. Th is concept was derived from the inability of the CNS to 1) form lymph organs a llowing for lymphatic drainage of CNS antigen to peripheral immune organs, 2) expr ess detectable levels of ma jor histocompatibility complex (MHC), a molecule required for T cells to recogn ize their cognate antigen, and 3) reject grafted foreign tissue (Carson et al., 2006). Moreover, the presence of a blood brain barrier (BBB), a structural boundary composed of endothelial ce lls separated by tight junctions, restricts the passage of blood-borne molecules to nutrients necessary for CNS maintenance while facilitating the transport of metabolites and excitatory neurot ransmitters considered toxic out of the CNS and was thought to prevent peripheral immune cell entry to the CNS (Han and Suk, 2005; Hickey et al., 1991; Ohtsuki, 2004). It is now appreciated that CN S immune privilege refers not to the absolute exclusion of peripheral immune responses but rather to uniq ue mechanisms that protect the complex and delicate tissue of the CNS from the intrinsic cons equences of inflammatory reactions (Carson et al., 2006). The brain and spinal cord, confined w ithin a an inelastic skull and vertebral column, respectively, have limited capacity to tolerate th e swelling that is associated with inflammation Moreover, inflammation in the CNS can induce ne uronal cell death and re generation. Thus, the removal and/or addition of neurons that are part of critical circ uitry may be detrimental. The BBB is restrictive but not impermeable to the entry of T lymphocytes even under nonpathological conditions (C ose et al., 2006; Hickey et al., 1991) The presence of perivascular macrophages at the BBB interface, however, perfor m immune regulatory functions that serve as 14

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a primary point of regulation, providing a hosti le environment for pathogens as well as for peripheral immune cells (Aloisi, 2001; Williams et al., 2001). Moreover, the constitutive expression of MHC proteins in the CNS rema ins low under normal conditions thus further restricting the ini tiation of autoimmune responses but can be upregulated following injury, with aging, and also during specific times during neurodevelopment (Boulanger, 2004; Corriveau et al., 1998; Goddard et al., 2007). While the rando m circulation of T cells in the normal CNS occurs at low levels, the persistence and accumula tion of T cells in the compromised CNS occurs when T cells detect their cognate MHC molecule expressed on an antig en presenting cell (APC) (Hickey et al., 1991). Following extravasation to the CNS parenchyma, T cells have the potential to interact with a variety of cells. Astrocytes and microglia ha ve been been shown to produce various cytokines that can impact T cell function and activation (Aloisi et al., 2 000). The two glial cell types differ, however, in their ability to function as APCs, with micr oglia being more efficient in their capacity to present antigen. Microglia, the re sident immune cells of the CNS, phagocytose foreign pathogens and neuronal debris. Moreov er, MHC molecules that are constitutively expressed by microglia can be upregulated following CNS in sult or infection. (Kreutzberg, 1996). Conversely, T cells can regu late the function of microglia by promoting their activated phenotype (Aloisi et al., 2000). The recent finding that neurons possess the ability to upregulate the expression of T cell regulatory molecules in vitro suggests that T cells may also directly interact with neurons (Liu et al., 2006). Context-Dependent Effects of T Cells in the Compromised CNS Although T cells have been shown to mainta in CNS health under normal conditions, their presence can exert effects that are both detrimenta l and beneficial in the co mpromised CNS. It is well-agreed upon that the pr esence of T cells in CNS can lead to deleterious effects, resulting in 15

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the demyelination, axonal damage, and neuronal lo ss that has been seen in some forms of CNS disease and injury (). Under certain experime ntally-induced conditions, however, T cells have been shown to confer neuroprotection to injure d neurons. Studies across various animal models of injury have demonstrated th e potential of T cells to promote neuronal survival, remyelination, and functional recovery, regardless of the status of the BBB (Bieber et al., 2003; Hofstetter et al., 2003; Schwartz and Hauben, 2002; Serpe et al., 2000; Serpe et al., 2002). Despite these findings, there rema in discrepancies regarding the ability of T cells to exert neuroprotection. The finding by Hauben et al. th at passive and active immunization to CNS antigen improves recovery following spinal cord injury is equivocal, with a different study demonstrating adverse effects following spinal cord contusion using similar immunization protocols (Hauben et al., 2000; J ones et al., 2004). Moreover, T cells appear to worsen acute damage following aseptic cerebral injury, as indicated by the greater extent of tissue damage and increased apoptosis seen in r ecombinase activating gene-2 kn ockout (RAG-2 KO) mice lacking mature, functional T and B cells (Fee et al., 2003 ). Whether the apopt osis was specific to neuronal or glial cells was not examined. Taken together, these studies clearly demonstrate the need to define the conditions in the CNS that drive T cells to act in a positive vs. negative manner. Facial Nerve Axotomy Model The well-characterized facial nerve axotomy model is commonly used to study the molecular mechanisms underlying neuronal and a xonal regeneration following peripheral nerve injury. The facial nerve projec ts from the motor neurons of the facial motor nucleus (FMN) to the facial musculature and contro ls various functions, including the eyeblink reflex and whisker response (Moran and Graeber, 2004; Serpe et al., 2002). The location of the facial nerve in the rodent model and a schematic of the facial nerv e axotomy model is shown in Figure 1-1A and 116

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1B, respectively. Peripheral nerv e injury induces a re trograde response that results in various cellular and molecular changes a ffecting the neuronal cell bodies (Makwana and Raivich, 2005). These changes are thought to occur as part of the regeneration program of the neuron and may help to maintain its survival following injury (Moran and Graeber, 2004). The experimental virtues of the facial nerv e axotomy model are manifold. Because the facial nerve is injured at its exit from the stylomastoid foramen, the BBB remains intact, thus preventing the trafficking of non-specific cells into the brain. Mo reover, conditions of regeneration and degeneration can be examined in the same type of lesion model by inducing injuries of varying degrees of severity. Nerve cr ush injury, the mildest form of injury, results in the regeneration of damaged axons within an intact neural sheath intact. Sheer transection of the nerve results in an intermediate form of injury and variable rates of ne rve regeneration (Moran and Graeber, 2004; Raivich et al., 2004). Nerve re section, considered the most severe form of nerve injury, involves removing a portion of the nerve to prev ent reconnection and produces profound neuronal loss and atrophy. Finally, as will be discussed in detail below, a notable feature of the model is the site-specific microglia l activation and T cell trafficking that occurs in the injured FMN. The close physical apposition of the various cells of interest T cells, microglia, and neurons renders the model ideal for studying their interactions. Neuroimmune Response in the Injured FMN One of the earliest changes to occur in the FMN following nerve axotomy is the activation and proliferation of microglia. Changes in mi croglial reactivity occur as early as one day following axotomy, where the highly ramified morphology of resting microglia become stout and deramified and with the peak of prolifera tion occurring at 3 days post-axotomy (Moller et al., 1996; Raivich et al., 1999). Moreover, microglial activation is accompanied by an upregulation of MHC I expression (Raivich et al., 1998). Over time, the presence of neuronal 17

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death in the FMN following axotomy results in th e formation of microglia l phagocytic clusters, which represent a dead or dying neuron being engulfed by group of micr oglial cells and have been used as an indirect marker of neuronal death. The peak rate of the neuronal death response in the FMN following axotomy has been shown to occur at 14 days. In addition to producing a variety of factors that can be toxic to neurons, including immune complement proteins, and nitric oxide (NO), microglia have been shown to expres s a number of factors that may be essential for neuronal survival (Banati et al., 1993; Batchelor et al., 1999; Chamak et al., 1994; Giulian, 1999). In a landmark study, Raivich and colleagues demonstrated that T cells enter brain parenchyma and specifically traffic to the site of the injured FMN following nerve axotomy (Raivich et al., 1998). Notably, the trafficking of T cells to the injured FMN occurred in the presence of an intact BBB. The initial phase of T cell infiltration between 2-4 days postaxotomy was followed by a more intense phase that peaked at 14 days post-axotomy. The two phases of infiltration appear to be mediated by specific cytokines, where interleukin-6 (IL-6), macrophage colony-stimulating factor (MCSF) is thought to mediate the initial antigenindependent response to axotomy and interleukin-1 (IL-1) and tumo r necrosis factor alpha (TNF) affect the later, antigen-inde pendent phase (Galiano et al., 2001; Raivich et al., 2003; Raivich et al., 1998; Streit et al., 1998). The trafficking of T cells to the injured FMN is a speciesdependent phenomenon, as the T cell response following axotomy is more prominent in mice compared to rats. Interestingly, these differences may be attributed to the extent of neuronal loss that occurs as a result of axotomy in these tw o species, where mice generally exhibit greater neuronal loss compared to rats (Moran and Graeber, 2004). 18

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Role of T Cells Following Facial Nerve Axotomy T cells have been shown to prevent the initial neuronal loss or slow the rate of neurodegeneration in the FMN follo wing facial nerve axotomy (Serpe et al., 1999; Serpe et al., 2000). It was shown that severe combined immunodeficient ( scid ), which lack functionally mature T and B cells, exhibited a greater rate of neuronal loss following f acial nerve transection than wild-type mice and that immune reconstitution of scid mice by adoptive transfer of normal splenocytes restored the rate of neuronal loss to wild-type levels. Interestingly, by 10 weeks post-axotomy, the latest time point examined in that study, the levels of neuronal survival appeared to converge between the two groups of mice, suggesting th at the ability of T cells to rescue neurons was transient. Similarl y, it was shown in a different study that scid mice exhibited greater neuronal de ath, as demonstrated by the increased number of microglial phagocytic clusters, in the injured FMN than wild -type mice at 14 days post-transection (Petitto et al., 2003). Additionally, functional recove ry of the whisker response was delayed in scid mice following facial nerve crush injury (Serpe et al., 2002). That immunode ficiency exacerbates neuronal survival was confirmed in recombinas e activating gene-2 (RAG-2 KO) mice, a similar mouse model deficient in periphe ral T and B cells (Serpe et al., 2000). Subsequent studies suggest that inter actions between CD4+ T cells and MHC2+ microglia are important in conferring neuroprotection in the injured FMN (Byram et al., 2004). By comparison, there was no clear associa tion between endogenous T cell responses and measures of neuronal survival and functional recovery following facial nerve axotomy in other studies. Studies in transgenic mice with marked decreases in T cell trafficking to the injured FMN resulted in no changes in the le vel of neuronal survival or in the rate of nerve regeneration (Galiano et al., 2001; Kalla et al., 2001; Raivich et al., 2003; Raiv ich et al., 2002; Werner et al., 2001). Moreover, despite marked decreases in T cell trafficking to the injured FMN in 19

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interleukin-15 knockout (IL-15 KO) mice compared to wild-type mice, neuronal death levels were comparable (Huang et al., 2007). Nota bly, Ankeny and Popovich (2007) showed that immunization with T cells primed to either CNS or non-CNS antigen prior to facial nerve transection in mice exacerbated neuronal loss. Taken together, these conflicting data suggest that there may be intrinsic differences be tween T cell responses derived from adoptively transferred cells, cells primed in vitro, and endogenous T cell responses, differences in the physiology of mice completely devoid of peripheral immune cells and those that have some level of endogenous cells, and/or methodological differen ces (i.e., surgery protocol, background strain of animals). 20

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21 A B Figure 1-1. Location of the facial nerve in rodents (A) and schematic of the facial nerve axotomy paradigm (B) (Moran and Graeber, 2004).

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CHAPTER 2 MATERIALS AND METHODS Animals All animals used in the following studies were cared for in accord ance to the NIH Guide for the Care and and Use of Laboratory Animals. Mice were housed in microisolator cages under specific pathogen free conditions. The foll owing strains of mice were used in this study: C57BL/6 (Jackson Laboratories, Bar Harbor, ME) C57BL/6 (Taconic, Hudson, NY) 129 (Taconic) B6x129 (F1) (Taconic) B6/RAG-2 KO (Taconic) 129/RAG-2 KO (Taconic) Animal Surgery Animals were anesthetized at a rate of 4% isoflurane, and maintained at a rate of 2% isoflurane. For nerve crush injuries, the main br anch of the facial nerve was exposed and microtipped forceps were used to apply compression to the nerve for a period of 10 seconds. For nerve transection injuries, the main branch of th e facial nerve was exposed and cut. For nerve resection injuries, the main branch of the faci al nerve was exposed and a portion of the nerve removed to prevent reconnection. The whiske r response was checked immediately following surgery to ensure complete paralysis. For the double injury paradigm described in Chapter 4, the right facial nerve was exposed and transected in sensitized mice while naive mice received a sham transection (nerve was exposed but not transected). Sixty-six days follo wing the initial surgery, th e left facial nerve was exposed and transected in both groups. In the fu nctional recovery study, the left facial nerve was exposed and crushed 70 days afte r the initial su rgery. Mice were sacrificed at 14 days postsecond injury. 22

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For the re-injury paradigm described in Ch apters 5 and 6, we compared two groups of mice, referred to as chronic resection + sham re -injury and chronic re section + re-injury. Both groups received a resection of the right facial nerve. Ten w eeks later, the right facial nerve was re-exposed and in the chronic resection + re-injury group, th e neuroma that had formed at the proximal nerve stump was removed. In the chronic resection + sham re-injury group, the neuroma remained intact. Both groups were sacrificed at 14 days post-second surgery. For animal perfusion, mice were anesthetized by intraperitoneal in jection of a 0.5 mg/ml ketamine cocktail solution (ketamine/xylazine/acep romazine in a 3:3:1 ratio) and were perfused transcardially with 4% paraformaldehyde (PF, Fisher) or 1 X phosphate buffered saline (PBS). Brains were collected, post-fix ed in 4% PF for 2 hours at room temperature, and cryoprotected by immersion in 30% sucrose (Fis her) overnight at 4C. Follow ing cryoprotection, brains were snapfrozen in isopentane (Fisher) and stored at -80C. Light Immunohistochemistry Using the ambiguus nucleus and the facial ne rve root as the starting and ending points, respectively, approximately forty 15um sections were cut throughout the caudal-rostral extent of the facial motor nucleus. Sections were collected on Superfrost/Plus slides (Fisher) and stored at -80C. For immunohistochemistry, tissue sections were incubated in normal goat serum (NGS) (Vector) for 1 hour at room temperature follo wed by overnight incubation with the primary antibody of interest at 4C (Table 1). Prior to incubation with the prim ary antibodies, CD4 and CD8, saline perfused tissue was post-fixed in zince fixative overnight. Sections were washed in 1 PBS after each incubation step. Visualizati on of the primary antibodies was performed by incubation of sections in goat anti-rat secondary antibody (1:2 000, Vector Labs) for 1 hour at room temperature followed by incubation in avid in-peroxidase conjugates (1:500, Sigma) for 1 23

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hour. No signal was obtained with each of the primary or secondary antibodies alone. The chromagen reaction was revealed by incubation in 3,3 diaminobenzidine (DAB)-H2O2 solution (Sigma; 0.07% DAB/0.004% H2O2). Sections were counterstained with cresyl violet, dehydrated in ascending alcohol washes, clea red in xylenes, and coverslipped. Table 1. Summary of primary antibodies used. Antigen Antibody Dilution Cellular IR Source CD3 CD3 (17A2, RtPC) 1:500 T lymphocytes Pharmingen CD4 CD4 (L3T4, RtPC) 1:500 T lymphocytes microglia Pharmingen CD8 CD8 (H35-17.2, RtPC) 1:500 T lymphocytes Pharmingen CD11b CD11b (5C6, RtPC) 1:500 Microglia Serotec MHC II I-A/I-E (C-18, RtPC) 1:500 Microglia Pharmingen NeuN NeuN (MsMC) 1:500 Neurons Chemicon Rt, Rat; Ms, Mouse; PC, polyclonal; MC, monoclonal Nissl stain Eight representative sections throughout the FM N were stained with cresyl violet (Sigma) following immunohistochemistry. Slides were placed in cresyl violet for 10-20 minutes, rinsed in distilled water, dehydrated in ascending alcohol washes, cleare d in xylenes, and coverslipped. 24

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Assessment of Functional Recovery Mice were scored for the intensity of th e whisker response each day following crush injury for 14 days, using a scale ranging from 0-3 that was previ ously described by Raivich et al. () (Raivich et al., 2004). A score of 0 represents complete paralysi s while a score of 3 represents complete recovery. Each mouse was placed in a clear plexiglass box and the whisker response was recorded for a period of 2 minutes using a Panasonic high definition digital camera. Mice were scored each day by 2 independent investig ators and the number of days to reach each recovery score was compared between groups. Image Analysis Optical density of CD11b staining in the in jured FMN was measured in 8 sections per animal using the MCID image analysis software The average ratio of CD11b staining intensity of the injured to uninjured FMN was calculated for each animal and subject groups were compared using ANOVA. Quantification and Statistical Analysis The number of CD3+ T cells or CD11b+ microglial phagocytic clusters was quantified in sections throughout the FMN by an experimenter under blind conditions. Eight sections per mouse (approximately 1/5 of the entire FMN) we re used to assess each variable. Mean counts per section were calculated for st atistical analysis and analysis of variance (ANOVA) was used to make comparisons between subject groups. Where applicable, Fishers least significance difference test was used to make pair-w ise post-hoc comparisons between groups. Neuronal survival and neuronal cell size were quantified using ImageJ software (National Institutes of Health). For th e assessments of neuronal surviv al, the number of Nissl-stained neurons in the injured FMN containing a nucleolus that were greater than 20 um in diameter 25

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26 were counted and expressed as a percentage of the number of neurons in the contralateral, uninjured FMN (% neuronal survival). Neuronal cell size was measured in 3 representative sections of the FMN each spaced 90 m apart. The medial sub-nucleus, which is innervated by the auricular branch of the facial nerve, remain s uninjured and was excluded from our analyses (McPhail et al., 2004).

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CHAPTER 3 ENDOGENOUS T CELL AND MICROGLIA L RESPONSE TO FACIAL NERVE AXOTOMY: EFFECT OF GENETIC BA CKGROUND AND THE RAG-2 KO GENE Introduction Emerging data suggest that injury-induced neuroglial reactivity may be genetically mediated (Lidman et al., 2003; Olsson et al., 2000; Piehl et al., 1999). In response to facial nerve axotomy, microglia are the glial cell type that undergo mitosi s in the injured FMN and are thought to play a critical role in motor neuron regeneration (Gr aeber et al., 1998; Streit, 2002). Robust differences in astroglial reactivity and overall MHC I expression have been described between inbred mouse strains following facial nerve axotomy, where high and low phenotypic responses are seen in C57BL/6 (B6) and 129 mi ce, respectively (Lidman et al., 2002). Although measures of microglial reactivity were not compared between th ese high and low reactive inbred mouse strains, other rese arch indicates that microglial respon siveness to facial nerve transection also appears to be influenced by gene tic background (Werner et al., 2001). Several lines of evidence suggest that leve ls of microglial reactiv ity induced by facial nerve axotomy may be modulated, in part, by peripheral T cells that migrate to the injured facial motor nucleus. In support of this, we have shown in a previous study that measures of microglial cell reactivity induced by facial nerve axotomy were modified si gnificantly by the presence of T cells in the injured FMN of interleukin-2 knockout mice (Petitto et al., 2003). Interactions between T cells and microglia may be critical in mediating neuroi mmunological processes associated with neuronal regeneration in mi ce (Byram et al., 2004; Moran and Graeber, 2004). Once thought to be detrimental to the CNS, the presence of peripheral T lymphocytes in the CNS have been shown to be neuroprotective following cer tain types of injury to the brain (Hauben et al., 2000; Moalem et al., 1999). In the mouse facial nerve axotomy model, T cells infiltrate the CNS through an intact blood brain barrier (BBB) and home to aff ected motoneurons (Moran and 27

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Graeber, 2004; Raivich et al., 1998). T lymphoc ytes appear to confer neuroprotection upon a selective population of facial motoneurons as severe combined immunodeficient ( scid ) mice, which lack mature T and B cells, show decreased neuronal survival following nerve injury when compared to wild-types (Jones et al., 2005a; Serpe et al., 1999). Adoptive transfer of functional T cells into scid mice restores the neuroregenerative capac ity of these animals to the levels of wild-type mice (Serpe et al., 1999; Serpe et al ., 2000). Given the degree of neuroprotection conferred by T cells in the injured FMN that was It was previously demonstrated that B6 and 129 mice exhibited high and low levels of astroglial reactivity and MHC1 expression, respectively, following facial nerve transection (Lidman et al., 2002). In this study, we compared the T cell and microglial response following facial nerve transection in B6 and 129 mice to test the hypothesis that these stra ins would also exhibit high and low levels of axotomy-induced T cell infiltration in the injured FMN and that these differences would be associated with change s in microglial reactivity (Lidman et al., 2002). Both strains of mice display the H2b haplotype, thus controlling for MHC genetics. To examine these hypotheses, we compared the following axotomy-induced dependent variables in four strains of mice, B6, 129 and recombinase activ ating gene-2 knockout (RAG2 KO) mice on their respective background strains. In addition, we followed the inheritance pattern of the aforementioned neuroimmune measures in the F1 generation produced by an outcross of B6 and 129 mice. Since RAG-2 KO mice lack mature T a nd B cells due to their inability to undergo V(D)J recombination, comparisons made between these mice w ith their respective background controls allowed us to determine if the peripheral response (i.e., T cell trafficking to the injured FMN) modifies the central response (i .e., microglial activity, neuronal death). 28

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Results Strain-dependent Differences in the T Cell Response to Injured Facial Motor Neurons As shown in Figure 3-1, the number of T cells in the FMN following facial nerve transection was greater in B6 compared to 129 mice. Because T cell counts in the 129 mice were at zero, accurate variance estimates could not be generated. Thus, T cell counts from these subject groups were not subjecte d to parametric or nonparametric statistical analysis to avoid violating mathematical assumptions used in these two inferential statistics models. Representative photomicrographs of the T cell re sponse in the injured FMN are shown in Figure 3-2. Note the robust T cell response in the inju red FMN of B6 (Figure 3-3A) compared to 129 mice (Figure 3-3B). The lack of T cells in th e FMN of 129 mice could not be attributed to a deficient source of peripheral T cells, as CD3+ T cells were present in the spleen (data not shown). Quantitative counts of CD3+ cells in the injured FMN of th e F1 cross are shown in Figure 3-1. Again, because the 129 st rain did not display CD3+ T cells, accurate variance estimates could not be assessed for statisti cal analyses. It is apparent, ho wever, that the level of T cell infiltration in the F1 cross is comparable to that of the C57 mice and markedly different from the 129 mice. Effect of Strain Differences on the Microglial and Neuronal Death Response to Injured Facial Motor Neurons To determine whether the T cell response infl uences microglial reactivity, we compared the number of MHC2+ microglia in the injured FMN between C57 and 129 mice. As shown in Figure 3-3, the 129 mice exhibited signi ficantly greater numbers of MHC2+ microglia than the B6 mice [F(1,10)=5.561; p<0.05]. The absence of T cells did not impact the level of MHC2+ 29

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microglia in either the B6 or 129 strain, as seen in the comparisons between each RAG-2 KO strain and their respec tive background strain. We also compared the nu mber of perineuronal CD11b+ microglial phagocytic clusters, an indirect measure of neuronal death, in B6 and 129 mice. As shown in Figure 3-4, significantly higher numbers of phagocytic microglial clusters were present in the injured FMN of B6 mice than in 129 mice [F(1,10)=14.072; p<0.01]. Image an alysis revealed no di fferences in overall CD11b staining intensity in the rati o of the optical density measure of the injured vs. uninjured FMN between the strains. Effect of RAG-2 Gene Deletion on the Microglial and Neuronal Death Response to Axotomy As expected, CD3+ T cells were not detect able in the injured FMN of RAG-2 KO mice on either background (data not shown). As shown in Figure 3-2, the number of MHC2+ microglial counts between the B6-RAG2 KO and the 129-RAG2 KO mice revealed a straindependent difference, as 129-RAG2 KO mice possessed significantly higher cell counts than B6RAG2 KO mice [F(1,10)=9.461; p<0.05] Photomicrographs of MHC II+ microglia in B6-RAG2 KO and 129-RAG2 KO mice are shown in Figures 1G-1H, respectively. A comparison of MHC II+ microglia in B6-RAG2 KO mice and B6 wild -type mice showed no significant differences (Figures 3-2). Similarly, the number of MHC II+ microglia did not differ between 129-RAG2 KO mice and 129 wild-type mice. Representative photomicrographs are shown in Figure3-3E to Figure 3-3F. In Figure 3-4, the number of mi croglial phagocytic clusters wa s significantly greater in the B6-RAG2 KO mice than in the 129-RAG2 KO mice [F(1,10)=7.628; p<0.020] but did not differ between the RAG2 KO animals of each strain compared to their respective background controls. Previously we had found that B6scid mice exhibited substantially greater neuronal 30

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loss than B6 mice at 14 days postaxotomy (Petitto et al., 2003). Since we did not observe the expected increased loss of motoneurons in either RAG2 KO strains compar ed to their respective background controls, we compared B6-RAG1 KO mice with B6 mice to examine whether the effects due to the loss of the RAG-1 gene were si milar to those seen with the loss of the RAG-2 gene or the SCID mutati on. Quantification of CD11b+ microglial phagocytic clusters between B6RAG1 KO mice and its wild-type control re vealed no significant differences (data not shown). Discussion In this study, we observed high and low levels of T cell trafficking to the injured FMN in B6 and 129 mice, respectively. The marked differences T cell infiltratio n were not correlated with differences in overall microglial activity, as demonstrated by MHC2 expression by microglia and image analysis of CD11b staining intensity. The high and low patterns of T cell infiltration in B6 and 129 mice, respectivel y, were, however, positively correlated with differences in astroglial reactivity and MHC1 expr ession previously demonstrated in these mice (Lidman et al., 2002). Since astrocytes play a role in maintaining the BBB and have been implicated in the recruitment of T cells, it is plau sible that the increased astrocytic reactivity that was observed previously in C57 mice may allow T cells greater access to the injured FMN. Contrary to our hypothesis, a greater endogenous T cell respon se in the axotomized FMN of C57 mice was not associated with less neur onal death. Although C57 mice exhibited more prominent T cell trafficking to the injured FMN compared to the 129 strain, they also exhibited a significant increase in the number of microglial pha gocytic clusters compared to the 129 strain. While we did not compare long-term neuronal su rvival between these two strains of mice, a previous study by Raivich et al. showed that neuronal loss was 30% and 20% in C57 and 129 mice, respectively, at 28 days post-transection. That a greater T cell response was associated 31

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with greater levels of neuronal death and less neuronal survival in dicates that T cells may be a response to the degree of neuronal loss. This no tion is supported by the observation that facial nerve axotomy generally results in more prof ound neuronal cell loss in a dult mice than rats, which typically exhibit less neuronal loss in th e absence of a notable T cell response following facial nerve axotomy (Graeber a nd Moran, Graeber et al., 1990, Streit and Kreutzberg, 1988). Alternatively, greater neuronal death and subseq uent loss may be a result of the increased presence of T cells. Other compensatory mechanisms, such as the increased expression of MHC2 by microglia observed in the 129 mice, may account for the reduced neuronal death levels observed in those mice. Under certain conditions, microglia have been shown to produce brain derived neurotrophic factor (BDNF), a potent survival factor for motor neurons (Batchelor et al., 1999; Serpe et al., 2005). Interestingly, the absence of peripheral T cel ls in RAG-2 KO mice of either background strain was not associated with changes in the le vels of neuronal death or measures of microglial reactivity, as demonstrated by the number of MHC2+ microglia. This finding is in contrast to the studies discussed previously th at demonstrated decreased neur onal survival in RAG-2 KO mice following facial nerve axotomy (Serpe et al., 2000). The differential outcomes may reflect methodological differences in assessing neuronal survival and loss, where surviving neurons were quantified by Serpe et al. and dead neurons being cleared by microglia were assessed in the current study. The identification of the genes involved in regulating the T cell and microglial response following CNS injury may be informative since th e interaction between these two events appears to mediate neuroprotection following injury to mo tor neurons. Moreover, T cells appear to promote the antigen-presenting phenotype of micr oglia by upregulating the expression of MHC2 32

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on microglia and influencing microglial proliferation (Aloisi et al., 2000, Carson, 2002; Sedgewick et al., 1998). Conversely, microglia have been shown to alter T cell function. Thus, it is important to determine whether those ge nes regulating the peri pheral response (i.e., infiltrating T cells) can infl uence the central response (i.e., microglial activation, neurodegeneration), which may be influenced by a separate set of genes. To begin addressing this issue, we compared the neuroimmune res ponse in the B6x129 F1 generation with the straindependent responses of the parent al strains, B6 and 129. We showed that the expression of T cell infiltration and the neuronal death response were dominant phenot ypes, as T cell levels in the injured FMN of the F1 generation resembled the B6 strain while levels of microglial phagocytic clusters resembled the 1 29 strain. By contrast, both parental strains contributed to the phenotype for MHC2 expression by microglia. Simila r patterns of inheritance were observed in the F1 generation of two rat strains with demons trated differences in the neuroimmune response following nerve root avulsion and suggest that th e genetic regulation of T cells and microglial MHC2 expression following injury is conserved across species and injury models. Moreover, that the complete absence of T cells in RAG-2 KO mice resulted in no differences in MHC2 expression by microglia or in th e neuronal death response when co mpared to wild-type controls, suggests that the genetic regulation of the centr al response to injury may be independent of peripheral immune responsiveness. 33

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Figure 3-1. Comparison of the number of CD3+ T cells in the injured FMN between B6, 129, and B6x129 (F1) mice. Each bar represents the meanS.E.M. of 6 mice/group. 34

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* Figure 3-2. Comparison of MHC2+ microglia in the axotomized FMN between subject groups. Each bar represents the mean S.E.M. of 6 mice/group. *p<0.05. 35

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Figure 3-3. Representative photomicrographs of CD3+ T cells and MHC2+ microglia in the FMN of B6, B6/RAG-2 KO, 129, and 129/RAG2 KO mice 14 days af ter facial nerve transection. A-H x220. 36

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37 Figure 3-4. Comparison of pe rineuronal CD11b+ microglial pha gocytic clusters in the axotomized FMN between subject groups. Each bar represents the mean S.E.M. of 6 mice/group. *p<0.01, **p<0.05.

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CHAPTER 4 T CELL MEMORY IN THE FACIAL MO TOR NUCLEUS: E FFECT ON NEURONAL SURVIVAL AND FUNCTIONAL RECO VERY FOLLOWING INJURY Introduction An important feature of T lym phocytes is their ability to en code long-term memory to a previously encountered antigen such that re-exposure to the antigen results in a response that is greater in magnitude and more rapid (Ahmed and Gray, 1996; Rogers et al., 2000). Although T cell memory responses to pathogens in the pe riphery are well-chara cterized, it was unknown whether the immue system of immunologically unmanipuated mi ce could be sensitized to neuronal injury in the brain and ex hibit memory when the same form of injury is induced later in life. Using the facial nerve axotomy model, we tested the hypothesis that previous injury to the FMN on one side of the brain woul d elicit a more robust T cell res ponse when a second injury is induced in the contralateral FMN later in adul thood. For this study, we compared two groups of mice, referred to here as sensi tized and nave. For the firs t surgery, the right facial nerve was exposed and the main branch was transected in sensitized mice. In nave mice, the main branch of the facial ne rve was exposed but not transected. Ten weeks later, the contralateral facial nerve was exposed and tran sected in both groups. The experi mental paradigm is depicted in Figure 4-1. We compared sensitized and nave mice at 14 days post-second injury for differences in the number of 1) CD3+ T cells, 2) perineuronal micr oglial phagocytic clusters, a measure of neuronal death, and 3) MHC2+ microglia. Since T cells appear to provide neuroprotection to axotomized facial motor ne urons under certain conditions in this injury model, a secondary hypothesis we sought to test was that the predicted increase in T cell trafficking to the injured FMN of sensitized mice would be associated with greater levels of neuronal survival. To test this hypothesis, we co mpared long-term neuronal survival at 49 days following the second injury in a different cohort of sensitized and nave mice. Neuronal survival 38

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was examined at 49 days post-axotomy in this study because of the substantial loss that has been shown to occur at this time point, allowing us to detect the predicted increase as well as potential decrements in neuronal survival (Serpe et al., 2000). A second aim of the study was to determine wh ether T cell memory is associated with improved functional recovery. Beca use nerve transection results in variable rates of recovery, we used a modified version of the double injury paradigm where the injury was a crush instead of a transection injury. The modified paradigm is shown in Figure 4-6. As discussed in Chapter 1, the facial nerve innervates muscles involved in the whisker and eyeblink response. Functional recovery of the whisker response was assessed by scoring the intensity of the whisker response for each mouse using a scale ranging from 0-3, where 0 represents complete paralysis and a 3 represents full recovery. Results Effect of Prior Exposure to Neuronal Injury on the T Cell and Microglial Response to Repeated Injury To determine whether prior exposure to ne uronal injury can elic it a T cell memory response to subsequent injury, we compared the number of CD3++ T cells in the injured FMN of sensitized and nave mice at 14 days following th e second injury, as shown in Figure 4-2A. As mentioned previously, we performe d the transection of the contra lateral (left) nerve 66 days following the first transection (righ t nerve) in the sensitized mice, as Raivich et al. (1998) have shown that T cells are cleared from the injured FMN by this time. This finding was confirmed by our lab where we examined animals at 50 days po st-axotomy and could not detect T cells in the injured FMN (data not shown). In sensitized mice, there was nearly a two-fold increase in the number of CD3+ T cells in the injured FMN compared to nave mice [F(1,30)=12.61, p<0.01]. There was no significant sex effect. Figure 4-3 shows photomicrographs of representative 39

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sections of the FMN following axotomy of the cont ralateral nerve in nave and sensitized mice. Note the robust T cell response in sensitized mice compared to nave mice. By 49 days postaxotomy, the number of T cells in the injured FMN of both subjec t groups was markedly reduced (1 2 CD3+ T cells/section) with no significant differe nces in the number of T cells between groups. An occasional T cell was observed in the uninjured FMN (control side) of nave mice. To determine whether the increased T cell resp onse observed in sensitized mice results in an increase in microglial activati on, we compared the number of MHC2+ microglia in the injured FMN between subject groups at 14 days post-axotom y, as shown in Figure 4-2B. The number of MHC2+ microglia did not significantly differ between subject groups. By day 49 post-axotomy, MHC2 expression on microglia was not apparent in either group. MHC2 positivity could not be detected in the uninjured FMN (c ontrol side) of nave mice. There were no differences in the intensity of CD11b staining in the injured FM N between subject groups (data not shown). Effect of T Cell Memory in the Injured FMN on the Neuronal Response to Facial Nerve Transection Measures of neruonal outcome were examin ed at two distinct time points following axotomy. In Figure 4-4, we compared the number of neurons undergoing cell death, as represented by the number of CD11b+ perineuronal microglial phagocyt ic clusters in the injured FMN of sensitized and nave mice at day 14 pos t-axotomy and showed that the number of dead/dying neurons identified by counting micr oglial phagocytic clusters did not differ significantly between subject groups. At day 49 post-axotomy, we were able to detect only an occasional neuron undergoing cell death. Microglia l phagocytic clusters were undetectable in the uninjured FMN (control side) of nave mice. NeuN immunoreactivity in the injured FMN also did not differ between subject groups at 14 days post-axotomy (data not shown). 40

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To examine the relationship between T cell me mory and long-term neuronal survival, we also quantified the number of Nissl-stained neuronal cell bodies at 49 days following the second surgery in a different cohort of na ive and sensitized mice. Since sensitized mice were injured in both FMN, assessments of % neuronal survival (inj ured vs. uninjured FMN) could not be made. Thus, comparisons were made between the abso lute number of neurons/section in the injured FMN of sensitized and nave mice. Although ther e was a significant decrease in the number of neurons counted in the injured compared to unin jured side, there were no significant differences in the number of Nissl-stained neurons between se nsitized and nave mice, as shown in Figure 45A. Since neurons have been shown to shrink significantly by 11 weeks post-injury, we also assessed the cross-sectio nal area of motor neurons in three representative sections (each 90um apart) as an additional measure of neuronal st atus. In Figure 4-5B, the groups did not differ significantly in this measure. Moreover, neuronal cell size in th e injured FMN of nave mice did not decrease significantly compared to the uninju red FMN, suggesting that neurons have not yet begun to shrink at this time point or th at nerve reconnection may have occurred. Effect of T Cell Memory in the Injured FMN on Functional Recovery Following Facial Nerve Crush In Figure 4-7A, sensitized mice showed an earlie r onset of recovery a nd reached a score of 1 by 3.28.18 days compared to 4.63.32 days in nave mice [F(1,13)=11.92, p<0.01]. There was no significant difference in the number of days for both groups to reach a score of 2 or 3. In Figure 4-7B, the overall rates of recovery for bot h groups are depicted with differences in the rates of recovery occu rring early between 3-4 days post-crus h (arrow). Between 6-14 days postcrush, the rates of recovery be tween groups were comparable. To determine whether the early onset of modest recovery in sensitized mice was associated with a T cell memory response in the FMN fo llowing crush injury, we compared the T cell 41

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response in naive and sensitized mice that reached a score of 3 by day 14. As shown in Figure 48A the number of T cells in the injured FMN following crush injury in nave and sensitized mice was 3.75.90 and 8.30.88 T cells/section, respectively [F(1,13)=5.20, p<0.05]. In Figure 4-8B, the presence of mi croglial phagocytic clusters wa s negligible in both subject groups. Discussion Our findings suggest that peri pheral T cells can encode long-term T cell memory to prior exposure to neuronal injury and respond more robustly to a similar form of CNS injury elicited later in adulthood. Sensitized mice given prior exposure to neuronal inju ry exhibited nearly a two-fold increase compared to naive mice. Although Raivich demonstrated that the T cell response in the FMN at 3 days post-injury was not affected by previous inju ry to the contralateral side, the spacing between injuries in that experiment was 11 days (Raivich et al., 1998). By contrast, the injuries in our studi es were separated by a period of 10 weeks, allowing for greater potential for T cell sensitization to occur. Though T cell migration into the CNS has been examined more extensively in in fection, little is known about whet her long-lived T memory cells can be generated to endogenous brain antigens. Interactions between T cells and APCs are required for antigen-specific T cell responses. Wher eas nave T cells appear to interact with antigen presenting cells in lymphoid tissues, antigen experienced T cells exhibit memory that enables them to interact with antigen presenting cells in non-l ymphoid tissues that are typically associated with lymphoid organs where T cells first encountered antigen (Campbell and Butcher, 2002; Masopust et al., 2001; Mora et al., 2003; Weninger et al., 2002; Williams and Butcher, 1997). In keeping with what is known from the immunological literature with regards to the facial nerve axotomy model in mice, initial antigen experience of effector T cells may be acquired in the draining cervical lymph nodes, and subsequently MHC2-bearing microglia may 42

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present antigen to experienced T cells at the site of injury in the inju red FMN (Byram et al., 2004; Olsson et al., 1992). Given th e considerable time period betw een the injury that induced sensitization and the contralateral injury later in life, long-lived me mory T cells may reside in the splenn, rather than the draining lymph node. The vibrissa motor cortex has been shown to send bilateral projections to both facial motor nuc lei in rats (Grinevich et al., 2005). Although unlikely, an alternative explanation for the increased T cel l response following the second axotomy in the sensitized mice could be attributab le to some form of supramotor activation that affected the contralateral facial motor nucleus, and in turn leadi ng to an upregulation of factors (e.g., chemokines, chemoattractive cytokines) responsible for T cell homing to the FMN (Raivich et al., 1998). Contrary to our initial hypothe sis, the presence of T cell me mory in the injured FMN of sensitized mice was not associated with changes in the rate of neuronal death at 14 days postaxotomy, as shown by the number of microglial pha gocytic clusters, or in the level of neuronal survival at 49 days post-axotomy. Moreover, neuronal cell size, which has been shown to decrease significantly following resection, was not impacted by the presence of increase T cell responses in the injured FMN (McP hail et al., 2004). In fact, we did not detect significant cell shrinkage following transection when compared to ne urons in the contralatera l, uninjured side. It is possible that the T cell memory response in the sensitized mice, alt hough greater in magnitude, was not sufficient to increase neuronal survival Alternatively, the num ber of T cells responding to neuronal injury in naive mice may be suffi cient to provide the necessary neuroprotection afforded by infiltrating lymphocyt es, and additional cells above such a critical threshold (e.g., levels seen in nave mice) may not confer adde d benefit. The findings in this study corroborate with those in Chapter 3 as well as with fi ndings by Raivich et al. in which there was no 43

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association between greater T cell response and improvements in measures of the rate of neuronal death or long-term neuronal survival (2001). A previous study by Yoles et al. showed that the survival of ganglion cel ls was increased when optic nerve crush injury was preceded by spinal cord contusion injury ( 2001). It is important to note, however, that experimentallyinduced injuries in itiated within the CNS, su ch as that described in the Yoles study, result in significant BBB breakdown, allowing nonspecific cells from the peri phery to traffic to the CNS (Pan W, 2001, Raivich G, 2004, Schnell, L, 1999). Our studies used a peripheral nerve injury model where T cells have been shown to traffi c across an intact BBB (R aivich et al., 1998). With regards to functional recovery following facial nerve crush, we observed a modest recovery effect following crush injury in sensit ized mice that was associated with a two-fold increase in the number of T cells trafficking to the injured FMN. Contrary to our hypothesis, however, the time to full recovery did not differ between subject groups. Although the appearance of weak whisker movement occurred be tween 3-5 days post-crush in sensitized mice, approximately 1.5 days earlier than what was obser ved in naive mice, the modest recovery effect was unlikely due to axonal regeneration. Previous ly, it was shown that wh isker reinnervation in the facial nerve crush model is no t apparent until 9 days post-crus h, which is consistent with the second phase of recovery that we observed in both groups between 8-9 days post-crush (Werner et al., 2001). Moreover, by 4 days post-crush, it was shown that th e fastest growing axons at the injured site extended approximately 6-7 mm, a dist ance that would be unlikely to bridge the gap between the proximal and distal ends of the cr ushed nerve. Although the spontaneous recovery could have been due to the degeneration-i nduced release of neurotransmitters at the neuromuscular junction, we speculate that the effect seen at 3 days post-crush may have been due to the early sprou ting of spared axons to their targ et. In studies where fluorogold was 44

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injected distal to the site of injury following facial nerve crus h, few fluorogold-positive neurons were observed as early as 3 days post-crush (Kam ijo et al., 2003). It wa s suggested that nerve compressions of different durati on and magnitude can produce crush injuries of varying severity, resulting in a mixed population of damaged and spared axons (K obayashi et al., 2003). While the regeneration of damaged axons may be unlikel y at early post-injury time points, the early sprouting of spared axons in th e injured FMN is plausible. W ith the whisker response being crucial for sensory processing in the mouse, the early onset of recovery that we observed in sensitized mice may, for example, be beneficial to their survival in the wild. Moreover, the association between T cell memory and functional recovery may be more significant in severe models of CNS injury, in which spontaneous re covery occurs despite the failure of damaged axons to regenerate (Barritt et al., 2006; Bradbury et al., 2002; Gage et al., 1983a; Gage et al., 1983b). 45

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nave sensitized L R L R DAY 0 DAY 66 DAY -T cells 80 -microglial phagocytic clusters DAY 115 -neuronal survival Figure 4-1. Schematic of the double injury paradigm. 46

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Figure 4-2. Quantification of CD 3+ T cells (A) and MHC+ microg lia (B) in the injured FMN of nave and sensitized mice at 14 days post-axotomy. Each bar re presents the mean S.E.M. of 15 (nave) and 17 (sensitized) mice. *p<0.01 nave sensitized Figure 4-3. Immunohistochemistry for CD3+ T cells in the in jured FMN of nave (A) and sensitized (B) mice. Note the robust T cel l response in the injured FMN of sensitized mice compared to nave mice. 47

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Figure 4-4 Quantification of CD 11b+ microglial phagocytic clus ters in nave and sensitized mice at 14 days post-axotomy. Each bar repr esents the mean S.E.M. of 15 (nave) and 16 (sensitized) mice. Figure 4-5. Quantitative cell counts (A) and average neuronal cell area (B) in the injured FMN of nave and sensitized mice at 49 days pos t-axotomy. Each bar represents the mean S.E.M. of 7 (nave) and 9 (sensitized) mice. *p<0.05 48

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nave sensitized L R L R DAY 0 Figure 4-6. Schematic of the modified double injury paradigm. Figure 4-7. Comparison of functi onal recovery in naive and sensitized mice. The number of days required for each group to reach each be havioral score is depicted in Fig. 2A, where each bar represents the mean S.E.M. of 8 nave and 7 sensitized mice. In 2B, the overall rates of recovery are shown for nave (solid) and sens itized (dashed) mice, with differences between groups indicated by the arrow. *p<0.01 DAY 70 DAY 84 -T cells -microglial phagocytic clusters -score functional recovery using a scale from 0-3 for 14 days 49

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50 Figure 4-8. Quantification of CD3+ T cells and CD11b+ microglial phagocytic clusters in the injured FMN of nave and sensitized mice at 14 days post-crush. For A, each bar represents the mean S.E.M. of 7 nave and 8 sensitized mice. For B, each bar represents the mean S.E.M. of 5 nave and 3 sensitized mice. *p<0.05

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CHAPTER 5 EFFECT OF INJURY SEVERITY ON TH E RATE AND MAGNITUDE OF THE T CELL AND NEURONAL DEATH RESPONSE FOLLOWING FACIAL NERVE AXOTOMY Introduction The effect of T cells in the central nervous system (CNS) appears to be contextdependent, as their presence has been shown to promote neuronal survival following certain types of injuries or, converse ly, contribute to CNS pathology, such as in experimental autoimmune encephalomyelitis (EAE) and inf ection (Byram et al., 2004; Martino and Hartung, 1999; Nau and Bruck, 2002; Schwartz, 2001; Serpe et al., 1999). Peripheral transection of the facial nerve in adult mice indu ces retrograde neuronal cell loss in the facial motor nucleus (FMN) that is accompanied by a site-specific infilt ration of T cells across an intact blood-brainbarrier to the injured motor ne urons (Moran and Graeber, 2004; Raivich et al., 1998). Although the trafficking of T cells to the injured FMN has been assumed to depend on injury severity, findings from independent studie s are conflicting. While the pres ence of T cells has been shown to be associated with substantial neuronal death in some studies, others have reported significant T cell trafficking following injury despite minima l neurodegeneration (Galiano et al., 2001; Ha et al., 2007; Ha et al., 2006; Ra ivich et al., 2003; Raivich et al ., 2002). Differences in animal models and methodology used in these studies may contribute to the disparate findings. Understanding the significance of T cells in relation to neuronal de ath may thus be critical to gain further insight regarding the bi-directional effects of T cells in the injured CNS. Although the time course of T cell accumulation in the FMN following facial nerve transection had been studied, it was unknown how the T cell response would be altered by other forms of facial nerve ax otomy that vary in injury severi ty (Raivich et al., 1998). Thus, the present study was designed to address whether the degree of neuronal cell death induced by the severity of peripheral nerve axotomy influences the T cell response in the injured CNS. To 51

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address these issues, we used two extreme variat ions of the facial nerv e axotomy model, nerve crush and resection, to compare inju ries that are of the same natu re (i.e., mechanical lesion) but that differ in their severity. A comparison of the T cell response between these two injuries, which differ considerably in their capacity for nerve regeneration and in the extent of neuronal loss, enabled us to examine T cell trafficking in relation to disparate levels of neuronal degeneration. We hypothesized that the differences in the level of neuronal injury induced by facial nerve crush versus resection would be associated with changes in the rate of accumulation and magnitude of the T cell response in the FMN. Comparisons were made cross time from 1-49 days post-injury in the FMN of C57BL/6 mice gi ven facial nerve crush or resection and the number of CD3+ T cells and CD11b+ microglial phagocytic clusters were quantified. In addition, we assessed the level of neuronal survival to determine whether differences in the level of neuronal death at the various time points reflected cumulative ne uronal loss over time. Results A Comparison of the Rate and Magnitude of the T Cell and Neuronal Death Response to Facial Nerve Crush and Resection The time course of the T cell and neuronal respon se to facial nerve crush and resection is shown in Figure 5-1. Neuronal death (dashed lin e) in the FMN for both treatment groups was not apparent at day 1 post-injury but increas ed to less than one microglial phagocytic cluster/section between days 3-7 post-injury. T cell infiltration (solid line) to the FMN in both treatment groups was minimal (<1 T cell/section) by day 1 post-injury and showed comparable increases of 3-4 T cells/section by day 7 post-injury. T cell infiltration in the contralateral, uninjured FMN across all time points was negligib le, with an average of less than 0.1 cells per 15 m section. Differences in the rate of accumulation and magnitude of the T cell response became apparent by 14 days post-injury, when the le vels of neuronal death reached their peak in 52

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both treatment groups. The number of CD11b+ microglial phagocytic clusters reached a maximal 0.5/section following facial nerve crush ( open circles, dashed line) and coincided with a peak number of 6.5.27 CD3+ T cells/section (open circles, solid line). By contrast, facial nerve resection induced a peak number of 2.54.45 CD11b+ microglial phagocytic clusters/section (closed circles, dashed line) by 14 days postinjury that was followed by a maximum number of 26.1.29 CD3+ T cells/section (closed circles, solid line) one week later at 21 days. Interestingly, there was a second modest increase in the T cell response between 21 and 28 days post-crush, where the average number of T cells rose from 1.63.85 cells/section at 21 days to 4.10.57 cells/section at 28 days. By 49 days post-injury, th e rate of neuronal death was decreased in both treatment groups and the number of T cells in the FMN declined to 0.19.06 cells/section (crush) a nd 2.50.78 cells/section (resect ion). In Figure 5-2A, Spearmans rank correlation coefficient ( ) revealed that th e number of CD11b+ microglial phagocytic clusters was highly co rrelated with th e number of CD3+ T cells when mice from all time points and both treatment groups were combined ( =0.68, p<0.01). Sim ilar significant values were obtained for nerve crush (Fig. 5-2B, =0.57, p<0.01) and nerve resection (Fig. 5-2C, =0.74, p<0.01) alone. Neuronal Cell Loss Following Facial Nerve Crush and Resection As seen in Figure 5-3 and as expected, neuronal cell loss at 49 days post-injury was significantly greater in mice that received faci al nerve resection compared to nerve crush [F(1,4)=20.853, p<0.05]. Discussion The findings in Chapter demonstrated that the magnitude and rate of T cell accumulation in the injured FMN were influenced by differences in the level of neuronal death induced by facial nerve crush and resection, two forms of mechanical injury to the facial nerve 53

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that result in mild and severe neuronal loss, respectiv ely. Interestingly, T cell and neuronal death responses were comparable in magnitude and followed a similar time course between 1-7 days post-injury, regardless of injury severity. Consistent with our hypothesis, greater T cell trafficking to the injured FMN was associated with higher levels of neuronal death, as demonstrated by the accumulation of microg lial phagocytic clusters, between 14-21 days following facial nerve resection. By contrast, facial nerve crus h induced minimal neuronal death that was accompanied by fewer numbers of T cel ls. Although Raivich et al. (1998) found an elevated plateau in the T cell response between 2-4 days that was followed by a more prominent peak at 14 days following facial nerve transection, we were unable to detect an early plateau in the T cell response in either nerve crush or res ection injuries, possibly due to the limited number of early time points assessed. Greater T cell trafficking to the injured FMN was associated with increased neuronal death and cumulative neuronal loss in mice that r eceived facial nerve resection. This finding is consistent with our data in Chapter 3, where greater T cell trafficking to the injured FMN was associated with the strain exhi biting greater neuronal death at 14 days post-injury (Ha et al., 2006)). That greater T cell infiltration to the injured FMN might exacerb ate neuronal cell death is unlikely, as we showed in Chapter 4 and others have shown that differences in the magnitude of the T cell response in the injured FMN were not associated with altered levels of neuronal death or long-term neuronal loss (Galiano et al., 2001; Ha et al., 2007; Huang et al., 2007; Raivich et al., 2003; Raivich et al., 2002). Here we found that the number of T cells correlated with the number of microglial phagocytic clus ters, suggesting that the T cell response to neuronal death is a graded event. The mechanisms that underlie this site-s pecific trafficking of T cells to the injured brain appear to involve the time-dependent expression of cytokines and cell 54

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adhesion molecules by injured neurons and microglia l phagocytic clusters (R aivich et al., 1998). The increased presence of microglial phagocytic clusters following facial nerve resection may facilitate the robust T cell response in the injured FMN. Howeve r, the correlation between the levels of neuronal death and T cells is reduced by the second increase in the T cell response observed at 28 days post-crush, which occurs in the absence of notable neuronal cell death. Interestingly, a comparison of findings from the cu rrent study with that of Raivich et al. (1998) revealed that the number of T cells in the in jured FMN following facial nerve transection in C57BL/6 mice, while higher than the response following nerve crush, was more comparable to levels induced by resection. Facial nerve transe ction results in nerve se vering with a potential for reconnection to occur and produces neuronal lo ss that is considered intermediate to that induced by facial nerve crush and resection (Mor an and Graeber, 2004; Raivich et al., 2004). While it would be expected that facial nerve tr ansection would induce a T cell response that is also intermediate in magnitude to that induced by facial nerve crush and re section, it is plausible that the two injuries would result in T cell responses of sim ilar magnitude but of different clearance kinetics, as nerve res ection and transection involve mech anical shearing of the neural sheath that differ in the potential for the nerve to reconnect. Future comparisons which include graded degrees of injury may be useful to fu rther characterize the T ce ll response to neuronal injury. The differences in the timing of the peak T cell response in relation to the peak of neuronal death between facial nerve crush and resection suggest that parameters of the T cell response may be directed by factors in addition to the level of neuronal cell death. The maximal T cell response coincided with peak levels of neuronal death at 14 days post-crush and is consistent with the time course previously descri bed following nerve transection (Raivich et al., 55

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1998). By contrast, for facial nerve resecti on, we found that the maximal T cell response occurred at 21 days post-injury, one week followi ng the peak rate of neuronal death. Moreover, we observed significant T cell infiltration in the injured FMN following facial nerve crush despite the presence of few microglial phagocytic clusters, which is consistent with other findings where T cells have been found to traffi c to the injured FMN despite the absence of neuronal death (Raivich et al., 2003). It is interesting to speculat e that the time course of the T cell response in the injured FM N may partly be dependent on the ability of the nerve to reconnect. Previously, it was shown that the decreased expression of NeuN in the FMN following facial nerve crush was transient in mice, where the return of NeuN expression coincides with the time when mice generally show a return of whisker function (McPhail et al., 2004b). That this loss of neuronal phenotype failed to return in mice that received facial nerve resection was suggested to be related to the inab ility of the nerve to reconnect. In the current study, the decline in the T cell response in the injured FMN between 14-21 days post-crush appears to be associated with the time when mice gain functio nal recovery of the whisker response. By contrast, the persistence of T cells in the injured FMN, as observed in this study for resection and in the transection studies by Raivich et al. (Raivich et al., 2004; Raivich et al., 1998), may be related to the absence of recovery due to permanent ne rve disconnection or a prolonged recovery period, as in th e case with nerve transection. We confirmed that the increased rate of neuronal death in res ected animals, as demonstrated by the by performing cell counts of Nissl stained neurona l cell bodies at 49 days post-injury, the latest time point assessed. It is important to note that ov erall neuronal death (as assessed by the number of microglia l phagocytic clusters across all time points) correlated with cumulative neuronal loss that occurred long-term as more cell death observed following facial 56

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nerve resection was associated w ith greater neuronal loss at 49 da ys post-resection, compared to nerve crush. Interestingly, the percentage of neurons taken up by phagocytic microglia did not account for the larger percentage of neurons lost at 49 days post-injury. With an average of 1700 neurons counted in the uninjured contralatera l FMN of our mice, cumulative neuronal loss by phagocytic microglia was approximately 4% and 16% following nerve crush and resection, respectively, while neuronal loss quantified by Nissl stain at 49 days post-injury was approximately 8% and 58% following nerve cr ush and nerve resecti on, respectively. The disparity between the two measur es of neuronal loss may be due to insufficient sampling of the number of microglial phagocytic clusters, as all post-injury time points were not accounted for. Alternatively, studies by McPhail et al. (2004a) suggest that follo wing facial nerve resection, the majority of injured neurons may reside in a shrunken and atrophic state, rendering them undetectable by Nissl staining. Re -injuring the facial nerve resu lted in a reversal of neuronal atrophy such that the number of countable neurons increased to 79% of the contralateral side. It is noteworthy that their finding translates to an actual loss of only 21%, which more closely matches the loss accounted for by phagocytic mi croglia in our study and provides further evidence that microglial phagocytic clusters ma y serve as an informative and conservative marker of neuronal death. In conclusion, our data demonstrate that the rate of accumulation and magnitude of T cells in the injured FMN is related to the leve l of neuronal death. Further understanding of the mechanisms by which T cells respond to different forms of neuronal inju ry may be useful in elucidating their role in CN S degeneration and injury. 57

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Figure 5-1. Temporal relationshi p between infiltrating CD3+ T cells (solid lines) and the number of CD11b+ microglia l phagocytic clusters (d ashed lines) in the FMN following facial nerve resection ( ) and crush ( ). For the T cell curve, each point represents the meanS.E.M. of 4 mice/treatment group except for days 5, 7, and 21 in the resection group, where n=3/time point. For the neuronal death curve, each point represents the meanS.E.M. of 4 mice/treatment group except for day 21 in the crush group (n=2) and days 1, 5, 7, and 21 in the resection group, where n=3/time point. 58

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59 Figure 5-2. Spearmans rank correlation analysis between the number of CD3+ T cells and CD11b+ microglial phagocytic clusters fo r both treatment groups combined (A), crush only (B), and resection only (C). E ach graph represents data pooled across all time points. Spearmans rank correlation coefficients ( ) are indicated and are significant in all cases (p<0.01). Figure 5-3. Comparison of neurona l survival in the FMN at 49 days following nerve crush and resection. Each bar represents the mean S.E.M. of 3 mice/group. *p<0.05

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CHAPTER 6 IMMUNODEFICIENCY AND REVERSAL OF NEUR ONAL ATROPHY: RELATION TO T CELLS AND MICROGLIA Introduction Increasing evidence suggests that in some forms of neuronal injury, neurons may not actually die following nerve injury but reside in an atrophic state, characterized by extreme cell shrinkage and a decreased abil ity to take up Nissl stain (McPhail et al., 2004a). Recent studies by McPhail et al. demonstrated that a population of facial motor neurons undergo a protracted period of degeneration or atrophy following a resec tion of the facial nerve in adult mice. Reinjuring the facial nerve stimulat ed a reversal in the atrophic status of the injured neurons, causing an increase in both their size and number. Reversal of neurona l atrophy has also been demonstrated in several different models of CN S nerve injury (Hagg et al., 1989; Kwon et al., 2002). The mechanisms that mediate this regenera tive response after a prolonged survival period remain unknown. Under normal conditions, the CNS is subjec t to continuous immune surveillance by low numbers of circulating periphera l T lymphocytes (Cose et al., 2006; Hickey et al., 1991). In pathogenic states such as experimental autoim mune encephalomyelitis (EAE) and infection, the presence of T cells in the br ain can have detrimental effect s (Martino and Hartung, 1999; Nau and Bruck, 2002), while in other contexts, T cells ha ve been shown to act in concert with glial cells to promote neuroregeneration (Byram et al., 2004; Martino a nd Hartung, 1999; Nau and Bruck, 2002; Raivich et al., 1998; Schwartz, 2003). Studies have de monstrated the effectiveness of T cells in preventing neuronal loss following injury (Armstrong et al., 2004; Jones et al., 2005a; Schwartz and Moalem, 2001). To date, res earch has focused on eluc idating the role of T cells in preventing initial neur onal death or slowing the rate of neurodegeneration and gradual neuronal loss following facial nerv e axotomy (Jones et al., 2005a; Serpe et al., 1999; Serpe et al., 60

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2000). Given that a substantial number of facial motor neurons undergo atrophy following axotomy and that the atrophied state of these neurons is reversible by re-injury T cells may also be involved in mediating the reversal of neuronal atr ophy following re-injury. In this study, we therefore used the re-injury model described prev iously by McPhail et al (McPhail et al., 2004a; McPhail et al., 2005) to test the hypothesis that th e reversal of motor neuron atrophy (i.e., increase in cell number and size) elicited by nerve re-injury would be impaired in immunodeficient recombinase activating gene-2 knockout (RAG-2 KO) mice, which lack mature T and B cells. Neuronal cell count s and mean cell size were compared in mice that received an initial resection of the facial nerve followed by a re-injury of the same nerve 10 weeks later versus mice that received only a single resectio n followed by a sham re-injury 10 weeks later. The re-injury paradigm is shown in Figure 6-1. In a landmark study, Raivic h et al. (Raivich et al., 1998) demonstrated that T cells cross an intact blood-brain-barrier (BBB) and home to degenerating neuronal cell bodies following peripheral transection of the facial nerve, and established that the peak of this response occurs at 14 days post-axotom y. Measures of neuronal survival were therefore assessed at 14 days after the 2nd surgery in both groups, which also allowed us to assess the peak T cell response in th e injured FMN. In Chapter 4, we showed that prior exposure to neuron al injury in the FMN early in adulthood induced a robust increase in T cell traffcking to the injured FMN, indicative of T cell memory, when the contralateral nucleus was injured later in adulthood (Ha et al., 2007). Inte restingly, this enhanced T cell trafficking to the injured FMN was not correlated with improve d neuronal survival. In teractions between T cells and microglia are important in the im mune-mediated improvement of motor neuron survival (Byram et al., 2004). That we did not see an improvement in ne uronal survival in the presence of a T cell memory response could be due to the possibility that the memory T cells 61

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encountered microglia in the c ontralateral FMN that were nave to injury and suggests that injury-experienced microglia may be needed to en code a type of memory that permits them to interact with memory T cells and mediate th e improvement in neuronal survival. Thus, secondary hypotheses we sought to test were whether prio r exposure to neuronal injury could elicit a T cell memory response when the same FMN is injured later in adulthood, and if the postulated increase in T cell homing to the FMN is correlated with the improvement in neuronal outcome measures in the current re-injury model. Studies documenting the presence of CD4+ and CD8+ T cells in the injured FMN are limite d (Ankeny and Popovich, 2007; Bohatschek et al., 2004; Liu et al., 2005), and none have been performed using this re section or re-injury model. To address the aforementioned T cell memory hypothesis and to assess the distribution of CD4+ and CD8+ T cells in the injured FMN, we compared the number of CD3+ T cells, CD4+ T cells, and CD8+ T cells in the FMN of 3 groups of WT mice. One group was assessed at 14 days post-resection but did not receive prior nerve inju ry and served as controls (acute resection). A second group received prior nerv e injury and was assessed at 14 days after a re-injury of the same nerve (chronic resection + re-injury). The third group was assesse d at 12 weeks after a single resection (chronic resection + sham). Finally, to determ ine whether prior injury alters the microglial response to re-injury, we also quantified the number of MHC2+ microglia in these subject groups. Results Effect of Immunodeficiency on the Reversal of Neuronal Atrophy We compared the effect of treatment (chroni c resection + re-injury vs. chronic resection + sham re-injury) on motor neuron survival in RAG-2 KO and WT mice. As seen in Figure 62A and as expected from the literature, motor neuron survival was significantly increased in chronically resected WT mice that received nerve re-injury compared to those that received sham 62

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re-injury [F(1,10)=6.083, p<0.05]. By contrast, the level of motor neuron survival did not differ between chronically resected RAG2 KO mice that received re-injury or sham re-injury. We also compared the effect of treatment on the cros s-sectional area of neurons measured in 3 representative sections throughout the FMN in RAG-2 KO and WT mice. As seen in Figure 62B, average cell size was significantly increased in chronically resected WT mice that received re-injury compared to those that received sh am re-injury [F(1,6)= 18.981, p<0.01]. By contrast, average cell size did not differ be tween chronically resected R AG-2 KO mice that received reinjury or sham re-injury. As seen in Figures 6-3A and 6-3B, binning of the neurons by cell size revealed a noticeable shift from smaller to larger cell sizes following nerve re-injury in WT mice. This shift in cell size following nerve re-injury was not apparent in the RAG-2 KO mice (Figures 6-3C and 6-3D). Figure 6 shows representative sections of the FMN from chronically resected WT and RAG-2 KO mice that received re-injury or sham re-inj ury. In Figures 6-4A and 6-4B, note the increase in the number and size of motor neurons in re-injured WT compared to sham re-injured WT mice. In Figure 6-4F, motor neurons remained shrunken or appeared to be lost to axotomy in RAG-2 KO mice that received re-injury. Effect of Nerve Re-Injury on the T Cell Response in the FMN To determine whether a T cell memory respons e is elicited following nerve re-injury, we compared the effect of treatment on the number of CD3+ T cells in the FMN of WT mice. Mice that did not receive prior nerve injury were assessed for their T cell response in the FMN at 14 days post-resection (acute resectio n) and were used as controls. As depicted in Figure 5A, there was a significant decrease in the number of CD3+ T cells in chronically resected mice that received re-injury [compare d to acute resection F(1,9)= 7.943, p<0.05]. There was also a significant decrease in the number of CD3+ T cells in chronically resected mice that received sham re-injury compared to acute resection [F(1,9)=11.577, p<0.01]. The number of T cells in 63

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the injured FMN was comparable between chronical ly resected mice that received re-injury or sham re-injury. By 12 weeks post-injury, there wa s an occasional T cell (<1 T cell/section) in the injured FMN of chronically resected mice that received sham re-injury. In Figures 6-5B and 6-5C, we quantified the number of CD4+ and CD8+ T cells, respectively, that were recruited to the FMN in acutely resected WT mice to determine whether there is a predominance of either T cell subtype in the FMN. The number of CD4+ and CD8+ T cells was comparable in the FMN of acutely rese cted mice. We also compared the number of CD4+ and CD8+ T cells in the FMN of chronically resect ed mice that received re-injury or sham re-injury versus the acute resection group to dete rmine whether re-injury alters their distribution in the FMN. ANOVA revealed a signifi cant decrease in the number of CD4+ T cells in chronically resected WT mice that received re -injury [F(1,8)=5.366, p<0.05] or sham re-injury [F(1,9)=15.771, p<0.01] compared to acute resection. The number of CD4+ cells did not differ between the chronic axotomy groups that received re-injury or sh am re-injury. Similarly, there was a significant decrease in the number of CD8+ T cells in chronically resected mice that received re-injury [F(1,9)=6.376, p<0.05] or sham re-injury [F(1,9)=7.621, p<0.05] compared to acute resection. The number of CD8+ T cells did not differ between the chronic axotomy groups that received re-injur y or sham re-injury. Effect of Nerve Re-Injury on the Microglial Response in the FMN To determine whether nerve re-injury alters microglial reactivity in the FMN, we compared the number of MHC2+ microglia between chronically resected WT mice that received re-injury and acutely resected WT mice. As shown in Figure 6-6, although there were fewer MHC2+ microglia following re-injury in chronica lly resected mice that received re-injury compared to acute resection mice, the gr oups were not statistically significant. ANOVA revealed a significant decrease in the number of MHC2+ microglia in chroni cally resected WT 64

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mice that received sham re-injury when compared to those that received re-injury [F(1,9)=16.771, p<0.01] or acute re section [F(1,8)=13.236, p<0.01]. Discussion We demonstrated that immunode ficiency prevented the re-i njury-induced reversal of neuronal atrophy in RAG-2 KO mice. Although th e number and size of injured motor neurons were not increased following nerve re-injury in RAG-2 KO mice, the expe cted increase in both measures was observed in re-injured wild-type mi ce. The degree of improvement in measures of neuronal survival, however, was not as robust as the improvement seen in the study by McPhail et al., (2004), likely because we performed our assessments at 14 days instead of 7 days following re-injury and suggests that the regenerative effect following re -injury is transient. It is possible that neuronal regenerati on did occur in RAG-2 KO mice but that the survival of those neurons was unsustainable due to immunodeficiency. In a previous study, the role of T cells in delaying neuronal loss was found to be time-de pendent, with a substantial reduction in neuroprotection seen between 4 and 10 weeks postaxotomy (Serpe et al ., 2000). Interestingly, the level of neuronal survival between RAG-2 KO and wild-type mice at 12 weeks post-resection was comparable, suggesting that the eventual fate of injure d neurons in immunodeficient and wild-type animals is the same. Contrary to our initial hypot hesis, we did not observe an increased number of T cells, indicative of T cell memory, in the re-injured FM N of wild-type mice. In fact, we failed to observe a notable T cell response in the re-i njured FMN of these mice, despite a marked improvement in neuronal survival. This finding is in contrast to the double injury model described in Chapter 4 in which the second inju ry was performed on the contralateral nerve, where sensitized T cells were exposed to neur ons that were naive to injury and undergoing degeneration. Although T cell memory is typically characterized by gr eater T cell responses 65

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following re-exposure to antigen, there may have been a functional enhancement of the few T cells present in the FMN, which allowed them to mediate the improvement in survival that was observed in the wild-type mice. Alternatively, T cells may have entered the FMN earlier than 14 days following re-injury to provide the n ecessary molecules (cytokines, chemokines, neurotrophic factors) to mediate neuroregeneration. That few T cells were observed in the FMN following re-injury does not preclude the possibility that T cells might exert their effects peripherally at the si te of the nerve injury. T cells can accumulate at the nerve stump following certain forms of peripheral nerve injury (Kleinschnitz et al., 2006; Moal em et al., 2004). Moreover, facial motor neurons have been shown to increased their expression of the anti -apoptotic gene bcl-2 following induction of an inflammatory response in the facial muscles of th e rat, suggesting that facial motor neurons are able to respond to peripheral immune signals (Mariotti et al., 2001). Although unlikely, T cell tolerance induced in this re-inj ury model could have accounted for the lack of a prominent T cell response. Given that additional T cells did not appear to infiltrate the FMN following re-injury, it is plausible that T cells are not i nvolved in neuroregenerative proce sses (i.e., reversal of atrophy) and that their neuroprotective bene fit could be attributable to their actions following the initial injury where they may sustain the viability of injured neurons by maintaining them in an atrophied state. Current work underway in the lab will address this working hypothesis by using adoptive transfer strategies to determine the im portance of the timing of the T cell response in this re-injury model. Briefl y, RAG-2 KO mice were immune reconstituted by adoptive transfer at two distinct time points, prior to the first or second injuries, a nd were subjected to the re-injury paradigm described in Figure 6-1. It is pr edicted that RAG-2 KO mice immune reconstituted 66

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prior to the re-injury will be impaired in their regenerative capacity due to the lack of T cellassociated support at the time of the initial injury. An additional study nearing completion will attempt to detect the presence of atrophied ne urons by using the retrograde tracer True Blue, which was applied to the nerve stump at the time of facial nerve resection. It is predicted that wild-type mice will show greater True Blue-positive neuronal la beling in the injured FMN than RAG-2 KO mice. 67

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68 sham re-injury WEEK 0 WEEK 10 WEEK neuronal survival 12 T cells microglial phagocytic clcusters Figure 6-1. Schematic of the re-injury paradigm.

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Figure 6-2. Quantification of mean cell counts (A) and mean cell si ze (B) in chronically resected WT and RAG-2 KO mice that received nerve re-injury or sham re-injury. Each bar in A represents the S.E.M. of 6 WT mice/ treatment and the S.E.M. of 6 (chronic resection + sham) and 5 (chronic resection + re-injury) RAG-2 KO mice. Each bar in B represents the S.E.M. of 4 WT mice/treatment and the S.E.M. of 3 RAG-2 KO mice/treatment. *p<0.05, **p<0.01 69

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Figure 6-3. Facial motor neurons binned accordi ng to cell size following sham re-injury and reinjury in WT (A-B) and RAG2 KO (C-D) mice. Note that the distributio n of neurons in the injured FMN shifts from small to large cell sizes and is normalized to the contralateral uninjured side following re-inj ury in B6 but not in RAG-2 KO mice. 70

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Figure 6-4. Photomicrographs of Nissl stained facial moto r neurons in WT and RAG-2 KO mice. The facial motor nucleus is oriented so that the medial sub-nucleus is located on the right. There was a significant loss a nd shrinkage of neurons in WT and RAG2 KO mice that received a chronic resection + sham injury (12 weeks after initial resection; B & E) compared to their resp ective contralateral controls (A & D). Following nerve re-injury, the number and size of injured motor neurons were markedly increased in WT (C) but not in RAG-2 KO mice (F). 71

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72 Figure 6-5. Quantification of CD3+, CD4+, and CD 8+ T cells in the FMN of WT mice. For A and C, each bar represents the S.E.M. of 5 (acute resection) and 6 (chronic resection + re-injury, chronic resection + sham) mice. Fo r B, each bar represents that S.E.M. of 5 (acute resection, chronic rese ction + re-injury) and 6 (chr onic resection + sham) mice. *p<0.05, **p<0.01 (compared to acute resection) Figure 6-6. Quantification of MHC2+ microglia in the FMN of WT mice. Each bar represents the S.E.M. of 5 (acute resection, chronic resection + sham) and 6 (chronic resection + re-injury) mice. *p<0.01 (compared to acute resection and chronic resection resection + re-injury)

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CHAPTER 7 PERSPECTIVES Future Directions Although the facial nerve axotom y model has been useful fo r extending our knowledge of the role of T cells in one form of CNS injury, it will be important to determine whether T cells operate in a similar manner to other forms of inju ry in different regions of the CNS. One model of interest is the fimbria fornix transection mode l, which results in a retrograde degeneration of neurons and site-specific microglial activation in the medial septum (Hollerbach et al., 1998). A feature of the fimbria fornix transection paradigm that distinguishes it from the facial nerve axotomy model is that the injury is initiated wi thin the CNS. Thus, T cells would be operating under conditions where the integrity of the BBB is impacted. Moreover, the findings in Chapter 6 suggest that T cells may prevent the death of a particular population of neurons by promoting their atrophic status following injury. Given that medial septal neurons have been shown to atrophy following fimbria fornix transection, it would be interesting to examine whether immunodeficiency impairs the atrophy and subsequent regenerati on of injured medial septal neurons. Insight gained from th ese studies could have significan t relevance to neurodegenerative conditions that involve deficits in le arning and memory processes. The findings in Chapter 4 demonstrated that the presence of T cell memory in the injured FMN did not affect levels of neuronal survival. The methods used to quantify the number of surviving neurons in the studies of Chapter 4 failed to account for a population of neurons that have been shown to shrink and undergo atrophy follo wing facial nerve axotomy. In light of the findings in Chapter 6 that suggest that T cells promote the atrophy of injured neurons, the presence of T cell memory in the injured FMN may increase the number of neurons that atrophy and their subsequent ability to regenerate. By combining the double injury model used in 73

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Chapter 4 with the re-injury model used in Ch apter 6, the regenerative response (reversal of atrophy) can be compared in mice that are naive and sensitized to neuronal in jury. It is expected that the presence of T cell memory in sensitized mice will increase the number of neurons that atrophy such that re-injury elicit s a greater regenerative respons e (i.e., more atrophied neurons available to regenerate). The proposed method of detecti ng neuronal atrophy by retrograde labeling with True Blue may al so provide additional insight re garding T cell memory and its effect on atrophied neurons. Concluding Remarks The use of T cells in developing immune-bas ed strategies to treat neurodegenerative disease or injury holds much promise. Vacci nes designed to boost the T cell response in the CNS are being proposed to aid in the treatm ent of various types of CNS injury and neuropsychiatric and neurodegene rative disease (Schwartz, 2000; Schwartz and Hauben, 2002). By exploiting the intrinsic ability of T cells to seek out regions of neurodegeneration and damage, it would also be possible to engineer T cells to deliver neuropro tective or regenerationrelated genes vital to the survival of the neuron. Indeed, current treatment for certain types of cancer has taken advantage of the ability of T ce lls to recognize and destroy tumor cells. With regards to the delicate nature of the CNS, however, it is critical to approach these promising strategies with caution. As discussed in Chapter 1, T cells have the potential to exert detrimental or beneficial effects in the CNS, depending on context. Before moving forward with such strategies, it was important to address some of the fundamental questions in this work regarding the role of endogenous T cell resp onses in the injured CNS. Based on our findings, we propose that T cells respond to the neuronal death induced by injury and aid in promoting the long-term su rvival of the surrounding neurons by maintaining them in an atrophied state where they can be prompted to regenerate. As the interactions 74

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between T cells and microglia have been shown to promote neuronal surv ival following injury, it will be important to understand the potential mechanisms underlying this process. It has been shown that supernatant collected from cells from the cervical lymph nodes of axotomized wildtype mice and re-activated by anti-CD3 was able to rescue injured facial motor neurons when administered to RAG-2 KO mice (Serpe et al., 2005). The neuroprotective effect was diminished when the supernatant was incubate d with anti-BDNF, providing indirect evidence that BDNF produced by T cells aid in promoting su rvival. In relation to our work, it is possible that interactions between T ce lls and microglia promote the atrophy of neurons by mediating growth factor production in the injured FMN. In addition, the molecular underpinnings of these interactions must be addressed. Recent studies suggest that T ce lls may influence the expression of pituatary adenylate cyclase activating pol ypeptide (PACAP) in injured motor neurons following facial nerve axotomy (Armstrong et al., 2004; Zhou et al., 1999). It will be interesting to determine whether the expression of PACAP and other related genes in injured neurons aid in promoting their atrophied phenotype and whether increases in the level of endogenous T cells (i.e., T cell memory) augment such gene expression. It is noteworthy that our studi es used immunologically intact mice, where the interactions between T cells, microglia, and injured neurons were examined under physiologically relevant conditions. Prior to this work, th e information regarding the role of T cells in the facial nerve axotomy model was gained from studies that used adoptive transfer strategies to restore the peripheral immune system of immu nodeficient mice. While these strategies have served as useful tools for the intitial identification of T cell-mediated neuroprotection in the facial nerve axotomy model, potential confounds are introduced with adoptive tr ansfer methods where T cells have been shown to undergo significant pro liferation and exhibit an activated, memory 75

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phenotype long after transfer. Thus, it is importa nt to consider whethe r these findings hold in immunologically intact mice. In our studies of intact mice, enhanced T cell responses did not result in the profound improvements in neurona l survival that were reported by Jones and colleagues. Our findings did, however, shed li ght on an elusive population of neurons that appear to be impacted by the actions of T cells Using immunodepletion strategies, it will be important in future studies to fu rther elucidate the different popul ations of T cells that mediate neuroprotection. In the studies of Chapter 4, T cell memory was elicited using a double injury model in which T cells were exposed and primed in vivo to endogenous CNS antigen. It is noteworthy that the presence of T cell memory in the injured FM N was associated with a modest recovery of function and did not worsen measures of neurona l survival and death. By contrast, several studies have used immunization protocols in whic h T cells were primed to myelin basic protein (MBP) prior to injury, in an effo rt to boost T cell responses to in jury (Hauben et al., 2000; Jones et al., 2004; Jones et al., 2002; Jones et al., 2005b). The iden tification of the specific CNS antigen to which T cells respond following neur onal injury is curren tly unknown. The finding that immunization with T cells primed to CNS antigen results in the marked improvement of functional recovery and neuropathology following spin al cord injury has not been replicated in independent studies. In fact, a study that followed the same i mmunization and injury protocol showed that aspects of recovery and neuronal outcome were exacerbated. These findings raise questions regarding potential intrinsic differences between T cell responses derived from experimental vs. endogenous sources of antigen. These and other relevant questions regarding antigen specificity should be the focus of future studies. 76

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Generally, the profound presence of inflammatory infiltrates in the CNS is thought to be predictive of severe neuropathology. As dem onstrated by the findings in Chapter 5, the magnitude of the T cell response was related to the severity of peripheral nerve injury and resulting neuronal loss. It woul d be hasty to assume, however, that greater T cell responses in the CNS result in exacerbated pathology. In Chap ter 6, we demonstrated that T cells may affect the long-term survival of atrophied neurons, sugges ting that actions of T ce lls in the injured CNS may be more enduring than previously t hought (Jones et al., 2005a). Notably, injured rubrospinal neurons have been shown to survive in an atrophied state for up to one year (Kwon et al., 2002). Subsequent treatm ent with BDNF applied at the site of the neuronal cell bodies reversed the atrophy and promoted the extension of the chronically injured axons into peripheral nerve grafts. Thus, treatment strategies th at suppress overall immune responses following certain forms of CNS injury may be disadvant ageous by preventing a normal and necessary response to tissue damage which could have the potential to provide l ong-term neuroprotection. A more fine-tuned approach in the treatment and management of these injuries would be to inhibit aspects of the immune response known to cause damage while permitting the beneficial aspects of the response. In conclusion, our studies provide the impetus for further investigation into the role of endogenous T cell responses in the compromised CNS. Understand ing the physiological role of T cells in the CNS under different conditions wi ll provide information regarding the importance of context in driving the benefici al vs. detrimental aspects of the T cell response. Insight gained from these studies can be used to guide the de velopment of immune-based treatment strategies that promote the repair and regene ration of damaged CNS tissue. 77

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Jones, T.B., Ankeny, D.P., Guan, Z., McGaughy, V ., Fisher, L.C., Basso, D.M., Popovich, P.G., 2004. Passive or active immunization with mye lin basic protein impairs neurological function and exacerbates neuropathology after spin al cord injury in rats. J Neurosci 24, 3752-3761. Jones, T.B., Basso, D.M., Sodhi, A., Pan, J.Z., Hart R.P., MacCallum, R.C., Lee, S., Whitacre, C.C., Popovich, P.G., 2002. Pathological CN S autoimmune disease triggered by traumatic spinal cord injury: implications for autoimmune vaccine therapy. J Neurosci 22, 2690-2700. Jones, T.B., Hart, R.P., Popovich, P.G., 2005b. Molecular control of physiological and pathological T-cell recruitm ent after mouse spinal cord injury. J Neurosci 25, 6576-6583. Kalla, R., Liu, Z., Xu, S., Koppius, A., Im ai, Y., Kloss, C.U., Kohsaka, S., Gschwendtner, A., Moller, J.C., Werner, A ., Raivich, G., 2001. Microglia and the early phase of immune surveillance in the axot omized facial motor nucleus: impaired microglial activation and lymphocyte recruitmen t but no effect on neuronal survival or axonal regeneration in macrophage-colony stim ulating factor-defic ient mice. J Comp Neurol 436, 182-201. Kamijo, Y., Koyama, J., Oikawa, S., Koizumi, Y. Yokouchi, K., Fukushima, N., Moriizumi, T., 2003. Regenerative process of the facial nerve: rate of regeneration of fibers and their bifurcations. Neurosci Res 46, 135-143. Kleinschnitz, C., Hofstetter, H.H., Meuth, S.G., Braeuninger, S., Sommer, C., Stoll, G., 2006. T cell infiltration after chronic constriction injury of mouse sciatic nerve is associated with interleukin-17 expression. Exp Neurol 200, 480-485. Kobayashi, S., Koyama, J., Yokouchi, K., Fukushima, N., Oikawa, S., Moriizumi, T., 2003. Functionally essential neuronal population of the facial moto r nucleus. Neurosci Res 45, 357-361. Kwon, B.K., Liu, J., Messerer, C., Kobayashi, N. R., McGraw, J., Oschipo k, L., Tetzlaff, W., 2002. Survival and regeneration of rubrospinal neurons 1 year after spinal cord injury. Proc Natl Acad Sci U S A 99, 3246-3251. Lidman, O., Fraidakis, M., Lycke, N., Olson, L., Olsson, T., Piehl, F., 2002. Facial nerve lesion response; strain differences but no involvement of IFN-gamma, STAT4 or STAT6. Neuroreport 13, 1589-1593. Lidman, O., Swanberg, M., Horvath, L., Broma n, K.W., Olsson, T., Piehl, F., 2003. Discrete gene loci regulate neurodegeneratio n, lymphocyte infiltration, and major histocompatibility complex class II expr ession in the CNS. J Neurosci 23, 9817-9823. 81

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86 BIOGRAPHICAL SKETCH A New Orleanian by birth, Grace was transplanted to Tampa, FL where she spent most of her childhood. The Florida sun was not enough, how ever, to keep her from venturing to the chilly northeast where she attended Boston University. Although she initially dabbled in the field of biomedical engineering, Grace soon rea lized that neuroscience was her calling. After four years in Boston, Grace decided to return home where she completed her graduate studies at the University of Florida in the laboratory of Dr. John Petitto. With her husband Abe, Grace relishes the company of their fa mily, friends, two dogs, Lyle and Fl etcher, and cat, Nala. In her free time, Grace can be found enjoying a variet y of activities, among which running, reading, and cooking are her favorite pastimes.