UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Motoneuron response to axonal injury McPhail, Lowell Thomas 2004

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_2004-931560.pdf [ 13.83MB ]
Metadata
JSON: 831-1.0091794.json
JSON-LD: 831-1.0091794-ld.json
RDF/XML (Pretty): 831-1.0091794-rdf.xml
RDF/JSON: 831-1.0091794-rdf.json
Turtle: 831-1.0091794-turtle.txt
N-Triples: 831-1.0091794-rdf-ntriples.txt
Original Record: 831-1.0091794-source.json
Full Text
831-1.0091794-fulltext.txt
Citation
831-1.0091794.ris

Full Text

MOTONEURON RESPONSE T O A X O N A L INJURY by LOWELL THOMAS McPHAIL  B . S c , University of British Columbia, 1992 M . S c , University of British Columbia, 1998  A THESIS S U B M I T T E D IN P A R T I A L F U L F I L M E N T OF T H E REQUIRMENTS FOR D E G R E E OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES THE D E P A R T M E N T OF Z O O L O G Y , We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH C O L U M B I A May 2004 © Lowell T McPhail, 2004  ABSTRACT Axonal injury in the spinal cord of the central nervous system (CNS) in higher vertebrates results in the atrophy or death of the injured motoneuron population, contributing to a failure in functional regeneration. In contrast, following an axonal injury in the peripheral nervous system (PNS) motoneurons typically survive and i f given the appropriate conditions, regenerate their axons back to their target muscles. Therefore, analysis of the survival and regeneration of PNS motoneurons in response to injury may increase our understanding of injury in the CNS and thus facilitate a treatment for CNS injuries. Rodent facial motoneurons are an excellent model to examine the effect of PNS axon injury as it is a well-defined system that is readily accessible to experimental manipulation. In this thesis I utilized this well established model of peripheral nerve injury to reveal several new findings related to the motoneuron response to nerve injury. Facial motoneurons display a differential susceptibility to injury depending on the developmental age of the animal. Following facial nerve injury in the neonatal rat the majority of the motoneurons die by an apoptotic mechanism; whereas, in the adult rat the majority of the motoneurons survive a similar injury. One possible explanation for this age dependant survival in response to injury is the level of survival-promoting factors such as the inhibitor of apoptosis proteins, N A I P and XIAP. However I found that the survival of adult compared to neonatal facial motoneurons is not due to the level of expression of these two inhibitory apoptotic proteins (NAIP and XIAP). There has also been a long standing belief that chronic nerve injury of mouse facial motoneurons results in the death of the majority of these neurons. However, I observe that many more chronically injured mouse facial motoneurons survived in an atrophied state that had been previously reported. This discrepancy is due in part to the difficulty in identifying atrophied neurons using Nissl stains or other neuronal phenotypic markers such as NeuN that are affected by axotomy leading to an underestimation of the surviving population. I determined that in addition to the increased survival of these neurons, i f chronically injured mouse facial motoneurons are subjected to a second nerve injury, the motoneurons re-express genes related to regeneration. Furthermore, I determined that the neuroma that forms at the end of a proximal stump following chronic nerve resection may be a source of factors that are capable of controlling this gene re-expression within the motoneurons. Finally, despite their atrophy, I demonstrated that chronically injured mouse facial motoneurons not only survive and re-express regeneration related genes, but are able to regrow their axons if provided with a suitable environment such as a pre-degenerated nerve graft.  ii  TABLE OF CONTENTS ABSTRACT  ii  T A B L E OF C O N T E N T S  iii  LIST OF FIGURES  vii  LIST OF A B B R E V I A T I O N S  viii  S T A T E M E N T OF O R I G I N A L CONTRIBUTIONS ACKNOWLEDGEMENTS  x xi  CHAPTER 1 GENERAL INTRODUCTION  1  Overview  2  Advantages of the facial motoneuron model over other injury models  2  The facial motoneuron model of PNS injury  3  Anatomy of facial nucleus  4  Facial nerve injury paradigms  9  Motoneurons response  13  Glial response  15  Regeneration of facial motoneurons  17  Age, species and gender differences  20  Chapter overviews and hypotheses  27  CHAPTER 2 - ENDOGENOUS EXPRESSION OF INHIBITOR OF APOPTOSIS PROTEINS IN FACIAL MOTONEURONS OF NEONATAL AND ADULT RATS FOLLOWING AXOTOMY  29  SUMMARY  30  INTRODUCTION  31  MATERIALS A N D METHODS  33  Animal care  33  Facial nerve axotomy  33  RT-PCR, Southern Blot  34  In Situ Hybridization  34  Western Blot  35  Immunohistochemistry  36  RESULTS  37 iii  RT-PCR analysis revealed changes in NAIP and X I A P m R N A after axotomy  37  In Situ Hybridization for NAIP and X I A P mRNA  37  NAIP and X I A P Protein expression as analyzed by Western blot  42  Immunohistochemistry for NAIP and XIAP in neonatal and adult facial motoneurons  42  DISCUSSION  50  CHAPTER 3 - AXOTOMY ABOLISHES NEUN EXPRESSION IN FACIAL BUT NOT RUBROSPINAL NEURONS 53 SUMMARY  54  INTRODUCTION  55  MATERIALS AND METHODS  57  Animal care  57  Rat facial nerve resection  57  Mouse facial nerve resection or crush  57  Mouse rubrospinal axotomy  58  Tissue preparation  58  Immunohistochemistry  58  NeuN immunoreactivity analysis  59  Western Blot  59  RESULTS  61  Loss of NeuN immunoreactivity following facial nerve resection  61  Transient loss of NeuN immunoreactivity following facial nerve crush  66  Maintenance of NeuN immunoreactivity following rubrospinal axotomy  66  DISCUSSION  71  CHAPTER 4 - AXONAL RE-INJURY REVEALS THE SURVIVAL AND REEXPRESSION OF REGENERATION ASSOCIATED GENES IN CHRONICALLY AXOTOMIZED ADULT MOUSE MOTONEURONS  74  SUMMARY  75  INTRODUCTION  76  MATERIALS A N D METHODS  78  Animal care  78  Re-injury of chronically axotomized mouse facial motoneurons  78  iv  Preparation of tissues  78  In situ hybridization  79  GFAP and F480 immunohistochemistry  80  Photomicrograph preparation  81  RESULTS  82  A second axon injury reverses the cell body atrophy in chronically axotomized mouse facial motoneurons  82  GAP-43 and a-tubulin in situ hybridization  88  Retrograde tracing and immunohistochemistry  91  DISCUSSION  94  CHAPTER 5 - GENE EXPRESSION IS REPRESSED BY TARGET DERIVED FACTORS FROM THE NEUROMA FOLLOWING CHRONIC RESECTION INJURY  97  SUMMARY  98  INTRODUCTION  99  MATERIALS AND METHODS  101  Animal care  101  Mouse facial axotomy (chronic)  101  Blockade of axonal transport from neuroma  101  Mouse peripheral nerve graft (chronic and acute)  102  Preparation of tissues  103  In situ hybridization  103  RESULTS  104  Axon re-injury increases the number of countable motoneurons  98  Motoneuron size following injection of GDNF or colchicine  104  GAP-43 and a-tubulin ISH  107  Growth of the axons of acutely and chronically axotomized facial motoneurons  114  DISCUSSION  117  v  CHAPTER 6 CONCLUSIONS AND FUTURE DIRECTIONS  120  Conclusion  121  Survival of neonatal and adult motoneurons  121  Survival of chronically injured mouse motoneurons  122  Motoneuron identification  123  Cell body response  125  The neuroma  126  Multiple injuries  128  Nerve graft  129  REFERENCES  131  vi  LIST OF FIGURES Figure 1.1 Rodent facial nerve  6  Figure 1.2 Facial motoneuron subgroups  8  Figure 1.3 Apoptosis pathways  2  Figure 2.1 NAIP and X I A P Southern blot  39  Figure 2.2 NAIP and X I A P ISH  41  Figure 2.3 NAIP Western blot  45  Figure 2.4 NAIP immunohistochemistry  47  Figure 2.5 Immunohistochemistry and Western blot for X I A P  49  Figure 3.1 NeuN immunoreactivity following facial nerve resection  63  Figure 3.2 NeuN Western blot and ChAT immunoreactivity  65  Figure 3.3 NeuN immunoreactivity following facial nerve crush  68  Figure 3.4 NeuN expression following rubrospinal tract axotomy  70  Figure 4.1 Chronically resected mouse facial motoneurons  85  Figure 4.2 Mouse facial motoneuron counts and size  87  Figure 4.3 GAP-43 and a tubulin ISH  90  Figure 4.4 GFAP immunohistochemistry  93  Figure 5.1 Survival of chronically injured mouse facial motoneurons  106  Figure 5.2 Motoneuron size; saline, GDNF and colchicine  109  Figure 5.3 GAP-43 ISH; saline, GDNF and colchicine  111  Figure 5.4 a tubulin ISH; saline, GDNF and colchicine  113  Figure 5.5 Motoneuron counts and size following nerve graft  116  vii  ABBREVIATIONS  35S-dATP Sulphur isotope 35 labeled deoxy-Adenosine Triphosphate AIF Apoptosis Inducing factor Apaf-1 Apoptotic protease activating factor B C A Bicinchoninic Colorimetric Acid BDNF Brain Derived Neurotrophic Factor BIR Baculoviral Inhibitor Repeat CAP-23 Cortical-Associated Protein of 23 kDa C A R D Caspase Recruitment Domain cDNA Complementary Deoxyribonucleic Acid CGRP Calcitonin Gene Related Peptide ChAT choline acetyltransferase CNTF Ciliary Neurotrophic Factor CNS Central Nervous System CSF Cerebrospinal Fluid D A B Diaminobenzidine DD Death Domain DED Death Effector Domain F A D D Fas Associated Death Domain F G FluoroGold FMNs Facial Motoneurons GAP-43 Growth Associated Protein 43 GDNF Glial Derived Neurotrophic Factor GFAP Glial Fibrillary Acidic Protein GIF Growth Inhibitory Factor HIAP1 and 2 Human Inhibitory Apoptosis Protein 1 and 2 HRP Horseradish Peroxidase IAP Inhibitory Apoptosis Protein IC Intracranial IGF Insulin Growth Factor ISH In Situ Hybridization LIF Leukaemia Inhibitory Factor viii  mRNA Messenger Ribonucleic Acid NAIP Neuronal Apoptosis Inhibitory Protein NGF Nerve Growth Factor NeuN Neuronal Nuclear NF200 Neurofilament 200 N M D A N-methyl-D-aspartate NOS Nitric Oxide Synthase PBS Phosphate-Buffered Saline PFA Paraformaldehyde PCR Polymerase Chain Reaction PNS Peripheral Nervous System R N A Ribonucleic Acid R N A i Ribonucleic Acid Interference ROS Reactive Oxygen Species RT-PCR Reverse Transcription Polymerase Chain Reaction SDS-PAGE Sodium Dodecyl Sulphate Poly-Acrylamide Gel Electrophoresis SF Stylomastoid Foramen TBST Tris Buffered Saline with 0.5% Tween 20 TNF Tumor Necrosis Factor T U N E L terminal deoxynucleotidyltransferase dUTP nick-end labeling XIAP X-linked Inhibitory Apoptosis Protein  ix  STATEMENT OF ORIGINAL CONTRIBUTIONS This thesis contains work that has been previously published or submitted for publication.  McPhail, L.T., Oschipok, L.W., Liu, J., and Tetzlaff, W. (2004). Gene expression is repressed by target derived factors from the neuroma following chronic resection injury. Submitted to Experimental Neurology. McPhail, L.T., Fernandes, K.J.L., Chan, C.C.M., Vanderluit, J.L. and Tetzlaff, W. (2004). Axonal re-injury reveals the survival and gene re-expression of chronically axotomized adult mouse motoneurons. Experimental Neurology (In Press).  McPhail, L.T., McBride, C.B., McGraw, J., Steeves, J.D. and Tetzlaff, W . (2004). Axotomy abolishes NeuN expression in facial but not rubrospinal neurons. Experimental Neurology.  185(1) 182-190. McPhail, L.T., Vanderluit, J.L., McBride, C.B., Oschipok, L.W., Crocker, S.J., X u , D., Thompson, C.S., Liston, P., Holcik, M . , Robertson, G.S. and Tetzlaff W. (2003). Endogenous expression of Inhibitor of Apoptosis Proteins in facial motoneurons of neonatal and adult rats following axotomy. Neuroscience. 17. 567-575  The thesis author Lowell McPhail was the primary researcher for all the results presented in the above articles. Technical expertise with radioactive probes was provided by L Oschipok. and K Fernandes and surgical assistance by J Vanderluit and J Liu. Neuron counting by C Chan. S Crocker, D Xu, C Thompson, P Liston and G Roberson produced the NAIP and XIAP R N A probes. C. McBride and J McGraw provided immunohistochemistry expertise. ISH quantification in Figure 4.3 is from a collaboration with K Fernandes (Fernandes, 2000).  The above statements and assessments of work performed for this thesis by the author and collaborators are stated as above.  Wolfram Tetzlaff, M D , PhD  x  ACKNOWLEDGEMENTS I would first like to thank my parents for their unwavering love and support throughout this endeavor and to whom this thesis is dedicated. I thank my supervisor Wolfram Tetzlaff for giving me the freedom to pursue my interests, yet being there to guide me. I have learned from your passion for scientific inquiry, your experience and knowledge. I would also like to thank the members of my committee, Dr Auld, Dr O'Conner Dr Roskams and Dr Steeves for their time and guidance. The most important aspect of any place is the people. I have been very fortunate during my graduate studies to be a part of a great group of individuals in the Tetzlaff, Ramer and Steeves laboratories. I am very thankful to all these friends and colleagues including; Carmen Chan, Maggie Hampong, Andrew Gaudet, Koroush Khodarahmi, Brian Kwon, Clarrie Lam, Jie Liu, Victoria MacDermid, Angela Magel, Loren Oschipok, Leanne Ramer, David Stirling and Bonnie Tsang. I am indebted to Jackie Vanderluit for her patience and generosity in teaching me everything in the beginning, to Jamie Borisoff and Ward Plunet for their insightful, yet often irrelevant and meaningless conversations, to Chris McBride for always laughing at my jokes and tirelessly editing my work, to John McGraw for betting me who would finish their thesis first, to Egidio Spinelli for always keeping me guessing and to Gordon Hiebert for getting me out of the lab on sunny weekends. Mostly I want to thank my partner Beverly Chua for her love and encouragement, she is my life.  xi  Chapter 1 General introduction  1  Overview Axonal injury in the spinal cord of the central nervous system (CNS) in higher vertebrates results in the atrophy or death of the injured neuron population, contributing to a failure in functional regeneration. In contrast, following an axonal injury in the peripheral nervous system (PNS) in most instances the motoneurons survive the injury. In addition, PNS neurons can, i f given the appropriate conditions, regenerate their axons back to their target muscles (reviewed in Fu and Gordon 1997). Clearly, examining the processes involved in the PNS motoneuron response to injury and the signals that control them may be beneficial to our understanding of the CNS and thereby facilitate a treatment for CNS injury. The PNS motoneuron response to injury is a multifactor event including changes at the axonal injury site as well as at the neuron soma. This response to injury poses a number of interesting questions. First, what are the factors that contribute to the survival of motoneurons following axonal injury? Second, what are the signals as a result of an axonal injury that mediate the motoneuron response to axonal injury? To address these questions I chose a well characterized model of PNS nerve injury, axotomy of the rodent facial motor nerve (cranial nerve VII).  Advantages of the facial motoneurons over other injury models The facial motoneuron model of regeneration has numerous advantages over several other models used to examine the response to injury. For example, the topographic organisation of the facial nucleus is of experimental value. Axonal injury to only the large branch results in over 90% of the motoneurons injured while the ventral medial group, the axons in the posterior auricular branch, remain uninjured and therefore serve as an internal control. The facial nucleus is a homogeneous motoneuron population with no evidence of interneurons (Travers 1985). In addition, facial motoneurons are a circumscribed population, making dissection of the motoneurons feasible for molecular and biochemical analysis (PCR and Western blot). The hypoglossal nucleus (cranial nerve XII), consists of approx 3500 neurons that innervate muscles of the tongue. However, unlike facial motoneurons, hypoglossal motoneurons differ in their size, shape and dendritic arborization depending on their myotopic subdivision (reviewed in Travers 1985). Furthermore, in hypoglossal motoneurons, some dendrites extend to the contralateral nucleus and also extend up to 1mm in length to the adjacent reticular formation, the solitary tract and the motor nucleus of the vagus. There is even some evidence for a small population of interneurons in the hypoglossal nucleus (reviewed in Travers 1985). These afferent connections, the presence of interneurons, differential motoneuron size, axon collaterals and 2  dendritic pattern may all influence the motoneurons response and therefore the interpretation of data obtained from hypoglossal nerve injuries. Spinal motoneurons, such as those that extend into the brachial plexus and sciatic nerves are also used extensively as PNS models for motoneuron regeneration. However, there are a number of draw backs with these models. First, sciatic nerves are a mixture of both sensory and motor fibers. This can be problematic in chronic experiments as the sensory component of the nerve is also injured resulting in scratching, chronic pain and even autophagy of the affected limb. Second the affected motoneuron population is dispersed within several spinal cord segments. This makes the physical dissection of the axotomized population of neurons for biochemical analysis technically challenging. In addition, the ventral horn also consists of numerous interneurons with connections to the contralateral side. Again these factors limit the variety of experiments that can be performed in the spinal motoneuron model.  The facial motoneuron model of PNS injury The facial motoneurons innervate the facial musculature and control whisker movement in many animals. Facial motoneurons have been extensively used as a model to study astrocyte and microglia activation as well as the associated glia and neuronal changes in response to motoneuron injury (Streit and Graeber 1993). Understandably then, the facial nucleus has also become one of the most often studied models for testing the in vivo effects of neurotrophic factors on axonal regeneration. One of the first documented uses of the facial model of axotomy was by F. Nissl where, in 1890, he presented his findings on the motoneuron response to axotomy in rabbits at a conference (reviewed in Cammermeyer 1955). More recently, using neonatal rats or mice, the facial model has become a popular paradigm for investigating axotomy induced apoptotic motoneuron death (Lowrie and Vrbova 1992, Soreide 1981, Vanderluit, et al. 2003, Vanderluit, et al. 2000). The facial motoneuron model of axonal injury has several advantages; it is a purely motor population, the axons are superficial, allowing easy access and manipulation, and also, axonal injury does not compromise the blood brain barrier of the motoneurons (Raivich, et al. 1998). Another attractive attribute of the facial model is the versatility of the response of the motoneurons to various injuries. For example, in neonatal rodents facial nerve injury leads to the apoptotic death of the majority of motoneurons within a few days (Lowrie and Vrbova 1992, Soreide 1981). In contrast, in the adult, the motoneuron response to nerve injury is both injury and species specific. For instance, avulsion injury, or cutting of the axons close to their cell 3  bodies results in massive motoneuron loss, hence it is an excellent model of adult motoneuron death in the rodent (Aperghis, et al. 2003, Soreide 1981). Anastomosis or nerve graft experiments enable analysis of the regenerative environments of the axons and rates of axonal growth. Nerve ligature facilitates the study of axonal transport to and from the motoneuron cell body. Nerve crush provides an opportunity to study not only axonal growth rate, but also gene expression changes during regeneration and subsequent target reconnection specificity. Finally, resection injury (permanent target loss) results in the atrophy or death of a proportion of the motoneuron population (more in mice than rats) and enables investigations involving a slower more prolonged degeneration of motoneurons, characteristic of some degenerative diseases.  Anatomy of facial nucleus The facial nucleus is a well defined homogeneous population of motoneurons located in the ventral part of the pons. The facial nucleus consists of approximately 4500 motoneurons in the adult rat. In the facial nucleus, the motoneurons are divided into six subdivisions: lateral, dorsolateral, ventral intermediate, dorsal intermediate, ventromedial and dorsomedial subgroups (Fig. 1.1). The general somatotrophic organization is a topographical organization, as determined by retrograde tracing, whereby within the subgroups dorsal muscles of the face are represented dorsally and ventral muscles are represented ventrally. There is also a rostro-caudal organization whereby the caudal muscles, those near the ear, are in the medial subgroups and the more rostral whisker muscles are in the lateral subgroups (reviewed in Travers 1985). The dendrites of the motoneurons extend in all directions, and although there are some limited projections into adjacent subgroups, for the most part, dendrites remain within their respective subgroups and project both rostral and caudal within the facial nucleus with an average combined dendritic tree length in the rat of 17,650 mm (Friauf 1986). The facial nerve of a rodent exits the skull via the stylomastoid foramen (behind the ear) where it bifurcates into the posterior auricular branch innervating the back of the ear. After a small distance the nerve again divides into the anterior auricular, the zygomatic, innervating eye muscles, and then the buccal and mandibular branches that all innervate the mouth, proboscis and vibrissae (Fig. 1.2). A l l neurons within the facial nucleus are motoneurons; and no evidence of interneurons or axon collaterals (Travers 1985). The facial motoneurons, although separated into distinct subgroups which have a myotopic organization do not differ in their cytoarchitecture. However, in the cat a few of the motoneurons in the facial nucleus do not innervate the muscles of the face but rather project to the cerebellar flocculus (reviewed in Travers 1985). 4  Figure 1.1 Facial motoneuron nucleus subdivisions Motoneurons are divided into six subdivisions: lateral (L), dorsolateral (DL), ventral intermediate (VI), dorsal intermediate (DI), ventromedial (VM) and dorsomedial (DM) subgroups (A). Application of the retrograde tracer Fast Blue to the large branch of the facial nerve results in an absence of labeling in motoneurons of the ventromedial subgroup which innervate the posterior auricular branch (B). Note that the Fast Blue and Neurotrace images are the same section.  5  Figure 1.2 Facial motoneuron nerve branches The rodent facial nerve exits the skull via the stylomastoid foramen then bifurcates into the posterior auricular branch innervating the back of the ear. The nerve again divides into the anterior auricular, the zygomatic, innervating eye muscles, and then the buccal and mandibular branches innervating the mouth, proboscis and vibrissae. Arrow heads indicate the two sites of injury used in this thesis. Image modified from (Kamijo, et al. 2003).  7  8  In the rodent, the control of muscles for whisker movement occupy about one third of the motor cortex (reviewed in Hattox, et al. 2002). Motoneurons of the facial nucleus receive afferent input from the midbrain, pons and the medulla. In the cat, afferent input from the midbrain (superior colliculus) has been associated with ear movement in response to sound. Other more diffuse projections from the midbrain are related to such behaviors as blinking, vocalizing and emotional facial musculature movements. Electrical stimulation of the contralateral red nucleus can even produce movement of the whiskers and eyelid, thus providing a conduit for cortical input to the facial motoneurons (reviewed in Travers 1985). However, more recent work suggests that electrical stimulation in the red nucleus does not reliably result in whisker movements (Isokawa-Akesson and Komisaruk 1987), therefore a role of the red nucleus in whisker movements remains uncertain. Projections from the parabrachial regions of the pons, which have second order vagal input, have been suggested to be involved in sniffing, vibrissae movements and breathing, all movements associated with the coordination of respiratory and facial movements. For the most part, afferent inputs from the medulla are derived from the reticular formation. These areas receive cutaneous input from the muscles that the facial nerve innervate and, therefore, are in large part responsible for the facial and ear reflexes in response to sensory stimuli (reviewed in Travers 1985). In addition, neurons within the reticular formation are involved in several other rhythmic motor tasks such as mastication and licking (reviewed in Buttner-Ennever and Holstege 1986). A more detailed examination of the whisker representation in the motor cortex and afferent connections to the facial nucleus is provided by Hattox and colleagues (Hattox, et al. 2002).  Facial nerve injury paradigms There are a number of different types of axon injury paradigms including; transection, resection, anastomosis, crush, avulsion and ligature. In facial motoneurons each of these injures has its own unique motoneuron response and experimental advantages. A nerve transection is the complete severing of the connective tissue surrounding the axons including the endoneurium, epineurium and perineurium. There are two types of transection injuries, resection and anastomosis. A resection is simply a transection with the additional removal of a distal segment of the nerve, thus preventing the axons from reconnecting with the distal stump and their targets. A transection and the subsequent reconnection of the two opposing ends of the nerve, either by glue or sutures, to facilitate target reconnection is termed an anastomosis. Nerve crush is the compression of the nerve, usually with forceps, severing the axolemma, pushing the axoplasm 9  from the crush site into the two opposing ends of the nerve. Like a transection, a crush results in the complete severing of the axons, but unlike a transection, a crush injury leaves the majority of the endoneurium, and all the perineurium and epineurium intact facilitating more accurate target reconnection. A facial nerve avulsion involves the exposure of the nerve at the stylomastoid foramen and then sustained traction of the nerve until it is severed closer to the neuron soma, near the rootlet /brainstem junction (Aperghis, et al. 2003). The ligature technique involves tying off of the nerve with a suture to prevent both anterograde and retrograde axonal transport. This enables the analysis of transported molecules within the axons, both in the proximal and distal sections of the nerve relative to the ligature.  Transection/resection  A proximal transection/resection of the rat facial nerve close to the brainstem compared to a more distal transection outside the stylomastoid foramen (SF), results in significantly more motoneuron loss (74%) in the intracranial (IC) injury group compared to the more distal injury group (28%>) at 28 days post transection (Mattsson, et al. 1999). No difference in astrogliosis is seen between IC and the more distal SF. However, there is more intense staining in the facial nucleus for the macrophage marker EDI at 28 days post axotomy in the IC group compared to the SF group, likely a consequence of the increased motoneuron death within the IC group (Mattsson, et al. 1999). The differential survival in these two transections is likely due to the differences in trophic support, and may also be a result of the axonal stump environment of the IC versus SF transactions. Intercranial transection allows for the uptake of cerebrospinal fluid (CSF) which is not the case with the more distal SF transection (Mattsson, et al. 1999). The significance of the more proximal environment and the precise role of the CSF as a death factor in this injury model are not yet clearly defined.  Anastomosis  Following transection and immediate anasotomosis of the facial nerve results in little, i f any, motoneuron death, even at 112 days post injury in the rat (Guntinas-Lichius, et al. 1994). The activation of microglia, astrogliosis and synaptic stripping all occur following anastomosis as in the resection model. However, the extent and duration of the changes are attenuated following anastomosis (Guntinas-Lichius, et al. 1994). In humans, head and neck trauma can often lead to a loss of facial muscle function due to the associated facial nerve injury. If the reconnection of the two ends of the facial nerve is impossible due to excessive damage, surgeons 10  often perform a hypoglossal nerve to facial nerve anastomosis. This procedure produces some functional recovery of facial muscle control and even limited facial expressive movement such as smiling (reviewed in Chen, et al. 2000). In guinea pigs, immediate hypoglossal to facial anastomosis increases regeneration of the facial nerve compared to if the procedure is delayed by 3 months (Chen, et al. 2000). In contrast, in the rat the longer the anastomosis is delayed, up to 56 days, the better the regeneration in the facial nerve (Guntinas-Lichius, et al. 1997). Despite efforts to facilitate functional recovery using facial to facial anastomosis, this procedure can often produce dysfunctional reflexes and aberrant facial movements due to the incorrect reinnervation of target muscles. Reinnervation of the vibrissae following facial to facial anastomosis in the rat removes the somatotopic organization seen in control uninjured animals. In addition, only 40% of the facial motoneurons succeed in connecting with their original targets and there is a great deal of polyneuronal innervation or hyperinnervation, i.e. the motoneurons innervate more than one target (Dohm, et al. 2000, Tomov, et al. 2002). Although numerous studies in the CNS have demonstrated the inhibitory nature of extracellular matrix proteins, the local application of collagen, laminin, fibronectin or tenascin the rat facial nerve did not reduce the redundant axonal branching (Dohm, et al. 2000). However, functional blocking antibodies to neurotrophic factors reduced sprouting in the facial nerve (Streppel, et al. 2002). In addition, transplanted olfactory mucosa reduced branching and promoted functional recovery in rat facial motoneurons following anastomosis (Guntinas-Lichius, et al. 2002).  Crush  Facial nerve crush is often used to study the effects of axonal injury on the regeneration of motoneurons. The crushing of the nerve results in complete axonal damage yet unlike a transection, a crush injury preserves most of the endoneurium and all of the perineurium and epineurium which provides a physical structure for axon guidance. Neither the number nor the duration the crush significantly alters the outcome of a nerve crush, as long as the perineurium and epineurium remain intact and there is no ipsilateral whisker movement for the first few days following injury (Bridge, et al. 1994). Facial nerve crush results in little, if any, motoneuron cell death in the adult rat or mouse. However, in the neonatal rodent, facial nerve crush results in the death of the motoneurons as does a transection injury (Kuzis, et al. 1999, Lowrie and Vrbova 1992, Soreide 1981). Following facial nerve crush in the adult mouse, the first indication of functional recovery is detected at 8 days post injury and is completed by 11 days post injury (Kamijo, et al. 2003). However, retrograde labeling of the motoneurons is not returned to pre 11  injury levels until 16 days post injury, suggesting that the motoneurons have varying growth rates (Kamijo, et al. 2003). In an effort to determine the efficacy of target re-innervation following a crush injury, researchers used a fluorescent reporter to view individual axons in a live mouse to determine the exact muscle fibers that an axon originally innervated. Using this technique it was demonstrated that after a nerve crush but not a nerve transection, the axons grew back to, and made functional connections with, the exact muscle fibers they originally innervated (Nguyen, et al. 2002). Using the facial model, Kamijo and colleagues (2003) found that in comparison to an anastomosis, a crush injury resulted in less excessive axonal branching of the motoneurons, supporting the concept for the requirement of a physical guidance structure to facilitate correct target re-connection.  Avulsion  At one month post avulsion of the facial nerve there is a significant loss (75%) of the motoneurons in the adult rat (Aperghis, et al. 2003, Soreide 1981). In the neonatal rat one week after facial nerve avulsion, there is 86% motoneuron loss (Aperghis, et al. 2003, Johnson 2001). In neonatal animals, this motoneuron loss is a result of death via an apoptotic mechanism as evidenced by both T U N E L (terminal deoxynucleotidyltransferase dUTP nick-end labeling) and caspase 3 staining in the dying motoneurons. However, neither T U N E L nor caspase 3 staining was observed in the adult animals (Aperghis, et al. 2003). The most likely mechanism associated with this reduction in survival is the reduced trophic support from an injury so proximal to the neuron soma. Although the list of the factors released at the injury site is still incomplete, this concept was supported by the effects of viral application of trophic factors. Trophic factors, such as B D N F and GDNF, which are normally produced following axonal injury by peripheral Schwann cells (reviewed in Fu and Gordon 1997), increase the survival of the facial motoneurons following an avulsion injury (Sakamoto, et al. 2003). The application of an adenoviral vector encoding growth inhibitory factor (GIF) to avulsed rat facial motoneurons also increased their survival and decreased nitric oxide synthase (NOS) activity, suggesting that the avulsed motoneurons are undergoing oxidative stress (Sakamoto, et al. 2003). Furthermore, both immuno-suppressants and calcium blockers enhance the survival of avulsed hypoglossal motoneurons (Tao and Aldskogius 1998). A full understanding of the mechanisms involved in avulsion induced motoneuron death remains to be elucidated.  12  Ligation  Studies as early as 1948 began to investigate axonal transport using the nerve ligature technique (reviewed in Bisby 1982). The ligature technique is mostly used for the analysis of retrograde and anterograde transported molecules in the proximal and distal sections of the nerve. However, in many instances this method is used to prevent the reconnection of the axons to their targets, but unlike a resection injury, a ligature maintains the perineurium continuity. Although there are some instances of the use of the facial nerve in transport studies (Tetzlaff, et al. 1989), more often it is used to prevent regeneration of the facial axons back to their original targets (Mattox, et al. 1988). In the rat or mouse, the sciatic nerve is a more technically suitable model for axonal transport studies due to its larger size compared to that of the facial nerve.  Motoneuron response The response of PNS neurons to axonal injury was initially principally conceptualized as that of the transformation from synaptic function to a growth phase (reviewed in Cragg 1970). However, despite the increased expression of a number of developmental genes such as tubulin, this is a distinct state and is not simply a recapitulation of development (reviewed in Bisby 1995). One of the hallmarks of peripheral nerve injury is the neuronal cell body response. The cell body response encompasses several morphological changes to the neuron soma, the most prominent of which is chromatolysis. Chromatolysis is the degradation and disintegration of the large aggregations of rough endoplasmic reticulum, a process resulting in the dispersal of Nissl substance and an alteration in the staining pattern observed using Nissl stains (Lieberman 1971). In rat facial motoneurons, the process of chromatolysis begins as early as 8 hours following any type of axonal injury and lasts over 112 days (Guntinas-Lichius, et al. 1996). Chromatolysis, like many other injury induced biochemical and morphological changes in facial motoneurons, is dependant upon the severity of the injury: Nerve crush results in few changes, nerve transection greater changes and nerve avulsion the most severe (Soreide 1981). Another morphological feature that is altered in response to facial nerve injury is the motoneuron soma size and nucleus volume. In both nerve transection and crush paradigms there is an initial increase in the size of the motoneuron soma, which then begins to return to normal with reconnection of the targets and the subsequent functional recovery (Vaughan 1990). However, i f target reconnection is prevented in the rat, the volume of the motor nucleus declines to 60% of the uninjured side by 112 days. This reduction in overall volume of the facial nucleus, accompanied by the associated  13  motoneuron loss results in a constant neuron density within the facial nucleus (Guntinas-Lichius, etal. 1994). In addition to the morphological changes at the neuron soma as a result of axonal injury, there are several changes in the expression of genes coding for growth associated proteins, cytoskeletal proteins, transcription factors, signaling molecules and homeostatic proteins (reviewed in Fernandes and Tetzlaff 2000). In the PNS, axonal injury is often correlated with the expression of regeneration associated genes, such as tubulin and GAP-43 (reviewed in Fernandes and Tetzlaff 2000). Following axonal injury in the CNS, these changes are often less intense, more transient or fail to occur, as is the case when the axonal injury is very distal from the cell body (Fernandes, et al. 1999). One of the best characterized growth associated proteins is GAP43. GAP-43 is anterogradely transported in the injured axon and interacts with several other growth cone proteins thus regulating actin dynamics (reviewed in Benowitz and Routtenberg 1997, Caroni 2001). GAP-43 expression is high during axonal development and during PNS regeneration (Schreyer and Skene 1993, Tetzlaff, et al. 1991), while most neurons show low levels in adulthood. Over-expression of GAP-43 is not sufficient for regeneration of CNS neurons, but does increase axonal sprouting and can override some of the inhibitory elements of myelin (Buffo, et al. 1997, Gianola, et al. 2004). However, if over-expressed in conjunction with other GAPs like CAP23, greatly enhances the growth propensity of dorsal root neurons (Bomze, et al. 2001). Interestingly, GAP-43 knockout mice have normal neurite formation and growth rate but their axonal path finding is impaired suggesting that GAP-43 is not entirely necessary for all axonal growth (Donovan, et al. 2002, Shen, et al. 2002, Strittmatter, et al. 1995). Despite many years of study, the signals involved in the regulation of the cell body response to axonal injury still remain largely unknown. The most generally accepted model is that axon injury stimulates the cell body response by removing a factor that is normally derived from the axon target and retrogradely transported to the neuron soma (Cragg 1970). Although the concept of retrogradely transported factors influencing the cell body response was initially put forward by Santiago Ramon y Cajal, a clear demonstration of this was the application of the axonal transport blocker colchicine to post ganglionic nerves that induced a motoneuron response (Cajal 1991, Purves 1976). Interestingly, numerous studies indicate that multiple control mechanisms must be in operation. For example, sprouting at the axon terminals of spinal motoneurons begins within a few hours of axotomy, which is much too fast to be accounted for by axonal transport, and sprouting occurs even i f the axons are also separated from their soma (Brown and Lunn 1988). This suggests that there must be control mechanisms for regeneration 14  within the cut axon tip that are not mediated by the cell body. The onset of changes at the cell body in facial motoneurons has also been shown to occur very rapidly following injury. The activation of ribosomal R N A transcription in the motoneurons begins within as little as 30 minutes post injury, whereas the retrograde transport of a neuronal tracer takes at least 3 hours (Huppenbauer, et al. 2001, Kinderman, et al. 1998). Some factors that regulate the motoneuron response to injury are retrogradely transported as a result of axon injury. These factors are derived from non neuronal cells such as Schwann cells at the site of injury, including; ciliary neurotrophic factor (CNTF), nerve growth factor (NGF) and glial cell-line derived neurotrophic factor (GDNF) (Henderson, et al. 1994, Heumann, et al. 1987, Meyer, et al. 1992, Naveilhan, et al. 1997, Sendtner, et al. 1992, Seniuk, et al. 1992, Stockli, et al. 1991). Another injury signal is that produced by the injury itself. This injury signal, although not yet fully characterized, is believed to be electrically propagated because of its immediate effects (reviewed in Ambron and Walters 1996). Initial somata changes are detectable in the motoneurons as early as 15 minutes post axotomy (Povelones, et al. 1997), which is more than ten times the rate of fast axonal transport. Other signals are only within the distal stump. For instance, the expression of the low affinity neurotrophin receptor p75 is only upregulated if the axons are permitted to grow into the distal stump. Blockade of axonal transport or the inhibition of regeneration using a ligature does not increase p75 expression (Bussmann and Sofroniew 1999). The contribution of each of these signals to the response of motoneurons to injury is poorly understood.  Glial response The activation of astrocytes, as measured by their production of glial fibrillary acidic protein (GFAP), is a hallmark of the response to injury in the brain and spinal cord. The astrocyte reaction to facial nerve lesion in a mouse was initially observed in 1955 by Cammermeyer, who found a slight change in the size of astrocyte nuclei size over the course of 100 days post injury (Cammermeyer 1955). These activated astrocytes produce a variety of neurotrophins, oligodendrocyte and microglial growth factors and proteins that function in survival and activation of the surrounding neurons and glia. (reviewed in Eddleston and Mucke 1993). In addition, reactive astrocytes also increase their expression of N-methyl-D-aspartate receptor (NMDA), perhaps in response to an increased requirement for glutamate uptake (Popratiloff, et al. 1996). The astrocyte response to motoneuron injury is very rapid. In less than one hour of an axon injury, there is an increased expression of the astrocyte gap junction protein connexin 43 (Rohlmann, et al. 1994). Within 24 hours post axotomy there is an increase in the 15  expression of G F A P m R N A and protein in activated astrocytes within the rat facial nucleus (Tetzlaff, et al. 1988). Following resection of the rat facial nerve (to prevent target reconnection), expression of GFAP remains elevated in the facial nucleus for up to one year (Laskawi and Wolff 1996). However, the long term expression of GFAP around mouse facial motoneurons is not known. The astrocytic response to injury is a non proliferating one in adult rat facial motoneurons, however, astrocytes do actively divide and hypertrophy in the neonatal rat following axon injury (Graeber, et al. 1998, Graeber, et al. 1988). The astrocyte response in the adult consists of two distinct phases. In the first phase there is a thickening of the astrocyte processes which are immunoreactive for GFAP. Astrocytes are normally located within the neuropil, but during the second week of injury the astrocytes move to a more perineuronal position when the displacement of the axotomized neurons from their synaptic terminals begins (Blinzinger and Kreutzberg 1968, reviewed in Graeber and Kreutzberg 1988). The second phase begins around three weeks post axotomy, becomes maximal around five weeks and persists for several months. In this phase, the astrocytes form long thin lamellar processes which completely surround, thus enveloping the neurons resulting in a complete separation of the neurons from their afferent axon terminals (Graeber and Kreutzberg 1988). The various stages of the microglial response to facial nerve axotomy are similar to, and therefore relevant to those seen in numerous other nervous system pathologies (Streit and Graeber 1993), thus underlying the extensive use of the facial model in the study of the glia response to injury. Using H-thymidine incorporation studies, microglia were identified as the 3  population of dividing cells within the adult facial nucleus after axotomy (Graeber, et al. 1988, Kreutzberg 1966, as reviewed in Streit and Graeber 1993). Several studies have shown a correlational relationship between the microglial response and regeneration. However, the infusion of a mitotic inhibitor did not affect the rate of regeneration of hypoglossal motoneurons following a nerve crush (Svensson and Aldskogius 1993). Both activated microglia and macrophages have been identified in the newborn rat facial nucleus as early as one day post injury (Graeber, et al. 1998). In the adult facial nucleus, no macrophages are evident and only after several weeks following axotomy are there phagocytic microglia. This is likely in response to the increased motoneuron mortality in the neonatal model that requires macrophages to remove the dead motoneurons. Although the exact mechanism responsible for the induction of the glia response in injured facial motoneurons is unknown, it is suspected to be related to the glycoprotein interleukin-6 (IL-6) (Streit, et al. 2000). IL-6 belongs to a family of neurokines including Leukemia Inhibitory Factor (LIF) and CNTF. In IL-6 knockout mice both GFAP 16  expression in astrocytes and microglia activation are decreased (Galiano, et al. 2001, Klein, et al. 1997).  Regeneration of facial motoneurons  In response to nerve injury, the axons of the PNS undergo Wallerian degeneration. Wallerian degeneration is a required process that removes, recycles and provides a permissive environment for the growth of axons. Following axotomy, both the axon and the myelin degenerate in the distal nerve stump. Inside the basal lamina tubes surrounding the nerve fiber are dividing Schwann cells forming structures termed endoneurial tubes or the bands of Biingner (reviewed in Fawcett and Keynes 1990, Hallpike 1976). In an elegant experiment, Friede and colleagues, regulated the influx of cells into chambers containing degenerating nerves, demonstrating that macrophages are key to the process of myelin phagocytosis and the proliferation of the Schwann cells (Beuche and Friede 1984, Scheidt and Friede 1987). Following axotomy, the axonal response to injury begins within a few hours. In the proximal stump, the axons degenerate back to the first node of Ranvier and then several sprouts from each axon often originate from the node as a result of a gap left by the retracting Schwann cells. The sprouts then begin to grow down through the bands of Biingner towards their targets. Although each axon may produce several axons, only a small proportion of these survive depending on target contact (reviewed in Fawcett and Keynes 1990). If the growing sprouts are prevented from reaching the distal nerve stump by a resection injury, the sprouts are either stunted or turn back on themselves and form a swollen tangled mass at the distal end of the, proximal stump, termed a neuroma (reviewed in Bisby 1995, Devor 1995). Although the regenerating environment consists of myelin and axonal debris, basal lamina, collagen and macrophages, the crucial component of successful regeneration in the PNS are the Schwann cells. Schwann cells play a dual role in the regeneration of injured axons; they express cell adhesion molecules on the surface of their outer membrane and also produce trophic factors (reviewed in Bunge 1993). The basal lamina produced by Schwann cells contains molecules such as laminin and fibronectin that are both promoters of neurite growth in vitro (reviewed in Fawcett and Keynes 1990). The neurite promoting activity of Schwann cells is likely to be in part due to adhesion molecules since antibodies to L l / N g - C A M , N-cadherin and integrins inhibit their neurite growth promoting effects (reviewed in Fawcett and Keynes 1990). The importance of Schwann cell-derived diffusible factors promoting neurite outgrowth was initially suggested 17  by Cajal, who observed little attraction to the distal nerve stump as a result of killing the Schwann cells with chloroform (Cajal 1991). This concept was again demonstrated in vivo: i f a nerve graft is first frozen, killing Schwann cells but leaving the basal lamina, there is no axonal growth. If Schwann cells enter the freeze-killed graft, axonal growth proceeds (Gulati 1988, Hall 1986, Ide, et al. 1983). However, i f the invasion of host Schwann cells into the previously frozen grafts is prevented by cytotoxins, the axons fail to enter the graft (Hall 1986). Within 24 hours of axonal injury, Schwann cells begin to decrease their expression of mRNA for myelin related proteins (LeBlanc and Poduslo 1990). In addition, Schwann cells begin to increase their production of neurotrophic factors such as N G F (Heumann, et al. 1987), B D N F (Meyer, et al. 1992), insulin-like growth factor (Kanje, et al. 1991), LIF (Curtis, et al. 1994) and G D N F (Naveilhan, et al. 1997). Following axonal injury, the normally high level of CNTF begins to decrease in Schwann cells (Sendtner, et al. 1992). Immediately upon injury CNTF is released from injured Schwann cells which may function as a temporary support factor, whose role may then be taken over by the production of other trophic factors (Sendtner, et al. 1992). Unlike CNTF, LIF expression increases in injured Schwann cells and is retrogradely transported back to the neuron cell body (Curtis, et al. 1994). Interestingly, only in the double knockout mouse for both LIF and CNTF is there a large reduction in survival of facial motoneurons following axotomy (Sendtner, et al. 1996). Despite the increase in NGF, the tyrosine kinase receptor for NGF in sensory neurons declines in response to injury (Verge, et al. 1992). Functional blocking antibodies to N G F have little effect on regeneration but NGF appears to be necessary for the proliferation and migration of non-neuronal cells (reviewed in Bisby 1995) . In contrast, the increase in B D N F in Schwann cells, although slower than for NGF, is accompanied by an increase in its receptor expression in facial motoneurons (Kobayashi, et al. 1996, Meyer, et al. 1992). The increase in expression of GDNF in the distal nerve stump peaks at around 7 days post injury (Naveilhan, et al. 1997). GDNF is retrogradely transported to the motoneurons and exogenous application can greatly attenuate the injury-induced decline in choline acetyltransferase in the facial nucleus, as well as increase sciatic nerve regeneration (Naveilhan, et al. 1997, Yan, et al. 1995). In addition, as a result of injury Schwann cells upregulate their expression of several receptors including the low affinity N G F receptor p75 (Taniuchi, et al. 1986) and the neuregulin receptors erbB2-erbB4 that again decline following axonal regeneration (reviewed in Hall 1999). Indeed, the significance of Schwann cells and their role in the regenerative potential of PNS motoneurons has been exemplified by the use of  18  peripheral nerve grafts or transplanted Schwann cells in an effort to bridge CNS injuries (reviewed in Richardson, et al. 1980, Zompa, et al. 1997). The next stage of the neuron response to injury is the regeneration phase. Following a nerve crush injury and to a lesser extent a transection and anastomosis, regeneration and functional recovery are obtained in facial motoneurons. Analysis and quantification of functional recovery of whisker movement is possible following axonal injury in the adult rodent facial model. There are two types of musculature responsible for movement of the large vibrissae of the adult rat. One type of muscle moves the entire mystacial pad and the other moves the individual hair follicle. The most rostral vibrissae lack the individual hair follicle musculature, therefore only the caudal most vibrissae should be used for functional recovery analysis (Dorfl 1982). The vibrissae, under normal conditions, are in a rostral erect position. Contraction of the muscle attached to the follicle pulls the base of the hair caudally and the vibrissae rostrally. Caudal movement is achieved by the passive elastic properties of the surrounding tissue (Dorfl 1982). Therefore injury to the facial nerve results in the absence of movement as well as the maintenance of the vibrissae in the caudal position (Tomov, et al. 2002). For functional recovery analysis, Tomov and colleagues (2002) devised a sophisticated method that included 5 parameters of whisking biometrics. Following axonal injury the vibrissae movements were filmed from above the rat. Using frame by frame analysis of the film, the authors measured vibrissae protraction, which is the forward movement in degrees from a line running rostrocaudal and perpendicular to a line between the rat's two orbits. Vibrissae frequency and amplitude of protraction, angular velocity and acceleration were also measured (Tomov, et al. 2002). Interestingly, in this experiment, facial to facial anastomosis was performed in normal and blind rats. In blind rats functional recovery was better than that of the sighted rats suggesting that the forced use and behavioral demand requirement of the blind rats played a major role in their recovery (Tomov, et al. 2002). In addition, if the contralateral trigeminal nerve is axotomized at the same time as the facial nerve is anastomosed, the accuracy of target reinnervation is increased from 27% to 41% and recovery of whisking is improved. Therefore a lack of sensory input on the uninjured side due to the trigeminal nerve transection and subsequent forced overuse of the injured facial motoneurons contributed to the more rapid recovery and improved accuracy of re-innervation (Skouras, et al. 2002).  19  Age, species and gender differences Age and species The injury response of facial motoneurons differs depending on the age of the animal. Facial motoneurons of neonatal rats and mice begin to die within 24 hours as a result of axonal injury (Lowrie and Vrbova 1992, Soreide 1981). In contrast, the majority of facial motoneurons in the adult rat survive injury (Guntinas-Lichius, et al. 1994). The increased susceptibility to axotomy induce motoneuron death in the neonate has been attributed to the increased dependence of immature neurons on target derived factors (Lowrie and Vrbova 1992). In axotomized neonatal facial motoneurons, this death occurs via an apoptotic mechanism (de Bilbao and Dubois-Dauphin 1996, Rossiter, et al. 1996). In the nerve crush model of injury in the neonate, there is a strong correlation between the age of the animal and survival of the motoneurons. Examining the age related response to nerve crush at a 30 day post crush endpoint, Kruzis and colleagues found that a crush induced a massive motoneuron loss in mice that were less than one week old at the time of injury. Starting at one week of age there was a significant increase in number of motoneurons surviving, as well as a decline in the rate of motoneuron loss (Kuzis, etal. 1999). Cell death can occur via a passive process, termed necrosis, or by an active, regulated process termed apoptosis. Following application of a noxious insult (axonal injury) death by necrosis involves the disruption of membrane integrity resulting in the influx of water and calcium ions culminating in cellular lysis (reviewed in Farber 1990). hi contrast to necrosis, apoptosis occurs via highly regulated subcellular signaling pathways and results in a stereotyped sequence of cellular demise with a distinct morphology including membrane blebbing, condensation of chromatin onto the inner side of the nuclear membrane and finally the pinching off of membrane bound bodies (Kerr, et al. 1972). Apoptosis can be triggered by a variety of stimuli, but it is also a normal occurrence during development. Within various regions of the developing vertebrate nervous system over 85% of the neurons are removed by apoptosis before or just following birth (Oppenheim 1991).This is an important process for ensuring neuron to target specificity during development (Oppenheim 1991). Apoptotic stimuli appear to offset the equilibrium between members of several families of proteins acting as apoptosis regulators. Although there are many stimuli that induce apoptosis, most function through one of two main pathways; via death receptor signaling or through the mitochondrial activation of apoptotic inducing molecules. Both pathways, however, converge on 20  a single family of proteolytic enzymes called caspases. Caspases regulate many of the morphological changes that define apoptosis, including chromatin condensation and D N A fragmentation which culminate in the degradation of the cell. Caspases are involved in both inflammation and apoptosis as a result of injury or noxious stimuli. The caspases involved in apoptosis can be divided into two broad classes; those which participate directly with the disassembly of the cell (effectors) and those that initiate the disassembly of the cell in response to a pro-apoptotic signal (initiators). Caspases all share a similar structure, substrate specificity and an amino acid sequence which contain an active cystine site (Nicholson and Thornberry 1997). Caspases are initially produced as inactive proenzymes (procaspases) ranging from 30 to 50 kD in size. The proenzymes are each comprised of an NH2-terminal domain, a large subunit (~20 kD) and a small subunit (~10 kD). Activation of the procaspases involves the cleavage and separation of the three domains followed by the formation of a heterodimer consisting of the small and large subunit. Two heterodimers then associate to form a tetramer having two independent active sites spanning the large and small subunits (reviewed in Thornberry and Lazebnik 1998). In addition to an absolute requirement for cleavage after an Asp residue, the recognition of at least four amino acids NH2terminal to the cleavage site is required, although this site differs among the caspases (Thornberry, et al. 1997). However, not all proteins having the specific sequence are cleaved by caspases, implying that other structural factors must play a role in substrate recognition. This high specificity for substrate recognition is seen during apoptosis where only a distinct population of proteins is cleaved (Thornberry and Lazebnik 1998). Several types of stimuli can result in the mitochondria's activation of caspases including mild cytotoxic stimuli and trophic factor withdrawal. Three general mechanisms of mitochondrial related cell death are known; 1) disruption of the electron transport chain and ATP production 2) alteration of the cellular reduction-oxidation potential and 3) the release of factors that activate members of the caspase family (Green and Reed 1998). Although a drop in ATP production as a consequence of the disruption in the electron transport chain is foreseeable, it happens relatively late in the apoptotic process. In addition, downstream apoptotic events have a requirement for A T P ; therefore, it is unlikely that the reduction in A T P induces the apoptotic process. The generation of reactive oxygen species (ROS) by the mitochondria also occurs late during an apoptotic event after caspase activation, leaving the functional role of ROS in mediating apoptosis unclear (reviewed in Green and Reed 1998). Factors released from the mitochondria mediate the activation of the initiator and then the effector caspases. Several 21  molecules act upstream of the mitochondria to regulate entry into the apoptotic pathway, which subsequently converges on the activation of death effector caspases. One of the most prominent families of these upstream regulator molecules is the Bcl-2 family, which consists of both pro and anti-apoptotic members. Although the exact mechanisms controlling the mitochondrial release of pro-apoptotic factors remains unclear, several of the factors released have been identified including apoptosis inducing factor (AIF), apoptotic protease activating factor (Apaf1) and cytochrome c. AIF can process caspase 3 but its exact mechanistic role in apoptosis remains unclear (Susin, et al. 1996). Apaf-1, cytochrome c and A T P oligomerise and activate caspase 9 (an initiator caspase) via a caspase recruitment domain (CARD) interaction (Srinivasula, et al. 1998). Caspase 9 can then activate the effector caspase 3 (Hakem, et al. 1998) (Fig. 1.3). Caspase 3 in turn, acts upstream of factors such as D N A fragmentation factor (Liu, et al. 1997) and/or the caspase-3-activated deoxyribonuclease (Enari, et al. 1998), which are involved in executing the changes in nuclear morphology characteristic of apoptotic cell death. One of the central regulators of the apoptotic death of facial motoneurons is caspase 3, which increases in its pro (inactive) form in adult and neonatal motoneurons following axotomy; however, the functionally active form of caspase 3 is only present in the axotomized motoneurons of newborn rats (Vanderluit, et al. 2000). In contrast to the activation of caspase 9 by cytotoxic stimuli, another initiator caspase, caspase 8, is involved in apoptosis through its involvement with the activation of death receptors of t he t umor n ecrosis f actor (TNF) s uperfamily, o f w hich t he t wo m ost c haracterized a re t he Fas/Apol and TNF1 receptors (Ashkenazi and Dixit 1998). A l l the death receptors contain sequence termed the death domain (DD) which mediates the receptors function in apoptosis. In response to receptor-ligand binding there is a clustering of the receptors facilitating the binding to the DD of an adapter protein called F A D D (Fas-associated death domain) with its own DD.  22  Figure 1.3 Apoptosis Pathways Death receptor and mitochondrial activation pathways of apoptosis. Adapted from (McBride, et al. 1999).  23  Death receptor signaling leading to caspase activation  Pro-apoptotic Signal  o  Death stimuli leading to mitochondrial activation of caspases  \  i  /  Altered ratio of pro- to anti-apoptotic Bcl-2 family members  CD  rr  Mitochondrial release of caspase-9 and cyto c  00 i  CD CO  QJ  Apoptosome Formation  CO CO  o o  D_  Oligomerization of long prodomain containing procaspases  cytochrome c Apaf-1 Procaspase-9  cp  d) co Q.  co  CO  Activated Initiator Caspase  lo crmA  • Hrocaspase-3  1  Caspase-3  | |-  Activated Effector Caspase  1  Morphological changes associated with apoptosis  24  NAIP XIAP c-IAP-1 c-IAP-2 survivin  F A D D also contains a region within its caspase recruitment domain (CARD) termed the death effector domain (DED), which binds to the D E D on pro-caspase 8 (reviewed in Ashkenazi and Dixit 1998). The oligomerization of F A D D with caspase 8 results in the self activation of caspase 8 via self cleavage. The caspase 8 pathway then converges with the mitochondrial pathway with caspase 3 (Muzio, et al. 1998) (Fig. 1.3). In addition, caspase 8 can cleave Bid, a pro-apoptotic Bcl-2 family member, resulting in the translocation of Bid from the cytosol to the mitochondria. The mitochondria can then release its apoptotic factors thereby further amplifying the apoptotic cascade (Li, et al. 1998). Facial motoneuron loss in the adult rat is a non apoptotic slow form of neuronal degeneration in which neuron loss occurs over several weeks (Moran, et al. 2001). In adult rats, following nerve resection to prevent target reconnection, one third of the facial motoneurons degenerate and are lost over several weeks, and the surviving ones remain in an atrophied state (Guntinas-Lichius, et al. 1994). Similarly, in the adult mouse, nerve transection leads to a progressive atrophy and loss of the majority (up to 75% loss) of the facial motoneurons if the axons are prevented from reconnecting to their targets (Torvik and Skjorten 1971). This loss, however, is dependent on the specific mouse strain, age of the animals, post injury time and the neuronal counting method employed (Ferri, et al. 1998, Hottinger, et al. 2000, Serpe, et al. 2000). The factors responsible for the differing survival rates between rats and mice and between strains are unknown at this time. The response to injury and the subsequent regeneration has been demonstrated to have age related changes. This may relate to the delayed Wallerian degeneration in aged animals because of a diminished Schwann cell reaction (reviewed in Verdu, et al. 2000). A comprehensive study on the morphological changes associated with a nerve crush injury in young (3 month) and old (15 month) rat facial motoneurons revealed that although there was a slight delay in functional recovery in the older animals, no difference in soma, nucleus or nucleolus size was detected up to 4 days post injury between the animals (Vaughan 1990). After the 4 day time point, however, a slightly prolonged nucleus response and an attenuated nucleolus response were detected in the older animals. B y 28 days post crush, both the nucleus and nucleolus were similar in both age groups (Vaughan 1990). In the mouse, a reduction in the number of uninjured facial motoneurons is only seen in very old animals (28 months) (Sturrock 1988). Interestingly, a recent study on the effects of age and diet on motoneuron survival found that although diet had no affect on the loss of motoneurons following avulsion injury, those rats that were given a restricted diet had 50% less facial motoneurons at two years of age compared 25  to the age matched ad libitum group (Aperghis, et al. 2003). This finding is in contrast to the common view that dietary restriction prolongs the life of the entire organism. Despite evidence that brain injury produces an enhanced glial response in older rats (Kyrkanides, et al. 2001), in the facial model, no such age dependant changes are detectable as a result of axonal injury (Hurley and Coleman 2003). When young (6 months) or old (two years) Fisher 344 and Wistar rats were compared, only in the older Wister rats was the facial motoneuron loss slightly increased. There was, however, a large difference between the two strains of rats, with the Wister rats having more motoneuron loss following nerve injury than the Fisher rats. There were also significantly fewer motoneurons in the uninjured facial nucleus of the older Wistar rats. However, in this same experiment the expression of the neuropeptides calcitonin gene related peptide (CGRP) and galanin or the neurotrophic factor receptors trk-C and the alpha sub-unit of the ciliary neurotrophic factor receptor showed no significant difference in expression in the surviving motoneurons of the young or old rats of either strain (Johnson and Duberley 1998). These and other rodent strain differences are becoming increasingly relevant to the study of nervous system injury. For example, many mouse strains respond differently in neurotrauma research paradigms such as the Morris water maze, Kainic acid induced hippocampal cell death, reperfusion injury and adult neurogenesis (reviewed in Steward, et al. 1999). Gender The gender of the animal can also influence the facial motoneuron response to axonal injury. Many cranial nuclei including facial motoneurons, have androgen receptors (Yu and McGinnis 1986). Early work demonstrated that resection of the facial nerve resulted in twice as much motoneuron loss in female or castrated male rats compared to gonad intact male rats (Yu 1988) . Testosterone treatment ameliorates the loss in females, but only in young females (Yu 1989) . Testosterone treatment in males is intriguing. In young males, exogenous testosterone treatment results in increased motoneuron loss, similar to that seen in castrated males. In older males however, testosterone treatment had no effect on motoneuron loss as result of axotomy (Yu 1990). Testosterone treatment also has no effect on survival of motoneurons in early postnatal rats following axotomy (Yu and Cao 1992). In the hamster however, the rate of regeneration of the facial nerve in female hamsters is greater than that of males, and treatment of the males with testosterone increased the rate of functional recovery following facial nerve crush (Kujawa, et al. 1989). In a later study, in situ hybridization analysis of ribosomal R N A expression as a result of axotomy or axotomy plus testosterone treatment in both male and female hamsters revealed that testosterone treatment augmented ribosomal R N A expression 26  following axotomy at later time points in the females compared to the males (Kinderman and Jones 1994). Testosterone treatment in male hamsters can augment P III tubulin mRNA expression (Jones, et al. 1999) and can also attenuate the expression of G F A P in castrated male hamsters (Coers, et al. 2002). The results of these studies highlight the need for caution on the use of mixed sex or strain groups of experimental animals in addition to age matched controls, especially in long term chronic paradigms.  Chapter overviews and hypothesises The majority of adult facial motoneurons survive axonal injury whereas the majority of neonatal motoneurons die via an apoptotic mechanism as a result of axonal injury. In Chapter 2 of this thesis I hypothesized that the differential susceptibility to axonal injury induced  death in the neonatal rat compared to adult rat facial motoneurons was due to the endogenous expression of the inhibitors of apoptosis proteins; neuronal apoptosis inhibitory protein (NAIP) and the X-linked inhibitor of apoptosis (XIAP). To test this, mRNA expression, using polymerase chain reaction (PCR) and in situ hybridization were performed following nerve transection in the neonate and adult rat. In addition, the protein expression of these two apoptotic inhibitors was characterized as result of injury using western blot analysis and immunohistochemistry. NeuN (Neuronal Nuclei) was initially discovered as a result of attempts to identify novel proteins involved in the regulation of neuronal phenotype. Recently, the NeuN antibody has been used in several areas of study as a specific marker to identify neuronal populations despite the fact that it binds to an as yet uncharacterized antigen/antigens (Mullen, et al. 1992). Despite recent success in our lab using NeuN as neuronal specific marker to differentiate atrophied chronically axotomized rubrospinal neurons from glia (Kwon, et al. 2002), my attempts to use the marker in axotomized facial motoneurons failed as a result of reduced expression of NeuN in the axotomized motoneurons. In Chapter 3 I hypothesized that the NeuN antigen was  differentially regulated as a result of axonal injury in the PNS (facial motoneurons) and CNS (rubrospinal neurons). To examine this, in Chapter 3 I analyzed NeuN immunoreactivity in adult rodent facial motoneurons (a PNS model) following nerve resection or crush and in rubrospinal neurons (a CNS model) after lesion of the dorsal, lateral funiculus at the cervical level of the spinal cord. The specificity of the NeuN antibody was confirmed using Western blot. Previous work in our laboratory determined that contrary to previous reports of the death of 50% of axotomized rubrospinal motoneurons that these motoneurons do not die but rather 27  survive up to one year, although in a severely atrophied state (Kwon, et al. 2002). Therefore, in Chapter 41 hypothesized that chronically axotomized adult mouse facial motoneurons like  rubrospinal neurons do not die but rather remain in an undetectable atrophied state. To test this, chronically axotomized (resected) mouse facial neurons were subjected to another axon injury ten weeks after the first injury. One week after the second axon injury, the size and number of countable motoneurons was determined and the mRNA expression of two regeneration associated genes; GAP-43 and a tubulin were analyzed. This work was performed in two strains of mice, Balb/c and C57BL/6-C3H. Following a nerve resection injury the axons are prevented from reaching their targets and eventually form a neuroma at the distal end of the proximal nerve stump. The results in Chapter 4 of this thesis determined that the subsequent removal of the neuroma initiated a hypertrophy of the cell bodies and the re expression of GAP-43 and tubulin mRNA. In light of these findings, in Chapter 5 I hypothesized that in addition to the positive factors produced  at the injury site that the neuroma itself may function in the regulation of gene expression in chronically axotomized mouse facial motoneurons via the production of target-like repressive signals. To test this, GDNF or the axonal transport blocker colchicine was injected proximal to the neuroma in chronically resected facial nerve of CD-I mice. Facial motoneuron size and the expression of GAP-43 and a tubulin were analyzed in all three conditions. In addition, to determine the regenerative potential of chronically injured motoneurons, a degenerated peripheral nerve graft was sutured onto the nerve stump of chronically transected mouse facial motoneurons. The number of regenerating motoneurons growing into the nerve graft in the chronic animals was compared to an age matched group of acutely injured mice with a similar nerve graft.  28  Chapter 2 Endogenous expression of Inhibitor of Apoptosis Proteins in facial motoneurons of neonatal and adult rats following axotomy  29  Summary  The inhibitor of apoptosis protein (IAP) family members inhibit cell death resulting from a variety of apoptotic stimuli. However, the endogenous expression of neuronal IAPs following axonal injury has not been thoroughly examined. Neonatal facial motoneurons are highly susceptible to axotomy induced apoptosis, whereas adult facial motoneurons survive axotomy. I hypothesized that the endogenous expression of IAPs may be involved in the differential susceptibility of adult and neonatal facial motoneurons to axonal injury. In this Chapter, I examined the expression of two endogenous IAPs, NAIP (Neuronal Apoptosis Inhibitory Protein) and X I A P (X-linked Inhibitory Apoptosis Protein), in adult and neonatal rat facial motoneurons following axotomy. Analysis using RT-PCR and in situ hybridization indicated that NAIP mRNA was increased in neonatal facial nuclei 24 hours post axotomy. In the adult, NAIP mRNA expression increased at 1, 3, 7 and 14 days post axotomy, while little change in the expression of X I A P m R N A was detected at any age or time point time point analyzed. Interestingly, immunohistochemistry using antibodies for NAIP and X I A P , revealed the level of these proteins was higher in the neonatal motoneurons when compared with the adult. Furthermore, immunohistochemistry and western blot for NAIP revealed, in contrast to the observed increase in NAIP mRNA, a decline in the expression of NAIP protein following axotomy in the adult, whereas no change in NAIP protein was detected in neonatal facial motoneurons. X I A P protein, as analyzed by immunohistochemistry and western blot, remained unchanged by axotomy in neonatal motoneurons and adult motoneurons. These results indicate differential expression and/or turnover of IAPs in neonatal versus adult facial motoneurons, and suggest the level of IAP expression alone is not an indicator of cell fate following axotomy.  30  Introduction The Inhibitor of Apoptosis Proteins (LAPs) were initially discovered in Baculoviruses where they function to prevent apoptosis of the infected host cells (Birnbaum, et al. 1994, Clem, et al. 1991, Clem and Miller 1994, Crook, et al. 1993). Several IAPs were subsequently identified in vertebrates, including XIAP, (X-linked inhibitor of apoptosis), NAIP, (Neuronal Apoptosis Inhibitory protein), HIAP1 and 2, (Human Inhibitor of Apoptosis proteins 1 and 2), Survivin, and Livin that all share homology within a region referred to as the Baculoviral Inhibitor Repeat (BIR) domain (Deveraux, et al. 1999, Holcik, et al. 2001). The BIR domains interact with caspases, which are proteases that play a central role in the execution of the apoptotic cell death program (Deveraux, et al. 1998, Deveraux, et al. 1997, Nicholson and Thornberry 1997). For example, the BIR2 domain of X I A P has been reported to directly inhibit caspases 3 and 7 (Chai, et al. 2001, Huang, et al. 2001), whereas the BIR3 domain is specific to caspase 9 (Deveraux, et al. 1999, Sun, et al. 1999). NAIP appears to be a specific inhibitor of caspases 3 and 7 (Maier, et al. 2002, X u , et al. 1997). IAPs may also influence cell survival via caspase independent mechanisms (Mercer, et al. 2000). For example, the expression of X I A P can modulate the activity of the transcription factor NF-kB, resulting in its translocation to the nucleus (Hofer-Warbinek, et al. 2000) and is involved in the induction of c-Jun N-terminal kinase activity (Sanna, et al. 1998). The antiapoptotic effects of N A I P against various apoptotic stimuli have also been extensively demonstrated in vivo (Liston, et al. 1996, Simons, et al. 1999, X u , et al. 1997). Indeed, virally mediated over-expression of NAIP or X I A P in the CNS prevents the death of hippocampal neurons following ischemia (Xu, et al. 1999, Xu, et al. 1997). In axotomized sciatic motoneurons of neonatal rats, viral or neurotrophin mediated over-expression of X I A P , NAIP or HIAP1 or 2 increases motoneuron survival (Perrelet, et al. 2002, Perrelet, et al. 2000). The role of NAIP as a regulator of neuronal survival has also recently been emphasized in mice with deletion of the NAIP-1 gene resulting in an increased susceptibility of hippocampal neurons to cell death following injection of kainic acid (Holcik, et al. 2000). The ability of NAIP and X I A P to prevent apoptosis in vivo prompted us to examine the endogenous expression of these IAPs in the facial motoneurons of neonatal rodents, an axotomyinduced model of neuronal death. The advantage of this model is that the facial nucleus consists of a compact, circumscribed population of neurons within the brainstem. This allows for microdissection and subsequent analysis of mRNA and protein expression. Following axotomy of the peripheral nerve, facial motoneurons of neonatal rodents begin to die within 24 hours, whereas 31  the majority of facial motoneurons (FMNs) of adults survive this particular injury (Lowrie and Vrbova 1992, Soreide 1981). In axotomized neonatal FMNs, death occurs by apoptosis that is most abundant around 28 hours after injury (de Bilbao and Dubois-Dauphin 1996, Rossiter, et al. 1996). Application of the caspase inhibitor, Y V A D - C H O , to axotomized FMNs of newborn mice reduces the number of apoptotic (TUNEL-positive) neurons by 32% at 24 hrs post-injury (de Bilbao and Dubois-Dauphin 1996). The limited specificity of peptide caspase inhibitors, however, has left the confirmed roles of specific caspases in the axotomy induced death of neonatal motoneurons unresolved (Garcia-Calvo, et al. 1998, Thornberry, et al. 1997). Recently our laboratory reported that embryonic and neonatal FMNs expressed high levels of pro-caspase 3 mRNA and full length caspase 3 protein, while levels were low in adult FMNs, which survive axotomy (Vanderluit, et al. 2000). Caspase 3 expression increased in the adult and neonatal motoneurons following axotomy; however, caspase 3 activation occurred only in the axotomized FMNs of newborn, but not adult, rats (Vanderluit, et al. 2000). The importance of caspase 3 in this model was further emphasized by the finding that the death of neonatal FMNs following axotomy is delayed in caspase 3 knock-out mice (Vanderluit, et al. 2000). In the present study, I hypothesized that expression of the endogenous regulators of apoptosis, NAIP and XIAP, may be involved in the differential susceptibility of newborn versus adult F M N s to peripheral nerve axotomy. I therefore analyzed the expression of NAIP and X I A P in F M N s of newborn versus adult rats following a facial nerve axotomy.  32  Materials and Methods Animal care Experiments were approved by the University of British Columbia's Animal Care Committee in accordance with the Canadian Council on Animal Care Guidelines. Sprague Dawley rats (both sexes of neonates; for adults males only (250-300g)) were obtained from the University of British Columbia's Animal Care Center, Vancouver, B C . The rats were maintained in a 12hr light/dark cycle and provided rodent chow and water ad libitum.  Facial nerve axotomy Within hours of birth (PO) neonatal rats were anaesthetized by hypothermia on ice (0°C). The branches of the left facial nerve were axotomized as they exited the stylomastoid foramen, the wound closed with sutures, the animals warmed on a heating pad and returned to their mother. The neonates were then sacrificed at 24 hours post surgery with a lethal injection of chloral hydrate (900 mg/kg). Adult male rats were anaesthetized with an intraperitoneal injection of a mixture of ketamine hydrochloride (72 mg/kg) (Bimeda-MTC, Cambridge, ON) and xylazine hydrochloride (9.1 mg/kg) (Bayer Inc, Etobicoke, ON). The branches of the left facial nerve were axotomized at the stylomastoid foramen and the wound closed with wound clips (Michel, Fine Science Tools, Vancouver, BC). At 12hours, 1, 3, 7 and 14 days post surgery the rats were killed with a lethal injection of chloral hydrate (900 mg/kg). For Reverse Transcription Polymerase Chain Reaction (RT-PCR) or Western blot, the brainstems of both adult and neonate rats were removed, fresh frozen on dry ice and the facial nuclei subsequently micro-dissected. Animals used for immunohistochemistry and in situ hybridization were perfused with 0.9 % phosphate-buffered saline (PBS) followed by cold 4 % paraformaldehyde in PBS (pH 7.4). Brainstems were removed and left to post-fix overnight in 4% paraformaldehyde, cryoprotected for 2 days in PBS containing 12, 16 and 22% sucrose concentrations, then frozen in dry ice cooled isopentane. Cryostat sections, (12 um thick) through the facial nucleus of the adults were collected and placed on Superfrost Plus slides (Fisher Scientific, Houston, TX) for in situ hybridization. For immunohistochemistry cryostat sections, (14 um thick) of adult and neonatal facial motoneurons were placed on the same slide to enable direct comparisons of protein levels. A l l experiments were performed in triplicate.  33  RT-PCR, Southern Blot Total R N A was extracted from pooled control and axotomized adult (n=4) and neonate facial (n=10) nuclei from each time point using TRIzol (Life Technologies, Gaithersburg, MD) and the protocol provided by the manufacturer. The sample was then DNase treated and reverse transcribed into c D N A with RT Superscript (GTBCO/BRL, Gaithersburg, MD) enzyme and nonspecific oligo dT primers. P C R was performed as described previously (Kobayashi, et al. 1996), with the following modifications: NAIP cDNAs were selectively amplified by PCR using 22 mer primers located at base pairs 26-47 (5'-AGG T A A A A G G G A C A C TGT G C A G-3') and at 213-192 (5'-CTC C C G C T A C A T G A A G A A A T C C-3') of the rat NAIP mRNA (Aegera). X I A P cDNAs were selectively amplified by PCR using 21 mer primers located at base pairs 120 (5'-TCT GGT G T G A G T TCT G A T AGG-3') and at 252-232 (5'-TGG A T A C C A CTT A G C A T G CTG-3') of the rat X I A P mRNA (Aegera). PCR amplification was initially performed at 20-35 cycles (30 sec at 94°C, 1 min at 50°C, 1 min at 72°C). Amplification of cDNAs were found to be within the linear range at 25 and 29 cycles for X I A P and NAIP respectively and were used in subsequent comparisons of the controls and axotomized facial nuclei. PCR for Cyclophilin, (Mearow, et al. 1993), was performed as a control to ensure that equal amounts of input c D N A was analyzed. Serial dilution PCR amplifications with 25, 12.5, 6.25 and 3.12 ng of input c D N A were performed in 50 pi reactions as described in Kobayashi, et al. (1996). P C R products were visualized on 2% agrose gels using ethidium bromide and blotted onto membranes (Zeta-Probe, Bio-rad, Hercules CA.). A 50 mer oligonucleotide, corresponding to the NAUVXIAP m R N A and internal to the respective primers and therefore within the amplified P C R product, was end-labeled with  S-dATP using terminal transferase (GIBCO  BRL) as described in (Fernandes, et al. 1998). Membranes were hybridized for 18 hours with the labeled 50 mer in solution then washed as described in (Sambrook, et al. 1989). Membranes were exposed to x-ray film (BioMax M R , Eastman Kodak, Rochester, N Y ) for 2-4 hours.  In situ hybridization The template for the NAIP riboprobe was generated by RT-PCR from rat hippocampal R N A using a primer directed against nucleotide sequences conserved in mouse and human NAIP (Xu, et al. 1997). The X I A P riboprobe template was constructed using a c D N A fragment (275 nt) spanning exons 2 and 3 was RT-PCR amplified from total rat R N A using forward (5'-TCT GGT G T G A G T TCT G A T AGG-3') and reverse (5'-CTC A T C C A A T A G G T A TTT G C A C3') primers. The fragment was then cloned into the pcDNA3 expression vector. The sequence of 34  the fragment was confirmed by oligonucleotide sequencing. Both sense and antisense  P-labeled  riboprobes were transcribed from the linearized template using the MAXIscript in vitro transcription kit (Ambion Inc. Austin, TX). Specific activities ranged from 0.6 to 1.2xl0 c.p.m. 7  per pi. Cryostat sections (12 urn thick) were rinsed, dehydrated and then incubated in hybridization buffer containing a sense or antisense NAIP or X I A P riboprobe (2 x 10 c.p.m. per 6  150 pi per slide) overnight at 50°C. Sections were then treated with ribonuclease A (10 pg/ml) at 35°C for 30 minutes and washed in 2X SSC for 30 minutes. After drying, slides were exposed to x-ray film (Kodak B I O M A X M R , Eastman Kodak, Rochester, N Y ) for 3 days. After developing the film, slides were dipped in Kodak NTB-2 photographic emulsion, diluted 1:1 in distilled water at 42°C, dried and exposed for three weeks at 4°C. The slides were then developed in Kodak D-19, fixed in Kodak fixer, rinsed and counterstained with 0.5% Toluidine blue in 1% sodium tetraborate, diluted 1:100 in distilled water. The slides were dehydrated in ethanol and coverslipped.  Western Blot Tissue from dissected facial nuclei (n=4 for each-time-point) were homogenized on ice in 0.01M Tris buffer at pH 7.0 with protease inhibitors (Roche Molecular Biochemicals, Laval, QU). Protein concentrations were determined using a bicinchoninic colorimetric acid (BCA) assay (Pierce Chemical Company, Rockford, IL). A total of 15 pg of protein was loaded into each well of a 7.5% (NAIP) or 12% (XIAP) SDS-PAGE gel and subjected to electrophoresis. The protein was then transferred electrophoretically to a P V D F membrane (Sigma Aldrich, Oakville, ON) overnight at 4°C. Membranes were then blocked at room temperature for 1 hour in 5% Blotto (5% (w/v) milk powder) in 0.01M Tris Buffered Saline with 0.5% Tween 20 (TBST). The membranes were incubated overnight at 4°C in an affinity purified NAIP rabbit polyclonal antibody (1:2000 dilution), which has been previously shown to selectively recognize NAIP protein (Xu et al., 1997b) or a X I A P rabbit polyclonal antibody (1:3000 dilution) (R&D Systems Minneapolis, M N ) . Membranes were washed three times in 0.5% Blotto in TBST and then incubated for 1 hour in an HRP-labeled anti rabbit antibody (1:2000) (Boeringer chemicals, USA). Finally the membranes were developed using chemiluminecense for 5 minutes as per the manufacturer's instructions (Boeringer chemicals, USA) and then exposed to autoradiographic film (Kodak Biomax, Eastman Kodak Company, Rochester N Y ) . Membranes were stripped, blocked and re-probed with an antibody for actin (ICN Biomedicals, Costa Mesa, CA.) to determine equal loading of samples. Densiometric quantification of the protein bands for the 35  NAIP western blot was determineded using Sigma Scan (SPSS Inc. Chicago IL). The ratios of band intensity of axotomy vs control for NAIP and actin were compared using a Students t test.  Immunohistochemistry Glucose oxidase-diaminobenzidine-nickel (DAB) and Fluorescent immunohistochemistry were performed on slides containing both adult (7 day post axotomy) and neonatal (24 hour post axotomy) tissue using the X I A P rabbit polyclonal antibody (R&D Systems Minneapolis, M N ) and the affinity purified rabbit polyclonal NAIP antibody (Xu, et al. 1997). For D A B , endogenous peroxidase activity was blocked by washing sections for 10 minutes in a 1:4 solution of 0.01M PBS containing 0.3% hydrogen peroxide and MeOH, followed by three washes in PBS. Sections were then blocked for one hour at room temperature in a solution of 5% normal serum in PBS containing 0.1 % Tween 20 (TBST). The sections were then incubated at 4°C with the NAIP antibody (1:2000) or X I A P (1:1000) for 48 hours in 0.01M TBST. Subsequently, sections were rinsed in TBST then incubated overnight at 4°C with biotinlabeled donkey anti-rabbit secondary antibody (1:200; Jackson ImmunoResearch Laboratories Inc, West Grove, PA) for D A B or with Alexa 488 goat anti-rabbit IgG conjugate secondary antibody (1:200; Molecular Probes, Inc, Eugene, OR), followed by 3 washes in PBS. The fluorescent immunohistochemistry slides were then coverslipped in PBS-glycerol. For D A B , the sections were incubated with a Biotin/Avidin system (Vector Laboratories, Inc. Burlingame, CA) for 30 minutes at room temperature, then washed 3 times in 0.01M PBS. The D A B method as described in the manufactures protocol was used to visualize the reaction and the sections mounted in Entellan (BDH Inc, Toronto ON).  36  Results RT-PCR analysis revealed changes in NAIP and XIAP mRNA after axotomy. To determine i f axotomy induced a change in the level of N A I P and X I A P mRNA expression, I performed RT-PCR analysis on control and axotomized facial nuclei from adult and neonatal rats. Cyclophilin m R N A expression, used as internal control, remained unchanged in the adult and neonatal facial nuclei following axotomy (Fig. 2.1-bottom). Serial dilution RTPCR followed by DNA-blotting of the PCR product revealed that axotomy induced an increase in NAIP mRNA expression in the neonate at one day and in the adult at 1 and 7 days post axotomy (Fig. 2.1-top). This elevation in NAIP mRNA expression in the adult was still evident at 14 days post axotomy (data not shown). In contrast to NAIP mRNA, X I A P m R N A expression was not obviously altered by axotomy in the adult or neonate (Fig. 2.1-bottom). mRNA expression in the neonates was not studied at later time-points since many of the neurons are undergoing apoptosis on days 2-3 (Rossiter, et al. 1996). Because of the limited number of samples which can be run on each gel, comparisons can only be made within gels and not between time points or ages. Therefore, differences between contralateral sides are technical in nature and should not be interpreted as an effect of axotomy.  In situ hybridization for NAIP and XIAP mRNA. Examination of m R N A expression in facial motoneurons of adult by in situ hybridization (ISH) corroborated the findings of the RT-PCR analysis. X-ray film autoradiography of the hybridized adult brainstem sections revealed a pronounced increase in NAIP ISH signal on the axotomized side of the facial nuclei 3 days post-injury (Fig. 2.2A). Only a faint increase in X I A P ISH signal was seen in some sections (Fig. 2.2B). Sections of the adult facial nucleus, 7 days post axotomy, stained with Toludine blue showed the concentration of silver grains to lie predominately over the neuronal cell bodies; however, some ISH was detectable in the smaller glia cells (<10pm) (Fig. 2C-F). NAIP mRNA expression in the motoneurons was increased in the axotomized adult facial motoneurons (Fig. 2.2C) compared with the un-injured control side (Fig. 2.2D). However, little change in XIAP expression was detected following axotomy (Fig. 2.2E, F). The Ribonuclease A treatment, which reduced the Toludine staining of the cell bodies, made quantification of the ISH impractical because of the inability to accurately outline the neuronal cell bodies.  37  Figure 2.1 N A I P and X I A P Southern blot Serial dilution RT-PCR for NAIP and X I A P mRNA in neonatal and adult axotomized and control facial nuclei. NAIP mRNA increased in the facial nuclei one day after axotomy in the neonate and in the adult at 1 and 7 days post axotomy (top). Axotomy resulted in little change in X I A P m R N A levels in the neonate and adult facial nuclei (bottom). No change in cyclophilin m R N A was detected following axotomy at any time point examined (bottom).  38  39  Figure 2 . 2 N A I P and X I A P ISH In situ hybridization of adult tissue sections for NAIP and X I A P m R N A in FMNs following axotomy. Low power magnification of the facial nucleus from adult 3 day postaxotomy sections demonstrated a large increase in NAIP m R N A (A) on the axotomized side (left side) compared to the level of X I A P mRNA which was only slightly affected by axotomy (B). Toluidine Blue staining and brightfield microscopy of adult 7 day post axotomy sections revealed that the increase in NAIP mRNA is localized predominately within the larger motoneurons, although the much smaller glia cells (<10um) do appear positive for NAIP ISH signal (C, D). X I A P m R N A is also localized mainly within the motoneurons (E, F). RNase treatment resulted in limited staining of the cell bodies with Toluidine Blue. Scale bar (C-F) = 25 um.  40  NAIP  XIAP  41  NAIP and XIAP protein expression as analyzed by Western blot I analyzed the expression of NAIP and X I A P protein by western blotting to determine i f changes in m R N A resulted in changes in protein expression. Total protein (15 pg per lane) pooled from micro-dissected facial nuclei of adults (n=4), revealed reduced levels of NAIP protein on the axotomized side compared with the control, uninjured side (Fig. 2.3). At 12 hours, 24 hours, 7 and 14 days post-axotomy, there was a marked reduction in NAIP protein (150 kD band) on the axotomized side. The polyclonal antibody for NAIP did recognize smaller protein bands (50, 20 and 10 kD) on the western blot. However, there was no obvious increase or decrease in any of the bands other than the 150 kD band for NAIP, nor were there any additional smaller bands on the axotomized side. A small reduction in protein of the loading control actin was also evident on the axotomized side. However, the reduction in actin was relatively small at all time points when compared with the reduction in NAIP protein. In addition, actin immunohistochemistry of facial motoneurons revealed little change following axotomy (data not shown) despite the known increase in mRNA (Tetzlaff, et al. 1991). This reduction in NAIP protein in adult FMNs, as analyzed by western blot, was reproduced independently in two separate laboratories (G.S., Robertson, Ottawa). Furthermore, densiometric quantification of the protein bands at all time points of the western blot revealed a significant difference in the average ratio of axotomy to contralateral of 0.57 ± 07 S E M for NAIP and 0.84 ± 06 S E M for actin (p<0.05). Therefore, unlike mRNA expression, which in the adult remains elevated for several days, NAIP protein expression is reduced in the axotomized side after injury. Western blot of X I A P protein revealed no obvious change in the adult motoneurons at 7 days post axotomy (Fig. 2.5E).  Immunohistochemistry for NAIP and XIAP in neonatal and adult facial motoneurons Using both D A B and fluorescent-immunohistochemistry the level of NAIP expression was determined in adult and neonatal facial motoneuron tissue sections placed on the same slide to allow for simultaneous processing. Immunohistochemistry using both methods revealed that the endogenous level of NAIP protein appeared much higher in the neonate than the adult (Fig. 2.4). Using fluorescent-immunohistochemistry, with the same intensity light source, the difference in NAEP immunoreactivity in the adult and neonate was clearly apparent (Fig. 2.4AD). Performing DAB-immunohistochemistry under the optimal conditions for the neonatal tissue (Fig. 2.4G, H), there was virtually no immunoreactivity detectable in the adult facial motoneurons (Fig. 2.4E, F). In the adult facial nuclei, fluorescent-immunohistochemistry 42  revealed a decrease in NAIP protein levels on the axotomized side 7 days after injury when compared with the uninjured side (Fig. 2.4A, B). At 24 hours post axotomy in neonatal facial motoneurons there was no detectable change in NAIP immunoreactivity using either fluorescentimmunohistochemistry (Fig. 2.4C, D) or D A B (Fig. 2.4G, H). DAB-immunohistochemistry for X I A P under the optimal conditions for the neonatal tissue (Fig. 2.5C, D), revealed little XIAP immunoreactivity in the adult facial motoneurons (Fig. 2.5 A , B), whereas the neonatal motoneurons had a much higher endogenous level of XIAP protein (Fig. 2.5C, D). Little change in XIAP immunoreactivity was detected as a result of axotomy in adult or neonatal facial motoneurons.  43  Figure 2.3 N A I P Western blot Western blot of NAIP protein expression in adult facial nuclei following peripheral ner axotomy. Axotomy results in a decrease in the expression of NAIP protein starting at 12 hours and continues to decline for 14 days. A slight reduction in the loading control (actin) is evident on the axotomized side; however, this reduction remains constant at all time points examined. Quantification of band intensity revealed a significant decline in the ratio of axotomy to contralateral for NAIP (0.57 + 07 SEM) compared to actin (0.84 + 06 SEM) (p<0.05).  44  12 hr Axo  Con  7 Day  24 hr Axo  Con  Axo  Con  14 Day Axo  Con 150 kD  Actin  ^^^^ ^^^^ ^gii^ ^^^^^^ ^^^fe ^^^^^  ^^^^^  45  ^^^^  r  ^^^F'  42 kD  Figure 2.4 N A I P immunohistochemistry Immunohistochemistry for NAIP protein in adult and neonatal facial motoneurons following peripheral nerve axotomy. The endogenous expression of NAIP protein is substantially higher in neonatal facial motoneurons (C, D), compared to those of the adult (A, B). In adults at 7 days post axotomy, fluorescent immunohistochemistry revealed that NAIP protein expression is reduced on the axotomized side compared to the uninjured contralateral side (A, B). In neonatal facial motoneurons there is little change in NAIP protein expression at 24 hours post axotomy (C, D). The difference in neonatal motoneuron number is a result of a few more motoneurons in that particular picture and is not a situation where more are NAIP immuno positive. Using D A B immunohistochemistry on sections placed on the same slide, NAIP protein expression is almost undetectable in the adult (E, F) at exposure levels optimum for neonatal tissue (G, H). Scale bars = 50 um.  46  Axotomy  Contralateral  47  Figure 2.5 Immunohistochemistry and western blot for XIAP Immunohistochemistry and western blot for X I A P protein in adult and neonatal facial motoneurons following peripheral nerve axotomy. Using D A B immunohistochemistry on sections placed on the same slide, XIAP protein expression is almost undetectable in the adult (A, B) at exposure levels optimum for neonatal tissue (C, D) indicating higher endogenous XIAP protein levels in the neonate motoneurons. Western blot of X I A P protein expression in adult facial nuclei 7 days following peripheral nerve axotomy corroborate immunohistochemistry data that axotomy results in a little change in the expression of X I A P protein in adult facial motoneurons (E). Scale bar = 50 pm.  48  49  Discussion Here I describe, for the first time, changes in expression of NAIP and X I A P mRNA and protein expression within the facial nucleus of adult and neonate rats following axotomy. Studies have demonstrated, in vivo, the anti-apoptotic effects of NAIP and X I A P when introduced by viral vectors (Perrelet, et al. 2000, X u , et al. 1999). More recently it was shown that NAIP and XIAP are essential for the glial cell-derived neurotrophic factor (GDNF) mediated protective effects of motoneurons following injury (Perrelet, et al. 2002). However, little is known about the endogenous expression and regulation of these anti-apoptotic proteins in adult and newborn rat facial motoneurons following axonal injury. I hypothesized that these two apoptotic inhibitor proteins may play a role in the differential survival of adult and neonatal FMNs following axotomy. Immunohistochemical expression analysis indicated a distinct difference in the baseline level of NAIP and X I A P protein in the facial motoneurons of the adult versus the neonate. In contrast to my initial expectations, the level of endogenous NAIP and XIAP protein were higher in neonatal motoneurons, which do not survive axonal injury, compared with adult motoneurons, which do survive axonal injury (Lowrie and Vrbova 1992, Soreide 1981). Furthermore, immunohistochemistry revealed a decline in NAIP expression at 7 days post axotomy in the adult whereas no change was seen in neonatal FMNs at 24 hours following axotomy. In contrast, NAIP mRNA expression in adult and neonatal F M N ' s increased following facial nerve axotomy. Axotomy of the facial nerve resulted in little change in X I A P mRNA or protein in the neonate or adult. In agreement, a recent study utilizing semi-quantitative RT-PCR also determined that X I A P m R N A is not up-regulated in adult mouse FMNs 48 hours after axotomy (Schweizer, et al. 2002). Contrary, at 4 days post axotomy, a significant decrease in XIAP immunoreactivity is detected in neonatal sciatic motoneurons (Perrelet, et al. 2002). This difference is likely explained by the differing time course of axotomy-induced motoneuron death in these two systems. Axotomized neonatal facial motoneurons begin to undergo apoptosis as early as 2 days post injury and the majority are gone by 7 days (Lowrie and Vrbova 1992) whereas neonatal sciatic motoneurons survive from 1 to 4 weeks post injury (Perrelet, et al. 2000). A n interesting and somewhat unexpected finding in the present study is the higher level of endogenous NAIP and X I A P in the neonatal facial motoneurons compared with the adult. In addition, NAIP m R N A increased following axotomy in the neonate, yet FMNs die following axonal injury in the neonate (Lowrie and Vrbova 1992). Increased IAP levels using adenoviral vectors or the application of neurotrophins at the time of injury provide protection against 50  apoptosis following axotomy in sciatic motoneurons in the neonatal rat (Perrelet, et al. 2002, Perrelet, et al. 2000). Thus, it appears that in neonatal FMNs, the endogenous increase in NAIP mRNA and relatively high level of NAIP and X I A P protein is insufficient to prevent apoptosis. Previous studies have also demonstrated higher IAP protein expression in rat neonatal brains than in adult brains (Korhonen, et al. 2001). The high level of caspase 3 in the neonate might explain the requirement for such a high level of IAP expression. Caspase 3, which is a target of the IAPs and expressed an order of magnitude higher in the neonatal FMNs, has a major role in the death of FMNs following axotomy in the neonate (Vanderluit, et al. 2000). Therefore, higher levels of IAPs may be required in the neonate than the adult in an effort to form a balance between the pro and anti-apoptotic factors at this developmental stage. Later time points in the neonate were not analyzed because any reduction in NAIP or X I A P immuno staining at 48 hours post axotomy would likely be a result of the rapid onset of neuronal death at this time point (Lowrie and Vrbova 1992). In the present study there was a reduction in NAIP protein expression in adults following axotomy, as shown by immunohistochemistry and western blotting. The decline in protein expression was in contrast to the increase in NAIP mRNA expression following axotomy as demonstrated using both RT-PCR and in situ hybridization. In models of transient forebrain ischemia, an up-regulation of NAIP protein was observed in those neurons that survive (Xu, et al. 1997). The endogenous role of NAIP was further emphasized by showing that NAIP genedeleted mice are more susceptible to cell death following kainic acid administration (Holcik, et al. 2000). In contrast, in our model there was a decrease in NAIP protein in the adult motoneurons that survive axonal injury. NAIP can protect neurons in both a caspase 3 dependent and independent pathways (Mercer, et al. 2000) and the precise mechanism by which NAIP inhibits apoptosis in these models remains to be elucidated. There are several possible explanations for the protein versus m R N A discrepancy in the adult in the present study. It is conceivable that NAIP protein is cleaved by proteases resulting in the elimination of the antigen. Cleavage of XIAP, resulting in separation of the BIR1-2 domain from the BIR-3 domain has been demonstrated in cells undergoing apoptosis following Fas (CD95) receptor activation (Deveraux, et al. 1999). Therefore, it is possible that NAIP may either bind caspases or act as a caspase substrate and be subsequently cleaved, potentially rendering the NAIP protein unable to bind the antibody. If cleavage of the NAIP protein did occur, additional smaller immunoreactive bands on the western blot may have been expected on the axotomized side. However, additional smaller bands were not detected in the present study, 51  despite using a polyclonal antibody that could recognize many epitopes. IAPs have been shown to catalyze their own ubiquitination and subsequent degradation by proteasomes following apoptotic stimuli in vitro (Yang, et al. 2000). However, NAIP lacks the R I N G domain that is required by the IAPs in the ubiquitination process (Yang, et al. 2000). Therefore the possibility that the reduction of NAIP protein in FMNs following axotomy is a consequence of ubiquitination and proteolysis to prevent caspase activation is remote. Alternatively, following axotomy, NAIP protein could be increasingly transported from the neuronal cell bodies to the axons to inhibit caspase activation. Supporting this, Mattson and colleagues, (1998) have shown caspase activation in synapses and dendrites of neurons following apoptotic stimuli. In addition, there is an increase in the activated form of caspase 3 in axons of olfactory neurons following a bulbectomy (Cowan, et al. 2001). Therefore, studies are required to investigate the activation of caspase 3 in the axons of FMNs and if NAIP or XIAP is transported into the axons following axotomy. The present study demonstrates the differential expression of NAIP and X I A P protein in adult versus neonate FMNs. Previous findings in our laboratory implicate caspase 3 as a principal death effector following neonatal facial axotomy (Vanderluit, et al. 2000). The precise mechanism by which the IAPs function to regulate apoptosis following axotomy in neonatal and adult facial motoneurons remain to be elucidated. Therefore, future studies using gene deletion mice, or the use of antisense techniques to reduce the expression of X I A P or NAIP protein expression following axotomy may provide further clues to the exact role of these proteins in adult and neonatal rodent FMNs.  52  Chapter 3 Axotomy abolishes NeuN expression in facial but not rubrospinal neurons  53  Summary M y initial attempts to use the pan neuronal marker NeuN in an effort to identify injured mouse facial motoneurons failed as a result of a large reduction in NeuN staining in injured facial motoneurons. In this Chapter, I analyzed NeuN immunoreactivity in adult rodent facial motoneurons (PNS model) following nerve resection or crush and in rubrospinal neurons (CNS model) after lesion of the dorsal, lateral funiculus at the cervical level of the spinal cord. The NeuN antibody, which binds to a poorly characterized antigen/antigens is increasingly being used in several areas of study as a specific marker to identify neuronal populations. However, despite the increasing reliance on NeuN as a pan-neuronal marker, changes of NeuN expression following axonal injury have not yet been examined. I found that peripheral nerve resection in the rat and mouse resulted in an almost complete loss of NeuN immunoreactivity in facial motoneurons by 3 days post injury and remained absent at 28 days post resection despite the survival of the neurons as evidenced by neuronal tracing. These results were confirmed with western blot. In the peripheral nerve crush model of injury there was an initial decline in NeuN immunoreactivity in facial motoneurons, but unlike the resection model, NeuN immunoreactivity began to return within 7 days post injury and returned to the un-injured level of expression by 28 days. In contrast, axotomy in the CNS model resulted in little decline in NeuN immunoreactivity in the rubrospinal neurons, even after 28 days post axotomy. These results indicate that NeuN expression in response to axonal injury is different in separate neuronal populations (PNS and CNS) and that care must be taken when addressing cell survival based on NeuN staining alone.  54  Introduction Identification of neurons in injured neuronal populations can be problematic due to severe neuronal atrophy, (Kwon, et al. 2002) which can result in the underestimation of the surviving neuronal population. Traditionally, Nissl stains have been used to obtain estimates of total neuron number. However, due to the injury induced-shrinkage of their somata, Nissl stained neurons can be difficult to differentiate from surrounding glia. Recent work by Kwon and colleagues demonstrated using a combination of methods to distinguish neurons from glia that contrary to previous work, rubrospinal neurons do not die after axonal injury; rather, they remain in a severely atrophied state for at least one year (Kwon, et al. 2002). Several studies have attempted to use neuronal retrograde tracing techniques to circumvent these problems of neuronal identification. However, retrograde tracing has technical limitations, such as efficacy of transport and in some instances the application of the tracer may itself produce an injury. As an alternative method for identifying neuronal populations following injury, many recent studies have attempted to employ immunohistochemical techniques using antibodies to neuron-specific proteins. However, phenotypic markers themselves are often affected by axonal injury. For example, following lesion of the fimbra-fornix, septal neurons lose their expression of the neurotransmitter enzyme choline acetyl transferase (ChAT), a phenomenon initially, but incorrectly, interpreted as neuronal death (Naumann, et al. 1994). Increasingly, the NeuN antibody is being used as a specific marker to identify populations of neurons. These studies have been performed in several areas of interest, including embryogenesis, neurogenesis, ischemia and neurotrauma (Jongen-Relo and Feldon 2002, Kwon, et al. 2002). The NeuN (Neuronal Miclei) antibody was initially described by Mullen et al. (1992) as a result of attempts to identify novel proteins involved in the regulation of neuronal phenotype. The appearance of NeuN is coincident with neuronal differentiation and the neuron's exit from the cell cycle (Mullen, et al. 1992). NeuN immunoreactivity has been detected in several neuronal populations with the exception of Purkinje cells, olfactory bulb mitral cells and retinal photoreceptor cells (Mullen, et al. 1992). The NeuN antibody, which binds to an as yet uncharacterized antigen/antigens, exhibits concentrated staining in the nuclei and diffuse cytoplasmic staining in neurons, and is associated with 3 to 4 bands in the 46 to 48 kDa range on western blots (Mullen, etal. 1992). Despite the increasing reliance on NeuN as a pan-neuronal marker, changes of NeuN expression following axonal injury have not been examined. In the present study, I compared NeuN immunoreactivity over time in the peripheral nervous system (PNS) and central nervous 55  system (CNS) following axotomy. NeuN immunoreactivity was analyzed in rodent facial motoneurons (PNS model) following nerve resection or crush of the facial nerve and in rubrospinal neurons (CNS model) after an axotomy at the cervical level of the spinal cord. The results indicate that NeuN expression is differentially regulated in separate neuronal populations (PNS and CNS) and in different injury paradigms following axonal injury, and that care must be taken when addressing cell survival based on NeuN immunoreactivity alone.  56  Materials and Methods Animal care Experiments were approved by the University of British Columbia's Animal Care Committee in accordance with the Canadian Council on Animal Care Guidelines. Male Sprague Dawley rats (n=7), (250-300g) and male CD-I mice (n=55) (6-8 weeks of age) were obtained from the University of British Columbia's Animal Care Centre, Vancouver, B C . The rats and mice were maintained on a 12hr light/dark cycle and provided rodent chow and water ad libitum.  Rat facial nerve resection Adult rats were anaesthetized with an intraperitoneal injection of ketamine hydrochloride (72 mg/kg; Bimeda-MTC, Cambridge, ON) and xylazine hydrochloride (9 mg/kg; Bayer Inc, Etobicoke, ON). The large branch of the left facial nerve was axotomized distal to its exit from the stylomastoid foramen and a 3-5 mm segment of the distal portion of the nerve was removed to prevent the regeneration of the axons to their targets. A small piece of Gelfoam (Upjohn, Kalamazoo, MI) soaked in 5% Micro Ruby in distilled water (dF^O) (dextran, tetramethylrhodamine, 3000 Mwt; Molecular Probes, Eugene, OR) was placed on the proximal nerve stump. The wound was then closed with wound clips (Fine Science Tools, Vancouver, BC). At 7 days post surgery the rats were killed with a lethal injection of chloral hydrate (900 mg/kg).  Mouse facial nerve resection or crush Adult mice were anaesthetized with an intraperitoneal injection of ketamine hydrochloride (135 mg/kg) and xylazine hydrochloride (6.5 mg/kg). The large branch of the left facial nerve was axotomized distal to its exit from the stylomastoid foramen and a 2-3 mm segment of the distal portion of the nerve was removed to prevent the regeneration of the axons to their targets. A small piece of Gelfoam soaked in 5% Fluro-Gold (Flurochrome Inc, Englewood, CO) in dH^O or 5% Micro Ruby (Molecular Probes) in dH^O was placed on the proximal nerve stump and the wound closed with wound clips. In an additional set of animals, the facial nerve was exposed as mentioned above and the large branch of the nerve was crushed twice for a period of 5 seconds with #5 forceps (Fine Science Tools). In two animals the nerve was exposed and Gelfoam soaked in Fluro-Gold solution was placed directly on the nerve without a resection or crush injury. At 1, 3, 7, 14 or 28 days post surgery, the mice were killed as described above. 57  Mouse rubrospinal axotomy Adult mice were anaesthetized as above and the vertebral column was exposed at the C3C4 level. A right side laminectomy was performed to expose the spinal cord, a small opening was made in the dura immediately lateral to the dorsal blood vessel and a lesion of the right lateral dorsal funiculus was performed using micro-iris scissors. A small piece of Gelfoam soaked in 5% Fluro-Gold in dH^O was placed into the injury site and the wound closed with wound clips. At 1, 7, and 28 days post surgery, the mice were killed as described.  Tissue preparation: Animals used for immunohistochemistry were perfused with 0.9 % phosphate-buffered saline (PBS) followed by cold 4 % paraformaldehyde in PBS. Brainstems were removed, left to post-fix overnight in 4% paraformaldehyde, cryoprotected in a PBS solution containing 22% sucrose and frozen in dry ice-cooled isopentane. For western blot analysis, the brainstems of both mice and rats were removed, fresh frozen on dry ice and the facial nuclei micro-dissected.  Immunohistochemistry The brains were cryosectioned at 14 pm from the caudal to rostral portion of the facial or rubrospinal nucleus. To control for immunohistochemical variability, sections from randomized treatment groups were placed on the same Superfrost Plus slide (Fisher Scientific, Houston, TX). Sections were then blocked for one hour at room temperature in a solution of 5% normal serum in PBS containing 0.1 % Triton X-100 (Sigma-Aldrich) and then incubated with the NeuN monoclonal antibody (1:100) (R&D Systems Minneapolis, M N ) overnight at 4°C. Subsequently, sections were rinsed in PBS, then incubated for 2 hours at room temperature with Alexa 488 goat anti-rabbit IgG conjugate secondary antibody (1:200; Molecular Probes, Inc, Eugene, OR). To identify all motoneurons within the facial nucleus in the facial crush model (without tracer) the slides were also incubated with NeuroTrace (1:300 Molecular Probes), a fluorescent Nissl stain, during the secondary antibody application. To confirm the efficacy of the injury in the facial crush model, immunostaining for ChAT, which declines in facial motoneurons following a crush was performed. Slides were prepared as above, incubated overnight at room temperature with the ChAT antibody (1:200; Sigma-Aldrich) and washed in PBS prior to signal amplification with a Tyramide Signal Amplification Kit as per the manufacture's instructions (PerkinElmer Life Sciences, Boston M A ) . A l l slides were then coverslipped in PBS-glycerol.  58  NeuN immunoreactivity analysis For quantitative analysis of NeuN immunoreactivity following immunohistochemistry for NeuN (outlined above), 3 tissue sections, each a minimum of 42um apart to prevent multiple analysis of individual neurons, from each animal (n=3 animals per time-point, except 28 day rubrospinal, n=2) were randomly selected. Images of the NeuN immunoreactivity for both the injured and the non-injured contralateral, as well as the neuronal tracer or Nissl stain on the injured side were captured using Northern Eclipse software (Empix Inc, Mississauga ON) and a digital camera mounted on a Zeiss microscope (Carl Zeiss, Toronto ON). To enable equal comparisons, care was taken to ensure that both injured and contralateral images were acquired using the same exposure and light intensity settings. Intensity threshold levels of NeuN immunoreactivity within the neurons were determined in Sigma Scan Pro software (SPSS Inc. Chicago, IL) on the injured and non-injured images simultaneously using the non-injured side as the intensity baseline. The NeuN image was then superimposed onto the tracer image and the area of NeuN immunoreactivity within tracer labeled neurons relative to the area occupied by the tracer was quantified. A l l quantifications were done by an individual blind to the treatment groups. Following injury, neurons were often observed to have only partial expression of NeuN immunoreactivity making the establishment of an objective counting method difficult. Therefore, data for NeuN immunoreactivity of the neurons are presented as a percent of total area occupied by the tracer within the neurons as opposed to total number of NeuN positive neurons, which would have failed to detect the more subtle changes in NeuN immunoreactivity. In the facial model, the medial group of motoneurons, which was not injured and maintains its NeuN expression, was used as an additional internal control to eliminate the possibility that reductions in NeuN were of a technical nature rather than a result of the injury. The medial group, however, was excluded from all analysis as were all areas of the image outside the facial nucleus. In the facial nerve crush model, the NeuN immunoreactivity data were presented as a percent of total area occupied by the Nissl stain within the neurons rather than the retrograde tracer. Statistical significance between time points was determined using a student's t test with Sigma Stat software (SPSS Inc.), p< 0.05 were considered significant.  Western Blot For western blot analysis, all branches of the facial nerve were resected and the animals were killed at 7 days post injury. Tissue from dissected facial nuclei, n=4 for each-time-point for rats and n=10 for mice, were homogenized on ice in 0.01M Tris buffer p H 7.0 with protease 59  inhibitors (Roche Molecular Biochemicals, Laval, QU). Protein concentrations were determined using a bicinchoninic colorimetric acid (BCA) assay (Pierce Chemical Company, Rockford, IL). A total of 15 pg of protein was loaded into each well of a 7.5% SDS-PAGE gel and subjected to electrophoresis. The protein was then transferred to a P V D F membrane (Sigma Aldrich). Membranes were then blocked at room temperature for 1 hour in 5% blocking solution (5% milk powder in 0.01M Tris Buffered Saline with 0.5% Tween 20). The membranes were incubated overnight at 4°C with the NeuN monoclonal antibody (1:500 dilution). Membranes were washed three times in 0.5% blocking solution, then incubated for 1 hour with HRP-labeled anti mouse antibody (1:2000) (Boeringer chemicals, USA). The membranes were developed using an E C L chemiluminecense kit (Amersham Biosciences, Piscataway, NJ) and exposed to autoradiographic film (Kodak Biomax, Eastman Kodak Company, Rochester N Y ) .  60  Results Loss of NeuN immunoreactivity following facial nerve resection Facial nerve resection in mice results in little decline of NeuN immunoreactivity at one day post injury but an almost complete elimination of NeuN immunoreactivity by 3 days (Fig. 3.1). The level of NeuN remained low at 7, 14 and 28 days post injury (Fig. 3.1). To test for a possible mouse strain-specific response, these results were confirmed in Balb-C and C57/BL6 mice strains (data not shown). In addition, facial axotomy in the adult rat also induced a rapid decline in NeuN immunoreactivity at 7 days post injury (data not shown, earlier time points were not examined). Application of the neuronal retrograde tracer Fluro-Gold onto un-injured facial nerve was used as a baseline measurement of NeuN to tracer overlap prior to axotomy. Quantification of the level of NeuN immunoreactivity as a proportion of the area occupied by tracer (i.e. in identified, injured neurons) is presented in Fig. 3.1 (bottom). At one day post resection, NeuN immunoreactivity exhibited a small but non significant decline to 45.1% (± 7.4 SD) of un-injured controls. However, a significant decline in NeuN compared to un-injured controls was detected at 3 days (1,6% ± 1.0 SD), 7 days (2.1% ± 0.8 SD), 14 days (3.5% ± 1.3 SD) and 28 days (2.0% ± 0.5 SD) post injury (pO.Ol) (Fig. 3.1, histogram). Western blot analysis of 7 day resected and contralateral (un-injured) protein samples from mouse and rat facial motoneurons confirmed the decline of NeuN immunoreactivity following resection. In both the rat and mouse, a reduction of two of the four bands associated with NeuN and an almost complete elimination of the two larger bands following resection of the facial nerve was evident (Fig. 3.2A).  61  Figure 3.1 NeuN immunoreactivity following facial nerve resection Facial nerve resection resulted in a modest decline of NeuN antigen level at one day post injury but an almost complete elimination of NeuN immunoreactivity by 3 days, which was maintained at 28 days post injury. Quantification of the level of NeuN immunoreactivity as a proportion of the area occupied by tracer revealed significant declines in NeuN compared to tracer alone were detected at 3, 7, 14 and 28 days post injury compared to un-injured controls (histogram, ± SD). * denotes statistical significance (p<0.01). Scale Bar 100 pm  62  ) 63  Figure 3.2 NeuN Western blot and ChAT immunoreactivity Western blot analysis of 7 day resected and contralateral un-injured protein samples from mouse and rat facial motoneurons demonstrated a reduction in both the rat and mouse of two of the smaller four bands and an almost complete elimination of the two larger bands (A). ChAT immunoreactivity 3 days post crush is almost completely eliminated except for those motoneurons in the uninjured dorsomedial subgroups (B). Scale bar =100 urn.  64  Mouse Res  Con  Rat Ras  left-ChAT  . - ?*.. J  «-» Con  right-ChAT  *i  * jr' . . .  .  11 • \« ^  a  £ ^  « * ^ ' *»4 * • '• j , » » . :  .  ^* ** • •  left-NissI  jight-NissI  65  —  »  Transient loss of NeuN immunoreactivity following facial nerve crush In the crush model of axonal injury, the initial decline in NeuN was comparable to that seen following resection. The efficacy of the crush was confirmed by the absence of whisker movement on the ipsilateral side at 3 days post injury and by the loss of ChAT immunostaining in the earlier time points (Fig. 3.2B). NeuN immunoreactivity was measured in Nissl stained uninjured facial motoneurons and used as the control level of expression. The percent overlap of NeuN to Nissl stain was less than that seen in the NeuN vs tracer because, unlike the Fluro-Gold, the Nissl stain also stains the glia cells. No statistically significant change in NeuN immunoreactivity was detected at 1 day post injury compared to the un-injured controls (Fig. 3.3). At 3 days post injury there was a significant decline (6.9% + 1.8 SD) compared to the one day time point (p<0.05). At 7 days post injury, however, there was a return of the NeuN immunoreactivity on the injured side (24.6% ± 6.4 SD) (Fig 3). The return of NeuN immunoreactivity continued at 14 days (27.6% ± 7.5 SD) and by 28 days (35.5% ± 5.8 SD) post crush it is similar to the un-injured control (Fig. 3.3, histogram).  Maintenance of NeuN immunoreactivity following rubrospinal axotomy Axotomy of the rubrospinal tract at the cervical level of the mouse spinal cord resulted in little change in NeuN immunoreactivity in the cell bodies of rubrospinal neurons (Fig. 3.4). At 1, 7 or 28 days post axotomy the neurons exhibited little change in NeuN immunoreactivity when compared to the un-injured contralateral nuclei. This was in stark contrast to the almost complete elimination of NeuN immunoreactivity the facial motoneurons following resection starting as early as 3 days following axotomy. The rubrospinal neurons did, however, exhibit axotomy induced atrophy as has been previously reported (Kobayashi, et al. 1997, Kwon, et al. 2002), indicating they had indeed been injured. Quantification of NeuN immunoreactivity clearly demonstrated the maintenance of the NeuN in rubrospinal neurons following injury, with no statistical differences detectable compared to one day (Fig. 3.4, histogram).  66  Figure 3.3 NeuN immunoreactivity following facial nerve crush Little change in NeuN immunoreactivity was detected at 1 day post facial nerve crush. At 3 days post injury there was a decline in NeuN immunoreactivity compared to un-injured controls. At 7 days post injury, however, there was a return of the NeuN immunoreactivity in facial motoneurons on the injured side. The return of NeuN immunoreactivity continued and by 28 days post crush, it was similar to the un-injured contralateral side. Quantification of the level of the NeuN immunohistochemistry (histogram, ± SD) revealed a significant decline at 3 days post crush. An increase was detected at 7 days post injury, and continued to increase at 14 and 28 days post crush, at which times they were not significantly different from the 1 day time point. * denotes statistical significance (p<0.05). Scale Bar 100 urn  67  Nissl stain  Nissl  1 day  3 day  7 day  NeuN  1 4 day  28 day  68  Figure 3.4 NeuN expression following rubrospinal tract axotomy Axotomy of the rubrospinal tract at the cervical level of the mouse spinal cord resulted in little change in NeuN immunoreactivity in the injured rubrospinal cell bodies. At 1, 7 or 28 days post axotomy, the rubrospinal neurons exhibited little change NeuN immunoreactivity compared to the un-injured contralateral nuclei. Quantification of NeuN immunoreactivity clearly demonstrated the maintenance of the NeuN in rubrospinal neurons with no significant change following injury (histogram, ± SD). Scale Bar 50 pm  69  Discussion The results of this study revealed a differential response of the neuron-specific marker, NeuN, to axonal injury in the CNS and PNS. NeuN expression was dramatically reduced in the PNS following facial nerve injury; whereas, its expression was maintained in the CNS following lesion of the rubrospinal tract. In addition, the nature of the PNS injury (crush vs resection) itself elicited a differential response of NeuN immunoreactivity within facial motoneurons. After resection of both the rat and mouse facial motoneurons, there was a near complete and long term loss of the NeuN expression, as demonstrated by both immunohistochemical and western blot analysis. In contrast, crushing the mouse facial nerve induced a transient decline in NeuN immunoreactivity that returned to initial levels by one week post injury. Fluorescent Nissl stain and retrograde tracers demonstrated the persistence of injured facial motoneurons, despite their near complete loss of NeuN. The increased responsiveness of NeuN immunoreactivity in axotomized neurons of the PNS compared to those of the CNS may relate to differences in the cellular response of both the injured neuron and the injury site environment. For example, peripheral nerve injury results in the removal of target (muscle) derived factors, and a concomitant increase in trophic molecules released by Schwann cells associated with the injured axons (reviewed in Fernandes and Tetzlaff 2000). Such factors are believed to play a role in many of the m R N A and protein changes exhibited by injured peripheral neurons. The cell body response to axonal injury also differs in these two populations of neurons. For example, in the PNS, axonal injury is often correlated with the expression of regeneration associated genes, such as actin, tubulin and proteins expressed at the growth cone including GAP-43 and CAP-23 (Cortical-Associated Protein of 23 kDa) (reviewed in Fernandes and Tetzlaff 2000). Following axonal injury in the CNS, these changes are often less intense, more transient or fail to occur, as is the case when the axonal injury is very distal from the cell body (Fernandes, et al. 1999). Thus, the differential expression of NeuN detected in the PNS and CNS as a result of axonal injury could be regulated by factors specific to the neuron soma and/or the axonal environment. The different pattern of NeuN expression in facial motoneurons following two different injuries is also intriguing and may be related to the neurons ability to reconnect with their targets following resection versus crush injury. In the resection model, reconnection of the axons to their targets is prevented by the removal of a small segment of the nerve. In contrast, the crush model allows the re-growing axons to reach their targets within a week in the mouse model and 12-14 days in the rat model (Miller, et al. 1989). The transient decline and re-expression of NeuN in the 71  crush model suggests that target derived factors may regulate NeuN expression. Thus, inability of the axons to reach their targets in the resection model could underlie the sustained decline in NeuN expression. The differential loss of NeuN between the CNS and PNS injury models and between the resection and crush model in the PNS may not simply be a consequence of target reconnection. It is tempting to speculate that injury-induced changes in NeuN expression correlate with the injured neuron's regenerative state. Axotomized rubrospinal neurons, which mount a minor, transient regenerative response (Tetzlaff, et al. 1991), exhibited no change in NeuN expression. In contrast, both resected and crushed facial motoneurons, which initiate aggressive regenerative responses (Tetzlaff, et al. 1991), exhibited a dramatic decline in NeuN expression. This loss of NeuN expression remained unchanged following resection, which prevents target reconnection and maintains the motoneurons' regenerative response. However, NeuN expression returned to control levels following facial nerve crush at a time that correlates well with the reconnection of the crushed axons to their targets, when the regenerative response declines (Ferri, et al. 1998). Further work will be required to determine a potential causal relationship between the regenerative state of injured neurons and NeuN expression, which, given its nuclear localization, could conceivably be involved in the regulation of gene expression (Mullen, et al. 1992). Regardless of the mechanisms regulating NeuN expression, an implication of the present findings is that the use of NeuN as a single specific phenotypic marker to assess the fate of injured neurons may lead to erroneous conclusions regarding the state of injured neuronal populations. In this respect, NeuN may be similar to choline acetyl transferase (ChAT), which also decreases in response to axonal injury. Fimbria-fornix lesion, for example, leads to a massive reduction in ChAT immunoreactivity in neurons of the septum, a phenomenon that was initially interpreted as cell loss (reviewed in Naumann et al., 1994). However, when later timepoints were analyzed, it became apparent that ChAT immunoreactivity recovered, indicating the injured neurons survived the injury and transiently down-regulated their ChAT phenotype (Naumann, et al. 1994). Thus, loss of phenotypic markers such as ChAT and NeuN, when used to identify neurons following axonal injury does not necessarily reflect the amount of cell loss; rather, it may reflect the physiological state of the neuron. Interestingly, the use of histochemical staining methods, like Nissl staining, often fail to reveal reliable numbers when applied after injury, as evidenced following chronic axotomy of rubrospinal neurons. Previous studies using Nissl stains concluded that 40-50% of rodent rubrospinal neurons died within 4 months of axonal injury 72  (Houle and Y e 1999, Mori, et al. 1997). However, using NeuN staining in conjunction with retrograde tracing and Nissl stain, we found no evidence for rubrospinal neuron death (Kwon, et al. 2002). In this instance, and in contrast to facial motoneurons, NeuN appears to be a reliable indicator of neuronal survival following axonal injury. In light of NeuN's widespread use as a pan-neuronal marker and its differential susceptibility to axonal injury in different neuronal populations, further characterization of NeuN appears warranted. Furthermore, this demonstration of axonal injury-induced loss of NeuN immunoreactivity from surviving peripheral neurons indicates caution must be taken regarding the use of a single phenotypic marker such as NeuN as a method to detect the presence of neurons following experimental treatments.  73  Chapter 4 Axonal re-injury reveals the survival and gene re-expression of chronically axotomized adult mouse motoneurons  74  Summary  Recently, our laboratory reported that chronically axotomized rubrospinal neurons survive for up to one year in an atrophied state. This finding contrasted previous work suggesting the death of up to 50% of the neurons over time. In the adult mouse, the majority of facial motoneurons appear to be lost as a result of chronic nerve resection. In this Chapter, I sought to determine if chronically resected adult mouse facial motoneurons, like rubrospinal neurons, survive in an atrophied state. To test this hypothesis, I asked whether a second nerve injury, ten weeks after an initial nerve resection, could stimulate a regenerative cell body response. After chronic resection (10 weeks), mouse facial motoneurons underwent atrophy resulting in a loss of countable neuronal cell bodies. In addition, the motoneurons failed to maintain their initial increase in expression of GAP-43 and a-tubulin mRNA. Re-injury of 10 week chronically resected facial motoneurons by the removal of the neuroma reversed the atrophy of the cell bodies, increased the percentage of identifiable cell bodies from 36% of contralateral to 79% in C57BL/6-C3H mice and from 28% of contralateral to 40% in Balb/c mice. Moreover, the reinjured motoneurons displayed an increase in GAP-43 and oc-tubulin m R N A expression. The results of this study indicate that a second axon injury stimulates regenerative cell body responses in chronically resected mouse facial motoneurons and suggest previous studies using this model may have overestimated the number of dying motoneurons.  75  Introduction Following axotomy of the facial nerve, facial motoneurons of neonatal rats and mice die within a few days by an apoptotic mechanism (Lowrie and Vrbova 1992, Soreide 1981, Vanderluit, et al. 2000). In contrast, in adult rats following a nerve resection, in which a portion of the nerve is removed to prevent target reconnection, only one third of the facial motoneurons are lost over a period of several weeks (Guntinas-Lichius, et al. 1994). In adult mice, nerve resection leads to progressive atrophy and the loss of the majority of the facial motoneurons. However, this loss is dependent on the mouse strain, age of the animal, and the post axotomy time (Ferri, et al. 1998, Hottinger, et al. 2000, Serpe, et al. 2000). Several lines of evidence have demonstrated the effectiveness of various treatments resulting in prolonged survival of mouse facial motoneurons. Many of these treatments have utilized transgenic, viral over-expression or the exogenous application of trophic factors that are normally produced or induced as a result of injury such as Glial Derived Neurotrophic Factor (GDNF), Ciliary Neurotrophic Factor (CNTF) and numerous cytokines associated with the immune system (Hottinger, et al. 2000, Schweizer, et al. 2002, Serpe, et al. 2000). In the central nervous system (CNS), previous studies concluded that 40-50% of rodent rubrospinal neurons were lost within 4 months of axonal injury (Houle and Ye 1999, Mori, et al. 1997), although the precise mechanism of death was never demonstrated. Recently, our laboratory provided evidence that rubrospinal neurons survive axotomy and remain in an atrophied state for up to one year (Kwon, et al. 2002). In support of this concept, earlier work in the septum demonstrated that the application of nerve growth factor (NGF), even after a 95 day delay, reversed the axotomy induced atrophy and lead to the re-appearance of most of the injured medial septum cholinergic neurons (Hagg, et al. 1989). A clear demonstration of a mechanism of death, to account for all of lost neurons in many adult models of axotomy, remains to be shown. In addition, the number of neurons lost as a result of injury is confounded by the protracted fashion by which they are lost and the technical difficulty detecting atrophied neurons. Therefore, I hypothesized that following chronic nerve resection, mouse facial motoneurons, like rubrospinal neurons, are rendered undetectable because of their extreme atrophy and are therefore assumed dead. To test this, I re-injured chronically resected facial motoneurons by removing the neuroma and counted the number of surviving motoneurons one week later (11 weeks post first injury). These motoneuron counts were compared to the number of surviving mouse facial motoneurons at 11 weeks post resection 76  injury (no second injury). Axonal regeneration in the adult mammalian peripheral nervous system (PNS) is correlated with the expression of several genes, including the cytoskeletal protein tubulin and the axonal growth cone protein GAP-43 (reviewed in Fernandes and Tetzlaff 2000). Therefore, to investigate the response of chronically injured motoneurons to a second axon injury, I analysed and compared the mRNA expression of two regeneration associated genes, GAP-43 and a-tubulin.  77  Materials and Methods Animal care Adult C57BL/6-C3H, Balb/c and CD-I mice were used in this study. The mice (8-12 weeks of age) were obtained from the University of British Columbia Animal Care Facility. Animals were housed in a 12h: 12h light:dark cycle and provided with standard rodent diet and water ad libitum. A l l experiments were performed in accordance with the guidelines of the Canadian Council for Animal Care and local animal care committees.  Re-injury of chronically axotomized mouse facial motoneurons Adult mice were anaesthetized with an intraperitoneal injection of a mixture of ketamine hydrochloride (135 mg/kg, Bimeda-MTC, Cambridge, ON) and xylazine hydrochloride (6.5 mg/kg, Bayer Inc, Etobicoke, ON). The large branch of the left facial nerve was axotomized approximately 3-4 mm distal from the stylomastoid foramen and a 2-3 mm segment of the distal portion of the nerve was removed to prevent the regeneration of the axons to their targets. The skin incision was then closed with silk sutures. Over the subsequent ten weeks, no mouse reestablished whisker movements ipsilateral to the nerve resection. Ten weeks following nerve resection, the chronically axotomized mice were re-anaesthetized and the resected nerve reexposed. In each case, the proximal nerve stump had formed a neuroma, and had not regenerated. Following visual inspection of the neuroma, the wound was immediately closed in one half of the experimental animals ("chronic axotomy" group). In the other half, the proximal nerve stump was re-axotomized, the neuroma removed, and the wound closed ("chronic axotomy plus second axotomy" group). One week later (i.e., 11 weeks after the initial resection), both groups were killed with a lethal injection of chloral hydrate (900 mg/kg). To examine the extent of gene re-expression in neurons as a result of axotomy, a third axotomy was performed in another subset of Balb/c mice (n=4). The mice were maintained for an additional 10 weeks following the second axotomy and then a third axotomy with the removal of the neuroma was performed on two of the mice. One week following the third axotomy both groups of animals were killed.  Preparation of tissues Animals were trans-cardially perfused with phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA). The brainstems were removed, post-fixed 8-12 hours in PFA, and then cryoprotected in a 22% solution of sucrose in PBS. Cryoprotected tissues were then frozen 78  using isopentane cooled on dry ice. Brainstems were cryostat sectioned in a coronal orientation at a thickness of 14 pm. Sections through the facial nuclei were collected onto Superfrost Plus slides (Fisher Scientific, Houston, TX) and stored at -80°C. Different treatment groups were randomly arranged on the microscope slides to eliminate any systematic handling biases. Cellular morphology was examined by staining slides with 0.2% cresyl violet. Slides were first placed in distilled water for 2 minutes and then into the cresyl violet for 5-10 minutes. Slides were then rinsed in water and dehydrated in increasing concentrations of ethanol and then chloroform. The slides were then coverslipped using Entellan (BDH, Toronto, ON). Motoneuron numbers were assessed by counting the number of neuronal profiles containing a visible nucleus/nucleolus on every second 14 pm section throughout the length of the facial nucleus for each mouse. Cell sizes were measured by circling the motoneuron profiles using Northern Eclipse software (Empix Imaging, Mississauga, ON) in 3 sections per mouse (minimum 42pm apart). Motoneuron counts and cell sizes were expressed as a percentage of the contralateral and graphed as the mean and standard error of the mean. For counts and cell size measurements each treatment group consisted of 5 mice and significant differences were determined using a Students t-test (p<0.05).  In situ hybridization (ISH) ISH with oligonucleotide probes was used to detect changes in the levels of GAP-43 and a-tubulin mRNAs. The GAP-43 52mer (5'- G C A T C G G T A G T A G C A G A G C C A TCT C C CTC CTT CTT CTC C A C A C C A T C A G C AA-3') is 100% complementary to GAP-43 mRNAs in mouse (bases 364-415, GenBank accession no. J02809) (Cimler, et al. 1987). To measure atubulin expression in mouse tissues, a 45mer (5'-ATC G T G C G C T T G G T C T T G A T G G T G G C A A T G G C A G C A T T G A C A TCT-3') that was complementary to multiple a-tubulin isotypes, including ma2 (bases 1057-1101, GenBank accession no. M28727; 45/45 bases complementary) (Lewis, et al. 1985), ma6 (bases 1021-1066, GenBank accession no. M13441; 2 mismatches at bases 3 and 6 of oligonucleotide) (Villasante, et al. 1986), m a l (bases 1026-1071, GenBank accession no. M28729; 2 mismatches at bases 3 and 6 of oligonucleotide) (Lewis et al., 1985), and ma7 (bases 1017-1062, GenBank accession no. M13443; 3 mismatches at bases 3, 30, and 33 of oligonucleotide) (Villasante et al., 1986) was used. Oligonucleotide probes were end-labeled with S-dATP to an activity of at least 600,000 35  cpm/pl using the terminal transferase enzyme. Hybridization of radioactively labeled 79  oligonucleotides to tissue sections was performed using the protocol of Verge (Verge, et al. 1992). Kodak NTB2 photographic emulsion, diluted 1:1 with double distilled water, was used for autoradiography of hybridized sections. Exposure times were approximately 1-2 days for atubulin or 5-8 days for GAP-43. Autoradiographic ISH signals were analyzed as described previously in (Fernandes, et al. 1998). Quantifications were done for approximately 25-30 cells per facial nucleus (n=4), and was limited to those cells with a visible nucleus. Values were graphed as signal per neuron using a Login scale. For each probe, at least three sections more than 100 pm apart were quantified per animal. Only those neurons in the lateral portion of the facial nucleus were analyzed, as the medial group was not subjected to an axon injury. Identification of neuronal cell bodies was accomplished using 0.2% cresyl violet as outlined above.  GFAP and F480 immunohistochemistry To distinguish severely atrophied facial motoneurons from similar sized glia cells, in Balb/c mice, a small piece of GelFoam (Upjohn, Kalamazoo, MI) soaked in a 5% FluoroGold in distilled water (dH20) (Flurochrome Inc, Englewood, CO) was placed on the nerve stump at the time of the first injury. In addition, a small piece of GelFoam soaked in 5% Micro Ruby in distilled water (dH20) (dextran, tetramethylrhodamine, 3000 Mwt; Molecular Probes Inc, Eugene, OR) was placed on the proximal nerve stump at the time of the second axon injury to retrogradely label the neurons. Following tissue preparation (outlined above) the tissue sections were first blocked for one hour at room temperature in a solution of 5% normal serum in PBS containing 0.1 % Triton X-100 (Sigma-Aldrich, Oakville, ON). The sections were then incubated overnight at room temperature with the rabbit polyclonal antibody for Glial Fibrillary Acidic Protein (GFAP) (1:500; Dako, Glostrup Denmark) to identify astrocytes, the rat polyclonal antibody F480 (1:50; Serotec Inc., Raleigh NC) to identify microglia, or the mouse monoclonal antibody NF200 (Sigma-Aldrich, Oakville, ON) for the identification of the facial motoneuron axon tracts within the brainstem. Subsequently, sections were rinsed in PBS then incubated for 2 hours at room temperature with the appropriate host IgG secondary antibody conjugated to Alexa 488, anti- Cy3, or A M C A (1:200; Molecular Probes Inc, Eugene, OR), followed by 3 washes in PBS. To identify motoneurons within the facial nucleus in the animals without tracers, the slides were also incubated with NeuroTrace (1:300 Molecular Probes), a fluorescent Nissl stain, during the secondary antibody application. A l l slides were then coverslipped in PBSglycerol. Quantification of G F A P immunoreactivity was performed on 3 sections per animal 80  (n=3 per time point) by an individual blind to the treatments. Intensity of G F A P signal was measured in a 4700 p m area in all sections within the same lateral portion of the facial nucleus. 2  The same threshold values were used on both the injured and contralateral side (ensuring that the contralateral side had a value of zero). Intensity values are expressed as a percentage of the total (4700 pm ) area that was positive for GFAP. 2  Photomicrograph preparation Images were captured using a SPOT digital color camera (Diagnostic Instruments, Inc., MI) and standard fluorescent or darkfield/brightfield microscope settings. Montages of images were generated into figure plates using Adobe PhotoShop (Adobe Systems Incorporated, San Jose, CA). Any adjustments of brightness and intensity were made equally to all treatment groups.  81  Results A second axon injury reverses the cell body atrophy in chronically axotomized mouse facial motoneurons Eleven weeks following resection of the adult C57BL/6-C3H mouse facial nerve, the chronically axotomized facial nucleus displayed a decrease in the number and size of Nisslstained motoneuron profiles (Fig. 4.IB). Atrophied (shrunken) motoneurons were located within the intermediate and lateral sub-groups (right side on all images). Those motoneurons in the medial subgroups with axons extending to the auricular branch were not injured. Eleven weeks after nerve resection, relatively fewer motoneuron cell bodies were observed in the intermediate and lateral sub groups (Fig. 4.IB), and these stained only faintly with the Nissl stain, when compared to the normal facial nucleus contralateral to axotomy (Fig. 4.1 A). I hypothesized that mouse facial motoneurons, like rubrospinal motoneurons, do not die following a chronic injury, but rather remain in a severely atrophied state and thus escape detection. To test this prediction, a chronic axotomy model was used to permanently separate axons from their target musculature. At 10 weeks after the initial axotomy we performed another axon injury by removing the neuroma that had formed on the proximal stump. The appearance of the chronically axotomized C57BL/6-C3H mouse facial motoneurons was markedly altered by cutting the neuroma from the proximal nerve stump one week prior to the 11 week endpoint (Fig. 4.1, bottom). Specifically, the apparent motoneuron number, size, and Nissl staining intensity were all increased by re-injury of the chronically axotomized axons. Moreover, the motoneuron cell bodies appeared even larger than the non-injured motoneurons contralateral to axotomy (compare Fig 4.1, bottom and top). Since mice display significant strain differences in a variety of responses, including injury responses (Steward, et al. 1999), I repeated these experiments using Balb/c mice, and found a similar pattern of results albeit less pronounced than the C57BL/6-C3H. A l l identifiable motoneurons, with the exclusion of those in the uninjured dorsal medial group, in three tissue sections per animal were used for measurement of the mean cross-sectional motoneuron size (Fig. 4.2A). Measurement revealed a shrinkage in the average size of the identifiable chronically axotomized motoneuron cell bodies, to approximately 80% of contralateral in both C57BL/6-C3H and Balb/c strains. Re-injury of the chronically axotomized motoneurons reversed their atrophy significantly to 123% ± 7% (C57BL/6-C3H) (pO.Ol) or 95% ± 6% (Balb/c) of non-injured contralateral motoneurons. Since loss of chronically axotomized facial motoneurons has been reported in a variety of studies, I determined the number of identifiable chronically axotomized motoneurons that were 82  present after 11 weeks, with or without a second injury. I found that compared to the contralateral un-injured side 36% ± 5% (C57BL/6-C3H) or 28% ± 2% (Balb/c) of countable motoneurons remained in the chronically axotomized facial nucleus, in agreement with other studies(Serpe, et al. 1999, Serpe, et al. 2000). However, the number of identifiable motoneurons was significantly increased to 79% ± 4% (C57BL/6-C3H) or 40% ± 1% (Balb/c) of the contralateral side following a second axotomy (p<0.01). Data are summarized in Fig. 4.2B. Binning of the cell size data illustrates that the generalized shift towards smaller cell sizes in chronically axotomized C57BL/6-C3H mice (Fig. 4.2C) is completely reversed by the second injury (Fig. 4.2D). A similar, though less dramatic, effect is observed in the Balb/c background strain (Fig. 4.2E,F).  83  Figure 4.1 Survival of chronically injured mouse facial motoneurons Three Nissl stained facial nuclei are shown, with all images having the orientation of the medial on the left and the lateral on the right. There was a high degree of atrophy in neurons that projected axons into the chronically axotomized large branch of the facial nerve (right side of images), whereas neurons, on the lower left of all images that projected to the smaller nonaxotomized auricular branch appeared healthy. Chronically axotomized neurons that received a second axon injury appeared larger and more numerous (C) than chronically injured (B) or nonaxotomized neurons on the contralateral nucleus (A). Scale bar=100 pm  84  85  Figure 4.2 Motoneuron counts and size Quantification of mean cell body size of chronically axotomized motoneurons and chronically axotomized motoneurons that had a second axon injury is expressed as a percent of uninjured contralateral motoneurons (A). In C57/C3H mice, a second injury significantly increased the average cell body size from 80% to 123% of the contralateral un-injured side. In Balb/c mice a second axon injury had little effect on the size of chronically axotomized motoneurons from 84% to 95% of contralateral. In C57 mice a second axon injury significantly increased the number of countable motoneurons from 36% to 79% of contralateral (B). A second axon injury in Balb/c mice also significantly increased the average number of countable motoneurons from 28% to 40%. Analysis of cell size distributions of axotomized and contralateral facial motoneurons are shown in C-F. Chronic axotomy shifted the distribution of cell bodies to smaller sizes (C,D). In motoneurons receiving a second axotomy this shift was prevented (E,G). * Denotes statistical significance. (p<0.01) Scale bar 100 pm.  86  B  Mean facial motoneuron size  Mean facial motoneuron counts  • Chronic axotomy  140.0  • Chronic axotomy  • Chronic axotomy + 2nd injury  *  n t_ aj ro  • Chronic axotomy + 2nd injury  ro t_ c o o  C57  Balb/c  C57  D  C57 motoneuron size - Chronic Axotomy 50  Balb/c  C57 motoneuron size - 2nd Injury 50  • Contralateral  40  • Contralateral  40  • Chronic axotomy  30  ^ o  20  a  10  CD  • Chronic axotomy + 2nd injury  30 20  cr  10 -i  0  P  <  F  <  ?  <>* -**V  f  area um  f area |im  Balb/c motoneuron size - Chronic Axotomy  Balb/c motoneuron size - 2nd Injury  50 • Contralateral  40 -  • Contralateral  axotomy | Chronic axoton  T  I Chronic axotomy + 2nd injury  30 -  Jiliw  20 10 0 -  f J* f "  f  ^  f ffi*  ^  #  jjV* ^  ^ area  #  c*.  &  ^ #  "  um  area um  2  87  ^ •>* * V  GAP-43 and a-tubulin in situ hybridization To determine the expression of regeneration associated genes in these chronically injured C57BL/6-C3H motoneurons, sections through the facial nucleus were processed for GAP-43 and a-tubulin in situ hybridization. At ten weeks following facial nerve resection a-tubulin expression was similar in chronically axotomized mouse motoneurons (Fig. 4.3 A , upper right) and their contralateral controls (Fig. 4.3A, upper left), but was strongly up-regulated following a second axonal injury (Fig. 4.3A, lower right). Similarly, GAP-43 hybridization signals in the chronic axotomy (Fig. 4.3B, upper right) and in the contralateral (Fig. 4.3B, upper left) facial nuclei were comparable, but were highly up-regulated in chronically axotomized motoneurons that were re-axotomized (Fig. 4.3B, lower right). Quantification revealed that following a second axonal injury, the majority of chronically injured motoneurons expressed a 3 to 10 fold increase in a-tubulin (Fig. 4.3C, bottom), and a 10 to 100 fold increase in GAP-43 ISH compared to the contralateral controls (Fig. 4.3D, bottom). hi a subset of Balb/c mice a third axotomy was performed, ten weeks after the second injury (21 weeks after the first). Similar to the results above, at ten weeks post second axotomy, a-tubulin in situ hybridization was low, but increased one week after a third axotomy (Fig. 4.3E). GAP-43 m R N A expression was not analyzed in these mice.  88  Figure 4.3 GAP-43 and a-tubulin in situ hybridization Darkfield micrographs of a-tubulin and GAP-43 ISH in chronically and re-axotomized facial nuclei (A,B). Low levels of a-tubulin and GAP-43 signal are present in both the contralateral (left panels) and chronically axotomized animals. Chronically axotomized motoneurons that received a second axon injury had a marked up-regulation of a-tubulin and GAP-43 signal (A,B, bottom right panels). Quantification of a-tubulin ISH demonstrated a decline in chronically axotomized motoneurons compared to their contralateral non-injured counterparts (C, top). Following a second axonal injury, the majority of chronically injured motoneurons showed 3 to 10 times more a-tubulin signal than contralateral (C, bottom). ISH quantification of GAP-43 signal in chronically axotomized motoneurons displayed equally low levels as their contralateral non-injured counterparts (D, top); however, this was up-regulated between 10 and 100 fold by a second axonal injury (D, bottom). Eleven weeks post second axotomy, a-tubulin is reduced below the level of the contralateral side. Following a third axotomy again a-tubulin is increased (E). Scale bar 100 pm  89  a-tubulin  GAP-43 B !;  '* -V * :» * . - , ^ V* |  contralateral  _  qhrortic Ax  *  contralateral  ' V-*. contralateral  ^pntagfttoral  2  D  1.5 1  chronic &x +  .  2r6l 2tac *  2.5 1.5  -I  TV.,-1«I  ' • '  * .«< ?.*>' "* •  i  . ' •»',  chronic Ax  o°  0.5  0.5 0 »  -0.8  -0.5  600  -1 -1 5 CO  c  01 55  x  W  • Chronic axotomy  -2  o Contralateral  x  in  0.5  >  290?  a go, 4Q0  600  • Chronic axotomy o Contralateral  2.5 1 .5 0.5 -0.5  o 600  -1  -2  -2.5  «  1 4  -1.5  (0 cn  2  -0.5 -(  -1.5  400  c  1 .5  0  o  2Q0 • o o  '  400  600  -1.5 • Chronic axotomy + 2nd injury  • Chronic axotomy + 2nd injury -2.5  o Contralateral  Cell size (nm ) 2  Cell size (|im ) 2  2 n d axo + 11 weeks  o Contralateral  feb'rttialateral  Contralateral^  90  Retrograde tracing and immunohistochemistry The identification of atrophied neurons is made difficult in part because of standard neuronal markers such as Nissl stain's reduced efficacy to label atrophied neurons, thereby making the distinction between an atrophied neuron and a glia cell difficult. The use of the neuronal specific marker NeuN in the present study was impractical as NeuN immunoreactivity declines in axotomized facial motoneurons (Chapter 3). In an effort to circumvent this problem, FluoroGold was applied to the axotomized nerve stump at the time of the first injury with the aim to distinguish severely atrophied facial motoneurons from glia cells. In addition, Micro Ruby was applied to the proximal stump in those mice receiving a second axotomy. Eleven weeks after the first axotomy, the Fluoro-Gold (shown in blue) was only prominently visible in very small cells (<10pm in diameter) (Fig. 4.4A) with few neurons positive for both the Micro Ruby (red) and Fluoro-Gold tracers. A lack of co-localization of G F A P immunoreactivity (green) and Fluoro-Gold indicated that these very small cells were not astrocytes (Fig. 4.4C). Immunohistochemical staining for microglia with the mouse specific ED-1 antibody F480 clearly shows that these small Fluoro-Gold labeled cells were microglia and not atrophied neurons (Fig. 4.4D-F). Therefore, the Fluoro-Gold had been taken up by the phagocytic microglia over the eleven weeks, making the identification of atrophied chronically axotomized neurons using Fluoro-Gold impractical. Of interest however, was the strong and continued up-regulation of G F A P throughout the eleven weeks after axotomy (even in those mice that did not receive a second axotomy). This maintenance of G F A P expression within the facial nucleus was not strain specific as it was present in both the C57BL/6-C3H, Balb/c and a third strain, CD-I (data not shown). Quantification of G F A P expression revealed that following a single nerve resection, GFAP remained elevated at 77 days post injury and was comparable to 14 day post injury levels. There was no apparent increase in G F A P expression as a result of the second injury. In addition to the maintained GFAP expression in the nucleus of resected animals, very high levels of GFAP were also detected within the injured facial nerve tract in the brainstem compared to the virtually undetectable levels 10 weeks following a crush injury, a model in which the axons successfully regenerate (Fig. 4.4J-L).  91  Figure 4.4 Retrograde tracing and immunohistochemistry Eleven weeks post axotomy, Fluor^Gold applied at the time of the first injury was found to be predominately in small cells (A). Application of the retrograde tracer Micro ruby (red) at the time of the second injury and immunostaining for G F A P (green) shows that these Fluro-Gold (blue) positive cells were not neurons or astrocytes (B,C). Using an antibody F-480 for the mouse ED-1 antigen indicates that the majority of the Fluoro-Gold positive cells are microglia (D-F). Quantification of the intensity of GFAP staining in the facial nucleus of mice at 14 and 77 days following a single resection showed little change in G F A P expression over this time (G-I). GFAP imunostaining was also found to be elevated in the facial motoneuron axon tract within the brainstem at 11 weeks after resection (L) compared to a crush injury at 10 weeks (J,K). Scale bar 100 urn.  92  A  B f V  1  ^  it'  0  Fluro-Gold  GFAP  D  E  \  « *  F-480  Fluro-Gold G  H  f  H . \  GFAP in Nucleus  ]  •*  GFAP-14day  GFAP-11 week  J  K  NfF200  GFAP-crush-10 wk  93  Discussion In the present study, re-injuring the axons of chronically axotomized motoneurons reversed the atrophy of their cell bodies, increased the number of countable motoneurons and stimulated the re-expression of GAP-43 and a-tubulin. The expression of G F A P was maintained for at least 77 days post axotomy in the facial nucleus and in the axon tract (if the axons are prevented from reaching their targets). These data demonstrate that due to the extreme atrophy of chronically resected motoneurons, previous survival studies may have underestimated the true extent of the surviving mouse facial motoneuron population following resection injury. Unlike neonate motoneurons that undergo rapid and quantifiable apoptosis within days following axotomy (de Bilbao and Dubois-Dauphin 1996, Rossiter, et al. 1996, Vanderluit, et al. 2000) mature motoneurons initially survive axotomy, but then many undergo slow atrophy and are lost. Thus, the true extent of axotomy-induced motoneuron death is difficult to determine in adult rodents. M y data suggest that this atrophy may render many motoneurons undetectable by standard staining procedures, such as Nissl staining, as they become difficult to distinguish from surrounding glial cells. However, the robust effect produced by re-injuring the axons of chronically axotomized motoneurons revealed that there was greater motoneuron survival than previously recognized following prolonged axotomy (resection). While the number of countable Nissl-stained motoneuron cell bodies was not completely restored to normal levels by re-injury, these experiments clearly demonstrate that significant numbers of motoneurons survived in a highly atrophic state. The exact number of permanently lost motoneurons and the number of surviving motoneurons that did not respond to the second nerve injury and thus remain in an atrophied state are undetermined. The use of Nissl stains like cresyl violet for identifying atrophied populations of neurons fails to reveal the entire population. In rubrospinal neurons, only 89% of the population was identified using Nissl stain compared to the pan neuronal marker NeuN (Kwon, et al. 2002). Unfortunately, the use of NeuN in facial motoneurons is impractical as NeuN is reduced in facial motoneurons as a result of axon injury (Chapter 3). Therefore, a more effective neuronal staining technique is required to identify and determine the exact number of atrophied versus surviving facial motoneurons. Determination of the extent of cell death in the adult facial model is technically challenging. Traditional apoptotic cell death labeling methods, such as T U N E L , are ineffective in models where only a small percentage of cells die over a protracted time period because apoptotic cells usually remain detectable for only a few hours (Rossiter, et al. 1996). In the adult 94  rat, recent evidence has shown that following nerve resection injury, facial motoneurons are lost by a slow, non-apoptotic form of neuronal degeneration (Moran, et al. 2001). In the present study, application of the retrograde tracer FluoroGold to identify these atrophied neurons in the mouse resulted in the identification of some larger neuronal profiles, but also many small FluoroGold positive microglia immunoreactive for the mouse ED-1 antigen F-480. This finding is in accord with previous work in the rat demonstrating a portion of neurons prevented from reaching their targets do in fact die and are phagocytosed by microglia (Angelov, et al. 1995, Angelov, et al. 1996). However, there is another significant portion that survives in a highly atrophied state and becomes detectable again after a second axonal injury. M y attempts to identify the atrophic motoneurons using the neuron-specific marker NeuN, as was recently performed on atrophied rubrospinal neurons (Kwon, et al..2002), was unsuccessful in the present study due to the rapid decline in NeuN expression in axotomized facial motoneurons (Chapter 3). Astrocytes are thought to have a role in neuroprotection (Graeber and Kreutzberg 1988, Streit, et al. 1998). However, our data would suggest that the astrocytic response to injury is not a factor in the differential motoneuron susceptibility to injury in the mouse versus the rat as the GFAP expression appears to be similar (i.e. maintained long term) in both species (Laskawi and Wolff 1996). No reduction in G F A P was detected at 77 days compared to 14 days in the mouse facial nucleus. Although the C57BL/6-C3H and Balb/c mice motoneurons responded similarly with increased gene expression and an increase in cell size following a second axon injury, the response was less pronounced with regard to motoneuron counts in the Balb/c mice. Many mouse strains respond differently in neurotrauma research paradigms such as the Morris water maze, Kainic acid induced hippocampal cell death, reperfusion injury and adult neurogenesis (reviewed in Steward, et al. 1999). In contrast, Lidman and colleagues found that following facial nerve resection, no difference in GAP-43 mRNA or G F A P protein expression was discernable between the C57BL/6 and Balb/c (Lidman, et al. 2002). The exact mechanism underlining the strain difference observed here remains unresolved, but it demonstrates the importance in understanding the genetic background affect in animal models used in neurotrauma research. It appears that caution should be used in interpreting previous reports of adult mouse facial motoneuron death after axotomy, as they may represent an underestimation of the surviving population. Axotomy-induced changes in neuronal gene expression may be regulated either by signals that appear (positive signals) or disappear (negative signals) following an axonal injury. 95  For motoneurons, many "positive" factors are derived from Schwann cells at the site of injury. These include ciliary neurotrophic factor (CNTF), several neurotrophins and glial cell-line derived neurotrophic factor (GDNF) (Henderson, et al. 1994, Heumann, et al. 1987, Meyer, et al. 1992, Naveilhan, et al. 1997, Sendtner, et al. 1992, Seniuk, et al. 1992, Stockli, et al. 1991). The negative signals resulting from target disconnection appear to play particularly prominent regulatory roles in the expression of many genes. For instance, blockade of axonal transport prevents the target contact-induced down-regulation of GAP-43 in developing corticospinal neurons (Karimi-Abdolrezaee and Schreyer 2002), and is sufficient to induce Tai-tubulin and GAP-43 expression in non-axotomized adult neurons (Woolf, et al. 1990, Wu, et al. 1993). Together, these data support a model whereby following axotomy, the loss of retrogradely transported target-derived repressive factors (negative signal) increase gene expression and the appearance of factors, produced as a result of the injury, that stimulate gene expression are positive signals.  .  In the present study, a second axon injury following chronic nerve resection resulted in an increase of both GAP-43 and a tubulin mRNA. I speculate that the response observed here is a result of positive factors produced at the site of injury, as target derived factors have been absent for some time. However, the influences of the neuroma, or its removal, on gene expression are not known at this time. In addition, our understanding of the repressive and positive signals as a result of injury, their expression timelines and their exact roles in gene regulation is poorly understood. It is also unclear as to why, despite continued target deprivation, GAP-43 and a-tubulin mRNA expression decrease in chronically injured mouse facial motoneurons. The present study demonstrates that following chronic resection injury, a larger proportion of mouse facial motoneurons survive than previously estimated. The ability of chronically axotomized motoneurons to re-express GAP-43 and a-tubulin, even after a third axotomy, suggests that these motoneurons also retain their regenerative potential to respond to re-injury long after resection injury. The identity of the factors controlling the motoneuron response to axotomy and their expression profiles remain central questions.  96  Chapter 5 Target derived factors from the neuroma repress gene expression following chronic facial nerve resection  97  Summary  In Chapter 4 I revealed that following a chronic nerve resection, axon re-injury reversed the atrophy, increased the number of countable motoneurons and resulted in the re-expression of GAP-43 and a tubulin mRNA. In this chapter I questioned whether the neuroma, which forms at the distal end of the proximal nerve stump, has a role in regulating gene expression following chronic resection injury. Following ten weeks of chronic resection, the axonal transport blocker colchicine or glial derived neurotrophic factor (GDNF) was injected into the facial nerve proximal to the neuroma. The injection of GDNF or colchicine elicited a significant increase in GAP-43 but not a tubulin mRNA. This suggests that following a chronic resection, the newly formed neuroma is a source of target like repressive signals that when removed, gene expression is increased. I then analyzed the regenerative potential of chronically resected motoneurons. Mice without a previous nerve injury and those with a chronic resection (10 weeks post injury) had a pre-degenerated segment of sciatic nerve attached to the proximal nerve stump of their facial nerve. Axons from both the chronic and acute groups grew into the grafts, however, significantly more retrogradely labeled motoneurons were counted in the acute group compared to the chronic resection group. No difference in motoneuron cell size was observed between the two groups. Therefore, despite severe atrophy, surviving mouse facial motoneurons retain the propensity to extend their axons when provided with the appropriate environment such as a predegenerated peripheral nerve graft.  98  Introduction  As a result of axonal injury, changes in gene expression may be regulated by signals that appear (positive) or disappear (negative). Positive signals are factors produced or released at the site of injury including cytokines and trophic factors derived from non neuronal cells such as fibroblasts and Schwann cells. The prototypical example of a positive signal is Leukemia Inhibitory factor (LIF). LIF is produced and released from Schwann cells following injury and is responsible for many neuropeptide changes in dorsal root ganglia (DRG) neurons that are absent in LIF knockout mice (Matsuoka, et al. 1997, Sun and Zigmond 1996, Zigmond, et al. 1996). Loss of target derived factors (negative signals) have a prominent role in the control of gene expression. Target disconnection can remove a sustaining factor resulting in the decreased expression of a gene. For example, there is a decrease in neurofilament m R N A expression in injured dorsal root ganglion (DRG) neurons. However, intrathecal infusion of nerve growth factor (NGF) reverses the injury-induced reduction of neurofilament m R N A in D R G neurons with the high-affinity N G F receptors (Verge, et al. 1990). Treatment with brain derived neurotrophic factor (BDNF) or neurotrophin 4/5 (NT-4/5) can also reverse some of the effects of axonal injury in spinal motoneurons (Friedman, et al. 1995). Target removal (negative signals) can also function to increase gene expression by preventing the retrograde transport of gene repressing factors. For example, the increased expression of GAP-43 and tubulin mRNA in injured D R G neurons is prolonged if target re-connection is prevented (Bisby 1988, Jiang, et al. 1994). Treatment of the proximal nerve stump with NGF, a factor normally produced by sensory targets, inhibits the injury induced upregulation of GAP-43 (Mohiuddin, et al. 1999). In uninjured adult neurons, the blockade of axonal transport (loss of repressive signals) is sufficient to induce Tai-tubulin and GAP-43 expression (Smith and Skene 1997, Woolf, et al. 1990, Wu, et al. 1993). In Chapter 4,1 demonstrated that in chronically axotomized mouse (C57/BL6 and Balb/c) facial motoneurons, the removal of the neuroma reversed the atrophy, increased the number of countable facial motoneurons and resulted in the re-expression of GAP-43 and a tubulin mRNA. Based on these previous observations, I asked whether this response to a second injury is the result of the loss of repressive signals, or is a result of the release of positive signals (trophic factors) produced at the injury site. To test this hypothesis, ten weeks following nerve resection, the trophic factor glial derived neurotrophic factor (GDNF) (a positive signal) or the axonal transport blocker colchicine (to block the transport of repressive factors) was injected 99  proximal to the neuroma in an outbred strain of mouse, C D - I . The outbred CD-I mice strain was chosen in an attempt to generalize the response compared to the more distinctive inbred strains. GDNF or colchicine injection proximal to the neuroma of chronically axotomized mouse facial motoneurons significantly increased GAP-43 mRNA expression and motoneuron size compared to saline injection. These data suggest that the neuroma, becomes a source of target like repressive factors, and the removal of these factors, together with positive signals released at the injury site act in concert to regulate gene expression and motoneuron size. Interestingly, despite their severe atrophy, 10 weeks after a resection injury, mouse facial motoneurons retain the ability to extend their axons into a permissive environment such as a pre-degenerated peripheral nerve graft sutured onto the resected facial nerve stump.  100  Materials and Methods Animal care Experiments were approved by the University of British Columbia's Animal Care Committee in accordance with the Canadian Council on Animal Care Guidelines. Male CD-I mice (n=28) (6-8 weeks of age) were obtained from the University of British Columbia's Animal Care Centre, Vancouver, B C . The mice were maintained in a 12hr light/dark cycle and provided rodent chow and water ad libitum.  Mouse facial axotomy (chronic) Adult mice (n=10) were anaesthetized with an intraperitoneal injection of a mixture of ketamine hydrochloride (135 mg/kg) (Bimeda-MTC, Cambridge, ON) and xylazine hydrochloride (6.5 mg/kg) (Bayer Inc, Etobicoke, ON). The large branch of the left facial nerve was axotomized approximately 3 mm distal from the foramen and a 2 mm segment of the distal portion of the nerve was removed to prevent the regeneration of the axons to their targets and the wound closed with silk sutures. At 10 weeks post injury all the mice were re-anaesthetized and the injured nerve and newly formed neuroma examined. In the control group (one axotomy only) the wound was immediately closed. In the second injury mice, the neuroma was removed and the wound closed with wound clips (Michel, Fine Science Tools, Vancouver, BC). One week following the second surgery (11 weeks from first injury) the mice were killed with a lethal injection of chloral hydrate (900 mg/kg). Animals were perfused with 0.9 % phosphate-buffered saline PBS) followed by cold 4 % paraformaldehyde in PBS (pH 7.4). Brainstems were removed and left to post-fix overnight in 4% paraformaldehyde, cryoprotected for 2 days in a PBS solution containing 22% sucrose and then frozen in dry ice-cooled isopentane.  Blockade of axonal transport from neuroma In adult mice (n=12) the facial nerve was resected as described above for chronic axotomy. After ten weeks the mice were re-anesthetized and, taking care not to manipulate the neuroma, saline, glial derived neurotophic factor (GDNF) or colchicine was injected into the nerve stump, proximal to the neuroma by an experimenter blind to the treatment groups. The mice were divided into three groups: In the first group the mice received 1 pi of 0.01 M PBS. In the second group, 1 pi (50mM) (Murphy, et al. 1999), of the retrograde axonal transport inhibitor colchicine (Sigma-Aldrich, Oakville, ON). The third group received 1 pi (2pg/pl) (Boyd and Gordon 2003), of G D N F (gift from Regeneron Pharmaceuticals, Tarrytown, N Y ) . Five days later 101  the animals were killed as described earlier. The five day time-point was chosen as the efficacy of the blockade begins to decline after 5 days (Macfarlane, et al. 1997).  Mouse peripheral nerve graft (chronic and acute) Adult male mice (n=6) were anaesthetized and the facial nerve was axotomized as previously described (chronic group). Nine weeks post injury the mice were re-anaesthetized and the sciatic nerve was axotomized and a 2 mm segment was removed to ensure that the axons did not reconnect with their targets. At this same time an additional aged matched group of mice, which had not received a facial nerve axotomy, underwent the same sciatic axotomy (acute group). One week post sciatic injury both groups of mice were re-anaesthetized and a 7-9 mm section of the degenerated sciatic nerve was removed. In the chronic graft group the neuroma was excised and the degenerated sciatic nerve segment was sutured onto the proximal facial nerve stump. In the acute graft group the facial nerve axotomized as previously described and the sciatic nerve sutured onto the proximal facial nerve stump. Three weeks following the graft, the mice in each group were re-anesthetized and the distal 1 mm of the graft was axotomized and a piece of gel foam with a 5% solution of the retrograde neuronal tracer Fast Blue (Sigma-Aldrich, Oakville, ON) was applied to the distal graft stump. One week later (4 weeks following the initial graft) the mice were killed as described earlier.  Preparation of tissues Brainstems were cryostat sectioned from the caudal to rostral extent of the facial nucleus at a thickness of 14 um. Every section through the facial nuclei was collected onto Superfrost Plus slides (Fisher Scientific, Houston, TX). Different treatment groups were randomly arranged on the slides to eliminate any systematic handling biases. Cellular morphology was examined by staining slides with 0.2% cresyl violet. Slides were first placed in distilled water for 2 minutes and then into the cresyl violet for 5-10 minutes. Slides were then rinsed in water and dehydrated in increasing concentrations of ethanol and then chloroform. The slides were then coverslipped using Entellan (BDH, Toronto, ON). Nissl stained motoneuron numbers were assessed by counting the number of neuronal profiles containing a visible nucleus on every second 14 um section throughout the extent of the facial nucleus. Cell sizes were measured using Sigma Scan Pro software (SPSS Inc. Chicago, IL). Motoneuron counts and cell sizes were expressed as a percentage of the contralateral and statistical significance determined using Sigma Stat software (SPSS Inc, Chicago, IL). 102  To identify all motoneurons within the facial nucleus in the nerve graft model (with Fast Blue tracer) the slides were incubated with NeuroTrace (1:300 Molecular Probes), a fluorescent Nissl stain, for 5 minutes then washed in PBS and coverslipped. Following capture of both the Fast Blue and fluorescent NeuroTrace images, the Fast Blue positive neurons were identified and counted based on the criteria of being larger than 10pm in diameter, as the nucleus was not visible using this method. Counting of Fast Blue labeled motoneurons was performed on every sixth section from the most caudal to rostral portion of the facial nucleus. For fluorescent Nissl stained neurons, only those neurons with a distinguishable nucleus were analyzed for cell size within the area of the lateral and intermediate sub groups of the facial nucleus, ensuring exclusion of the uninjured medial groups.  In situ hybridization The GAP-43 52 and a-tubulin ISH probes used in this Chapter were those as previously described in Chapter 4. Probes were end-labeled with S-dATP using deoxynucleotide terminal 35  transferase according to a standard protocol (Giehl and Tetzlaff 1996). Tissue sections were hybridized tolO cpm of probe for 16-18h at 44°C. After drying, slides were dipped in Kodak 6  NTB-2 photographic emulsion, diluted 1:1 in distilled water at 42°C, dried, and exposed for 2 days (tubulin) and 8 days (GAP-43) at 4°C. Following development, slides were then dehydrated in a series of alcohols and stored at room temperature. For quantification of the hybridization signals the tissue sections were stained for 1 hour in a 0.01% solution of ethidium bromide, washed under cold running water for 20 minutes, and rinsed for 30 minutes in double-distilled water. This resulted in the fluorescent illumination of neuronal cell body profiles and intense staining of glial cells. The slides were dehydrated in ethanol and coverslipped. The area of the neuron soma occupied by autoradiographic ISH signal was quantified using Sigma Scan Pro software (SPSS Inc. Chicago IL). For the GAP-43 probe, at least two sections and three sections for the a tubulin, all more than 42 pm apart, to avoid analysis of the same neuron, were quantified per animal (n=4) in each group. Analysis was limited to those neurons with cell body profiles with a minimum diameter twice that of the average glia profile (10pm), as the nucleus was not visible. In each treatment condition the ISH signal/neuron was determined as a percentage of the total area of the neuron soma area occupied by ISH signal. Data for each treatment group are graphed in terms of their mean and standard error of the mean (SEM) of the proportion of the ISH signal in the contralateral nucleus for each treatment group. 103  Results Axon re-injury increases the number of countable motoneurons In our previous work we analyzed the response to a second axon injury in two inbred strains of mice, C57/B16 C3H and Balb/c. In the present study we used an outbred strain of mouse, CD-I that responded to a second axon injury similar to the C57/B16 C3H and Balb/c strains. At 11 weeks post injury the motoneurons in the chronic axotomy mice (middle panel, Fig. 5.1 A) were fewer and highly atrophied compared to the uninjured contralateral side (top panel Fig. 5.1 A). As a result of the second axotomy the atrophy was reversed and the motoneurons displayed increased Nissl staining and appeared more numerous (lower panel Fig. 5.1 A). Motoneuron counts on every second section through the facial nucleus plotted as a percentage of the contralateral side are shown in Fig. 5. IB. The second axon injury resulted in a significant increase in the number of countable motoneurons from 34.2% ± 2.2% S E M of contralateral in the chronically axotomized group, to 49.7% ± 5.2% S E M of contralateral in reaxotomized group. Measurement of motoneurons in the lateral and intermediate groups revealed that the average size of the chronically axotomized motoneurons was 75.0% ± 3.5% S E M of the contralateral side. The second injury resulted in an average size of 94.5% ± 8.5% S E M of the contralateral uninjured side (Fig. 5.B). Binning of the motoneuron size data demonstrate a trend to a more normal distribution in size in the animals receiving a second axon injury (Fig. 5.ID), compared to those in the chronic axotomy group (Fig. 5.1C).  Motoneuron size following injection of GDNF or colchicine To examine the effect of positive signals and the loss of repressive factors on motoneuron size, GDNF or colchicine was injected into the proximal nerve stump of chronically axotomized facial motoneurons (Fig. 5.2). Injection of GDNF resulted in a significant increase in motoneuron soma size to 113.4% ± 1.5% S E M compared to both saline and colchicine. Colchicine injection significantly increased motoneuron size to 98.2% ± 0.7% S E M compared to saline 86.6% ± 1.8% S E M . ( A N O V A , Pairwise Multiple Comparison Procedures (Holm-Sidak method) p<0.01).  104  Figure 5.1 Survival of chronically injured mouse facial motoneurons Axon re-injury in the CD-I mouse strain resulted in the reversal of neuronal atrophy and an increase in the number of countable motoneurons. A l l images have the medial group on the left and the lateral groups on the right. At 11 weeks the motoneurons in the chronic axotomy mice (middle panel) are fewer and highly atrophied compared to the uninjured contralateral side (top panel). In those mice that also a received a second axon injury the atrophy is reversed and the motoneurons appear more numerous (lower panel). Measurement of motoneuron size revealed that the average size of the chronically axotomized group was 74.0% ± 3.5% S E M of the contralateral side and 93.0% + 8.5% S E M as a result of a second injury. Motoneuron counts revealed a significant increase from 34.2% ± 2.2% S E M contralateral to 49.7% ± 5.2% S E M (*). t test, p<0.05. Binning of the motoneuron size data demonstrate a trend of increased neuronal size in the animals receiving a second axon injury.  105  106  GAP-43 and a-tubulin I S H To investigate whether the regulation of gene expression in response to a second injury is related to the loss of repressive factors from the neuroma, or is result of the release of positive factors at the injury site, we injected G D N F or colchicine into the facial nerve proximal to the neuroma. Compared to saline injection, GDNF produced a detectable increase in GAP-43 ISH (Fig. 5.3A-D), whereas the increase in GAP-43 ISH was even more pronounced following colchicine injection (Fig. 5.3E,F). Quantification of GAP-43 ISH signal indicated significant increases in GAP-43 ISH following the injection of GDNF (3.0 ± 0.54) or colchicine (5.4 ± 0.84) compared to saline 1.3 + 0.05 (Fig. 5.3G) ( A N O V A , Pairwise Multiple Comparison Procedures (Dunn's Method) p<0.05). GDNF or colchicine injection had little effect on a-tubulin ISH signal compared to saline (Fig. 5.4A-F). Quantification of the a-tubulin ISH signal did not reveal any significant change in a-tubulin following injection of G D N F (1.7 ±0.17) or colchicine (2.1 ± 0.18) compared to saline (1.8 ± 0.37) (Fig. 5.4G).  107  Figure 5.2 Motoneuron size Motoneuron size measurement following injection of saline, GDNF or colchicine into the proximal nerve stump of chronically axotomized facial motoneurons. Motoneuron size was significantly increased following injection of GDNF (113.4% ± 1.5%.SEM) or colchicine (98.2%o  ± 0.7% SEM) compared to the saline 86.6% ± 1.8% S E M (*). The increase in size from  the GDNF injection was also significantly more than the colchicine injection (#). A N O V A , A l l Pairwise Multiple Comparison Procedures (Holm-Sidak method) p<0.01.  108  A  B  Saline  Contralateral  C  D  GDNF E ^  Contralateral  Colchicine  F  *  Contralateral  _  *  Saline  GDNF  Colchicine  treatment (n=4, mean +/- SE)  109  Figure 5.3 G A P - 4 3 in situ hybridization Darkfield micrographs GAP-43 ISH signal in chronically axotomized facial nuclei 5 days post injection of saline, G D N F or colchicine (A-F). Compared to the contralateral uninjured side, saline injection had little effect on GAP-43 ISH signal (A,B). G D N F injection produced a detectable increase (C,D), whereas the colchicine's effect on GAP-43 ISH signal was even more pronounced (E,F). Quantification of the GAP-43 ISH signal as a proportion of the un-injured contralateral side (G), revealed a slight increase in signal (1.3 ± 0.05) following the injection of saline. Compared to the saline injection, the injection of either G D N F or colchicine produced statistically significant increases in GAP-43 ISH (3.0 + 0.54 and 5.4 ± 0.84) for GDNF and colchicine respectively (*). (Kruskal-Wallis One Way Analysis of Variance on Ranks (Dunns) pO.Ol). Scale bar 50 pm,  110  B  •"  •  '% •  • «...  »  M,'  Saline ^ C  iPV  Contralateral ^  *  D  Contralateral F  E  1 m  • "  j  s  Colchicine  Contralateral  G 7  g w IS  m o o "S c o t o CL o  6 5  I LL  Saline  GDNF  Colchicine  t r e a t m e n t (n»4, m e a n +/- SE)  111  Figure 5.4 a-tubulin in situ hybridization Darkfield micrographs a-tubulin ISH signal in chronically axotomized facial nuclei 5 days post injection of saline, GDNF or colchicine (A-F). Compared to the contralateral uninjured side, saline or GDNF injection had little effect on a-tubulin ISH signal (A-D). The injection of colchicine on a-tubulin ISH signal was slightly more pronounced. Quantification of the atubulin ISH signal as a proportion of the uninjured contralateral side (G), revealed a slight but statistically insignificant increase following the injection of saline (1.8 ± 0.37), GDNF (1.7 ± 0.17) or colchicine (2.1 ± 0.18). Scale bar 50 um  112  Motoneuron Size  Saline  GDNF  Colchicine  treatment (n=4, +J- SD)  113  Growth of the axons of acutely and chronically axotomized facial motoneurons. To evaluate the ability of the chronically axotomized mouse motoneurons to regenerate 10 weeks post axotomy, the neuroma was removed and a pre degenerated sciatic nerve was grafted onto the proximal stump. Fluorescent images of the retrograde tracer Fast Blue in the motoneurons in the chronic and acute group (Fig. 5.5A,B) revealed the growth of axons into the sciatic nerve graph. The corresponding fluorescent Nissl stain images (Fig. 5.5C,D) show that many of the motoneurons grew into the graft with the exception of those in the ventral medial and dorsal medial sub groups (bottom right on all images) that were not injured (auricular branch). Counting of Fast Blue labeled motoneurons (>10pm) was performed on every sixth section from the most caudal to rostral portion of the facial nucleus. Motoneuron counts indicate that significantly more motoneurons in the acute group (276 ± 7) compared to the chronic group (185 ± 27) (Fig. 5.5E, t test, p<0.05) grew into the nerve graft. Soma size measurement of Nissl stained motoneurons in the intermediate and lateral portions of the facial nucleus, expressed as a percentage of the contralateral, determined that there was no significant difference in motoneuron soma size between the acute (99.2 ± 1.9%) and chronic (91.7 ± 4.5%) groups.  114  Figure 5.5 Motoneuron counts and size following nerve graft Growth of the axons from acute and chronically axotomized facial motoneurons 4 weeks after the nerve graph (14 weeks after initial axotomy). The application of the retrograde tracer Fast Blue reveals the growth of axons into the sciatic nerve graph in both the acute and chronic resection groups (A,B). The corresponding fluorescent Nissl stain images (C,D) demonstrate the fact that the majority of the motoneurons grew into the graft with the exception of the ventral medial and a few in the dorsal medial sub group (bottom right on all images) that were not axotomized. Fast Blue positive motoneuron profile (>10 pm) counts indicated that significantly more motoneurons in the acute group (276 ± 7) compared to the chronic group (185 ± 27) grew into the graft (E). (*) t test, p<0.05. Measurement of soma size of the intermediate and lateral portions of Nissl stained motoneurons, expressed as a percentage of the contralateral, determined that there was not a significant difference between the acute (99.2% ± 1.9%) and chronic (91.7%o + 4.5%) motoneurons soma size. Scale bar 100 pm.  115  A  »f  B 4  «  f  *  **  «*  * J|  K  ^  #  *  0 m  «  •>  t •  *  %  ##  41  #'  d  Acute  Chronic  c  D  *  •  * .  t'fciuii  • .  J  *  *k  %.  *  H  -" •  »>  11 '• d', A •  . #  *  -% « v  '  •  • •« ; • s'  i *  •  *  g  &  i #  f*  *  jp  r i"«,  ,'• ,  .* "  *  Acute  Acute Chronic treatment (n=3, + / - S E M )  Acute Chronic treatment (n=3, +/- SD)  116  „*'  Discussion In the present study I investigated the relative roles of the neuroma and positive factors produced at the injury site, in the response of mouse facial motoneurons to a second axonal injury. Removal of the neuroma 10 weeks post injury resulted in reversal of atrophy and a significant increase in the number of countable neurons in the CD-I strain of mouse. The injection into the nerve stump of the axonal transport blocker colchicine or GDNF, proximal to the neuroma resulted in a significant increase, of GAP-43 m R N A expression, but not a tubulin mRNA. Furthermore, the injection of GDNF or colchicine significantly increased the size of the chronically injured motoneurons. Axons from both the acute and chronic resection groups extended axons into the pre-degenerated sciatic nerve graft, however, significantly more Fast Blue retrogradely labeled motoneurons were counted in the acute compared to the chronic group. Motoneuron soma size measurements indicated that the average size of the regenerating motoneurons was similar in both the chronic and acute groups. Previously I reported an increase in soma size and number of countable mouse facial motoneurons as a result of a second axon injury ten weeks after an initial nerve resection. In addition to an effect on soma size, re-injury also increased the expression of GAP-43 and a tubulin mRNA (Chapter 4). These previous experiments were performed in two inbred strains of mice C57/B16 C3H and Balb/c. In these strains of mice, the second axotomy resulted in significant increases in the number of countable motoneurons in both strains; however, there was a considerable difference between the two strains. In the present study an outbred strain of mice CD-I was used, a second injury increased the countable motoneurons from 34% to almost 50% of the contralateral un-injured side, an intermediate response between the C57BL/6 C3H and Balb/c. Several studies have demonstrated a strain specific response differences to a variety of neurological insults and in the animal's ability to perform cognitive tasks such as the Morris water maze (reviewed in Steward, et al. 1999). Although, a comparison of the C57BL/6J and Balb/c strains revealed no difference in GAP-43 mRNA and G F A P protein expression following facial nerve resection (Lidman, et al. 2002). Despite the variance in the extent of the response to a second axotomy, all three strains display a significant increase in the number of countable facial motoneurons following the second axon injury indicating that this is not a strain specific response. GAP-43 expression is initially up-regulated following injury but then is down-regulated following target connection of retinal ganglion neurons in the zebra fish (Bormann, et al. 1998). Blockade of axonal transport up-regulates the expression of both GAP-43 m R N A and a - l 117  tubulin in non axotomized adult motoneurons and D R G neurons (Karimi-Abdolrezaee and Schreyer 2002, Smith and Skene 1997, Wu, et al. 1993). The infusion of adult spinal cord extracts into postnatal rats results in a decline in GAP-43 m R N A expression in corticospinal neurons (Karimi-Abdolrezaee and Schreyer 2002). However, there is no reduction in GAP-43 expression when cerebellar tissue extracts are used, indicating that the spinal cord produces GAP-43 repressive factors (Karimi-Abdolrezaee and Schreyer, 2002). In the present study, the injection of colchicine into the nerve stump, just proximal to the neuroma resulted in a significant increase in GAP-43 m R N A expression. This suggests that repressive signals, perhaps like those in the muscle or spinal cord, are produced in the neuroma. This may explain the eventual down regulation of GAP-43 mRNA after chronic axotomy, despite continued target deprivation. I do not suggest however, that there is a complete supplement of target derived factors in the neuroma as motoneuron size remains well below uninjured levels, indicative of a lack of trophic support. The blockade of axonal transport resulted in an almost 5 fold increase in GAP-43, yet a tubulin mRNA expression was not significantly affected by the colchicine treatment. The differential regulation of these two genes is not surprising as GAP-43 and tubulin are likely to be regulated by a number of factors, some of which may act independently on either GAP-43 or tubulin, while others may act on both genes. For instance, the intravitreal administration of BDNF following axotomy of retinal ganglion cells increases GAP-43 but has no effect on T a i tubulin mRNA expression (Fournier, et al. 1997). Interestingly, my previous work revealed that complete surgical removal of the neuroma does increase the expression of a tubulin mRNA at 7 days post axotomy approximately 5 fold (Chapter 4). In the present study, no significant increase in a tubulin mRNA was observed following injection, yet m R N A expression in all three injections ranged from 1.7 to 2.1 fold. At ten weeks post resection, prior to the removal of the neuroma, a tubulin m R N A expression is below that of the contralateral side (Chapter 4). Thus in the present study, the injection itself likely produced a small amount of injury that may have resulted in the production of positive signals responsible for the small increase in tubulin expression in all groups. GDNF is one of the most potent neurotrophins for motoneurons, is upregulated in Schwann cells as a result of axonal injury and is retrogradely transported to the motoneuron cell bodies (Hammarberg, et al. 1996, Henderson, et al. 1994, Hoke, et al. 2000, Naveilhan, et al. 1997, Yan, et al. 1995). Furthermore, a decline in the expression of G D N F correlates with the failure of regeneration into chronically denervated nerves (Hoke, et al. 2002). In the adult mouse, 118  only GDNF and not other Schwann cell derived factors such as brain derived neurotrophic factor (BDNF), N G F or insulin growth factor (IGF-1) resulted in the hypertrophy of the motoneuron soma and increased survival as a result of avulsion injury (Li, et al. 1995). Here, the injection of GDNF proximal to the neuroma resulted in a significant increase in the size of the motoneuron soma and GAP-43 m R N A expression compared to both colchicine and saline. In addition, motoneuron size following saline injection (87.9%) was significantly increased over the chronic axotomy group (74.0%>), indicating a role for positive factors produced by the injection itself. Interestingly, colchicine significantly increased the average motoneuron size over that of the saline injection suggesting that repressive signals also regulate cell size. It appears that both positive and repressive signals regulate gene expression and motoneuron size, although to varying degrees. Taken together, these data suggest that maximum gene expression of both genes would only be obtained following both target removal and the presence of positive factors produced at the injury site. Significantly more motoneurons in the acute group versus the chronic resection group were labeled with Fast Blue indicating more axons grew into the peripheral nerve grafts. Interestingly, no significant difference in the average size of the regenerating neurons was detected, likely a result of positive factors produced within the pre-degenerated nerve graft in both treatment groups. The present results demonstrate that following a chronic axotomy in which target reinnervation is prevented, a second axon injury results in an increase in the number of countable facial motoneurons in the CD-I mice. Second, the neuroma itself may function as a source of target derived factors that act as repressive signals to regulate GAP-43 gene expression. Third, both positive signals and the loss of repressive signals may have a role in motoneuron hypertrophy as a result of axonal injury. Together, these data support the concept that numerous signals operate to influence gene expression following nerve injury. One is the loss of target derived factors (repressive signals), another is the production/release of factors at the injury site (positive signals). These signals appear to regulate gene expression in gene specific fashion, as only GAP-43 was significantly affected by the blockade of axonal transport from the neuroma. Lastly, despite their severe atrophy, chronically axotomized mouse facial motoneurons retain the propensity to extend their axons when provided with the appropriate environment such as a predegenerated peripheral nerve graft.  119  Chapter 6 Conclusions and future directions  120  Conclusion: In this thesis I have utilized a well established model of peripheral nerve injury, rodent facial motoneurons to reveal several new findings related to motoneuron survival in response to nerve injury. The survival of adult compared to neonatal facial motoneurons is not due to the level of expression of inhibitory apoptotic proteins, NAIP and X I A P . Furthermore, I have demonstrated that more chronically resected mouse facial motoneurons survive in an atrophied state than had been previously reported. This discrepancy is due in part to the difficulty in identifying atrophied neurons using Nissl stains or other neuronal phenotypic markers such as NeuN that are affected by axotomy leading to an underestimation of the surviving population. The neuroma that forms at the end of a proximal stump following nerve resection may be a source of factors that are capable of controlling gene expression in the neuron soma. Finally, despite their atrophy, chronically injured mouse facial motoneurons are able to re-grow their axons if provided with a suitable environment such as a pre-degenerated nerve graft. The findings of this thesis have increased our understanding regarding the response of the motoneurons to axon injury.  Survival of neonatal and adult motoneurons IAPs are thought to be key regulators of cell survival as a result of an apoptotic stimuli including axonal injury (Perrelet, et al. 2002, Perrelet, et al. 2000, X u , et al. 1999). Thus, in Chapter two, my finding on the endogenous expression of X I A P and NAIP in adult and neonatal facial motoneurons was contrary to my initial hypothesis. The survival of the majority of adult rat facial motoneurons vs the apoptotic death of the majority of neonatal motoneurons would suggest that the adults would have a higher endogenous expression of IAPs. However, in Chapter 2,1 describe, for the first time, changes in expression of NAIP and X I A P mRNA and protein within the facial nucleus of adult and neonate rats following axotomy. More specifically, the expression of NAIP and X I A P was greater in the neonates than the adults. Thus, it appears that in neonatal facial motoneurons, the relatively high levels of NAIP and X I A P protein is insufficient to prevent apoptosis following injury. The high level of caspase 3, which is a target of the IAPs and expressed an order of magnitude higher in the neonatal facial motoneurons, has a major role in the death of facial motoneurons following axotomy in the neonate (Vanderluit, et al. 2000). Therefore, it is likely that higher levels of IAPs are required in the neonate than in the adult to form a balance between the pro and anti-apoptotic factors at this developmental stage. More recently, the release of cytochrome c from the mitochondria was observed to occur prior to 121  the activation of caspase 3 in neonatal facial motoneurons as a result of axotomy (Vanderluit, et al. 2003). In addition to the release of cytochrome c from the mitochondria, there is a release of Diablo/Smac and Omi/HtrA2 which prevent the IAPs from inhibiting the capases (reviewed in Adams 2003). The microinjection of exogenous Smac and cytochrome c into sympathetic neurons, but neither alone, permits caspase activation and apoptosis (Deshmukh, et al. 2002). Therefore these factors may also be functioning to attenuate the high level of endogenous IAPs and thus facilitate apoptosis in this model. As mentioned in Chapter 2, in a neonatal model of axonal injury induced "neuron death, IAPs mediate the actions of GDNF, a potent survival factor for neonatal motoneurons (Perrelet, et al. 2002). However, at present little is known about IAPs in adult axonal injury models, likely due to the absence of apoptotic death in adult axotomy models. O f interest would be R N A interference (RNAi) or conditional knockout experiments in the adult to reduce the expression of IAPs in injured and non-injured motoneurons. Second, perhaps the increased survival of resected facial motoneurons in rats versus mice is related to the expression of IAPs. Furthermore, as demonstrated in Chapter 5, the injection of GDNF increased GAP-43 expression and the size of chronically injured mouse motoneurons; it would be of interest to determine if these effects are mediated through the IAPs in this model of injury. These studies and those mentioned in the discussion in Chapter 2 are required to further our understanding of the function of IAPs in the adult.  Survival of chronically injured mouse motoneurons In Chapters 4 and 5 of this thesis I determined that a second axon injury increased the size and the number of countable mouse facial motoneurons. Errors in the determination of the exact number of surviving neuronal populations are often associated with the counting method. Numerous reviews have outlined these problems and possible solutions (reviewed in Guillery 2002). One such solution is a mathematical formula, the Abercrombie-based correction factor (Abercrombie 1946). This method corrects for the size of objects counted in relation to the thickness of the tissue section. This correction factor is used to determine the total number of objects in the area studied. When the objects are very small in relation to the section thickness this value is close to one. As the objects increase in size this value becomes smaller. In Chapters 4 and 5, the criterion used to identify a motoneuron for motoneuron counts was the motoneuron nuclei.  122  The average diameter of a rat facial motoneuron nucleus is approximately 18pm and 8pm for a mouse (Ashwell and Watson 1983, Vaughan 1990). At 7 days post nerve crush injury in a rat there is a 30% increase in nucleus size (Vaughan 1990). Although there is evidence for a slight increase in the average size of several neuronal nuclei as a result of axon injury, reports show no increase in other neurons including those of mouse facial motoneurons (Lieberman 1971). Assuming there was a 25% change in the size of the mouse facial motoneuron nuclei; this would approximate the mean nucleus size to 10pm in the second axotomy group and 6pm in the chronic group. Using the Abercrombie-based correction factor (T/T+h) where T is the section thickness and h is the object thickness, the correction factors would be: non-injured contralateral side 0 .64, chronic 0.70 and the second injury group 0.58. If these correction factors are applied to the present data and a statistical analysis applied (t-test, p<0.05), the results of all three mouse strain counts remain significantly different (percent contralateral ± SEM). For C57; 44.1 ± 6.1, 64.4 ± 4.2, Balb/c; 30.6 ± 1.9, 36.4 ± 1 and CD-I; 33.7 ± 2.2, 49.7 ± 5.2 for chronic and chronic plus a second injury respectively. A n increase in nucleus size has not been previously reported and was not observed in the present studies. Measurement of the nuclear diameter in all identifiable nuclei in three sections per animal (n=5) in the CD-I mice revealed no statistical difference in average diameter (pm ± SEM): Chronic axotomy, 8.2 ± 0 . 2 1 , contralateral, 8.9 ± 0.32. Second axotomy, 8.1 ± 0.15, contralateral 8.3 ± 0.35. Therefore, I am confident that the increases in motoneuron number described in this thesis are a result of an increased number of identifiable motoneurons and not simply related to counting errors due to object size change. Although the number of countable motoneurons was not completely restored to normal levels by re-injury, these experiments clearly demonstrate that significant numbers of motoneurons survived in a highly atrophic state. The exact number of permanently lost motoneurons and the number of surviving motoneurons that did not respond to the second nerve injury and remain in an atrophied state are undetermined.  Motoneuron identification Initially, in an effort to identify chronically injured mouse facial motoneurons I used the pan neuronal marker NeuN. This lead to the findings presented in Chapter 3 where I demonstrate a large reduction in the protein expression of NeuN in facial motoneurons and relatively little change in the level of NeuN expression in rubrospinal neurons following injury. The differential 123  mRNA and protein expression in the PNS and CNS of a particular gene as a result of axon injury invariably leads to speculation that the gene in question has a role in the process of regeneration. However, numerous other possibilities exist; NeuN is concentrated within the nucleus suggesting that it is a transcription factor. Whether NeuN is directly related to regeneration or its regulation is simply a by product of another pathway is just one of the questions that remain to be answered regarding NeuN. Obviously, the first question is what is NeuN? To answer this, the isolation and determination of the protein sequence of NeuN is required. The next step would be to elucidate the function of NeuN. Function blocking antibodies, knock out mice or the transgenic/viral over expression of NeuN would be starting points to help ascertain the exact role of NeuN. Any attempt to speculate on the function of NeuN prior to these experiments would simply be conjecture. Despite the unknown function of NeuN, of importance at present is the limitation of NeuN as a pan neuronal marker in PNS and that caution must be taken regarding the use of a single phenotypic marker such as NeuN as a method to detect the survival of neurons, most notably those of the PNS following axonal injury. Using stereological based methods to determine the exact number of neurons, our laboratory determined that following a chronic rubrospinal axotomy the neurons do not die but rather remain in an atrophied state (Kwon, et al. 2002). However, as previously mentioned, the use of Nissl stains enables the detection of only 89% of the population compared to the pan neuronal marker NeuN (Kwon, et al. 2002). Unable to use NeuN to identify chronically atrophied mouse facial motoneurons because of NeuN's decline in this model following injury, in Chapter 4 of this thesis I applied FluoroGold (FG) to the motoneurons at the time of injury. Unfortunately, this technique was also unsuccessful, as the F G was taken up by phagocytic microglia. More recently, in an attempt to determine the efficacy of Nissl stains in chronic mouse facial motoneurons, I performed an experiment not previously described in this thesis. In this study I retrogradely labeled facial motoneurons with a topical application of the tracer F G onto the facial nerve. The first group consisted of non-injured control mice, the second group consisted of 10 week chronically resected mice (n=3 per group). Using gel foam, the F G was applied directly to the outer surface of the large branch of the facial nerve in both groups, taking care not to cause further damage to the nerve. One week later the mice were killed and the facial nucleus sectioned. Digital images of the F G labeled motoneurons were collected on 8 sections (every 5 ) per mouse, and the same sections were then stained with the Nissl stain, cresyl violet, th  and digital images collected. Neurons within a circumscribed region of the intermediate and lateral subgroups were then counted in both the Nissl stained and F G digital images by an 124  individual blind to the experimental paradigm. Expressed as a ratio of Nissl to F G labeled motoneurons, a significantly higher percentage of Nissl stained motoneurons was counted in the non-injured control group 92.6% (±2.6 SEM) compared to the chronic resection group 82.7% (±1.8 SEM) (t-test p<0.05). These results suggest that as is the case with chronic rubrospinal neurons, Nissl stain fails to label the entire population of chronically injured mouse facial motoneurons. However, this is only a preliminary study as a larger number of experiments are needed to confirm these findings. For instance, the efficacy of the F G labeling needs to be confirmed. F G labeling on the treatment side should be expressed as a percentage of the uninjured contralateral side after F G labeling to eliminate intra animal variability. In addition, in the uninjured paradigm, the use of NeuN or another neuronal phenotypic marker to confirm that the criterion selected for F G profiles (>10 pm in diameter) is specific to neurons. Obviously this can only be preformed on the un-injured side as NeuN declines in injured facial motoneurons. Should the use of F G in this paradigm prove technically unassailable i.e., every F G profile is indeed a neuron and every neuron is labeled with FG, a more accurate determination of the amount of survival following chronic resection will be possible.  Cell body response The association of axotomy-induced genes with regeneration was initially demonstrated in dorsal root ganglion neurons. Injury of the central branch fails to increase growth associated genes and the axons fail to regenerate. In contrast, injury to the peripheral branch increases gene expression and the central projecting neurons are then capable of regeneration (Richardson and Issa 1984). A more direct demonstration of the requirement of gene expression for regeneration was illustrated by Bomze and colleagues (Bomze, et al. 2001). Increased axonal outgrowth was observed as a result of the over-expression of both GAP-43 and CAP-23 compared to minimal regeneration using the individual genes (Bomze, et al. 2001). This study by Bomze and colleagues underscores the need to investigate, simultaneously, more than one gene product. This can be accomplished using microarrays, which are becoming increasingly more prevalent in the area of regeneration. For example, in our laboratory, using microarrays, studies are underway examining mRNA in rubrospinal and facial motoneurons in response to injury. The data from these microarrays may provide a starting point to begin investigating novel genes that may function in the differential regenerative potentials of the PNS and CNS as a result of axonal injury. Although microarrays may provide candidate genes, the expression of a gene as a result of injury does not necessary imply that it is of functional relevance. Indeed, in many knockout 125  mice, the loss of function results in no phenotypical or regenerative effect or in some cases the regenerative failure is confounded by the associated developmental defects. For example, in vivo, GAP-43's role in regeneration was determined to be one of guidance rather than neurite extension as shown in vitro. In GAP-43 deficient mice axon growth rate is normal, but the axons fail to grow to their proper targets (Strittmatter, et al. 1995). Furthermore, the results of gain of function experiments are often plagued by the fact that in the normal situation, PNS regeneration may already be at its maximum and therefore any further increases are undetectable. For example, in hamster facial motoneurons, testosterone increases the rate of regeneration in males but little in females, perhaps because regeneration is already at a maximal rate in the females (Jones 1993). With the exception of the present studies and those of Kwon and colleagues (Kwon et al., 2002), little is known regarding gene expression in chronically axotomized neurons. Despite the limitations, a microarray would allow the comparison between chronically resected versus non injured motoneurons. Second, analysis of chronically resected motoneurons that are subjected to a second axon injury compared to an acutely resected population would be of interest to determine the genes responsible for hypertrophy. In addition, microarrays of chronically injured neurons that have been provided with a pre-degenerated peripheral nerve graft to profile gene expression during the axon growth phase would be of value. These could provide some insight into the gene expression profiles of chronically axotomized neurons and would be a starting point for further investigation into the control mechanisms responsible for changes in gene expression following axonal injury.  The neuroma I found the neuroma and its regulation of gene expression to be one of the most intriguing of all the findings in this thesis. In 1968, Watson demonstrated that following an initial increase, R N A expression returned to baseline levels if hypoglossal axons are prevented from reaching their targets (Watson 1968). More importantly, Watson showed that the removal of the neuroma initiated a re-expression of R N A synthesis, demonstrating that the removal of the peripheral target influence is not necessary for all aspects of the cell body response (reviewed in Lieberman 1971). At the time, Gragg postulated that neurons were full of a repressive substance and that following injury the repressor substance leaked out with the axoplasm. This reduction in repressor substance reduced its effective concentration and resulted in R N A synthesis (Cragg 1970). Therefore, the finding of Watson's regarding the removal of the neuroma was interpreted 126  to be a result of a loss of a repressor substance in the axoplasm (Cragg 1970). However, ligation and axonal transport blockade experiments later supported the loss of target derived factors concept over that of a of repressor substance in the axoplasm. Nevertheless, the findings in Chapters 4 and 5 support Watson's concept that the neuroma is involved the regulation of motoneuron gene expression, albeit likely via a mechanism other than loss of axoplasm. The exact mechanism of action and a complete inventory of these factors within the neuroma responsible for the m R N A expression response remain undefined. Again, a microarray would be an excellent starting point to identify potential factors responsible for this regulation. The neuroma and a section of un-injured nerve could serve as the two treatment groups. Further confirmation and investigation into the exact cell source of any m R N A expression changes using ISH would be required, followed by protein expression analysis. In addition to the use of microarrays to analyze gene expression, several mechanistic studies could also be performed. For instance, if the fully formed neuroma is in fact functioning to down regulate GAP-43 and tubulin, then would the neuroma from a chronically resected nerve transplanted onto an acutely resected nerve attenuate gene expression in the newly injured motoneuron? Another option would be to use an in vitro preparation, for example, exposing the neurites in a neuron culture to chronic a neuroma homogenate. In such an experiment, I would anticipate that the factors present within the neuroma homogenate would attenuate gene expression and or perhaps even neurite outgrowth/length. A third option is to crush the nerve just proximal to the neuroma to determine i f gene expression is attenuated sooner than a freshly resected nerve. In this instance, the axons would reach the source of repressive signals (the neuroma) much sooner than their original targets (muscle) in a normal nerve crush paradigm. Hence, mRNA expression of GAP-43 or tubulin should be reduced to uninjured levels more quickly in the neuroma group. Finally, a fourth option is the topical application (not injection) of the retrograde transport blocker colchicine proximal to the neuroma prior to a crush or surgical removal of the neuroma. Under these conditions, if the neuroma is in fact functioning as a source of repressive target derived like factors; again GAP-43 would be up regulated but not tubulin. The rational for these additional experiments is to corroborate the results in Chapters 4 and 5 that indicate tubulin is regulated more by positive factors than repressive factors in the chronic model of injury. In these proposed experiments the expression of tubulin should not be up-regulated following topical colchicine application or the subsequent removal of the neuroma. In this instance the transport of the positive factors would be blocked and therefore tubulin expression should not change. However, if there is an increase in tubulin under these 127  circumstances then this would suggest that tubulin is regulated by signals that are not axonally transported and therefore, likely to be electrically propagated signals. Electrical stimulation, for as little as one hour, proximal to the site of injury increases the regenerative capacity of motoneurons (Al-Majed, et al. 2000). This effect from electrical stimulation is suggested to originate via the cell body as tetrodotoxin blockade of action potentials to the cell body eliminates any effects of the electrical stimulation (Al-Majed, et al. 2000). Furthermore in vitro evidence suggests that the up regulation of immediate early genes and neurite outgrowth are associated with the depolarization induced calcium entry (Kocsis, et al. 1994). Obviously then i f tubulin became up-regulated in this topical colchicine application paradigm then action potential blockade or calcium blocker experiments (nimodipine) (Tao and Aldskogius 1998), would be the next step for the confirmation of this concept of electrically propagated signals regulating tubulin expression in this model. Before undertaking extensive work to understand the exact mechanism by which the neuroma acts to regulate the neuron response, Cragg pointed out one important consideration that must first be made. This involves the possible innervation of muscles adjacent to the neuroma by the transected axons. If the axons are indeed innervating adjacent muscle fibers, then the results observed by removal of the neuroma would involve the removal of target derived factors from the muscles and may not directly implicate the neuroma (Cragg 1970). In an attempt to address this issue, I resected mouse facial nerves and inserted the proximal stumps into small tubes, and the distal ends of the tubes were then sealed. Unfortunately, at 11 weeks post resection, examination of the injury site revealed that the tubes had detached from the nerves. If the axons are innervating adjacent muscle fibers, one might expect the motoneurons to appear similar to those on the contralateral un-injured side. Therefore, I would speculate that adjacent muscle innervation is unlikely as the majority of the resected mouse facial motoneurons remain in a highly atrophied state.  Multiple injuries Numerous treatments resulting in increased survival of injured mouse facial motoneurons have utilized transgenic, viral over-expression or the exogenous application of trophic factors normally produced or induced as a result of injury such as GDNF, C N T F and numerous cytokines (Hottinger, et al. 2000, Schweizer, et al. 2002, Serpe, et al. 2000). In Chapter 4 of this thesis I utilized a second axon injury (removal of the neuroma) to increase the expression of these factors to reverse the atrophy and increase gene expression in the chronically injured 128  motoneurons. In Chapters 4 and 5 of this thesis, I examined the m R N A expression of GAP-43 and a tubulin in C57 C3H and Balb/c and CD-I mouse strains following chronic resection of facial motoneurons and in response to a second and even a third axon injury. As mentioned, there are several reports of strain differences and there remains a large amount of work to elucidate these strain differences. Recently, gene chip analysis of DRGs from Harlan and Sprague Dawley rats demonstrated that 19-22% of the total genes changes were regulated in a strain specific manner after a peripheral injury (Valder, et al. 2003). However, of central interest to me, are not strain differences, but rather questions regarding the response of the facial motoneurons to multiple facial nerve injuries. Does the level of gene expression differ or is it the same (GAP-43 and tubulin) in mouse facial motoneurons after each successive injury? To answer this, quantification of ISH for GAP-43 and a tubulin m R N A would be required using age and strain matched mice processed together. Another question pertaining to the multiple injury paradigm is the distance of the motoneuron soma from the injury sites. In both rubrospinal and retinal ganglia cells the injury distance plays a prominent role in gene expression. In these models, only injuries close to the cell body elicit an increase in GAP-43 (Doster, et al. 1991, Fernandes, et al. 1999). In sympathetic neurons, distant injuries produce less of an increase in expression of T a 1 tubulin compared to more proximal injuries (Mathew and Miller 1993). In contrast, in D R G neurons GAP-43 expression is the same whether the injury is proximal or distal from the cell body (Liabotis and Schreyer 1995). D R G neurons are a unique population of neurons and their response to injury is dependant upon which of their axons are injured i.e., central vs peripheral (Schreyer and Skene 1993). In addition, following a sciatic nerve injury the expression of GAP-43 and tubulin in D R G neurons remains elevated, unlike facial motoneurons where the expression of these two declines within weeks of the injury (reviewed in Fu and Gordon 1997). In light of these differences between the facial and D R G models, there exists the possibility that injury distance from the neuron soma may in fact have a role in the facial motoneuron's response to injury, an issue that requires further study.  Nerve graft In Chapter 41 demonstrate that more chronically injured mouse facial motoneurons survive than had been previously reported. To determine the regenerative potential of these chronically injured motoneurons, in Chapter 5 I grafted a pre-degenerated sciatic nerve onto the chronically resected mouse facial motoneurons to provide a favorable environment for the axons to grow into. Significantly more motoneurons were counted in the acute group compared to the 129  chronic resection group. Thus, despite severe atrophy, surviving mouse facial motoneurons retain the propensity for regeneration if provided the appropriate environment. O f interest to me are the factors produced in the pre-degenerated nerve. I believe that this is an excellent model system to examine the trophic support requirements of chronically axotomized motoneurons, as the entire complement of factors is present in the graft. Treatments with the application of a single exogenous neurotrophin are limited by virtue of the fact that not only are the identities of all the factors not yet known, but exact timelines for administration and the precise neurotrophin concentration required have yet to be determined. For example, the application of B D N F and GDNF has a synergistic effect on promoting axonal regeneration of motoneurons. The number of motoneurons that grew into a chronically dennervated nerve was more with a combination of BDNF and GDNF than the arithmetic sum of the individual treatments (Boyd and Gordon 2003). In addition, B D N F acts in a dose dependant manner whereby a very high a dose can be detrimental to regeneration (Boyd and Gordon 2002). These data underscore the need for a more extensive characterization of the factors involved in PNS regeneration. A pre-degenerated peripheral nerve graft could be useful in examining the full extent of survival of chronically resected mouse facial motoneurons as the entire complement of factors/signals is available to the neurons. For instance, a pre-degenerated peripheral nerve graft could be transplanted onto the proximal stump at 9 weeks post chronic resection and the animals killed two weeks later. Motoneuron counts to compare survival to those of a single or second axotomy at 9 weeks without the nerve graft would be required. I propose that the predegenerated nerve graft would provide all the trophic support required for the motoneurons and that the surviving population would be more identifiable resulting in an increase in motoneuron counts, possibly above that seen with a second injury (without the graft). This proposed approach is in accordance with recent increases in combinatorial treatments in spinal cord injury. The manipulation of one gene or the treatment with one neurotrophic factor is unlikely to promote axonal regeneration leading to functional recovery in a system as complex as the mammalian CNS. Although, descriptions of gene expression and other details regarding the neuronal response to axonal injury and PNS regeneration have amassed a large collection of data over the past several years, many questions still remain. It is amazing to me that despite enormous technological advances, we are still deciphering the concepts initially set forth by Santiago Ramon y Cajal almost a century ago (Cajal 1991).  130  REFERENCES Abercrombie, M . , 1946. Estimation of nuclear population from microtome sections. Anat Rec 94. 239-247. Adams, J.M., 2003. Ways of dying: multiple pathways to apoptosis. Genes Dev 17. 2481-2495. Al-Majed, A . A . , Neumann, C M . , Brushart, T.M., and Gordon, T., 2000. Brief electrical stimulation promotes the speed and accuracy of motor axonal regeneration. J Neurosci 20. 2602-2608. Ambron, R.T., and Walters, E.T., 1996. Priming events and retrograde injury signals. A new perspective on the cellular and molecular biology of nerve regeneration. M o l Neurobiol 13.61-79. Angelov, D.N., Gunkel, A., Stennert, E., and Neiss, W.F., 1995. Phagocytic microglia during delayed neuronal loss in the facial nucleus of the rat: time course of the neuronofugal migration of brain macrophages. Glia 13. 113-129. Angelov, D.N., Neiss, W.F., Streppel, M . , Walther, M . , Guntinas-Lichius, O., and Stennert, E., 1996. ED2-positive perivascular cells act as neuronophages during delayed neuronal loss in the facial nucleus of the rat. Glia 16. 129-139. Aperghis, M . , Johnson, LP., Patel, N . , Khadir, A., Cannon, J., and Goldspink, G., 2003. Age, diet and injury affect the survival of facial motoneurons. Neuroscience 117. 97-104. Ashkenazi, A., and Dixit, V . M . , 1998. Death receptors: signaling and modulation. Science 281. 1305-1308. Ashwell, K.W., and Watson, C.R., 1983. The development of facial motoneurones in the mouse-neuronal death and the innervation of the facial muscles. J Embryol Exp Morphol 77. 117-141. Benowitz, L.I., and Routtenberg, A., 1997. GAP-43: an intrinsic determinant of neuronal development and plasticity. Trends Neurosci. 20. 84-91. Beuche, W., and Friede, R.L., 1984. The role of non-resident cells in Wallerian degeneration. J Neurocytol 13. 767-796. Bimbaum, M.J., Clem, R.J., and Miller, L.K., 1994. A n apoptosis-inhibiting gene from a nuclear polyhedrosis virus encoding a polypeptide with Cys/His sequence motifs. J. Virol. 68. 2521-2528. Bisby, M . , 1995. Regeneration of peripheral nervous system axons. In The Axon (S. G. Waxman, J. D. Kocsis, and P. K . Stys, Eds.), pp. 553-578. Oxford University Press.  131  Bisby, M . A . , 1982. Ligature Techniques. In Axoplasmic Transport (D. G. Weiss, Ed., pp. 437441. Springer-Verlag, Berlin; New York. Bisby, M . A . , 1988. Dependence of GAP43 (B50, F l ) transport on axonal regeneration in rat dorsal root ganglion neurons. Brain Res 458. 157-161. Blinzinger, K., and Kreutzberg, G., 1968. Displacement of synaptic terminals from regenerating motoneurons by microglial cells. Z Zellforsch Mikrosk Anat 85. 145-157. Bomze, H . M . , Bulsara, K.R., Iskandar, B.J., Caroni, P., Skene, J.H., Weber, J.R., and Smith, D.S., 2001. Spinal axon regeneration evoked by replacing two growth cone proteins in adult neurons. Nat Neurosci 4. 38-43. Bormann, P., Zumsteg, V . M . , Roth, L.W., and Reinhard, E., 1998. Target contact regulates GAP-43 and alpha-tubulin mRNA levels in regenerating retinal ganglion cells. J Neurosci Res 52. 405-419. Boyd, J.G., and Gordon, T., 2002. A dose-dependent facilitation and inhibition of peripheral nerve regeneration by brain-derived neurotrophic factor. Eur J Neurosci 15. 613-626. Boyd, J.G., and Gordon, T., 2003. Glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor sustain the axonal regeneration of chronically axotomized motoneurons in vivo. Exp Neurol 183. 610-619. Bridge, P.M., Ball, D.J., Mackinnon, S.E., Nakao, Y . , Brandt, K., Hunter, D.A., and Hertl, C , 1994. Nerve crush injuries—a model for axonotmesis. Exp Neurol 127. 284-290. Brown, M.C., and Lunn, E.R., 1988. Mechanism of interaction between motoneurons and muscles. Ciba Found Symp 138. 78-96. Buffo, A., Holtmaat, A.J., Savio, T., Verbeek, J.S., Oberdick, J., Oestreicher, A . B . , Gispen, W.H., Verhaagen, J., Rossi, F., and Strata, P., 1997. Targeted overexpression of the neurite growth-associated protein B-50/GAP-43 in cerebellar Purkinje cells induces sprouting after axotomy but not axon regeneration into growth-permissive transplants. J Neurosci 17. 8778-8791. Bunge, R.P., 1993. Expanding roles for the Schwann cell: ensheathment, myelination, trophism and regeneration. Curr Opin Neurobiol 3. 805-809. Bussmann, K . A . , and Sofroniew, M . V . , 1999. Re-expression of p75NTR by adult motor neurons after axotomy is triggered by retrograde transport of a positive signal from axons regrowing through damaged or denervated peripheral nerve tissue. Neuroscience 91. 273281.  132  Buttner-Ennever, J., and Holstege, G., 1986. Anatomy of premotor centers in the reticular formation controlling oculomotor, skeletomotor and autonomic motor systems. Prog Brain Res 64. 89-98. Cajal, S.R., 1991. Cajals Degeneration & Regeneration of the Nervous System. Oxford University Press, New York. Cammermeyer, J., 1955. Astroglial changes during retrograde atrophy of nucleus facialis in mice. J Comp Neurol 102. 133-150. Caroni, P., 2001. New E M B O members' review: actin cytoskeleton regulation through modulation of PI(4,5)P(2) rafts. Embo J 20. 4332-4336. Chai, J.J., Shiozaki, E., Srinivasula, S.M., Wu, Q., Dataa, P., Alnemri, E.S., and Shi, Y . G . , 2001 Structural basis of caspase-7 inhibition by XIAP. Cell 104. 769-780. Chen, Y.S., Hsu, C.J., Liu, T . C , Yanagihara, N . , and Murakami, S., 2000. Histological rearrangement in the facial nerve and central nuclei following immediate and delayed hypoglossal-facial nerve anastomosis. Acta Otolaryngol 120. 551-556. Cimler, B . M . , Giebelhaus, D.H., Wakim, B.T., Storm, D.R., and Moon, R.T., 1987. Characterization of murine cDNAs encoding P-57, a neural-specific calmodulin-binding protein. J Biol Chem 262. 12158-12163. Clem, R.J., Fechheimer, M . , and Miller, L.K., 1991. Prevention of apoptosis by a baculovirus gene during infection of insect cells. Science 254. 1388-1390. Clem, R.J., and Miller, L . K . , 1994. Control of programmed cell death by the baculovirus genes p35 and IAP. Mol. Cell. Biol 14. 5212-5222. Coers, S., Tanzer, L., and Jones, K.J., 2002. Testosterone treatment attenuates the effects of facial nerve transection on glial fibrillary acidic protein (GFAP) levels in the hamster facial motor nucleus. Metab Brain Dis 17. 55-63. Cowan, C M . , Thai, J., Krajewski, S., Reed, J . C , Nicholson, D.W., Kaufmann, S.H., and Roskams, A.J., 2001. Caspases 3 and 9 send a pro-apoptotic signal from synapse to cell body in olfactory receptor neurons. J. Neurosci. 21. 7099-7109. Cragg, B.G., 1970. What is the signal for chromatolysis? Brain Res 23. 1-21. Crook, N.E., Clem, R.J., and Miller, L.K., 1993. A n apoptosis-inhibiting baculovirus gene with zinc finger-like motif. J. Virol. 67. 2168-2174.  133  Curtis, R., Scherer, S.S., Somogyi, R., Adryan, K . M . , Ip, N . Y . , Zhu, Y . , Lindsay, R . M . , and DiStefano, P.S., 1994. Retrograde axonal transport of LIF is increased by peripheral nerve injury: correlation with increased LIF expression in distal nerve. Neuron 12. 191204. de Bilbao, F., and Dubois-Dauphin, M . , 1996. Acute application of an interleukin-1 betaconverting enzyme-specific inhibitor delays axotomy-induced motoneurone death. Neuroreport 7. 3051-3054. de Bilbao, F., and Dubois-Dauphin, M . , 1996. Time course of axotomy-induced apoptotic cell death in facial motoneurons of neonatal wild type and bcl-2 transgenic mice. Neuroscience 71. 1111-1119. Deshmukh, M . , Du, C , Wang, X . , and Johnson, E . M . , Jr., 2002. Exogenous smac induces competence and permits caspase activation in sympathetic neurons. J Neurosci 22. 80188027. Deveraux, Q.L., Leo, E., Stennicke, H.R., Welsh, K., Salvesen, G.S., and Reed, J . C , 1999. Cleavage of human inhibitor of apoptosis protein X I A P results in fragments with distinct specificities for caspases. E M B O J. 18. 5242-5251. Deveraux, Q.L., Roy, N . , Stennicke, H.R., Van Arsdale, T., Zhou, Q., Srinivasula, S.M., Alnemri, E.S., Salvesen, G.S., and Reed, J . C , 1998. IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases. E M B O J. 17. 2215-2223. Deveraux, Q.L., Takahashi, R., Salvesen, G.S., and Reed, J . C , 1997. X-linked IAP is a direct inhibitor of cell-death proteases. Nature 388. 300-304. Devor, M . , 1995. Abnormal excitability in injured axons. In The Axon (S. G. Waxman, J. D. Kocsis, and P. K . Stys, Eds.), pp. 530-552. Oxford University Press. Dohm, S., Streppel, M . , Guntinas-Lichius, O., Pesheva, P., Probstmeier, R., Walther, M . , Neiss, W.F., Stennert, E., Angelov, D.N., Tomov, T.L., Grosheva, M . , Schraermeyer, U., Skouras, E., Popratiloff, A., Rehm, K.E., Wewetzer, K., Azzolin, N . , Kazemi, S., Haas, C , Grothe, C , Wevers, A., Dramiga, J., Effenberger, K . , Klein, J., Hilgers, R.D., Rosenblatt, J.D., Gunkel, A., and Lichius, O.G., 2000. Local application of extracellular matrix proteins fails to reduce the number of axonal branches after varying reconstructive surgery on rat facial nerve. Restor Neurol Neurosci 16. 117-126.  134  Donovan, S.L., Mamounas, L.A., Andrews, A . M . , Blue, M.E., and McCasland, J.S., 2002. G A P 43 is critical for normal development of the serotonergic innervation in forebrain. J Neurosci 22. 3543-3552. Dorfl, J., 1982. The musculature of the mystacial vibrissae of the white mouse. J Anat 135. 147154. Doster, S.K., Lozano, A . M . , Aguayo, A.J., and Willard, M . B . , 1991. Expression of the growthassociated protein GAP-43 in adult rat retinal ganglion cells following axon injury. Neuron 6. 635-647. Eddleston, M . , and Mucke, L., 1993. Molecular profde of reactive astrocytes—implications for their role in neurologic disease. Neuroscience 54. 15-36. Enari, M . , Sakahira, H., Yokoyama, H., Okawa, K., Iwamatsu, A., and Nagata, S., 1998. A caspase-activated DNase that degrades D N A during apoptosis, and its inhibitor ICAD. Nature 391. 43-50. Farber, J.L., 1990. The role of calcium in lethal cell injury. Chem Res in Toxi. 3. 503-508. Fawcett, J.W., and Keynes, R.J., 1990. Peripheral nerve regeneration. Annu Rev Neurosci 13. 43-60. Fernandes, K. J. 2000. Signals regulating neuronal cell body response to axotomy, (PhD), University of British Columbia, Vancouver. Fernandes, K.J., Fan, D.P., Tsui, B.J., Cassar, S.L., and Tetzlaff, W., 1999. Influence of the axotomy to cell body distance in rat rubrospinal and spinal motoneurons: differential regulation of GAP-43, tubulins, and neurofilament-M. J Comp Neurol 414. 495-510. Fernandes, K.J., Kobayashi, N.R., Jasmin, B.J., and Tetzlaff, W., 1998. Acetylcholinesterase gene expression in axotomized rat facial motoneurons is differentially regulated by neurotrophins: correlation with trkB and trkC m R N A levels and isoforms. J Neurosci 18. 9936-9947. Fernandes, K.J., and Tetzlaff, W., 2000. Gene expression in axotomized neurons: identifying the intrinsic determinants of axonal growth. In Axonal regeneration in the central nervous system (N. A . Ingoglia, and M . Murray, Eds.), pp. 219-266. Marcel Dekker, Inc. Ferri, C.C., Moore, F.A., and Bisby, M . A . , 1998. Effects of facial nerve injury on mouse motoneurons lacking the p75 low-affinity neurotrophin receptor. J Neurobiol 34. 1-9. Fournier, A.E., Beer, J., Arregui, C O . , Essagian, C , Aguayo, A.J., and McKerracher, L., 1997. Brain-derived neurotrophic factor modulates GAP-43 but not T alpha 1 expression in injured retinal ganglion cells of adult rats. J. Neurosci. Res. 47. 561-572. 135  Friauf, E., 1986. Morphology of motoneurons in different subdivisions of the rat facial nucleus stained intracellularly with horseradish peroxidase. J Comp Neurol 253. 231-241. Friedman, B., Kleinfeld, D., Ip, N . Y . , Verge, V . M . , Moulton, R., Boland, P., Zlotchenko, E., Lindsay, R . M . , and Liu, L., 1995. B D N F and NT-4/5 exert neurotrophic influences on injured adult spinal motor neurons. J. Neurosci 15. 1044-1056. Fu, S.Y., and Gordon, T., 1997. The cellular and molecular basis of peripheral nerve regeneration. Mol. Neurobiol. 14. 67-116. Galiano, M . , Liu, Z.Q., Kalla, R., Bohatschek, M . , Koppius, A., Gschwendtner, A., Xu, S., Werner, A., Kloss, C.U., Jones, L.L., Bluethmann, H., and Raivich, G., 2001. Interleukin6 (IL6) and cellular response to facial nerve injury: effects on lymphocyte recruitment, early microglial activation and axonal outgrowth in IL6-deficient mice. Eur J Neurosci 14. 327-341. Garcia-Calvo, M . , Peterson, E.P., Leiting, B., Ruel, R., Nicholson, D.W., and Thornberry, N.A., 1998. Inhibition of human caspases by peptide-based and macromolecular inhibitors. J. Biol. Chem. 273. 32608-32613. Gianola, S., Rossi, F., Buffo, A., Holtmaat, A.J., Savio, T., Verbeek, J.S., Oberdick, J., Oestreicher, A . B . , Gispen, W.H., Verhaagen, J., and Strata, P., 2004. GAP-43 overexpression in adult mouse Purkinje cells overrides myelin-derived inhibition of neurite growth Targeted overexpression of the neurite growth-associated protein B-50/GAP-43 in cerebellar Purkinje cells induces sprouting after axotomy but not axon regeneration into growthpermissive transplants. Eur J Neurosci 19. 819-830. Giehl, K . M . , and Tetzlaff, W., 1996. B D N F and NT-3, but not N G F , prevent axotomy-induced death of rat corticospinal neurons in vivo. Eur. J. Neurosci. 8. 1167-1175. Graeber, M . B . , and Kreutzberg, G.W., 1988. Delayed astrocyte reaction following facial nerve axotomy. J Neurocytol 17. 209-220. Graeber, M . B . , Lopez-Redondo, F., Ikoma, E., Ishikawa, M . , Imai, Y . , Nakajima, K., Kreutzberg, G.W., and Kohsaka, S., 1998. The microglia/macrophage response in the neonatal rat facial nucleus following axotomy. Brain Res 813. 241-253. Graeber, M . B . , Tetzlaff, W., Streit, W.J., and Kreutzberg, G.W., 1988. Microglial cells but not astrocytes undergo mitosis following rat facial nerve axotomy. Neurosci. Lett. 85. 317321. Green, D.R., and Reed, J . C , 1998. Mitochondria and apoptosis. Science 281. 1309-1312. 136  Guillery, R.W., 2002. On counting and counting errors. J Comp Neurol 447. 1-7. Gulati, A . K . , 1988. Evaluation of acellular and cellular nerve grafts in repair of rat peripheral nerve. J Neurosurg 68. 117-123. Guntinas-Lichius, O., Angelov, D.N., Stennert, E., and Neiss, W.F., 1997. Delayed hypoglossalfacial nerve suture after predegeneration of the peripheral facial nerve stump improves the innervation of mimetic musculature by hypoglossal motoneurons. J Comp Neurol 387. 234-242. Guntinas-Lichius, O., Neiss, W.F., Gunkel, A., and Stennert, E., 1994. Differences in glial, synaptic and motoneuron responses in the facial nucleus of the rat brainstem following facial nerve resection and nerve suture reanastomosis. Eur Arch Otorhinolaryngol 251. 410-417. Guntinas-Lichius, O., Neiss, W.F., Schulte, E., and Stennert, E., 1996. Quantitative image analysis of the chromatolysis in rat facial and hypoglossal motoneurons following axotomy with and without reinnervation. Cell Tissue Res 286. 537-541. Guntinas-Lichius, O., Wewetzer, K., Tomov, T.L., Azzolin, N . , Kazemi, S., Streppel, M . , Neiss, W.F., and Angelov, D.N., 2002. Transplantation of olfactory mucosa minimizes axonal branching and promotes the recovery of vibrissae motor performance after facial nerve repair in rats. J Neurosci 22. 7121-7131. Hagg, T., Fass-Holmes, B., Vahlsing, H.L., Manthorpe, M . , Conner, J.M., and Varon, S., 1989. Nerve growth factor (NGF) reverses axotomy-induced decreases in choline acetyltransferase, N G F receptor and size of medial septum cholinergic neurons. Brain Res 505. 29-38. Hakem, R., Hakem, A., Duncan, G.S., Henderson, J.T., Woo, M . , Soengas, M.S., Elia, A., de la Pompa, J.L., Kagi, D., Khoo, W., Potter, J., Yoshida, R., Kaufman, S.A., Lowe, S.W., Penninger, J.M., and Mak, T.W., 1998. Differential requirement for caspase 9 in apoptotic pathways in vivo. Cell 94. 339-352. Hall, S.M., 1986. The effect of inhibiting Schwann cell mitosis on the re-innervation of acellular autografts in the peripheral nervous system of the mouse. Neuropathol Appl Neurobiol 12.401-414. Hall, S.M., 1986. Regeneration in cellular and acellular autografts in the peripheral nervous system. Neuropathol Appl Neurobiol 12. 27-46. Hall, S.M., 1999. The biology of chronically denervated Schwann cells. Ann N Y Acad Sci 883. 215-233.  Hallpike, J.F., 1976. Histochemistry of peripheral nerves and nerve terminals. In The Peripheral Nerve (D. N . Landon, Ed., pp. 605-665. Chapmand and Hall, London. Hammarberg, H., Piehl, F., Cullheim, S., Fjell, J., Hokfelt, T., and Fried, K . , 1996. GDNF mRNA in Schwann Cells and D R G Satellite Cells After Chronic Sciatic Nerve Injury. Neuroreport 7. 857-860. Hattox, A . M . , Priest, C.A., and Keller, A., 2002. Functional circuitry involved in the regulation of whisker movements. J Comp Neurol 442. 266-276. Henderson, C.E., Phillips, H.S., Pollock, R.A., Davies, A . M . , Lemeulle, C , Armanini, M . , Simmons, L., Moffet, B., Vandlen, R.A., Simpson, L.C., and et al., 1994. GDNF: a potent survival factor for motoneurons present in peripheral nerve and muscle. Science 266. 1062-1064. Heumann, R., Korsching, S., Bandtlow, C , and Thoenen, H . , 1987. Changes of nerve growth factor synthesis in nonneuronal cells in response to sciatic nerve transection. J Cell Biol 104. 1623-1631. Hofer-Warbinek, R., Schmid, J.A., Stehlik, C , Binder, B.R., Lipp, J., and de Martin, R., 2000. Activation of NF-kappa B by XIAP, the X chromosome-linked inhibitor of apoptosis, in endothelial cells involves T A K 1 . J. Biol. Chem. 275. 22064-22068. Hoke, A., Cheng, C , and Zochodne, D.W., 2000. Expression of glial cell line-derived neurotrophic factor family of growth factors in peripheral nerve injury in rats. Neuroreport 11. 1651-1654. Hoke, A., Gordon, T., Zochodne, D.W., and Sulaiman, O.A., 2002. A decline in glial cell-linederived neurotrophic factor expression is associated with impaired regeneration after long-term Schwann cell denervation. Exp Neurol 173. 77-85. Holcik, M . , Gibson, H., and Komeluk, R.G., 2001. X I A P : Apoptotic brake and promising therapeutic target. Apoptosis 6. 253-261. Holcik, M . , Thompson, C.S., Yaraghi, Z., Lefebvre, C.A., MacKenzie, A . E . , and Korneluk, R.G., 2000. The hippocampal neurons of neuronal apoptosis inhibitory protein 1 (NAIPl)-deleted mice display increased vulnerability to kainic acid-induced injury. Proc Natl Acad Sci U S A 97. 2286-2290. Hottinger, A.F., Azzouz, M . , Deglon, N . , Aebischer, P., and Zurn, A.D., 2000. Complete and long-term rescue of lesioned adult motoneurons by lentiviral-mediated expression of glial cell line-derived neurotrophic factor in the facial nucleus. J Neurosci 20. 5587-5593.  138  Houle, J.D., and Ye, J.H., 1999. Survival of chronically-injured neurons can be prolonged by treatment with neurotrophic factors. Neuroscience 94. 929-936. Huang, Y . H . , Park, Y . C . , Rich, R.L., Segal, D., Myszka, D.G., and Wu, H . , 2001. Structural basis of caspase inhibition by XIAP: Differential roles of the linker versus the BIR domain. Cell 104. 781-790. Huppenbauer, C.B., Tanzer, L., and Jones, K.J., 2001. Detection of retrogradely transported W G A - H R P in axotomized adult hamster facial motoneurons occurs after initiation of the axon reaction. J Neurocytol 30. 907-916. Hurley, S.D., and Coleman, P.D., 2003. Facial nerve axotomy in aged and young adult rats: analysis of the glial response. Neurobiol Aging 24. 511-518. Ide, C , Tohyama, K . , Yokota, R., Nitatori, T., and Onodera, S., 1983. Schwann cell basal lamina and nerve regeneration. Brain Res 288. 61-75. Isokawa-Akesson, M . , and Komisaruk, B.R., 1987. Difference in projections to the lateral and medial facial nucleus: anatomically separate pathways for rhythmical vibrissa movement in rats. Exp Brain Res 65. 385-398. Jiang, Y.Q., Pickett, J., and Oblinger, M . M . , 1994. Long-term effects of axotomy on beta-tubulin and N F gene expression in rat D R G neurons. J Neural Transplant Plast 5. 103-114. Johnson, LP., 2001. Rapid estimates of neuron number in the confocal microscope combined with in situ hybridisation and immunocytochemistry. Brain Res Brain Res Protoc 8. 113125. Johnson, LP., and Duberley, R . M . , 1998. Motoneuron survival and expression of neuropeptides and neurotrophic factor receptors following axotomy in adult and ageing rats. Neuroscience 84. 141-150. Jones, K.J., 1993. Recovery from facial paralysis following crush injury of the facial nerve in hamsters: differential effects of gender and androgen exposure. Exp Neurol 121. 133138. Jones, K.J., Storer, P.D., Drengler, S.M., and Oblinger, M . M . , 1999. Differential regulation of cytoskeletal gene expression in hamster facial motoneurons: effects of axotomy and testosterone treatment. J Neurosci Res 57. 817-823. Jongen-Relo, A . L . , and Feldon, J., 2002. Specific neuronal protein: a new tool for histological evaluation of excitotoxic lesions. Physiol Behav 76. 449-456.  139  Kamijo, Y . , Koyama, J., Oikawa, S., Koizumi, Y . , Yokouchi, K., Fukushima, N . , and Moriizumi, T., 2003. Regenerative process of the facial nerve: rate of regeneration of fibers and their bifurcations. Neurosci Res 46. 135-143. Kanje, M . , Skottner, A., Lundborg, G., and Sjoberg, J., 1991. Does insulin-like growth factor I (IGF-1) trigger the cell body reaction in the rat sciatic nerve? Brain Res 563. 285-287. Karimi-Abdolrezaee, S., and Schreyer, D.J., 2002. Retrograde repression of growth-associated protein-43 m R N A expression in rat cortical neurons. J Neurosci 22. 1816-1822. Kerr, J.F.R., Wyllie, A . H . , and Currie, A.R., 1972. Apoptosis: A basic biological phenomenon with wide ranging implications in tissue kenetics. Brit J. Cancer 26. 239-257. Kinderman, N.B., Harrington, C.A., Drengler, S.M., and Jones, K.J., 1998. Ribosomal R N A transcriptional activation and processing in hamster facial motoneurons: effects of axotomy with or without exposure to testosterone. J Comp Neurol 401. 205-216. Kinderman, N.B., and Jones, K.J., 1994. Axotomy-induced changes in ribosomal R N A levels in female hamster facial motoneurons: differential effects of gender and androgen exposure. Exp Neurol 126. 144-148. Klein, M . A . , Moller, J . C , Jones, L.L., Bluethmann, H., Kreutzberg, G.W., and Raivich, G., 1997. Impaired neuroglial activation in interleukin-6 deficient mice. Glia 19. 227-233. Kobayashi, N.R., Bedard, A . M . , Hincke, M.T., and Tetzlaff, W., 1996. Increased expression of B D N F and trkB m R N A in rat facial motoneurons after axotomy. Euro J. Neurosci. 8. 1018-1029. Kobayashi, N.R., Fan, D.P., Giehl, K . M . , Bedard, A . M . , Wiegand, S.J., and Tetzlaff, W., 1997. B D N F and NT-4/5 prevent atrophy of rat rubrospinal neurons after cervical axotomy, stimulate Gap-43 and T-alpha-1-tubulin mRNA expression, and promote axonal regeneration. J. Neurosci. 17. 9583-9595. Kocsis, J.D., Rand, M . N . , Lankford, K . L . , and Waxman, S.G., 1994. Intracellular calcium mobilization and neurite outgrowth in mammalian neurons. J Neurobiol 25. 252-264. Korhonen, L., Belluardo, N . , and Lindholm, D., 2001. Regulation of X-chromosome-linked inhibitor of apoptosis protein in kainic acid-induced neuronal death in the rat hippocampus. M o l Cell Neurosci 17. 364-372. Kreutzberg, G., 1966. Experimental neuropathology. Nervenarzt 37. 437-439. Kujawa, K . A . , Kinderman, N.B., and Jones, K.J., 1989. Testosterone-induced acceleration of recovery from facial paralysis following crush axotomy of the facial nerve in male hamsters. Exp Neurol 105. 80-85.  Kuzis, K., Coffin, J.D., and Eckenstein, F.P., 1999. Time course and age dependence of motor neuron death following facial nerve crush injury: role of fibroblast growth factor. Exp Neurol 157. 77-87. Kwon, B.K., Liu, J., Messerer, C , Kobayashi, N.R., McGraw, J., Oschipok, L., and 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. Kwon, B.K., Liu, J., Oschipok, L., and Tetzlaff, W., 2002. Reaxotomy of chronically injured rubrospinal neurons results in only modest cell loss. Exp Neurol 177. 332-337. Kyrkanides, S., O'Banion, M . K . , Whiteley, P.E., Daeschner, J . C , and Olschowka, J.A., 2001. Enhanced glial activation and expression of specific CNS inflammation-related molecules in aged versus young rats following cortical stab injury. J Neuroimmunol 119. 269-277. Laskawi, R., and Wolff, J.R., 1996. Changes in glial fibrillary acidic protein immunoreactivity in the rat facial nucleus following various types of nerve lesions. Eur Arch Otorhinolaryngol 253. 475-480. LeBlanc, A . C , and Poduslo, J.F., 1990. Axonal modulation of myelin gene expression in the peripheral nerve. J Neurosci Res 26. 317-326. Lewis, S.A., Lee, M . G . , and Cowan, N.J., 1985. Five mouse tubulin isotypes and their regulated expression during development. J Cell Biol 101. 852-861. L i , H., Zhu, H., X u , C.J., and Yuan, J., 1998. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell 94. 491-501. L i , L., Wu, W., Lin, L.F., Lei, M . , Oppenheim, R.W., and Houenou, L.J., 1995. Rescue of adult mouse motoneurons from injury-induced cell death by glial cell line-derived neurotrophic factor. Proc Natl Acad Sci U S A 92. 9771-9775. Liabotis, S., and Schreyer, D.J., 1995. Magnitude of GAP-43 induction following peripheral axotomy of adult rat dorsal root ganglion neurons is independent of lesion distance. Exp Neurol 135.28-35. Lidman, O., Fraidakis, M . , Lycke, N . , Olson, L., Olsson, T., and Piehl, F., 2002. Facial nerve lesion response; strain differences but no involvement of IFN-gamma, STAT4 or STAT6. Neuroreport 13. 1589-1593. Lieberman, A.R., 1971. The axon reaction: a review of the principal features of perikaryal responses to axon injury. Int Rev Neurobiol 14. 49-124.  141  Liston, P., Roy, N . , Tamai, K., Lefebvre, C , Baird, S., Cherton-Horvat, G., Farahani, R., McLean, M . , Ikeda, J.E., MacKenzie, A., and Komeluk, R.G., 1996. Suppression of apoptosis in mammalian cells by NAIP and a related family of IAP genes. Nature 379. 349-353. Liu, X.S., Zou, H., Slaughter, C , and Wang, X . D . , 1997. DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger D N A fragmentation during apoptosis. Cell 89. 175-184. Lowrie, M . B . , and Vrbova, G., 1992. Dependence of postnatal motoneurones on their targets: review and hypothesis. Trends Neurosci. 15. 80-84. Macfarlane, B.V., Wright, A., and Benson, H.A., 1997. Reversible blockade of retrograde axonal transport in the rat sciatic nerve by vincristine. J Pharm Pharmacol 49. 97-101. Maier, J.K., Lahoua, Z., Gendron, N.H., Fetni, R., Johnston, A., Davoodi, J., Rasper, D., Roy, S., Slack, R.S., Nicholson, D.W., and MacKenzie, A . E . , 2002. The neuronal apoptosis inhibitory protein is a direct inhibitor of caspases 3 and 7. J Neurosci 22. 2035-2043. Mathew, T.C., and Miller, F.D., 1993. Induction of T alpha 1 alpha-tubulin m R N A during neuronal regeneration is a function of the amount of axon lost. Dev. Biol. 158. 467-474. Matsuoka, I., Nakane, A., and Kurihara, K., 1997. Induction of LIF-mRNA by TGF-beta 1 in Schwann cells. Brain Res 776. 170-180. Mattox, D.E., Felix, H., Fisch, U . , and Lyles, C.A., 1988. Effect of ligating peripheral branches on facial nerve regeneration. Otolaryngol Head Neck Surg 98. 558-563. Mattsson, P., Meijer, B., and Svensson, M . , 1999. Extensive neuronal cell death following intracranial transection of the facial nerve in the adult rat. Brain Res Bull 49. 333-341. McBride, C.B., McPhail, L.T., and Steeves, J.D.S., 1999. Emerging therapeutic targets in caspase -dependent disease. Emerging therapeutic targets 3. 391-411. Mearow, K . M . , Kril, Y . , and Diamond, J., 1993. Increased N G F m R N A expression in denervated rat skin. Neuroreport 4. 351-354. Mercer, E.A., Korhonen, L., Skoglosa, Y . , Olsson, P.A., Kukkonen, J.P., and Lindholm, D., 2000. NAIP interacts with hippocalcin and protects neurons against calcium-induced cell death through caspase-3-dependent and -independent pathways. E M B O J. 19. 3597-3607. Meyer, M . , Matsuoka, I., Wetmore, C , Olson, L., and Thoenen, H . , 1992. Enhanced synthesis of brain-derived neurotrophic factor in the lesioned peripheral nerve: different mechanisms are responsible for the regulation of B D N F and N G F mRNA. J Cell Biol 119. 45-54.  142  Miller, F.D., Tetzlaff, W., Bisby, M . A . , Fawcett, J.W., and Milner, R.J., 1989. Rapid induction of the major embryonic alpha-tubulin mRNA, T alpha 1, during nerve regeneration in adult rats. J. Neurosci. 9. 1452-1463. Mohiuddin, L., Delcroix, J.D., Fernyhough, P., and Tomlinson, D.R., 1999. Focally administered nerve growth factor suppresses molecular regenerative responses of axotomized peripheral afferents in rats. Neuroscience 91. 265-271. Moran, L.B., Kosel, S., Spitzer, C , Schwaiger, F.W., Riess, O., Kreutzberg, G.W., and Graeber, M.B., 2001. Expression of alpha-synuclein in non-apoptotic, slowly degenerating facial motoneurones. J Neurocytol 30. 515-521. Mori, F., Himes, B.T., Kowada, M . , Murray, M . , and Tessler, A., 1997. Fetal spinal cord transplants rescue some axotomized rubrospinal neurons from retrograde cell death in adult rats. Expt. Neurol. 143. 45-60. Mullen, R.J., Buck, C.R., and Smith, A . M . , 1992. NeuN, a neuronal specific nuclear protein in vertebrates. Development 116. 201-211. Murphy, P.G., Borthwick, L.S., Johnston, R.S., Kuchel, G., and Richardson, P.M., 1999. Nature of the retrograde signal from injured nerves that induces interleukin-6 m R N A in neurons. J Neurosci 19. 3791-3800. Muzio, M . , Stockwell, B.R., Stennicke, H.R., Salvesen, G.S., and Dixit, V . M . , 1998. A n induced proximity model for caspase-8 activation. J. Biol. Chem. 273. 2926-2930. Naumann, T., Kermer, P., and Frotscher, M . , 1994. Fine structure of rat septohippocampal neurons. III. Recovery of choline acetyltransferase immunoreactivity after fimbria-fornix transection. J. Comp. Neurol. 350. 161-170. Naveilhan, P., ElShamy, W . M . , and Ernfors, P., 1997. Differential regulation of mRNAs for GDNF and its receptors Ret and GDNFR alpha after sciatic nerve lesion in the mouse. Eur J Neurosci 9. 1450-1460. Nguyen, Q.T., Sanes, J.R., and Lichtman, J.W., 2002. Pre-existing pathways promote precise projection patterns. Nat Neurosci 5. 861-867. Nicholson, D.W., and Thornberry, N.A., 1997. Caspases - Killer proteases. Trends Biochem Sci. 22. 299-306. Oppenheim, R.W., 1991. Cell death during development of the nervous system. Ann Rev Neurosci. 14. 453-501.  143  Perrelet, D., Ferri, A., Liston, P., Muzzin, P., Korneluk, R.G., and Kato, A . C . , 2002. IAPs are essential for GDNF-mediated neuroprotective effects in injured motor neurons in vivo. Nat Cell Biol 4. 175-179. Perrelet, D., Ferri, A., MacKenzie, A.E., Smith, G.M., Korneluk, R.G., Liston, P., Sagot, Y . , Terrado, J., Monnier, D., and Kato, A.C., 2000. IAP family proteins delay motoneuron cell death in vivo. Eur. J. Neurosci. 12. 2059-2067. Popratiloff, A., Kharazia, V . N . , Weinberg, R.J., Laonipon, B., and Rustioni, A., 1996. Glutamate receptors in spinal motoneurons after sciatic nerve transection. Neuroscience 74. 953958. Povelones, M . , Tran, K., Thanos, D., and Ambron, R.T., 1997. A n NF-kappaB-like transcription factor in axoplasm is rapidly inactivated after nerve injury in Aplysia. J Neurosci 17. 4915-4920. Purves, D., 1976. Functional and structural changes in mammalian sympathetic neurones following colchicine application to post-ganglionic nerves. J Physiol 259. 159-175. Raivich, G., Jones, L.L., Kloss, C.U., Werner, A., Neumann, FL, and Kreutzberg, G.W., 1998. Immune surveillance in the injured nervous system: T-lymphocytes invade the axotomized mouse facial motor nucleus and aggregate around sites of neuronal degeneration. J Neurosci 18. 5804-5816. Richardson, P.M., and Issa, V . M . , 1984. Peripheral injury enhances central regeneration of primary sensory neurones. Nature 309. 791-793. Richardson, P.M., McGuinness, U . M . , and Aguayo, A.J., 1980. Axons from CNS neurons regenerate into PNS grafts. Nature 284. 264-265. Rohlmann, A., Laskawi, R., Hofer, A., Dermietzel, R., and Wolff, J.R., 1994. Astrocytes as rapid sensors of peripheral axotomy in the facial nucleus of rats. Neuroreport 5. 409-412. Rossiter, J.P., Riopelle, R.J., and Bisby, M . A . , 1996. Axotomy-induced apoptotic cell death of neonatal rat facial motoneurons: time course analysis and relation to NADPH-diaphorase activity. Exp Neurol 138. 33-44. Sakamoto, T., Kawazoe, Y . , Shen, J.S., Takeda, Y . , Arakawa, Y . , Ogawa, J., Oyanagi, K., Ohashi, T., Watanabe, K., Inoue, K., Eto, Y . , and Watabe, K., 2003. Adenoviral gene transfer of GDNF, B D N F and TGF beta 2, but not CNTF, cardiotrophin-1 or IGF1, protects injured adult motoneurons after facial nerve avulsion. J Neurosci Res 72. 54-64.  144  Sakamoto, T., Kawazoe, Y . , Uchida, Y . , Hozumi, I., Inuzuka, T., and Watabe, K., 2003. Growth inhibitory factor prevents degeneration of injured adult rat motoneurons. Neuroreport 14. 2147-2151. Sambrook, J., Fritsch, E.F., and Maniatis, T., 1989. Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N Y , 9.31-9.62. Sanna, M.G., Duckett, C.S., Richter, B.W., Thompson, C.B., and Ulevitch, R.J., 1998. Selective activation of JNK1 is necessary for the anti-apoptotic activity of hILP. Proc Natl Acad Sci U S A 95. 6015-6020. Scheidt, P., and Friede, R.L., 1987. Myelin phagocytosis in Wallerian degeneration. Properties of millipore diffusion chambers and immunohistochemical identification of cell populations. Acta Neuropathol (Berl) 75. 77-84. Schreyer, D.J., and Skene, J.H., 1993. Injury-associated induction of GAP-43 expression displays axon branch specificity in rat dorsal root ganglion neurons. J. Neurobiol. 24. 959-970. Schweizer, U . , Gunnersen, J., Karch, C , Wiese, S., Holtmann, B., Takeda, K., Akira, S., and Sendtner, M . , 2002. Conditional gene ablation of Stat3 reveals differential signaling requirements for survival of motoneurons during development and after nerve injury in the adult. J Cell Biol 156. 287-297. Sendtner, M . , Gotz, R., Holtmann, B., Escary, J.-L., Masu, Y . , Carroll, P., Wolf, E., Brem, G., Brulet, P., and Thoenen, H., 1996. Cryptic physiological trophic support of motoneurons by LIF revealed by double gene targeting of CNTF and LIF. Current Biology 6. 686-694. Sendtner, M . , Stockli, K . A . , and Thoenen, H., 1992. Synthesis and locaization of ciliary neurotrophic factor in the sciatic nerve of the adult rat after lesion and during regeneration. J. Cell Biol. 118. 139-148. Seniuk, N . , Altares, M . , Dunn, R., and Richardson, P.M., 1992. Decreased synthesis of ciliary neurotrophic factor in degenerating peripheral nerves. Brain Res 572. 300-302. Serpe, C.J., Kohm, A.P., Huppenbauer, C.B., Sanders, V . M . , and Jones, K.J., 1999. Exacerbation of facial motoneuron loss after facial nerve transection in severe combined immunodeficient (scid) mice. J Neurosci 19. RC7. Serpe, C.J., Sanders, V . M . , and Jones, K.J., 2000. Kinetics of facial motoneuron loss following facial nerve transection in severe combined immunodeficient mice. J Neurosci Res 62. 273-278.  145  Shen, Y . , Mani, S., Donovan, S.L., Schwob, J.E., and Meiri, K.F., 2002. Growth-associated protein-43 is required for commissural axon guidance in the developing vertebrate nervous system. J Neurosci 22. 239-247. Simons, M . , Beinroth, S., Gleichmann, M . , Liston, P., Korneluk, R.G., MacKenzie, A.E., Bahr, M . , Klockgether, T., Robertson, G.S., Weiler, M . , and Schulz, J.B., 1999. Adenovirusmediated gene transfer of inhibitors of apoptosis protein delays apoptosis in cerebellar granule neurons. J Neurochem. 72. 292-301. Skouras, E., Popratiloff, A., Guntinas-Lichius, O., Streppel, M . , Rehm, K . E . , Neiss, W.F., and Angelov, D.N., 2002. Altered sensory input improves the accuracy of muscle reinnervation. Restor Neurol Neurosci 20. 1-14. Smith, D.S., and Skene, J.H.P., 1997. A Transcription-Dependent Switch Controls Competence of Adult Neurons For Distinct Modes of Axon Growth. J. Neurosci. 17. 646-658. Soreide, A.J., 1981. Variations in the axon reaction after different types of nerve lesion. Light and electron microscopic studies on the facial nucleus of the rat. Acta. Anat. 110. 173188. Soreide, A.J., 1981. Variations in the axon reaction in animals of different ages. A light microscopic study on the facial nucleus of the rat. Acta. Anat. 110. 40-47. Srinivasula, S.M., Fernandes-Alnemri, A . M . , and Alnemri, E.S., 1998. Autoactivationof procaspase-9 by Apaf-1-mediated oligomerization. Mol. Cell 1. 949-957. Steward, O., Schauwecker, P.E., Guth, L., Zhang, Z., Fujiki, M . , Inman, D., Wrathall, J., Kempermann, G., Gage, F.H., Saatman, K . E . , Raghupathi, R., and Mcintosh, T., 1999. Genetic approaches to neurotrauma research: opportunities and potential pitfalls of murine models. Exp Neurol 157. 19-42. Stockli, K . A . , Lillien, L.E., Naher-Noe, M . , Breitfeld, G., Hughes, R.A., Raff, M.C., Thoenen, H., and Sendtner, M . , 1991. Regional distribution, developmental changes, and cellular localization of C N T F - m R N A and protein in the rat brain. J Cell Biol 115. 447-459. Streit, W.J., and Graeber, M . B . , 1993. Heterogeneity of microglial and perivascular cell populations: insights gained from the facial nucleus paradigm. Glia 7. 68-74. Streit, W.J., Hurley, S.D., McGraw, T.S., and Semple-Rowland, S.L., 2000. Comparative evaluation of cytokine profiles and reactive gliosis supports a critical role for interleukin6 in neuron-glia signaling during regeneration. J Neurosci Res 61. 10-20.  146  Streit, W.J., Semple-Rowland, S.L., Hurley, S.D., Miller, R.C., Popovich, P.G., and Stokes, B.T., 1998. Cytokine m R N A profiles in contused spinal cord and axotomized facial nucleus suggest a beneficial role for inflammation and gliosis. Exp Neurol 152. 74-87. Streppel, M . , Azzolin, N . , Dohm, S., Guntinas-Lichius, O., Haas, C , Grothe, C , Wevers, A., Neiss, W.F., and Angelov, D.N., 2002. Focal application of neutralizing antibodies to soluble neurotrophic factors reduces collateral axonal branching after peripheral nerve lesion. Eur J Neurosci 15. 1327-1342. Strittmatter, S.M., Fankhauser, C , Huang, P.L., Mashimo, H., and Fishman, M.C., 1995. Neuronal pathfmding is abnormal in mice lacking the neuronal growth cone protein GAP-43. Cell 80. 445-452. Sturrock, R.R., 1988. Loss of neurons from the motor nucleus of the facial nerve in the ageing mouse brain. J Anat 160. 189-194. Sun, C , Cai, M . , Gunasekera, A . H . , Meadows, R.P., Wang, H., Chen, J., Zhang, H., Wu, W., Xu, N . , Ng, S.C., and Fesik, S.W., 1999. N M R structure and mutagenesis of the inhibitor-of-apoptosis protein XIAP. Nature 401. 818-822. Sun, Y . , and Zigmond, R.E., 1996. Leukaemia inhibitory factor induced in the sciatic nerve after axotomy is involved in the induction of galanin in sensory neurons. Eur J Neurosci 8. 2213-2220. Susin, S.A., Zamzami, N . , Castedo, M . , Hirsch, T., Marchetti, P., Macho, A., Daugas, E., Geuskens, M . , and Kroemer, G., 1996. Bcl-2 inhibits the mitochondrial release of an apoptogenic protease. Journal of Experimental Medicine 184. 1331-1341. Svensson, M . , and Aldskogius, H., 1993. Regeneration of hypoglossal nerve axons following blockade of the axotomy-induced microglial cell reaction in the rat. Eur J Neurosci 5. 8594. Taniuchi, M . , Clark, H.B., and Johnson, E . M . , Jr., 1986. Induction of nerve growth factor receptor in Schwann cells after axotomy. Proc Natl Acad Sci U S A 83. 4094-4098. Tao, R., and Aldskogius, H., 1998. Influence of FK506, Cyclosporin A , Testosterone and Nimodipine on Motoneuron Survival Following Axotomy. Restor Neurol Neurosci 12. 239-246. Tetzlaff, W., Alexander, S.W., Miller, F.D., and Bisby, M . A . , 1991. Response of facial and rubrospinal neurons to axotomy: changes in m R N A expression for cytoskeletal proteins and GAP-43. J. Neurosci. 11. 2528-2544.  147  Tetzlaff, W., Graeber, M . B . , Bisby, M . A . , and Kreutzberg, G.W., 1988. Increased glial fibrillary acidic protein synthesis in astrocytes during retrograde reaction of the rat facial nucleus. Glia 1. 90-95. Tetzlaff, W., Zwiers, H., Lederis, K., Cassar, L., and Bisby, M . A . , 1989. Axonal transport and localization of B-50/GAP-43-like immunoreactivity in regenerating sciatic and facial nerves of the rat. J. Neurosci. 9. 1303-1313. Thornberry, N.A., and Lazebnik, Y . , 1998. Caspases: enemies within. Science 281. 1312-1316. Thornberry, N.A., Ranon, T.A., Pieterson, E.P., Rasper, D . M . , Timkey, T., Garciacalvo, M . , Houtzager, V . M . , Nordstrom, P.A., Roy, S., Vaillancourt, J.P., Chapman, K.T., and Nicholson, D.W., 1997. A Combinatorial Approach Defines Specificities Of Members O f the Caspase Family and Granzyme B - Functional, Relationships Established For Key Mediators Of Apoptosis. J. Biol. Chem. 272. 17907-17911. Tomov, T.L., Guntinas-Lichius, O., Grosheva, M . , Streppel, M . , Schraermeyer, U . , Neiss, W.F., Angelov, D.N., Skouras, E., Popratiloff, A., Rehm, K . E . , Wewetzer, K., Azzolin, N . , Kazemi, S., Dohm, S., Haas, C , Grothe, C , Wevers, A., Dramiga, J., Effenberger, K., Klein, J., Stennert, E., Walther, M . , Hilgers, R.D., Rosenblatt, J.D., Gunkel, A., and Lichius, O.G., 2002. Transplantation of olfactory mucosa minimizes axonal branching and promotes the recovery of vibrissae motor performance after facial nerve repair in rats. Exp Neurol 178. 207-218. Torvik, A., and Skjorten, F., 1971. Electron microscopic observations on nerve cell regeneration and degeneration after axon lesions. I. Changes in the nerve cell cytoplasm. Acta Neuropathol (Berl) 17. 248-264. Travers, J., 1985. Organization and projections of the orofacial motor nuclei. In The Rat nervous system (G. Paxinos, Ed., pp. 111-128. Academic Press, Sydney. Valder, C.R., Liu, J.J., Song, Y . H . , and Luo, Z.D., 2003. Coupling gene chip analyses and rat genetic variances in identifying potential target genes that may contribute to neuropathic allodynia development. J Neurochem 87. 560-573. Vanderluit, J.L., McPhail, L.T., Fernandes, K.J., Kobayashi, N.R., and Tetzlaff, W., 2003. In vivo application of mitochondrial pore inhibitors blocks the induction of apoptosis in axotomized neonatal facial motoneurons. Cell Death Differ 10. 969-976.  148  Vanderluit, J.L., McPhail, L.T., Fernandes, K.J., McBride, C.B., Huguenot, C , Roy, S., Robertson, G.S., Nicholson, D.W., and Tetzlaff, W., 2000. Caspase-3 is activated following axotomy of neonatal facial motoneurons and caspase-3 gene deletion delays axotomy-induced cell death in rodents. Eur. J. Neurosci. 12. 3469-3480. Vaughan, D.W., 1990. Effects of advancing age on the central response of rat facial neurons to axotomy: light microscope morphometry. Anat Rec 228. 211-219. Vaughan, D.W., 1990. The effects of age on enzyme activities in the rat facial nucleus following axotomy: acetylcholinesterase and cytochrome oxidase. Exp Neurol 109. 224-236. Verdu, E., Ceballos, D., Vilches, J.J., and Navarro, X . , 2000. Influence of aging on peripheral nerve function and regeneration. J PeripherNerv Syst 5. 191-208. Verge, V . M . , Tetzlaff, W., Bisby, M.A., and Richardson, P . M . , 1990. Influence of nerve growth factor on neurofilament gene expression in mature primary sensory neurons. J. Neurosci. 10. 2018-2025. Verge, V . M . K . , Merlio, J.-P., Grondin, J., Ernfors, P., Persson, H., Riopelle, R.J., Hokfelt, T., and Richardson, P.M., 1992. Colocalization of N G F binding sites, trk mRNA, and lowaffinity N G F receptor m R N A in primary sensory neurons: responses to injury and infusion of NGF. J. Neurosci. 12. 4011-4022. Villasante, A., Wang, D., Dobner, P., Dolph, P., Lewis, S.A., and Cowan, N.J., 1986. Six mouse alpha-tubulin mRNAs encode five distinct isotypes: testis-specific expression of two sister genes. M o l Cell Biol 6. 2409-2419. Watson, W.E., 1968. Observations on the nucleolar and total cell body nucleic acid of injured nerve cells. J Physiol 196. 655-676. Woolf, C.J., Reynolds, M . L . , Molander, C , OBrien, C , Lindsay, R . M . , and Benowitz, L.I., 1990. The growth-associated protein GAP-43 appears in dorsal root ganglion cells and in the dorsal horn of the rat spinal cord following peripheral nerve injury. Neuroscience 34. 465-478. Wu, W., Mathew, T . C , and Miller, F.D., 1993. Evidence that the loss of homeostatic signals induces regeneration-associated alterations in neuronal gene expression. Developmental Biology 158. 456-466. Xu, D., Bureau, Y . , Mclntyre, D . C , Nicholson, D.W., Liston, P., Zhu, Y . , Fong, W.G., Crocker, S.J., Korneluk, R.G., and Robertson, G.S., 1999. Attenuation of ischemia-induced cellular and behavioral deficits by X chromosome-linked inhibitor of apoptosis protein overexpression in the rat hippocampus. J. Neurosci. 19. 5026-5033.  Xu, D.G., Crocker, S.J., Doucet, J.P., Stjean, M . , Tamai, K., Hakim, A . M . , and Ikeda, J.E., 1997. Elevation of neuronal expression of Naip reduces ischemic damage in the rat hippocampus. Nat. Med. 3. 997-1004. Xu, D.G., Korneluk, R.G., Tamai, K., Wigle, N., Hakim, A., Mackenzie, A., and Robertson, G.S., 1997. Distribution of neuronal apoptosis inhibitory protein-like immunoreactivity in the rat central nervous system. J. Comp. Neurol. 382. 247-259. Yan, Q., Matheson, C , and Lopez, O.T., 1995. In vivo neurotrophic effects of G D N F on neonatal and adult facial motor neurons. Nature 373. 341-344. Yang, Y., Fang, S., Jensen, J.P., Weissman, A . M . , and Ashwell, J.D., 2000. Ubiquitin protein ligase activity of IAPs and their degradation in proteasomes in response to apoptotic stimuli. Science 288. 874-877. Yu, W.H., 1988. Sex difference in neuronal loss induced by axotomy in the rat brain stem motor nuclei. Exp Neurol 102. 230-235. Yu, W.H., 1989. Administration of testosterone attenuates neuronal loss following axotomy in the brain-stem motor nuclei of female rats. J Neurosci 9. 3908-3914. Yu, W.H., 1990. Administration of testosterone fails to attenuate axotomy-induced motoneuron loss but results in castration-like effect in young male rats. Neuroendocrinology 52. 595599. Yu, W.H., and Cao, C.G., 1992. Testosterone fails to rescue motoneurons from axotomy-induced death in young rats. Neuroreport 3. 1042-1044. Yu, W.H., and McGinnis, M . Y . , 1986. Androgen receptor levels in cranial nerve nuclei and tongue muscles in rats. J Neurosci 6. 1302-1307. Zigmond, R.E., Hyatt-Sachs, H., Mohney, R.P., Schreiber, R.C., Shadiack, A . M . , Sun, Y., and Vaccariello, S.A., 1996. Changes in neuropeptide phenotype after axotomy of adult peripheral neurons and the role of leukemia inhibitory factor. Perspt. Dev. Neurobiol. 4. 75-90. Zompa, E.A., Cain, L.D., Everhart, A.W., Moyer, M.P., and Hulsebosch, C.E., 1997. Transplant therapy: recovery of function after spinal cord injury. J Neurotrauma 14. 479-506.  150  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0091794/manifest

Comment

Related Items