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Galectin-1 in injury and regeneration 2004

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GALECTIN-1 IN INJURY A N D REGENERATION by JOHN M C G R A W B. Sc. The University of British Columbia, 1994 M . Sc. The University of British Columbia, 1999 THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF T H E REQUIRMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in T H E F A C U L T Y OF G R A D U A T E STUDIES Department of Zoology We accept this thesis as conforming to the required standard T H E UNIVERSITY OF BRITISH C O L U M B I A July 2004 © John McGraw, 2004 11 ABSTRACT In higher vertebrates, injury to both sensory and motor axons within the central nervous system (CNS) results in permanent loss of sensation and paralysis. In contrast, peripheral nervous system (PNS) injuries are often followed by functional recovery. This disparity in the regenerative ability of the PNS and CNS is attributed to both the intrinsic gene response of injured neurons and the environment across which an injured axon must extend in order for successful regeneration to occur. Recent reports demonstrate that the carbohydrate-binding protein galectin (Gal)-l, which is expressed during the development of sensory and motor neurons and in the mature adult, is involved in regrowth of axotomized neurons following a peripheral nerve injury. Specifically, exogenous application of recombinant Gal l to injured peripheral nerves increased the rate of axonal growth into acellular grafts. However, the axotomy-induced changes of endogenous neuronal Gal l expression have not been examined. In this thesis, I demonstrate that axotomized neurons that were able to regenerate or initiate a growth response also had increased G a l l expression. Gal l returned to uninjured levels upon target re-innervation suggesting that the target may partially regulate Gal l expression. Furthermore, homozygous Gal l null mutant (-/-) mice showed an attenuated rate of functional recovery after a nerve crush. I also examined sensory responses of Gal l -/- mice, since Ga l l is expressed during sensory neuronal development as well as within the adult dorsal root ganglion (DRG). The absence of Gal l in the Ga l l -/- mouse led to an increased threshold for thermal nociceptive stimuli. This correlated with differences in nociceptive neuron proportions and their pattern of termination within the spinal cord. Taken together these data underscore an important contribution of endogenous Gal l to the regenerative process and to sensory neuronal development and/or maintenance. TABLE OF CONTENTS Abstract ii Table of Contents iii List of Figures v List of Tables vii Abbreviations viii Statement of Original Contributions ix Acknowledgements x Chapter 1: General Introduction 1 Traumatic Nerve Injuries 1 Limitations to CNS Regeneration 2 Lectins 11 Hypothesis, relevance and models 19 Chapter 2: Galectin-1 expression correlates with the regenerative potential of rubrospinal and spinal motoneurons 22 Introduction 23 Materials and Methods 25 Results 29 Discussion 39 Chapter 3: Regulation of Galectin-1 expression by axotomy in rat primary afferent neurons 43 Introduction 44 Materials and Methods 46 Results 50 Chapter 4: Increased Thermal-Pain Thresholds In Galectin-1 Null Mutant Mice: Correlation With Altered Nociceptive Primary Afferent Neuronal Number And Spinal Terminal Fields 80 Materials and Methods f. 83 Results 87 Discussion 104 iv Chapter 5: Endogenous motoneuronal Galectin-1 increases after axotomy and promotes functional recovery after facial nerve injury 108 Introduction 109 Materials and Methods I l l Results 113 Discussion 129 Chapter 6: General Discussion 134 Summary of Results 134 How does Gall promote axonal growth? 139 Consequences of an enhanced neuronal growth mode 146 Other Future directions 149 Overall Significance 151 Concluding remarks 151 References 152 V LIST OF FIGURES Figure 1.1 Structure of the galectin family 13 Figure 2.1 Gal l ISH signal in cervical spinal motor neurons 31 Figure 2.2 Gall-IR of spinal motor neurons 33 Figure 2.3 Gal l ISH signal in the red nucleus 35 Figure 2.4 Ga l l ISH signal in the red nucleus following BDNF treatment 37 Figure 3.1 Photomicrographs of Gal l in situ hybridization signal 51 Figure 3.2 Quantification of Gall autoradiographic signal in the DRG 53 Figure 3.3 Gall-IR and CGRP-IR in the DRG 54 Figure 3.4 Gall-IR and IB4-IR in the DRG 57 Figure 3.5 Gall-IR and NF200-IR in the DRG 59 Figure 3.6. Proportion of Gall-IR cells within the DRG following injury 61 Figure 3.7 Distribution of Ga l l - IR in the C7 dorsal horn 64 Figure 3.8 Gal l ISH in the dorsal horn following rhizotomy 66 Figure 3.9 Gal l ISH at the DREZ after rhizotomy 69 Figure 3.10 rhGall-Ox does not promote growth of NF200 and CTB labeled fibers 71 Figure 3.11 rhGall-Ox promotes growth of CGRP and W G A labeled fibers 73 Figure 4.1 Gal l -/- and wt sensory tests 88 Figure 4.2 Gall-IR in the C7 and C8 DRGs of Gal l wt mice 90 Figure 4.3 Gal l ISH signal in the DRG 94 Figure 4.4 CGRP and IB4 in Gal l -/- and wt mice 96 Figure 4.5 CGRP and IB4 in the dorsal horn of Gal l -/- and wt mice 98 Figure 4.6 c-fos-IR in the mouse dorsal horn after noxious temperature 100 Figure 4.7 NeuN-IR in the mouse dorsal horn 102 vi Figure 5.1 Gal l is expressed in uninjured facial motoneurons 114 Figure 5.2 Gal l ISH in Gal l wt mice after a nerve crush 116 Figure 5.3 Gal l ISH in CD-I mice after a nerve crush 119 Figure 5.4 Gal l ISH in CD-I mice after a nerve resection 121 Figure 5.5 Positive and negative signals regulate Gal l mRNA 124 Figure 5.6 Behavioural recovery following a facial nerve crush in Gal l wt and -/- mice 127 vii LIST OF TABLES Table 1. A summary of the diversity of sensory neurons within the D R G 7 Table 2. A summary of Gal l changes in S M N and RSN following axotomy 41 Table 3. A summary of changes in Gal l expression in sensory neurons following injury 77 Table 4. A summary of Gal l mRNA changes in Facial motoneurons 130 Table 5. A summary of changes observed in Gal l expression following injury 135 Abbreviations -/- homozygous null mutation -/+ heterozygous mutation A N O V A analysis of variance BBB blood brain barrier BDNF brain derived growth factor °C degree Celsius C cervical level Ca + + calcium ion CAP-23 cortical-associated protein - 23 kDa CGRP calcitonin gene-related peptide CNS central nervous system CNTF ciliary neurotrophic factor CRD carbohydrate recognition domain CSPG chondroitin sulfate proteoglycan CST corticospinal tract CTB cholera toxin sub unit B DREZ dorsal root entry zone DRG dorsal root ganglia E embryonic day E C M extracellular matrix E R K extracellular signal-regulated kinases EtBr ethidium bromide F-actin filamentous actin F A K focal adhesion kinase FGF fibroblast growth factor Gal l galectin-1 Gall-Ox oxidized galectin-1 Gall-Red reduced galectin-1 G A P - 43 growth associated protein - 43 kDa GDNF glial cell line-derived neurotrophic factor GFAP glial fibrillary acidic protein GFR GDNF receptor GTPase guanosine triphosphatases IB4 isolectin Bandeiraea simplicifolia IHC immunohistochemistry IL Interleukin IR immunoreactivity ISH in situ hybridization kDa kilodalton L lumbar pm micrometer M A P K mitogen-activated protein kinase M E K M A P K / E R K kinase mRNA messenger ribonucleic acid NF neurofilament NGF nerve growth factor NK-1 substance P receptor NOS nitric oxide synthase NT neurotrophin PB phosphate buffer PBS phosphate buffer saline PFA paraformaldehyde rh recombinant P post natal day PBS phosphate buffered saline PN peripheral nerve PNS peripheral nervous system R A G regeneration associated gene redox reduction oxidation R N red nucleus RST rubrospinal tract SCI spinal cord injury S E M standard error of the mean S M N spinal motoneuron T thoracic level thermo temperature-activated transient TRPs receptor potential ion channels Trk tropomyosin receptor kinase or tyrosine kinase TSA tyramide signal amplification W G A wheat germ aggulatin wt wild type IX STATEMENT OF ORIGINAL CONTRIBUTIONS This thesis contains material that has been submitted for publication: J. McGraw, L . W. Oschipok, J. Liu, G.W. Hiebert, C.F.W. Mak, H. Horie, T. Kadoya, J.D. Steeves, M.S. Ramer and W. Tetzlaff (accepted) Galectin-1 expression correlates with the regenerative potential of rubrospinal and spinal motoneurons. Neuroscience J. McGraw, L.T. McPhail, L . W. Oschipok, H. Horie, J.D. Steeves, F. Poirier, M.S. Ramer, and W. Tetzlaff (Submitted) Endogenous Galectin-1 increases after axotomy and promotes functional recovery after facial nerve injury. European Journal of Neuroscience J. McGraw, L. W. Oschipok, T. Kadoya, H. Horie, J.D. Steeves, W. Tetzlaff and M.S. Ramer (Submitted) Regulation of Galectin-1 expression by axotomy in rat primary afferent neurons. Neuroscience J. McGraw, A . D . Gaudet, L. W. Oschipok, H. Horie, J.D. Steeves, F. Poirier, W. Tetzlaff, and M.S. Ramer (will be Submitted) Increased Thermal-Pain Thresholds In Galectin-1 Null Mutant Mice: Correlation With Altered Nociceptive Primary Afferent Neuronal Number And Spinal Terminal Fields. Pain The thesis author, John McGraw, was the primary researcher for all the results presented in the above articles and this thesis. L.W. Oschipok provided expertise with in situ hybridization. J. L iu performed the surgeries and the tissue processing for the rubrospinal tract experiments. L.T. McPhail performed the all of the facial nerve surgeries and processed the CD-I mice tissue. F. Poirier generated the galectin-1 null mutant mice. T. Kadoya of Pharmaceutical Research Laboratory, Kirin Brewery Co. kindly provided recombinant oxidized galectin-1 protein and galectin-1 antibodies for use in these experiments. A . D . Gaudet, C.F.W. Mak, F. Zhang and S.J. Williams kindly provided technical assistance with computer tracings analysis. A l l work was performed under the supervision and experimental design provided by the thesis author. The above statements and assessment of work performed by the thesis author are correctly stated above. X ACKNOWLEDGEMENTS I would like to express gratitude to John Steeves, as my supervisor and leader of ICORD, who has created an environment that allowed me to collaborate with four laboratories that spanned three countries. Also, he has always provided astute insight into the neuroscience field and has always encouraged me to focus on a larger view of science. I must also thank my other supervisor Matt Ramer for all of his sensory supervision, encouragement and enthusiasm. As for Wolfram Tetzlaff, I must thank him for letting me give the galectin project a try and for all of his guidance both within the laboratory and around Tokyo's subway system. Also, I would like to thank Alaa El-Husseini and Alison Buchan, the other members of my supervisory committee, for their valuable contributions to my research project. Hidenori Horie cannot go unrecognized for all of his efforts with this galectin project. A l l of the people in ICORD made the laboratory environment a stimulating and fun place to work in, especially Jaimie Borisoff, Chris McBride and Lowell McPhail who were years of entertainment. In particular, Lowell for agreeing to our race and for making working late a little too easy and to Gordon Hiebert for reminding me about the important things in life. The British Columbia Neurotrauma Foundation should also be recognized for their financial support. I would also like to give special gratitude to my parents and sisters for their years of encouragement. I would like to express my deepest appreciation to my wife Jennifer for her unwavering support. 1 CHAPTER 1: GENERAL INTRODUCTION Traumatic Nerve Injuries Traumatic nerve injury, particularly in the central nervous system (CNS), often results in a permanent devastating disability. Two examples of traumatic nerve injuries are spinal cord injury (SCI) and brachial plexus injury. One of the most persistently pursued goals in the field of neuroscience is the full restoration of neuronal function after a traumatic SCI. This is due in part to the frequency with which such injury occurs, and its profound impact on those injured and on society. Approximately 3 in every 100,000 people living in developed countries sustain an SCI each year (Dincer et al., 1992; Levi et al., 1995; Nobunaga et al., 1999; Dryden et al., 2003). In Canada, 78% of these injuries occur in people between 15 and 34 years of age whose average life expectancy is almost equivalent to that of an able-bodied person (CPA, 2000). SCI and associated permanent loss of neurological function, in some cases followed by a lifetime of chronic pain, can be economically, socially and physically debilitating for those who sustain an SCI. There is also great financial cost; indeed, the total direct cost of SCI in Canada can amount to as much as $25 million over the lifetime of the injured person (Dijkers, 1997; Kennedy et al., 1997; McCol l et al., 1997; CPA, 2000; ICORD, 2003). Brachial plexus injury is another type of commonly occurring nerve injury involving damage to the complex of nerves innervating the arms. The brachial plexus has both a peripheral and centrally projecting branch. When injury occurs to the central branch, permanent monoplegia, anesthesia and often the onset of severe intractable pain of the upper extremities can result (Berman et al., 1998). In the United States, this type of injury occurs as a complication of childbirth in 3 out of every 1000 births (Dunham, 2003) and in 4 out of every 100,000 young healthy individuals as a result of accidents related to motorcycle and snowmobile use, or sustained during manual labour (Midha, 1997). It is significant that when injury occurs in the PNS component of the brachial plexus, the results can be less devastating. This is due in large part to one of the disparities between the central nervous system and the peripheral nervous system: regenerative ability. 2 Limitations to CNS Regeneration Since medical observations were first recorded, the most consistent and striking difference noted between the peripheral and central nervous systems has been that the permanent deficit that is a result of a traumatic CNS injury appears never to be restored, whereas either partial or full recovery is common after a peripheral nerve injury. Although the presence of this distinction remained "received wisdom" among medical professionals and scientists up until the early 20 t h century, scientific discoveries within the last century have begun to challenge this notion. In their landmark series of experiments published in 1928, F. Tello and S. Ramon y Cajal demonstrated growth into peripheral nerve grafts of a limited number of axotomized CNS axons. Although these experiments clearly disproved historical notions of the impossibility of CNS regeneration, their significance was not recognized until more than fifty years later, when they were revisited and expanded upon by Richardson and colleagues using more modern techniques. The efforts of Richardson and colleagues galvanized the field of neuronal regeneration research (Richardson et al., 1980; David and Aguayo, 1981; Richardson et al., 1982; Richardson et al., 1984). These elegant experiments clearly demonstrated that there is intrinsic growth capacity within the injured CNS when a growth- supportive environment is present, and laid the initial foundation for our present understanding of the processes required for axonal regeneration. Subsequent research in this field has focused on either the intrinsic growth capacity of injured neurons or the environment across which an injured axon must extend in order for successful regeneration to occur in the CNS (Tetzlaff and Steeves, 2000). This thesis sets out to examine whether the intrinsic neuronal expression of galectin (Gal)-l correlates with the growth potential of neurons and whether Gal l promotes neuronal regeneration. In order to arrive at this hypothesis I will first briefly summarize both the extrinsic (environmental) and intrinsic factors that inhibit axonal regeneration. Then I will examine the dorsal root ganglion (DRG) system in order to examine strategies to promote axonal regeneration and to describe the discovery of Gal l as a growth promoting protein. The introduction is concluded by a brief overview of my hypothesis and how it will be addressed in chapters 2-5. Finally Chapter 6 concludes with a general discussion of results and limitations of these experiments, potential mechanisms that may explain the reported results, and future studies. 3 Environmental inhibition of regeneration The work of Richardson and Tello demonstrated that glial (non-neuronal) cells and the associated extracellular matrix molecules of the adult CNS (commonly called the extrinsic environment) do not support regenerative growth. In contrast, during initial development of the CNS, axonal outgrowth must readily occur. This perceived shift from a growth- supportive environment during development to a growth-restrictive environment after development coincides with axonal myelination by glial cells called oligodendrocytes (Hasan et al., 1993), suggesting that myelin is a growth inhibitor. Myelin was one of the earliest characterized axonal growth inhibitors within the CNS (Caroni and Schwab, 1988a). In order to promote axonal growth by injured CNS axons, four main approaches have been employed to overcome myelin's growth-inhibiting effects. The earliest approach involved the use of functional blocking antibodies to promote regrowth and sprouting of the axotomized corticospinal tract (CST) (Schnell and Schwab, 1990; Bregman et al., 1995). These antibodies blocked specific myelin epitopes found at 35 and 250 kDa within a myelin fraction, or what is now thought of as Nogo (Caroni and Schwab, 1988b; Spillmann et al., 1998). Upon identification of the Nogo receptor, a second approach was to create specific receptor antagonists that also promoted CST regrowth (GrandPre et al., 2002). A third approach involves immunologically killing oligodendrocytes to reduce all myelin epitopes around the injury site. Through the use of complement proteins and oligodendrocyte-specific antibodies, some functional axonal growth and recovery was reported to be observed after partial or complete injury (Keirstead et al., 1995; Dyer et al., 1998). In the fourth and most novel approach, S. David and colleagues used a myelin vaccination approach, which elicits an immunological response against myelin in order facilitate growth or promote sparing of axotomized CST axons (Huang et al., 1999). These diverse in vivo approaches have all supported the notion that myelin contributes significantly to regenerative failure in the CNS. However, the idea that myelin is the principal barrier to regeneration in the CNS was recently challenged. Previous in vitro data demonstrated that myelin strongly inhibited DRG growth (Schwab et al., 1993). Davies and colleagues carefully transplanted dissociated 4 mouse dorsal root ganglion neurons into the heavily myelinated rat corpus collusm (Davies et al., 1997) and surprisingly, these neurons grew long distances. The mouse axons appeared to only stop growing in proteoglycan rich areas (Davies et al., 1997; Davies et al., 1999). Previous in vitro studies have demonstrated clearly that these proteoglycans inhibit DRG outgrowth and are a known component not only of the barrier between the PNS and CNS, but also of the astroglial scar (McKeon et al., 1991; Pindzola et al., 1993; Asher et al., 2001; Zhang etal., 2001). The astroglial scar forms after a penetrating injury to the CNS, and is comprised of various glial and other non-neuronal cells. In vertebrates, any major disruption of tissue such as dermis or CNS tissue often results in a fibrotic scar with associated extracellular matrix (ECM) molecules (Fitch and Silver, 1997a; Martin, 1997). This scar serves to quickly re-seal the wound by walling off the area from further damage and to restore homeostasis. Although the scar that develops following CNS trauma is beneficial in that it serves to contain the damaged area and restore the barrier between the periphery and CNS, it unfortunately also creates a barrier to regenerating axons (Davies et al., 1999). To overcome this regenerative obstacle, researchers have sought to reduce or remove the instigators of astrogliotic scar formation or the associated inhibitory E C M molecules (reviewed in McGraw et al., 2001). Perhaps the most promising results were observed when the proteoglycans at the scar site were enzymatically degraded. In axotomized ascending sensory fibers within the spinal cord, this treatment enhanced the growth into a predegenerated peripheral nerve graft in one model as well as increased both anatomical plasticity and functional recovery after a dorsal column crush (Bradbury et al., 2002). Although the reductions of inhibitory myelin epitopes or glial scar E C M molecules seem promising, only very limited axonal growth has ever been achieved to date. Accordingly other factors are likely to be involved that are actively inhibiting regeneration or that are required for regeneration but are absent. Examining a regenerating system, such as the PNS, gives further insight into the potential reasons for the lack of repair within the CNS. PNS development and regeneration The differing abilities of the CNS and PNS to regenerate are in part explained by the differing glial response to injury. In the injured CNS, oligodendrocyte myelin and astroglial 5 scar formation inhibit axonal regrowth. In stark contrast to the CNS, in a peripheral nerve injury, Schwann cells (the myelinating cells of the PNS) and macrophages actively reduce inhibitory barriers to neuronal repair. Schwann cells proliferate, retract their processes, and form linear arrays (bands of Biingner) in the distal segment in preparation for nerve regrowth (Ramon y Cajal, 1928; Bunge, 1987). In addition, by releasing proteases to degrade the E C M , Schwann cells actively degrade inhibitory proteoglycans (Fu and Gordon, 1997). Within the first few days after injury occurs, blood-borne macrophages invade the injury site and along with the resident macrophages, serve to remove growth-inhibitory myelin debris (Perry et al., 1987). These macrophages also attenuate protease release from Schwann cells. Excessive protease expression in Schwann cells could damage surviving neurons and thus must be regulated to prevent over degradation (La Fleur et al., 1996). The need for this macrophage response is clearly evident when macrophages are unable to clear debris. For example, in the C57BL/Wld s mutant mouse, delayed Wallerian degeneration occurs due to mutation in an ubiquitination factor (Conforti et al., 2000), in turn causing the failure of macrophages to clear axonal debris and inhibitory proteins (Perry et al., 1990; Chen and Bisby, 1993; Glass et al., 1993). A consequence of this mutation is delayed peripheral regeneration. As opposed to the glial cells in the CNS that form barriers to growth, non- neuronal cells within the PNS actively participate in neuronal regeneration by providing a more permissive growth environment. Schwann cells are not only involved in preparing the environment for peripheral growth, but also in providing as a source of neurotrophic factors that aid in neuronal survival and regeneration of the injured neuron. The discoveries of nerve growth factor (NGF) and its tropomyosin receptor kinase (Trk) A led to the identification of a family of neurotrophins and their respective receptors. Some time later, other members of the neurotrophin family include brain-derived neurotrophic factor (BDNF), neurotrophin (NT)-3, NT-4/5 and glial cell-line derived neurotrophic factor (GDNF), and their associated receptors (see table 1) where discovered (Levi-Montalcini, 1966; Barde et al., 1978; Kaplan et al., 1991; Lin et al., 1993). The DRG system has proven to be an excellent model for understanding the neurotrophins and their effects on neurodevelopment and repair, since an increasing amount of evidence 6 suggests that distinct populations of sensory neurons require different target-derived neurotrophins for survival during development and phenotypic maintenance in the adult. Rodent expression patterns of neurotrophic receptor mRNA and protein support the idea that neurons of different functional classes are regulated by different members of the neurotrophin family. For example, the NGF receptor TrkA localizes to 70-80% of all DRG neurons during development and in about 40% of medium and small-sized unmyelinated DRGs (Molliver et al., 1995; Molliver and Snider, 1997). Null mutations (-/-) to the NGF or TrkA gene led to a 70-80% decrease in sensory neuronal survival in the adult and a loss of pain sensation demonstrating that N G F signaling via the TrkA receptor is required for survival of these nociceptive neurons (Crowley et al., 1994; Smeyne et al., 1994; Silos- Santiago et al., 1995). However, as development proceeds, some neurons switch their neurotrophic factor requirement. Starting at embryonic (E) 15, continuing until postnatal (P) 7 and lasting until adulthood, half of NGF-dependent neurons lose their TrkA expression and begin to express RET, the signaling receptor for GDNF (Molliver and Snider, 1997; Molliver et al., 1997). This leads to an adult DRG population comprised of 40% TrkA and 30% Ret expressing neurons. Even though both of these populations are mainly small diameter C- fiber neurons responding to nociceptive stimuli, they overlap minimally. Although the specific functional nociceptive roles of these fiber populations remains elusive, that these fibers terminate in separate spinal laminae, and that they express different catalogues of molecules offer some indication of discrete functions. The third population of sensory neurons possessing medium to large somata are responsible for mechano- and proprioception, and express the NT-3 receptor, TrkC (McMahon et al., 1994). 10% of all DRG neurons express mRNA for the B D N F receptor TrkB in a population of cells that convey mechanoreceptive information (Mu et al., 1993; Wright and Snider, 1995; Carroll et al., 1998). 7 Sensory modality Neurotrophic dependence Expressed neurotrophic receptor Cell Phenotypic marker . * size •Hp nociceptors N G F TrkA CGRP galanin small 40 nociceptors GDNF RET, G F R a l GFRa2, GFRa3 IB4 P2X3 small 30 proprioceptors NT3 TrkC NF200 NPY large 25 mechanorecptors B D N F TrkB NF200 large 10-30 hair follicle receptor NT-4/5 TrkB med- large Table 1. A summary of the diversity of sensory neurons within the DRG. 8 After an axotomy, neurotrophins also play an important role in axonal regeneration. Following axonal injury, Schwann cells within the distal stump upregulate the expression of NGF, B D N F and NT-4 (Heumann et al., 1987; Meyer et al., 1992; Funakoshi et al., 1993; Anand et al., 1997). However this change in expression is not sufficient to compensate for the lack of target-derived factors and as a result, the injured D R G undergoes a number of changes (Heumann et al., 1987; Knyihar-Csillik et al., 1991). These involve the down- regulation of the peptides calcitonin gene-related peptide (CGRP) (Noguchi et al., 1990) and substance P (Nielsch et al., 1987), and the up-regulation of the peptides vasoactive intestinal polypeptide (Shehab and Atkinson, 1986), galanin (Hokfelt et al., 1987) and neuropeptide Y (Wakisaka et al., 1991). Furthermore, an up-regulation of the enzyme nitric oxide synthase (NOS) (Verge et al., 1992) and growth-associated protein (GAP)-43 (Woolf et al., 1990) are observed after axotomy. Together, these changes are thought to represent adaptive responses serving to reduce the deleterious effects of peripheral nerve damage and to promote survival and regeneration of the lesioned sensory afferents (Hokfelt et al., 1994). While peripheral nerve injury changes the expression of a variety of genes in the DRG, this response is attenuated or nonexistent after a rhizotomy (Chong et al., 1996; Andersen and Schreyer, 1999). Intrinsic gene response correlates with regeneration in the DRG The outcome of sensory nerve lesions may vary considerably, depending upon the extent and location of injured neurons. For instance, a lesion between the dorsal root ganglion and the periphery (axotomy) typically results in the most successful recovery (Fu and Gordon, 1997). In contrast, recovery never occurs after a rhizotomy, a lesion occurring between the CNS and the DRG (Carlstedt et al., 1988; Bradbury et al., 2000). Dorsal root axons regenerate at half the rate of injured peripheral processes (Wujek and Lasek, 1983; Oblinger and Lasek, 1984). This disparity in regenerative ability of the DRG is due, in part, to the differing gene responses elicited by these two injuries. For example, within one day of a peripheral axotomy the transcription factor c-jun is upregulated and remains elevated until target innervation (Leah et al., 1991; Herdegen et al., 1997). In contrast, a rhizotomy only elicits a slight increase in c-jun expression (Jenkins et al., 1993; Broude et al., 1997). Because of this 9 difference in regenerative ability, the DRG is an excellent system in which to examine both the changes in gene expression and regenerative ability of axotomized neurons in vitro and in vivo. In culture, D R G neurons extend long processes only after they have been axotomized in vivo 2-7 days prior to culture, whereas only short-branched processes occur without prior axotomy (Smith and Skene, 1997). The reversal of this axotomy-induced growth mode by transcriptional inhibitors suggests the requirement of mRNA synthesis for successful in vitro growth (Smith and Skene, 1997). These findings are confirmed in vivo. Axotomized ascending sensory fibers in the dorsal column do not sprout nor do they grow into a peripheral nerve graft. However, these fibers will both sprout or grow into a CNS tissue or a pre-degenerated peripheral graft if their peripheral process is axotomized one week before their central process is cut (Richardson and Issa, 1984; Richardson and Verge, 1987; Neumann and Woolf, 1999). Peripheral axotomy initiates many gene changes within the DRG, but a few well-characterized genes have garnered much of the attention. For example, GAP-43 (also known as F l , B-50 and neuromodulin) is used as a marker of an enhanced intrinsic growth state (Plunet et al., 2002). Usually only detected within subpopulations of neurons within the mature nervous system, GAP-43 increases when neurons enter a regenerative state (Skene, 1989; Schreyer and Skene, 1991; Tetzlaff et al., 1991; Chong et al., 1992; Fernandes et al., 1999). For example, rhizotomy in the D R G does not lead to changes in GAP-43, whereas, peripheral axotomy increases GAP-43 expression in neuronal somata from whence it is transported into the uninjured central branch of the nerve (Schreyer and Skene, 1991; Chong et al., 1994b). Thus this well-characterized protein is also used as a marker for regenerative growth ability within the CNS. Cervically axotomized rubrospinal neurons capable of limited growth into a predegenerated peripheral nerve graft have only a mild and transitory gene (GAP-43, T a i tubulin) response (Tetzlaff et al., 1991; Kobayashi et al., 1997). With this meager response, a small population of injured rubrospinal neurons (43 of a total of 1200 rubrospinal neurons) readily grows into a degenerated peripheral nerve (Kobayashi et al., 1997). Application of brain-derived neurotrophic factor (BDNF) to the red nucleus increases regenerative-associative gene expression and also increases the growth propensity of these injured axons as indicated by the increased number of neurons (131 out 10 of 1200 rubrospinal neurons) able to grow into the graft (Kobayashi et al., 1996; Kobayashi et al., 1997). The link between GAP-43 and growth was mainly correlational. GAP-43-deficient mice develop normally, but their neurons fail to grow in the proper pathways in the optic chiasm (Strittmatter et al., 1995). Furthermore, GAP-43 over-expressing mice fail to promote the growth of injured ascending dorsal column fibers into permissive PN transplants (Bomze et al., 2001). Only with the combined over-expression of cortical-associated protein (CAP)-23, another growth-associated protein, and GAP-43 were the ascending fibers able to grow into PN grafts. However, the growth that occurred was less than that of a priming lesion (Bomze et al., 2001). These experiments indicate that a multitude of intrinsic factors are required to achieve even a limited amount of growth within the CNS environment. DRG regeneration strategies Several strategies exist to enhance the injury response to rhizotomy. Exogenous growth factor application has had the most success in rescuing changes in neurochemistry, conduction velocity and gene expression after both axotomy and rhizotomy (Verge et al., 1995; Munson and McMahon, 1997; Munson et al., 1997; Bennett et al., 1998). Not surprisingly, specific neurotrophins affect those neurons expressing the appropriate receptors. For example, rhizotomized large-diameter sensory neurons expressing TrkC can regrow through the dorsal root entry zone (DREZ), the PNS-CNS interface for sensory neurons, to make functional connections through the exogenous application of NT-3 (Ramer et al., 2000). Likewise, G D N F promotes growth of the neurons expressing GFRct l ; however, BDNF has little effect even though 10-25% of the neurons express TrkB (Ramer et al., 2000). Importantly, regenerating neurons make appropriate and functional connections. For example, after rhizotomy, N G F treatment causes small diameter nociceptive fibers to regenerate back to the dorsal horn and thus allow rats to sense painful stimuli and respond appropriately (Ramer et al., 2000). NT3 treatment results in the restoration of proprioception (Ramer et al., 2002). Delaying NT3 treatment by 1 or 2 weeks after rhizotomy results in a considerable decrease in growth into the DREZ with no functional connections (Ramer et al., 2001b). These data indicate that there are many putative barriers to entry into the CNS. Research has begun to focus on the interplay of intrinsic and extrinsic factors limiting 11 regeneration but now time appears to alter the amount each one of these factors contributes to regenerative failure. In order to further elucidate other putative factors involved in neuronal repair, Horie et al (1999) used a DRG cell culture assay combined with cells derived from monkey kidney. Kidney cells secrete a wide variety of factors that it was hoped would play a role in axonal repair. Using COS 1 conditioned media in DRG explants, Horie et al observed that a 14.5 kDa fraction had some growth-promoting effect. After purification and sequencing, they identified the lectin, Galectin (Gall)- l as the growth-promoting protein. Using E. coli as expression vectors of human G a l l , recombinant (rh) G a l l was then obtained to create functional blocking antibodies. It was observed that Gal l protein expression was elevated at the injury site in both Schwann cells and neuron terminals. When rhGall-Ox was applied to axotomized rat sciatic nerves, an increase in neuronal growth into an acellular environment was noted, whereas Gal l functional blocking antibodies reduced the amount of growth when compared to control antibody application (Horie et al., 1999). A new class of proteins had been found to have growth-promoting effects within the DRG. Lectins Lectins are carbohydrate-binding proteins that recognize specific oligosaccharide structures and, most notably, cause agglutination and/or lysis of erythrocytes and leukocytes (termed "lectin activity") (Barondes, 1988). The earliest identification of a lectin was in 1872 when crystalline structures, termed Charcot-Leyden crystals, were grown from the sputum of asthmatics (reviewed in Kilpatrick, 2002). These crystals have been recently identified as galectin-10 (reviewed in Kilpatrick, 2002). Not until 1902 was the first lectin activity described (Flexner and Noguchi, 1902). Today the lectin family is classified on the basis of its members' dependence on Ca + + for binding ligands. The C-type lectins, of which there are seven subfamilies, require C a + + for carbohydrate binding, whereas the S-type, known as the galectins, bind independently of Ca** (Barondes et al., 1994b). Galectins Galectins are members of a highly conserved family of lectins widely distributed throughout the animal kingdom. These proteins occur in organisms ranging from viruses, plants, nematodes, and invertebrates to humans (Cooper, 2002). To date, fourteen mammalian 12 forms, galectin-1 through to 14 respectively, have been identified in a wide variety of tissues (Barondes et al., 1994b; Rabinovich et al., 2002). Of these galectins, only galectin-1, 3 and 5 were found biochemically and then cloned. The other galectins have been identified using sequence homology, since galectins share structural similarities in their carbohydrate recognition domain (CRD). Using similar sequence-based searching techniques within human genomic data, seven additional mammalian candidates have been identified that may be included in the galectin family (Cooper and Barondes, 1999; Cooper, 2002). Although similarities exist between galectins' CRDs, the overall structure linking the CRDs together further classifies the galectins into specific groups (Figure 1.1) (Hirabayashi and Kasai, 1993). Galectin-1, 2, 5, 7, 10, 11 and 13 are termed prototypical galectins because they exist as monomers or non-covalent homodimers consisting of two identical CRDs. Gal3 exists only as a monomer with two functional domains and Gal4, 6, 8, 9 and 12 exist as high order oligomers (Barondes et al., 1994b; Barondes et al., 1994a; Leffler et al., 2004). Gal l was first identified in electric eels in 1975 (Teichberg et al., 1975). It is one of the most extensively studied galectins since its expression occurs in most organs (Perillo et al., 1998) as well as a wide range of cells including placenta (Poirier et al., 1992) and muscle cells (Wasano et al., 1990) and neurons (Regan et al., 1986). 13 (A) CRD Repeats CRD I 1 CRD CRD ] r. * ^ link CP Galectins 1,2,5,7,10,11,13 Galectin-3 Galectins 4,6,8,9,12 ( B ) Figure 1.1 Galectin structures (A) Homologous carbohydrate recognition domain (CRD) present in all galectins. The primary protein structure is represented along the top row of the diagram. The quaternary structure is represented in the second row. Galectin-1 can exist either as a monomer or a homodimer indicated by the space between the two circular structures. (B) Structure of a human Gal l dimer. 40% of the galectin-1 protein is a |3-pleated sheet (arrows) and 10% a- helix with random coils (wire). 14 Galectin-1: structure Gal l exists as a monomeric or homodimeric protein comprised of subunits of 135-amino- acid long-chains, each such chain having a molecular weight of 14.5 kDa (Barondes et al., 1994b). The structure of a human Gal l monomer consists of |3-pleated sheets (40%), a- helixes (10%) and random coils. As a dimeric molecule, G a l l possesses two galactoside binding sites allowing either intramolecular or intermolecular crosslinking by binding more than one sugar residue. Gal l has been reported to be released from myogenic cells (Cooper and Barondes, 1990), Chinese hamster ovary cells (Cho and Cummings, 1995), human leukemia cell lines (Lutomski et al., 1997b), Schwann cells and dorsal root ganglions (Sango et al., 2004). G A L E C T I N - 1 : 2 R E D O X F O R M S Gal l exists in two known conformations, each of which has a different biological effect. These conformations are dictated by Gal l ' s reduction/oxidation (redox) state. Under reducing conditions, reduced Gal l - (Red) exists as a homodimer (Lobsanov et al., 1993). In the reduced conformation there are two CRD that facilitate both intra and wfermolecular binding (Liao et al., 1994; Perillo et al., 1998). This binding then promotes cellular adhesion, fasciculation and/or agglutination via |3-galactoside binding (Perillo et al., 1998). |3- galactoside binding is termed lectin activity. In vitro, lectin activity can be inhibited either by the addition of specific sugar residues that would competitively inhibit (3-galactoside binding or by G a l l oxidization (Tracey et al., 1992; Brewer, 2004). The change from a reduced to oxidized state was then termed "oxidative inactivation" (Tracey et al., 1992), yet this oxidative inactivation switched Gal l from allowing P-galactoside binding (lectin activity) to promoting neuronal outgrowth (Inagaki et al., 2000). When oxidized, Gal l changes conformation and exists only as a monomer. Examining Gal l ' s molecular structure gives further insight into how the switch from a homodimer to a monomer occurs. Within each Gal l subunit, six cysteines can form three intramolecular disulfide bonds (Cys 2-Cys 1 3 0, Cys^-Cys 8 8 and Cys^-Cys 6 0) (Gupta et al., 1996; Horie and Kadoya, 2000). When these bonds form, Gal l changes from a reduced state to an oxidized state (Gall-Ox) (Horie and Kadoya, 2000). Ga l l is believed to exist mainly in a reduced state within cells, since free radicals that can be generated in oxidizing environments can be deleterious to normal cellular 15 functions. When secreted, a proportion of the released Gal l becomes oxidized due to the oxidizing conditions in the extracellular environment. Galectin-1: location and secretion Although G a l l is present on the cell surface or E C M like all other galectins, it lacks a recognizable secretion sequence and accordingly does not pass though the standard Golgi/ER pathway. Instead, G a l l has an acetylated N-terminus, free sulfhydryls and lacks the glycosylation that is most often associated with cytoplasmic proteins. Cellular localization is predominately cytoplasmic with some nuclear staining, but is never observed inside the classic secretory pathways. Similar to that of fibroblast growth factor (FGF)-l and -2, the secretion of Gal l has been demonstrated though a novel non-classical secretory mechanism possibly through an A B C transporter (Cleves et al., 1996; Cleves and Kelly, 1996) and not via the classical vesicle-mediated exocytosis (Cooper and Barondes, 1990; Schafer et al., 2004). Galectin-1: non-neuronal tissue M U S C L E T I S S U E Gal l expression occurs in both striated and smooth muscle cells (Watt et al., 2004). In vitro studies demonstrate that the fusion of myoblasts is reduced if the cells originate from Gal l -/- mice (Poirier and Robertson, 1993). Striated muscle from Gal l -/- mice has more connective tissue than that of Ga l l wild type (wt) animals. After muscle injury, Ga l l wt muscle cells demonstrate an increase of Gal l protein expression. The fact that striated muscle in Gal l -/- mice does not regenerate as quickly as Gal l wt muscle tissue after injury suggests that the injury induced increase of G a l l expression in G a l l wt mice is involved in muscle regeneration (Watt et al., 2004). Therefore Gal l is important for both muscle development and repair after injury. K I D N E Y D E V E L O P M E N T Kidney cells are known to express G a l l , 3 and 9 (Hughes, 2002). Ga l l particularly is important for E C M and connective tissue organization (Wasano et al., 1990). There are no reports of differences in kidney development or organization between Gal l -/- and wt mice. 16 T C E L L REGULATION Research has identified immunomodulatory properties for G a l l . In three experimental autoimmune diseases, the T-cell-mediated autoimmune disease experimental encephalomyelitis (EAE), experimental autoimmune myasthenia gravis (EAMG) in rabbits and collagen induced rheumatoid arthritis, Gall either prevents the initiation of the disease as in the case with E A E or attenuates the disease's effects in the case of E A M G or rheumatoid arthritis (Levi et al., 1983; Offner et al., 1990; Rabinovich et al., 1999). Although the precise mechanisms (including Gal l ' s redox state) remain unclear, Ga l l is implicated in regulating immune activation through T cell apoptosis (Perillo et al., 1995; Nguyen et al., 2001; Zuniga et al., 2001). It is suggested that the reduction of active Ga l l is correlated with immune activation imbalance (Lutomski et al., 1997a). G a l l may also regulate immune responses under normal physiological conditions (Rabinovich et al., 2002). Ga l l expression occurs in immune-privileged sites such as the placenta and the eyes (Hirabayashi and Kasai, 1984; Iglesias et al., 1998; Ogden et al., 1998). At such sites, inflammation could have devastating consequences, so multiple factors ensure rapid elimination of inflammatory cells. Recently, FasL expression in sites of immune privilege has been shown to selectively kill T cells by apoptosis to maintain the immune barrier (Ferguson and Griffith, 1997a, b; Griffith and Ferguson, 1997; Stuart et al., 1997). Accordingly, Gal l expression might aid in maintaining immune barriers. C A N C E R In multicellular organisms, homeostasis is maintained through a balance of proliferation and death. Ga l l can limit T cell infiltration by inducing apoptosis, but Ga l l expression also occurs within constantly rejuvenating or growing tissues, such as smooth muscle, the adult olfactory system or other cells undergoing rapid growth such as tumour cells (Puche et al., 1996; Moiseeva et al., 2000; Camby et al., 2001). For example, the malignancy of astrocytomas, prostate carcinomas and metastatic pancreatic cancer correlates with high Gal l expression (Berberat et al., 2001; Camby et al., 2001; van den Brule et al., 2001). In one of the few experiments demonstrating a direct effect of G a l l on metastatic growth, the application of Ga l l anti-sense mRNA to cultured astrogliomas significantly reduced the cells' malignancy (Yamaoka et al., 2000). It is proposed that Ga l l changes the adhesive 17 properties of cell-cell and cell-matrix interactions of the tumours while attenuating the immune response to increase their ability to proliferate and spread. Galectin-1: neuronal expression The first publication relating to Gal l expression within the rat CNS was presented in 1986 (Regan et al., 1986). Using antibodies against a G a l l epitope, the authors reported G a l l (then termed RL-14.5) protein expression in 63% of primary sensory somata as well as expression in spinal motor neurons (Regan et al., 1986). The initial expression began at E13- 14 as these sensory neurons finished their final mitotic division and began their growth towards their targets within the dorsal horn of the spinal cord. When Gall-expressing neurons reached their targets, Ga l l expression remained elevated, albeit at lower levels (Regan et al., 1986; Hynes et al., 1990; St John and Key, 1999; Sango et al., 2004). In situ hybridization studies later revealed that the Gal l protein within the neurons reflects the synthesis by the neurons themselves and not the accumulation of the protein from other central or peripheral cells (Hynes et al., 1990). Ga l l expression was also observed in the olfactory bulb, cranial motoneurons and sympathetic ganglia (Hynes et al., 1990). However, although studies using Ga l l -/- mutant mice have offered further insight into this lectin's potential role in axonal growth and development, the precise role of G a l l during development remains elusive. Galectin-1: neuronal Junction Gal l expression occurs in a specific class of olfactory neurons. In the olfactory system, nerve fibers originating in the olfactory epithelium converge on spatially-defined glomeruli in the olfactory bulb (St John and Key, 1999). The ensheathing glial cells along this pathway synthesize and express Ga l l or laminin (Mahanthappa et al., 1994; Crandall et al., 2000). Although these two molecules associate in the same tract, the function of their interaction remains uncertain (Crandall et al., 2000). In vitro, Gal l promotes the fasciculation of primary olfactory neurons by facilitating the binding axon-axon and axon-matrix (such as laminin) interactions in an integrin-independent mechanism (Mahanthappa et al., 1994). Using Gal l - /- mice, generated by inserting a neo cassette containing a stop codon into the Gal l gene by homologous recombination in embryonic stem cells (Poirier and Robertson, 1993), the effects of Gal l on olfactory bulb development were examined. In the adult Gal l -/- mice, the 18 Dolichos biflorus agglutinin-binding neurons came close to their appropriate targets but failed to reach them and make proper connections (Puche et al., 1996). In these animals, immunohistochemistry revealed a normal distribution of Schwann cells, N C A M , and laminin at the olfactory bulb (Puche et al., 1996). These data suggested that Gal l may be involved in neuronal outgrowth and/or synaptic connectivity of specific neuronal populations and/or has a putative role in neuronal maintenance. Galectin-1: neuropathological conditions P A I N Partial nerve injury is frequently associated with hyperalgesia (increased pain sensitivity), allodynia (pain from non-noxious stimuli) and general ongoing pain. As previously indicated, high levels of G a l l are associated with increased growth and oxidized Ga l l promotes outgrowth from peripheral as well as central branches of DRG neurons in vitro (Horie et al., 1999; Berberat et al., 2001; van den Brule et al., 2001). Using a model for neuropathic pain, in the spinal nerve ligation model, Ga l l immunoreactivity increases in the dorsal horn (Camby et al., 2001; Imbe et al., 2003). Using a cDNA array to examine changes in the gene expression 2d-28d after a sciatic nerve axotomy reveals that Gal l mRNA increases as much as two to five times after injury at all time points examined (Xiao et al., 2002). The increase in Gal l expression elicits some neuropathic effects, since intrathecal administration of function-blocking Gal l antibodies attenuates mechanical hyperalgesia (Cameron et al., 1997; Imbe et al., 2003). In summary, Gal l correlates with increased excitation and sensitization of neurons in a neuropathic pain state, whereas reduction of G a l l activity reduces both the anatomical and functional changes associated with this increased activity. N E U R O N A L REGENERATION: G A L 1-OX The growth-promoting effects of Gal l were discovered using kidney cells combined with an in vitro DRG assay (Horie et al., 1999). Horie and colleagues demonstrated that recombinant Gall-Ox monomers, and not the reduced dimers, specifically promoted DRG outgrowth in vitro and accelerated injured peripheral nerve growth into silastic tubes in vivo (Horie et al., 1999). This work was later repeated using acellular autografts with the retrograde tracer FluoroGold applied at the ends of the grafts to label growing axons (Fukaya et al., 2003). For both D R G and spinal motor neurons, there were an increased number of FluoroGold-labeled somata with rhGall-Ox infusion as compared to vehicle alone treatment (Fukaya et al., 19 2003). Conversely, Ga l l functional-blocking antibodies significantly reduced the number of labeled DRGs and S M N when compared to control rabbit IgG application alone (Fukaya et al., 2003). In both of these experiments, the increased growth of axotomized neurons was closely associated with increased Gal l immunoreactivity in Schwann cells as well as increased migration into the tubes or autografts (Horie et al., 1999; Fukaya et al., 2003). In these experiments, only complete nerve resections attached to either a silastic tube or an acellular graft were used to examine nerve regeneration. This injury model prevents the assessment of functional regeneration through the use of specific sensory and motor assessment because the regenerating axons are unable to reach their targets. Accordingly, these experiments demonstrate that Gall-Ox increases the rate of neuronal regrowth in vitro and vivo, but not whether appropriate and functional connections are made. Hypothesis, relevance and models Although all of the results discussed above supported the hypothesis that exogenously- applied recombinant Gall-Ox promotes axonal regeneration in Gall-expressing neurons, I believed that the more basic questions of 1) whether neuronal Ga l l expression changed after axotomy or 2) whether the intrinsic G a l l expression had a role in axonal injury and/or regeneration had not been answered. After surveying the literature, / hypothesized that endogenous neuronal Gall expression influences the growth potential of axotomized neurons. Testing this hypothesis required a clinically relevant model that would further our understanding of regenerative neuronal processes in traumatic injury. Since most of the Gal l expression data indicated that both the S M N and DRG neuronal populations would yield some results, I chose the brachial plexus as a model system to attempt to examine Gal l expression in a medically relevant injury. Brachial plexus injuries are medically complex, due to associated anesthesia, paralysis and complex pain of differing degrees (Berman et al., 1998). Scientifically, these injuries are intriguing. Depending on the severity and location of injury, recovery of brachial plexus motor and sensory deficits varies considerably. Here, lesion classification can aid in determining functional outcome. Clinically, an upper limb rhizotomy with associated motor nerve avulsion or axotomy injury often results in one of three recognized syndromes: Duchenne-Erb or Erb's syndrome (with the levels C5-C6 being injured), Klumpke or 20 Duchenne-Aran syndrome (C8-T1 injured) or the most devastating when the roots from C5- T l are damaged. Increased understanding of regenerative failure may lead to strategies to promote regenerative success in rodents and will hopefully aid in better treatments and recovery for humans. Experimental outline addressing my hypothesis To ascertain whether Gal l expression correlates with growth propensity, I examined two well-established neuronal regeneration models. I compared changes in G a l l mRNA expression in axotomized S M N (which are known to successfully regenerate back to their targets), to the non-regenerating axotomized rubrospinal neurons. These results are presented in Chapter 2. To further support this hypothesis I then examined DRG neurons, since they have different regenerative responses depending on lesion location. Here I examined whether Ga l l expression changes between a peripheral axotomy (increased regenerative response) and a rhizotomy (limited or no regenerative response). Using rhGall, I artificially increased the available Gal l -Ox to determine whether this would facilitate growth into the CNS after rhizotomy. These results are presented in Chapter 3. Although the application of Gal l or functional blocking antibodies addresses questions about Gal l involvement in regeneration, it does not address the role of the endogenous production of Ga l l . Thus I have examined Gal l -/- mice and their possible functional deficits in both the naive and injured animals. Since Gal l has a putative role in neuropathic pain, Chapter 4 examines whether G a l l -/- mutant mice display differences in nociceptive behavior and sensory anatomy. Finally, in Chapter 5, using Gal l -/- mice, I tested whether the complete absence of Gal l changes the regenerative ability of neurons. Although the medial nerve of the brachial plexus is interesting to study because it contains sensory, motor and sympathetic components, it is also difficult to assess functional regeneration. Separating sensory and motor behavior responses can be potentially challenging. Accordingly, I chose to examine the facial nucleus, which primarily contains motoneurons. Since this nerve innervates the 21 mouse whisker pad, I examined the rate of functional recovery as determined by analyzing whisker movement between Gal 1 -/- and wild type mice. Overall, in the following four chapters I attempted to assess injury-induced changes in neuronal G a l l expression and whether these changes are important for functional regeneration. 22 CHAPTER 2: GALECTIN-1 EXPRESSION CORRELATES WITH THE REGENERATIVE POTENTIAL OF RUBROSPINAL AND SPINAL MOTONEURONS 23 Introduction A striking disparity exists between the regenerative ability of axons within the CNS, and neurons that project to the periphery. Extrinsic factors such as the glial response and the intrinsic gene expression of the injured neurons are implicated in the lack of regeneration within the CNS (Steeves and Tetzlaff, 1998; McGraw et al., 2001). In particular, axonal regeneration of peripherally axotomized motor neurons is associated with a cell body response that includes the increased expression of immediate-early genes, trophic factors, trophic factor receptors, neuropeptides and cytoskeletal proteins (Herdegen et al., 1992; Tetzlaff et al., 1994; Fernandes et al., 1999; Fernandes and Tetzlaff, 2000). Recently, exogenous application of rhGall-Ox has been shown to promote both the rate and success of axonal elongation of peripherally-projecting neurons; however, both the expression and role of the endogenous neuronal Ga l l expression in the CNS injury is unknown (Horie and Kadoya, 2000; Fukaya et al., 2003). Lectins are carbohydrate-binding proteins that recognize specific oligosaccharide moieties involved in promoting immune cell agglutination and cell signaling processes (Barondes, 1988). G a l l is a member of the galectin subfamily of lectin proteins whose sequence is conserved from Caenorhabditis elegans through to humans (Barondes et al., 1994b). The growth state of various tissues from this diverse range of organisms is tightly correlated with G a l l expression. For example, in adult rodents and mammals, G a l l expression occurs within tissues that constantly rejuvenate or grow, such as smooth muscle, the adult olfactory system, and tumour cells (Puche et al., 1996; Moiseeva et al., 2000; Camby et al., 2001). Within the developing nervous system, high levels of Ga l l expression are also observed (reviewed in Perillo et al., 1998; Moiseeva et al., 2000). In embryonic rats, Ga l l mRNA expression increases within the somata of spinal motoneurons (SMN) until they reach their target muscles, following which, expression is maintained at a lower level throughout adult life (Hynes et al., 1990). After a nerve injury, axons re-enter a growth mode to re-establish their connections. Ga l l mRNA increases within 6 to 24 hours in motoneurons following 24 facial nerve axotomy (Akazawa et al., 2004). Here I examined the expression of Gal l in regenerating and non-regenerating neurons. Specifically, I compared the Gal 1 mRNA level within the somata of injured S M N , a population that does regenerate to its peripheral targets (Nguyen et al., 2002), to that of injured rubrospinal neurons (RSN), a CNS neuronal population that does not typically exhibit axonal regeneration following spinal axotomy (Barron et al., 1975; Tetzlaff et al., 1994). I also assessed the effects of brain derived growth factor (BDNF) on Gal l expression within the red nucleus, since B D N F application to the red nucleus increases both the expression of genes associated with regeneration, and the ability of axotomized rubrospinal neurons to grow into a permissive peripheral nerve graft (Kobayashi et al., 1997; Kwon et al., 2002b). 25 Materials and Methods Surgery Adult male Sprague-Dawley rats (Charles River Breeding Laboratories, weight 200-250 g) were used. A l l surgery was performed in accordance with the Canadian Council for Animal Care and approved by the University of British Columbia Animal Care Committee. 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) and all surgery was under sterile conditions. Rubrospinal tract lesion Under anesthesia, 15 Sprague-Dawley rats underwent a transection of the dorsolateral funiculus at the level of the fourth cervical vertebra (C4), severing the rubrospinal tract unilaterally. The lesion was made with a pair of fine iris scissors. Six animals underwent intracranial implantation of a 28-gauge cannula into the vicinity of the rubrospinal neurons (6.3 mm posterior to bregma, 1.7 mm to the right of midline, and 6.5 mm deep to the dural membrane) as described previously (Kobayashi et al., 1997). An osmotic minipump (Alzet no. 2001, 1 pg/h, Palo Alto, CA) was connected to the cannula by silastic tubing. Three animals received 12 pl/d of BDNF (gift from Regeneron Pharmaceuticals, Tarrytown, NY) in a vehicle solution of 20 m M sterile PBS, 100 units of Penicillin/Streptomycin (Gibco B R L , Burlington, ON), and 0.5% rat serum albumin (Sigma-Aldrich, Oakville, ON) while three animals received vehicle alone. Spinal nerve lesions 12 (n=4 for each time point) rats were anesthesitized, and the spinal nerves were exposed on the left side from C6 to C8. Nerves were cut at their exit points from the spinal column and a 5mm section of peripheral nerve was removed. The wound was closed in layers with silk sutures. Perfusion I cryosectioning At either 7 or 14 days, animals were injected with a lethal dose of chloral hydrate and monitored. Upon the loss of nociceptive reflexes, animals were perfused intracardially with PBS followed by cold 4% paraformaldehyde. The brain and spinal cord were removed and the tissue post-fixed for 24 hours in 4% paraformaldehyde at 4°C. Tissue was cryoprotected in a 22% sucrose solution in PB. After cyroprotection, tissue was rapidly frozen in 26 supercooled 2-methylbutane and, later, 16 urn cryosections of brain or spinal cords were cut, cold-mounted onto glass slides (Superfrost plus) and stored at -80°C. Immunohistochemistry For Gal l immunohistochemistry of spinal motoneurons, slides were washed in 0.1m PBS for 20 minutes, after which rabbit anti-Gall (1:6000 in 0.1M PBS, 0.2% Triton X-100, and 0.1% sodium azide) was applied to the slides overnight. Two separate Gal l antibodies were used for the analysis showing identical results. One Ga l l antibody was generous gift from Dr. D. N . Cooper at the University of California San Francisco (Regan et al., 1986) and the other was previously generated (Horie et al., 1999). Both antibodies are shown to be specific for Gal l alone (Regan et al., 1986; Horie et al., 1999). The slides were then washed for 30 minutes in PBS and a blocking solution of 10% goat serum and 0.1% Triton-XlOO in PBS was added for 20 minutes at room temperature. The slides were then exposed to a donkey anti-rabbit secondary antibody (1:500, Jackson ImmunoResearch Inc, West Grove, PA) for one hour. A tyramide signal amplification step was then employed as per the manufactures instructions (PerkinElmer Lifescience, Boston, MA) . Cy3-conjugated Steptavidin (1:500, Sigma) was used to visualize the Gal l antibody. A fluorescent Nissl stain (Neurotrace, 1:200, Molecular Probes Inc. Eugene, OR) was then applied to visualize nissl substance. Slides were coverslipped with a 3:1 solution of glycerol: PBS. In situ hybridization The rat G a l l probe, corresponding to bases 393-443, was a 51-mer oligonucleotide complementary to the 3'-untranslated sequence of Gal l 5'-CAC T C A A A G G C C A C A C A C TTA A T C T T G A A G T C A C C A T C C G C C G C C A T G T A G - 3 ' (GenBank accession number NM019904). The probes were end-labeled with [ 3 3P]-dATP (Perkin-Elmer, Woodbridge, On) using deoxynucleotide terminal transferase according to a standard protocol (Kobayashi et al., 1997). Perfusion-fixed sections were hybridized to 1.2 x 106 cpm of probe for 16-18 h at 44°C. The slides were dipped in Kodak NTB-2 emulsion and exposed for 3 days (SMN) or 5 days (red nucleus). Slides were then dehydrated in a series of alcohols and stored at room temperature. Spinal cord sections were later re-hydrated in dH 20 for 1 hour and counterstained with Neurotrace (Molecular Probes. 1:200). For the red nucleus, sections were stained in 0.01% ethidium bromide (EtBr) for 1 hr and rinsed under running 27 H 2 0 for 1 hr. Slides were then dehydrated in a series of alcohols and coverslipped with Entallen (Fisher Scientific, Nepean, ON). Image analysis Determination of immunoreactivity within somata has been previously described (Ramer et al., 2003). Briefly, for spinal motoneuron immunohistochemistry, a fluorescent microscope (Axioskop, Carl Zeiss, Toronto, ON) was used to visualize chromophore labeled tissue, and images were captured using a digital camera (Carl Zeiss) in combination with Northern Eclipse software (Empix Inc, Mississauga ON). Neurotrace and Gal l images were taken of both the injured and the contralateral sides at the same image capture settings. Motoneuron cell bodies were outlined on the neurotrace image. The resulting drawn layer was then used to determine the staining intensity of Ga l l labeled profiles in the same double-labeled sections. An area in close proximity to the traced images but without neuronal cell bodies was also traced to determine background (non-specific) labeling. The intensity of this layer was subtracted from the motoneuron intensity values. To determine proportions of positively labeled motoneurons, the threshold for positive immunoreactivity was determined subjectively and recorded in each section so that variations in intensity could be accounted for on a section-by-section basis. Injured somata were compared to the uninjured contralateral somata. For in situ hybridization (ISH), Neurotrace (spinal motoneurons) or EtBr (rubrospinal neurons) and darkfield images of silver grains were taken of both the injured and the contralateral sides using a digital camera attached to a fluorescent microscope in combination with Northern Eclipse software. A l l images were analyzed with SigmaScan Pro 5 software (SPSS Inc, Chicago, IL). The percentage area occupied by silver grains of each neuronal cell body determined the somal grain area fraction. This was measured by outlining the individual neuronal cell bodies using the neurotrace or EtBr image and applying the resulting cell profile layer to the darkfield (silver grain) image. For each animal, the percentage area occupied by ISH signal per soma was determined for both the axotomized and contra lateral (uninjured) side. The difference between background autoradiographic signal and the mean ISH signal per soma was calculated. At least three sections, >100 pm apart, were quantified 28 per ventral horn or red nucleus, and the relative ISH signal was represented as a multiple of background. Statistics Quantification was performed blind with respect to the treatment groups. A l l data are represented as mean ± standard error of the mean (SEM), and all tested were carried out using SigmaStat 3.0 (SPSS Inc, Chicago, IL). Unless otherwise indicated, a Student's t-test was applied to the average area covered by ISH signal per somata to detect significant changes between axotomized and contralateral neurons and an A N O V A test was used to determine significant differences between groups. If significance was found then a post hoc Holm-Sidak test was used. Significance was assigned at P<0.05. 29 Results Spinal nerve axotomy increases motoneuron Gall mRNA and Protein Levels Axotomy of cervical spinal nerves from the 6 t h cervical (C6) to 8 t h cervical (C8) level was performed in order to examine changes in Gal l mRNA in spinal motoneurons, 7 and 14 days (n=4 animals per group) after injury. Spinal nerves were lesioned at their exit point from the spinal column to ensure that all motoneurons in a particular segment were completely severed. Seven days after axotomy, the average percentage cell soma area occupied by silver grains was 16.6 ±1 .7 (mean ± SEM) times greater than background on the injured side and 9.3 ± 0.9 on the contralateral uninjured side, representing a significant increase (p<0.05, Figure 2.1). 14 days after injury, this expression remained significantly elevated to 15.0 ± 2.1 times background signal on the axotomized side and 6.6 ± 0.8 on the contralateral uninjured side (p<0.05, Figure 2.1). Gal l immunohistochemistry on the same tissue revealed an increase in the proportion of motoneurons possessing high levels of G a l l staining intensity compared to the uninjured controls (Figure 2.2), and a cumulative sum chart (Figure 2.2 right inset) reveals a significant increase (as observed by a right shift of the curve) of the proportion of G a l l positive S M N soma at both 7 and 14 days after injury (p < 0.05 Kolmogorov-Smirnov test). BDNF reverses RSN axotomy induced decrease of Gall mRNA expression RSNs within the central projecting rubrospinal tract were transected at the C4 level, and the red nucleus was examined 3, 7 and 14 days after injury (n=3 animals per group). The expression of Ga l l mRNA in the uninjured red nucleus was lower than that of uninjured S M N as indicated by the longer (3 days for S M N , 5 days for RSN) exposure time required to obtain comparable silver grain densities. In contrast to SMNs, axotomized RSN exhibited a decreased Gal l mRNA expression. At 3 days post-lesion, the axotomized RSN mean silver grain proportional area was 10.2 ± 0.9 times greater than background compared to 11.2 ± 1.2 times background on the uninjured (contralateral) side (not significant, p>0.05, Figure 2.3). At 7 days post-lesion, the axotomized RSN somata had a significantly lower average silver grain proportional area of 7.7 ± 0.7 times background compared to 12.0 ± 0.8 on the uninjured (contralateral) side, (p<0.02, Figure 3). At 14 days post-lesion, there was an even greater decrease in Gal l average grain area fraction over individual somata from 9.7 ± 0.7 30 times background on the uninjured side to 5.3 ± 0.6 times background on the injured side (p<0.05, Figure 2.3). To test the hypothesis that Gal l is associated with regenerating systems, the regenerative response of RSNs was stimulated via infusion of BDNF into the vicinity of the injured RSNs (Kobayashi et al., 1997; Plunet et al., 2002). In the present study, 12 pg/day infusion of BDNF for seven days after injury resulted in a significant increase in the Ga l l silver grain proportional area, which was 9.0 ±1 .3 times background on the uninjured contralateral RSN compared to 13.9 ± 1.0 times background on the axotomized R S N (p<0.05, Figure 2.4). No significant differences in G a l l expression were observed between the axotomized and uninjured contralateral RSN in vehicle-treated control animals (Figure 2.4). However, between treatment group comparisons showed that there was a significant difference between the BDNF treated group and both the vehicle treated group (p=0.02, Figure 4) and untreated lesioned somata at 7d (p<0.02, Figure 2.3 and 2.4). The vehicle treatment effect has been previously described in the same paradigm when examining changes of GAP-43 mRNA within the red nucleus (Kobayashi et al., 1997). Sense oligonucleotides did not reveal any specific autoradiographic signal within SMN or the red nucleus. 31 Figure 2.1 Gall ISH signal in cervical spinal motor neurons The proportional area of motoneuron somata covered by silver grains was quantified 7 and 14 days (n=4 per group) after a C6-C8 spinal nerve lesion and compared to background signal. Seven days after axotomy the average proportional area was 16.6 ±1.7 (mean ± SEM) times greater than background on the injured side, which was significantly higher than the uninjured contralateral side of 9.3 ± 0.9 times greater than background (p<0.02). 14 days after injury, this expression remained elevated at 15.0 ±2.1 times greater than background on the axotomized side and 6.6 ± 0.8 on the contralateral uninjured side representing a significant increase (p<0.02). * indicates p<0.05, Scale bar = 50 microns. 32 33 Figure 2.2 Gall-IR of spinal motor neurons Seven and fourteen days after injury an increase in Gal l immunoreactivity was observed compared to the uninjured contralateral side. Histograms of immunoreactivity (0-255 grayscale) revealed a significant increase in Gal l immunoreactivity (graph on far right). Cumulative sum (Qsum) plots were used to determine statistical significance (chart inset far right, p< 0.05 Kolmogorov-Smirnov test). Scale bar = 50 microns 34 contralateral axotomized 0 1 2 3 4 5 S 7 B 9 10 1 1 1 2 intensity 35 Figure 2.3 Gall ISH signal in the red nucleus Gal l in situ hybridization signal in the red nucleus 3, 7 and 14 days (n=3 per group) after a transection of the dorsolateral funiculus (which includes the rubrospinal tract) at the fourth cervical level. At 3 days post lesion, the axotomized RSN had an average proportional area of somata covered by silver grains was 10.2 ± 0.9 (mean ± SEM) times greater than background compared to 11.2 ± 1.2 times background on the contralateral side (p>0.05, top panel). At 7 days post lesion, the axotomized RSN somata had a significantly lower average proportional area covered of 7.7 ± 0 . 7 times background compared to 12.0 ± 0.8 on the uninjured contralateral side (p<0.02, middle panel). At 14 days post lesion, there was a significant decrease in Gal l average grain area fraction over individual somas from 9.7 ± 0.7 times background on the uninjured side to 5.3 ± 0.6 times background on the injured side (p=0.01). * indicates p<0.05, scale bar = 50 microns. days post lesion 37 Figure 2.4 Gall ISH signal in the red nucleus following BDNF treatment Gal l in situ hybridization signal in the red nucleus seven days after transection of the dorsolateral funiculus (including the rubrospinal tract) at the forth cervical level, plus a one- week infusion of either vehicle or B D N F (n=3 per group) into the vicinity of the red nucleus. Here I show that after a 12 pg/day infusion of BDNF there is a significant increase in the average proportional area of somata covered by silver grains from 9.0 ± 1.3 (mean ± SEM) times background on the uninjured contralateral RSN compared to 13.9 ± 1.0 times background on the axotomized RSN (p<0.05, middle panel and histogram). No significant differences in G a l l expression were observed between the injured (8.4 ± 1 . 6 times background) and uninjured contralateral (8.6 ± 1.1 times background) RSN in vehicle-treated control animals (p>0.05, top panel and histogram). There was a significant difference between the B D N F treated group and the untreated lesioned somata at 7d compared (p<0.02 Figure 2.3) or to the vehicle treatment group (p=0.02). * indicates p<0.05). Scale bar = 50 microns. 38 uninjured axotomized vehicle BDNF 39 Discussion The repertoire of molecules whose expression increases in successfully regenerating neurons includes trophic factors, cytokines, cytoskeletal and guidance molecules (Fernandes and Tetzlaff, 2000; Kwon et al., 2002a). Changes in these molecules are thought to represent adaptive responses to reduce the deleterious effects of peripheral nerve damage and promote survival and regeneration of lesioned axons. Previous work has shown that high Ga l l expression correlates with both growing S M N and D R G neurons during neuronal development (Regan et al., 1986). Furthermore, in the adult, application of rhGall-Ox increases both the regenerative rate and success for both sensory and S M N regeneration (Regan et al., 1986; Fukaya et al., 2003). However, changes in the endogenous neuronal expression of Ga l l are currently unknown. Here, I examined whether endogenous Gal l expression changes following axonal injury, and how its expression correlates with the regenerative propensity of the injured neurons. A large increase in Gal l mRNA and protein occurs within the neuronal somata of S M N 7 and 14 days following peripheral axotomy. I then asked whether Gal l expression was correlated with the muted regenerative potential of CNS neurons in the well-characterized rubrospinal system. When injured at the cervical level, these neurons are unable to regenerate but can initiate a weak regenerative response (Tetzlaff et al., 1991). In contrast to S M N , Gal l mRNA expression decreased after axotomy. These results are similar to the expression profiles of regeneration-associated genes such as interleukin-6, which shows a decrease of mRNA in the red nucleus after axotomy but an increase in the facial nucleus after a peripheral injury (Streit et al., 2000). Unlike SMNs, the upregulation of genes associated with regeneration in RSNs is meager (Fernandes et al., 1999; Fernandes and Tetzlaff, 2000). This modest change in gene expression correlates with the limited ability of a small number of neurons to grow into a pre-degenerated peripheral nerve graft (Kobayashi et al., 1997). B D N F application to the red nucleus not only enhances the regeneration associated gene response of these axotomized neurons, but also leads to a three-fold increase in the number of RSNs able to grow into a peripheral nerve graft (Kobayashi et al., 1997). Similar to other regeneration-associated genes, Ga l l expression in injured RSNs increased in response to B D N F application. This is the first demonstration of Ga l l expression in the red nucleus, 40 injury-induced changes in Gal l expression within motoneurons, differential Ga l l responses to injury, and a neurotrophic factor-induced change in Ga l l mRNA expression. These data, as summarized in Table 2, indicate that Gal l mRNA expression strongly correlates with the regenerative potential of injured neuronal populations. Although the expression pattern of Gal l is suggestive of a significant effect in axonal regeneration, its precise role remains elusive. While previous reports show that the manipulation of exogenous Gall-Ox around a peripheral nerve injury site promotes both rate and success of peripheral nerve regeneration only recently has this mechanism begun to be elucidated (Horie et al., 1999; Horie and Kadoya, 2000; Fukaya et al., 2003). Gal l has been hypothesized to act as a cytokine and/or as an adhesion molecule. This potential dual role for Gal l may be attributed to the differing redox states in which G a l l can exist. Oxidized and reduced G a l l may have different functional roles within the nervous system. Previous studies have reported that exogenously applied, rhGall-Ox can promote DRG outgrowth and regeneration in vitro and in vivo, as well as spinal motoneuron regrowth in vivo (Horie et al., 1999; Horie and Kadoya, 2000; Fukaya et al., 2003). Conversely, application of anti-Gall antibody reduces the number of successfully regenerating SMNs and decreases DRG neurite outgrowth (Horie et al., 1999; Fukaya et al., 2003). Although the Gal l -Ox receptor has not been identified, recent observations indicate that Gal l binds to macrophages in vitro, leading to the release of an unidentified factor that facilitates both Schwann cell migration and DRG neurite outgrowth (Horie et al., 2004). This corresponds to the findings that recombinant oxidized galectin-1 (rhGall-Ox) promotes Schwann cell migration into an acellular graft or empty tube and that Schwann cells and peripherally projecting (vigorously regenerating) neurons contain Gal l protein (Horie et al., 1999; Fukaya et al., 2003). These data indicate that Gall-Ox may be acting as a cytokine via macrophages to promote axonal regrowth. 41 neuronal population Gall change following axotmm spinal motoneurons t red nucleus I red nucleus + vehicle <-» red nucleus + BDNF t Table 2. A summary of Gall changes in SMN and RSN following axotomy. 42 Neuronally expressed Gal has been shown to be released into the extracellular environment, where it is readily oxidized (Cooper and Barondes, 1990; Avellana-Adalid et al., 1994; Imbe et al., 2003; Schafer et al., 2003; Sango et al., 2004). Therefore the increased endogenous Gal l expression in axotomized S M N observed in this report could be released into the extracellular space, where it becomes oxidized, and potentially acts in a similar manner as applied rhGall-Ox. The released Gal l may facilitate peripheral glial cells to promote axonal regeneration, whereas the lack of specific signals within the CNS may contribute to regenerative failure of the injured RSN. Some G a l l is not released into the extracellular environment but remains within the cell, most likely in its reduced form. Here, Gal l may also influence the injury response through effects on second messenger cascades of neurotrophin signaling. Reduced Ga l l has recently been shown to stabilize the binding of the GTPase H-Ras to microdomains, resulting in the alteration of intracellular signaling cascades that may modify the neuronal trophic response (Paz et al., 2001; Elad-Sfadia et al., 2002). Accordingly, both redox states of Gal l may lead to an increase of neuronal growth propensity and motility. The results of the present study demonstrate a differential response of Gal l mRNA expression to axonal injury of PNS versus CNS projecting neuronal populations. Furthermore, increased expression of Ga l l correlates with the regenerative propensity of injured neurons. 43 CHAPTER 3: REGULATION OF GALECTIN-1 EXPRESSION BY AXOTOMY IN RAT PRIMARY AFFERENT NEURONS 44 Introduction Injury to the peripheral or central branches of primary sensory neurons illustrates the difference in regenerative capacity of the peripheral and central nervous systems. Axotomy of a sensory neuron's peripheral branch can result in successful anatomical and functional regeneration. This is in contrast to the complete absence of recovery observed if the axotomy occurs to the centrally projecting branch that terminates in the spinal cord. Both the extrinsic CNS environment and the intrinsic neuronal injury response contribute to this disparity (Steeves and Tetzlaff, 1998; Fernandes and Tetzlaff, 2000; McGraw et al., 2001). Axonal regeneration of peripherally axotomized sensory neurons is associated with a cell body response that serves to reduce the deleterious effects of the peripheral nerve damage and to promote survival and growth of the lesioned sensory afferents (Hokfelt et al., 1994). Recently, application of rhGall-Ox has been shown to promote both the rate and of axonal elongation of peripherally projecting neurons and the success of reinnervation; however, little is known regarding the changes in endogenous neuronal Ga l l expression after primary afferent axonal injury (Horie and Kadoya, 2000; Fukaya et al., 2003; Imbe et al., 2003). Gal l is one of 14 known members of the galectin family of fi-galactoside-binding proteins (Barondes et al., 1994b). Gal l is one of the most extensively studied galectins since its expression occurs in most organs as well as in a wide range of cells including neurons, placenta and muscle cells (Wasano et al., 1990; Poirier et al., 1992) (Regan et al., 1986; Perillo et al., 1998). High Gal l expression in these tissues correlates with cellular growth and rejuvenation. For example, in astrogliomas, elevated Gal l expression occurs in highly malignant tumors (Yamaoka et al., 2000; Camby et al., 2001). This metastatic growth was attenuated by application of anti-sense Gal l mRNA (Yamaoka et al., 2000). Within the developing nervous system, Gal l expression in dorsal root ganglia (DRG), spinal motor neurons, cranial and olfactory neurons begins after the last cell division occurs and remains elevated, albeit at lower levels, until their targets are reached (Regan et al., 1986; Hynes et al., 1990; St John and Key, 1999). These observations suggest that Gal l is associated with cellular growth. 45 Within the lumbar D R G almost all neurons display some Gal l - IR but the small diameter sensory neurons that express c-RET mRNA have the highest Gall-IR in the adult (Regan et al., 1986; Sato and Perl, 1991; Sango et al., 2004). Approximately 70% of all sensory afferents are small diameter neurons (Snider and McMahon, 1998). These small diameter nociceptive neurons can be further classified based on their neurochemistry. About half of the small diameter neurons are peptidergic since they express CGRP, whereas, the other half of these neurons are non-peptidergic neurons, bind IB4 and express the GDNF signaling receptor c-Ret (Chen et al., 1995; Molliver et al., 1997; Bradbury et al., 1998). Large-caliber axons carrying proprio- and mechanoceptive information are identifiable within the D R G by their expression of the large molecular weight neurofilament NF200 (Lawson et al., 1984). The distribution of Gal l expression in peptidergic (CGRP expressing), non-peptidergic (IB4 binding) and large diameter (NF200 expressing) neurons is investigated. Furthermore I examined the changes in Gal l protein and mRNA expression in these neurons after an axotomy and after a rhizotomy. The promotion of sensory fiber regrowth into the CNS after a dorsal rhizotomy was attempted through the application of rhGal-Ox. 46 Materials and Methods Surgery A total of 40 adult male wistar rats (University of British Columbia's animal care facility, weight 200-250 g) were used for this study. See Chapter 2 Materials and Methods for anesthetic details. To reduce post-operative pain and lessen blood flow to the muscle, 0.4 mL of 2% lidocaine with epinephrine (Vetoquinol, Quebec, QC) was then injected into the exposed superficial musculature around the spinal column. Rhizotomy or an axotomy of primary afferents were performed on the left side at the C5 to the 2 n d thoracic level (Tl). For the rhizotomy, details of the procedure are found in (Ramer et al., 2001b). Briefly, small pieces of vertebrae from C4-T2 were removed and the dorsal roots were exposed. The roots were crushed midway between the DRG and DREZ. For the axotomy, nerves exiting the spinal column at C5-T1 were cut and a 5mm section of nerve was removed. This ensured that all the peripheral nerves for a particular DRG were axotomized. Gall infusion Directly after a C5-T1 rhizotomy (as described above), some animals received either recombinant galectin-1 (described previously Horie et al., 1999), or vehicle delivered by 2 week osmotic minipumps (n=5 per group, 3 DREZ per animal analyzed, Alzet model 2002) (previously described in Ramer et al., 2000; Ramer et al., 2001b). To trace growing axons, the left median nerve of adult male Wistar rats was injected with a tracer cocktail containing 1% cholera toxin sub unit B (CTB, for the large diameter fibers, List Biological Inc, Campbell, CA) , 1% wheat germ aggulatin (WGA, both peptidergic and non-peptidergic nociceptive fibers, Vector Labs, Burlingame CA) 11 days after the rhizotomy. One or two weeks following the injury, the C5-T1 spinal cord was harvested. Perfusion I cryosectioning See Chapter 2, materials and methods for details In situ hybridization See Chapter 2 Materials and methods for ISH details. Slides were dipped in Kodak NTB-2 emulsion and exposed for 2 days (DRGs) or 4 days (dorsal horn and DREZ). Sections were counter stained with Nissl as described in Chapter 2. 47 lmmunohistochemistry For Gal l immunohistochemistry of DRGs, slides were washed in 0.1M PBS for 20 minutes, after which rabbit anti-Gall (1:4000) (Horie et al., 1999) and one of mouse anti-CGRP (1/2000, Sigma), mouse anti NF200 (clone N52, 1/500, Sigma), rabbit anti-GFAP (1/1000, Sigma) or IB4 (1/50, Sigma), goat anti-WGA (1/200, Vector Laboratories) was applied to the slides overnight. The slides were then washed for 30 minutes in PBS and a blocking solution of 10% goat serum and 0.1% Triton-XlOO in PBS was added for 20 minutes at room temperature. The slides were then exposed to a donkey anti-rabbit secondary antibody (1:500, Jackson ImmunoResearch Inc, West Grove, PA) for one hour. For G a l l immunohistochemistry, a tyramide signal amplification step was then employed as per the manufactures instructions (PerkinElmer Lifescience, Boston, M A ) . Cy3-conjugated Steptavidin (1:500, Sigma) was used to visualize the G a l l antibody and Alexa 488- conjugated donkey anti mouse (1/300, Jackson Immunological Research) or Alexa 488- conjugated donkey anti goat (1/300, Jackson Immunological Research) was used to visualize the other primary antibodies. Slides were coverslipped with a 3:1 solution of glycerol: PBS. DRG quantification-ISH DRG quantification was carried out as described by Ramer et al. (2001b), Bennett (2000) and in Chapter 2 materials and methods. Briefly, Nissl-stained DRGs cell bodies were outlined creating an image overlay. The percentage area occupied by silver grains determined the somal grain area fraction. The somal grain area fraction was measured by outlining the individual neuronal cell bodies using the neurotrace and applying the resulting cell profile layer to the darkfield image. For each animal, the percentage area occupied by ISH signal per soma was determined for both the axotomized and contra lateral (uninjured) side. The difference between background autoradiographic signal and the mean ISH signal per soma was calculated. Using recursive translation, a stereological counting method which reconstructs cell populations based on size-distribution of profiles (Rose and Rohrlich, 1988), the proportion of cells having a signal 5 times background was calculated. This threshold was arbitrarily chosen to observe changes in mRNA levels in different sub-populations within the DRG. At least three sections, >100 pim apart, were quantified per DRG. 48 DRG quantification-immunoreactivity DRG quantification was carried out as described by Ramer et al. (2001b). Briefly, labeled images of Gal l and either CGRP, IB4 or NF200 were imported into SigmaScan Pro 5.0 (SPSS Inc., Chicago, IL). The DRG cell bodies were outlined creating an image overlay. The average intensity and feret diameter of each object identified by the overlay was automatically measured. The threshold for immunopositivity for G a l l , IB4, CGRP and NF200 was determined by averaging three cell bodies in each section that were judged to be minimally positive. Using recursive translation, a stereological counting method which reconstructs cell populations based on size-distribution of profiles (Rose and Rohrlich, 1988), the proportion of cells expressing an antigen and the soma sizes were calculated. At least three sections, >100 pirn apart, were quantified per DRG. Dorsal horn quantification- Gall- immunoreactivity Dorsal horn quantification was carried out as described by Ramer et al. (2001a). Briefly, for each rat cervical spinal cord level, three Gall-IR images were imported into SigmaScan Pro 5.0 (SPSS Inc.) where a threshold was applied. Staining was measured along three non- overlapping 50-micron wide strips starting from uppermost border of grey matter and extending 450 microns ventrally. Measurements at each depth were averaged across sections in mice and mean ± S E M axonal density was plotted as a function of depth. For every 10 microns of depth, the average axonal density was determined. Using a student's t-test, differences between Gal wt and -/- mice were determined at 10-micron intervals. Dorsal horn quantification Gall-ISH At least two sections per spinal cord level were analyzed. Nissl and darkfield (silver grain) images were taken of both the injured and the contralateral non-injured side using a digital camera attached to a microscope (Carl Zeiss, Axioskop) in combination with Northern Eclipse software (Empix Inc, Mississauga ON). A l l images were analyzed with SigmaScan Pro 5 software (SPSS Inc.). The percentage area in the dorsal horns (see Figure 3.8E) occupied by silver grains determined the grain area fraction. Background autoradiographic signal was determined from the uninjured corticospinal tract. For each section, the percentage area in the dorsal horn occupied by ISH signal was determined for both the 49 axotomized and contralateral (uninjured) side (see Figure 3.8E). These data were expressed as percentage of the ISH background signal. DREZ ISH quantification Analysis of Gal l ISH in the DREZ was similar to the dorsal horn ISH analysis as previously described. Briefly at least two sections per DREZ were analyzed. Nissl images aided in determining the D R E Z boundary so that similar sizes of both PNS and CNS tissue could be delineated as indicated in Figure 3.91. A threshold was applied to the silver grain image to determine grain area fraction of both PNS and CNS tissue at the DREZ. DREZ tracer quantification Axonal regeneration into the CNS was quantified densitometrically as previously described in Ramer et al (2000; 2001b): a threshold was applied to each of three nonadjacent images from each DREZ at C7 or C8, and the axonal density for either NF200, CTB, CGRP or WGA was determined in the DREZ as delineated by GFAP-IR. Statistics Quantification was performed blind with respect to the treatment groups. A l l data is represented as mean ± standard error of the mean (SEM), and all tests were carried out using SigmaStat 3.0 (SPSS Inc). Unless otherwise stated, a Student's t-test was used to determine significance. A l l results are stated as mean ± standard error of the mean (SEM)Significance was assigned at p<0.05. 50 Results Changes in the Gall expression in the DRG following injury In the uninjured D R G 56.8 ± 5.6% (mean ± SEM) of neuronal somata had a grain area density 5 times greater than background (n=3, Figure 3.1 A , 3.2 A and 3.2 F). Immunohistochemistry revealed that only 47.5 ± 1.1% of somata were Gall-immunoreative (IR) (n=3, Figure 3.6 A and 3.6 F). Gall-IR was observed in both the cytoplasm and nucleus of sensory neurons (Figures 3.3 C, 3.4C, 3.5C). Of the Gall-IR somata 28.2 ± 2.4% were CGRP - IR (Figure 3.3A, histogram), 33.3 ± 1.77% were IB4 binding (Figure 3.4A, histogram) and 5.9 ± 0.2% were NF200 -IR (Figure 3.5A, histogram). 7 days following a rhizotomy, there were no significant changes in either Gal l mRNA expression (n=3, 64.5 ± 5.5% somata, Figure 3.IB, 2B and F), Gall-IR (Figure 3.6B and F) or co-localization with CGRP, IB4 or NF200 (Figure 3.3F, 4F, 5F) when compared to uninjured DRGs. Again at 14 days after a rhizotomy I did not observe any significant changes in either mRNA expression (n=3, 60.1 ± 4.2%, Figure 3.1C, 3.2C and 3.2D), Gall-IR (49 ± 1.8% somata, Figure 3.6C and 3.6D) or co-localization with CGRP, IB4 or NF200 (Figure 3.31, 3.41, 3.51) when compared to uninjured DRGs. After a spinal nerve lesion in which all the sensory fibers for the particular DRG were axotomized, there were significant increases in both Gal l mRNA and protein expression. I observed a significant increase in the proportion of somata with high silver grain density at 7 days to 82.4 ± 2.3% of all somata (n=3). This increase was mainly restricted to the large diameter cells (Figure 3.ID, 3.2D and F). Gall-IR also was increased at this time point to 65 ± 3.2% (n=3, Figure 3.6F) of all somata with some nuclei displaying greater Gall-IR than control levels (Figure 3.4M and O). Again this increase was mainly in the large diameter (NF200-IR) cells (Figure 3.6E) since 11.6 ± 2% of Gall-IR somata were also NF200 positive (Figure 3.4L) compared to 5.9 ± 0.2% of Gall-IR and NF200-IR cells (Figure 3.4C). There was also a significant decrease in CGRP and IB4 co-labeling with Gal l (4.4 ± 1.7%, Figure 3.3L and 23.1 ± 3.0%, Figure 3.4L respectively) as a result of the decrease in the overall CGRP-IR and IB4 binding of somata. 14 days after axotomy, these results did not significantly change from 7 days after axotomy. 51 Figure 3.1 Photomicrographs of Gall in situ hybridization signal A l l sections were counter stained with fluorescent Nissl stain (Figure 3.1, left column). In the uninjured DRG, silver grains could clearly be observed over cell soma (A and B). At 7 and 14 days after rhizotomy, there was no apparent change in the number of silver grains over cell bodies (D and F). However, at both 7 and 14 days after a spinal nerve lesion (peripheral axotomy), an increase in silver grains over somata was observed (H and J). Scale bar = 100 microns. 52 53 Figure 3.2 Quantification of Gall autoradiographic signal in the DRG A baseline of 5 times background (intense signal) silver grain signal was arbitrarily set as the threshold for somata positive for Gal l mRNA. In uninjured cervical D R G (C6-C8), 56.8 ± 5.6% (mean ± SEM) of the cells had 5 times greater signal than the background (F). High levels Gal l mRNA (A, black bars), as indicated by silver grains, were observed in somata of all sizes when compared to the total cellular population (A, white bars) but were predominately located in small diameter somata (A). Following a rhizotomy (rhiz), there was a small increase to 64.5 ± 5.5% at 7 days and 60.1 ± 4.2% at 14 days of the total number of cells with high proportion of silver grains (F). There appeared to be a slight increase in the larger diameter cells (grey bars) when compared to the uninjured control animals (black bars, B and C). Only after a spinal nerve lesion (axo) was there a significant increase in the number of cells highly expressing Gal l mRNA (F). There was a significant increase in silver grains both at 7 days after an axotomy (82.4 ± 2.3% of total cells), and at 14 days after axotomy (75.7 ± 6.2%) when compared to uninjured DRGs (F). In particular an increased number of larger diameter cells expressed G a l l mRNA (D and E). * indicates p<0.05 compared to uninjured animals. cell diameter (microns) _ F =2 §0.8 8 I -Bfo.e € „0.4 8.2 g-io.2 0.0 _ 55 Figure 3.3 Ga l l - IR and C G R P - I R in the D R G Photomicrographs of immunohistochemistry of G a l l and C G R P immunoreactivity of cervical DRGs. In the uninjured (control) DRG, 28.2 ± 2.4% of the Gall-immunoreactive (IR) somata also expressed CGRP (Figure 3.3 C). At 7 and 14 days after rhizotomy 27.0 ± 2.4% and 26.1 ± 2.3% of the Gall-IR somata expressed CGRP respectively (Figure 3.3F and I). After axotomy, only 6.5 ± 1.3% of Gall-IR cells expressed C G R P at 7 days and 4.4 ± 1.7% of Gal-IR somata also expressed CGRP at 14 days (Figure 3.3L and O). These are both significantly less than control values. Scale bar = 50 microns, * indicates p<0.001 when compared to uninjured DRGs.  57 Figure 3.4 Gall-IR and IB4-IR in the DRG Photomicrographs of immunohistochemistry of Gal l immunoreactivity and IB4-binding of cervical DRGs. In the uninjured (control) DRG, 33.3 ± 1.77% of the Gall-IR somata also bound IB4 (Figure 3.4 A,B,C) . At 7 and 14 days after rhizotomy 36.0 ± 1.1% and 34.5 ± 0.9% of Gall-IR somata bound IB4 respectively. After axotomy, 23.1 ± 3.0% of Gall-IR cells bound IB4 after 7 days and 18.9 ± 2.0 % of Gal-IR somata also bound IB4 at 14 days (Figure 3.4L and O). These are both significantly less than control values. Scale bar = 50 microns, * indicates p<0.001 when compared to uninjured DRGs. 58 59 Figure 3.5 Gall-IR and NF200-IR in the DRG Photomicrographs of immunohistochemistry of G a l l and NF200 immunoreactivity in cervical DRGs. In the uninjured (control) DRG, 5.9 ± 0.2% of the Gal l - IR somata also expressed NF200 (Figure 3.5 C). At 7 and 14 days after rhizotomy 6.4 ± 0 . 1 % and 7.1 ± 0.2% of the Gal l - IR somata expressed NF200 respectively (Figure 3.3F and I). After axotomy, there was an increase to 11.6 ± 2% of Gall-IR cells that expressed NF200 after 7 days and 12.0 ± 1.2% of Gal-IR somata also expressed NF200 at 14 days (Figure 3.3L and O). These were both significantly greater than control values. Scale bar = 50 microns, * indicates p<0.001 when compared to uninjured DRGs.  61 Figure 3.6. Proportion of Gall-IR cells compared to the total proportion of somata within the DRG. In the uninjured DRG, 47.5 ± 1.1% of somata were Gall-IR (6F). Gall-IR was mainly observed in both small and medium diameter cells (black bars, A) when compared to the entire population (white bars, A). While Gall-IR was found in cells of all sizes, a greater proportion of small diameter CGRP and IB4-positive cells expressed G a l l than large diameter NF200 positive cells. 7 days after rhizotomy, 52.0 ± 2.1% of cells were Gal l - IR and after 14 days 49 ± 1.8% of somata were Gall-IR. While these were not significantly different from control values there appeared to be an increase in the proportion of large diameter somata that had Gall-IR (C and D). 7 days after axotomy there was a significant increase in Gall-IR cells (65 ± 3.2%) and after 14 days 70.8 ± 4.3% of somata were Gall-IR. * indicates p<0.05 compared to uninjured DRGs. X Gall +ve cells all cells cell diameter (microns) o % 0.8 + i 0.6 CD "S 0.4 c o 1 0.2 Q. i . o.o / / / / / 63 At 14 days post-axotomy, 75.7 ± 6.2% of axotomized somata had high ISH levels (Figure 3.IE, 3.2E, 3.2F). 70.8 ± 4.3% of the somata were also Gall-IR (Figure 3.6E, F). Of these Gall-IR somata, 4.4 ± 1.7% were CGRP positive (Figure 3.30), 18.9 ± 2.0 % bound IB4 (Figure 3.40) and 12.0 ± 1.2% were NF200 positive (Figure 3.50). Overall few changes were observed in Gal l mRNA or protein expression after a rhizotomy but after an axotomy larger diameter NF200-IR increase their Gall-IR. Gall IHC expression in the dorsal horn Gall-IR was present in the uninjured dorsal horn (n=3, Figure 3.7A,C,E, D). Both 7 and 14 days following a rhizotomy there was a complete absence of Gal l - IR in the dorsal horn (Figure 3.7B, D). This data indicates that Gal l protein is normally anterogradely transported to primary afferent terminals fields in the dorsal grey matter. Following a peripheral axotomy, I observed a significant increase in Gall-IR deeper within the laminae (Figure 3.8F and H). This was most likely due to the increased Gal l expression in large-diameter somata (Figures 3.50, 6E). In the intact dorsal horn Gal l mRNA was not detectable (Figure 3.8). However, 7 and 14 days after rhizotomy silver grains were observed in the degenerating fiber tracts within the spinal cord (cuneate fasciculus and the medial dorsal horn where large diameter fibres invade the grey matter) indicating Ga l l mRNA expression (Figure 3.8). 7 days following a rhizotomy, these degenerating fiber tracts had 3.3 ± 0.2 times background and at 14 days it was 4.7 ± 0.3 times background which were significantly greater than the uninjured sides of 1.3 ± 0.2 and 1.2 ± 0.2 times background at 7 and 14 days respectively (p<0.05). After both 7 and 14 days following a rhizotomy, an increase in autoradiographic signal occurred. At 7 days following a rhizotomy there was an increase to 3.3 ± 0.2 times background compared to 1.3 ± 0.2% on the uninjured side. 14 days following a rhizotomy there was an increase to 4.7 ± 0.3% of background compared to the uninjured side of 3.3 ± 0.2% of background (Figure 3.8 A and C). 64 Figure 3.7 Distribution of Gall- IR in the C7 dorsal horn. Compared to the contralateral (uninjured) dorsal horn (A and C), there was a complete absence of Gall-IR at both 7 and 14 days after rhizotomy (B and D). At 7 and 14 days after a spinal nerve lesion (axotomy), Gall-IR was observed at increased depths in the dorsal horn (Figure 3.7F and H) when compared to the uninjured side (E and G). When Gall-IR is plotted against depth, the axotomy-induced shift in depth of Gall-IR is apparent at both 7 and 14 days (gray lines) in the dorsal horn ipsilateral to the uninjured contralateral side (black lines). At all points between the arrows, there is a significant difference in Gall-IR between axotomized and uninjured sides. Scale bar = 200 microns. 65 66 Figure 3.8 Gall ISH in the dorsal horn following rhizotomy Gal l mRNA significantly increased in the dorsal horn following a rhizotomy. Using a cervically spinal cord section one week after rhizotomy that has been stained for NF200, the major landmarks of the dorsal spinal cord can be identified (A). On the rhizotomized side (right side) the intact gracile fasciculus (GF), corticospinal tract (CST) and degenerating cuneate fasciculus (CF) and dorsal horn (DH) can clearly be seen. The arrows indicate the large-diameter axons entering the deeper lamina. The dotted area on the rhizotomized (right) and uninjured contralateral (left) side on the autoradiographic sections (B and C) were quantified for ISH signal and expressed as a percentage of background (uninjured CST tract). After a rhizotomy, the Gal l autoradiographic signal increased significantly to 3.3 ± 0.2 times background compared to 1.3 ± 0.2% on the uninjured side after 7 days and 4.7 ± 0.3% on the rhizotomized side 1.2 ± 0.2% on the contralateral side after 14 days. * Indicates p<0.05 when compared to the contra lateral side. Scale bar = 200 microns 67 A Mi te ; . > ... . ' • ''-•••«" • : \ ' / CF • • / • " \ / / • • DH _ ' CST B 7d c: • •—mm 14d T3 CZ 13 O i— o CO - Q W Q) E 6 5 4 3 2 1 X 0 CO • uninjured rhizotomy 7d 14d 68 Gall mRNA expression at the DREZ After a rhizotomy, an increase was observed in Gal l autoradiographic signal to 4.3 ± 0.5 times greater than the contralateral side after 7 days and 5.3 ± 1 . 0 after 14 days on the PNS side (n=3, Figure 3.9A,B and J). On the CNS side of the D R E Z , an increase in autoradiographic signal 1.2 ± 0.2 times the contralateral side after 7 days and 1.4 ± 0.1 after 14 days was observed (n=3, Figure 3.9E,F and J). Intrathecal Gall-Ox has little effect on central regeneration of rhizotomized offerents Gall-Ox has previously been demonstrated to increase the regenerative rate after a peripheral axotomy (Horie et al., 1999; Fukaya et al., 2003). To test whether Ga l l promotes sensory neuron growth into the CNS after a rhizotomy rhGall-Ox (10 pg/ml) was infused for 2 weeks directly after a cervical rhizotomy. There were no significant differences in NF200 staining central to DREZ between vehicle and Gal l infusions (n=5 per group, Figure 3.10). Staining for the large diameter neuronal tracer CTB showed a non-significant increased trend of staining between the two treatment groups. CGRP-IR in the D R E Z increased after Gal l treatment (n=5 per group, 0.016 ± 0.001% of DREZ) compared to vehicle alone (0.005 ± 0.0007%, Figure 3.11). Immunohistochemistry for the small diameter neuronal tracer W G A also revealed a slight significant increase in immunoreactivity for the Ga l l treated animals (0.006 ± 0.001% of area) compared to vehicle alone (0.002 ± 0.0002%). While these increases were significant, they were small. 69 Figure 3.9 Gall ISH at the DREZ after rhizotomy Gal l mRNA significantly increases in the PNS after a rhizotomy. Figure 3.9A, B, E and F are photomicrographs of autoradiographic signal (silver grains) following a Gal l ISH of the DREZ. Figures 9C, D, G and H are the same sections counter stained with fluorescent Nissl stain. Arrows indicate the DREZ boundary. Figure 3.91 is a schematic illustrating the PNS and CNS interface. After rhizotomy, the Gal l autoradiographic signal increased to 4.3 ± 0.5 times greater than the contralateral side after 7 days and 5.3 ± 1 . 0 after 14 days on the PNS side (n=3, Figure 3.9A,B and J). On the CNS side of the DREZ, the autoradiographic signal increased to 1.2 ± 0.2 times the contralateral side after 7 days and after 14 days, an increase of 1.4 ±0.1 (n=3, E, F and J). Scale bar = 100 microns, * indicates p<0.001 compared to the uninjured contralateral side. 7d 14d 71 Figure 3.10 rhGall-Ox does not promote growth of NF200 and CTB labeled fibers In triple-labeled images of G F A P (to delineate the DREZ), NF200 (large diameter fibers) and CTB (neuronal tracer), I observed that 1.0 ± 0.2% of the DREZ area (delineated by GFAP, see methods) was covered by NF200-IR after rhGall-Ox infusion: this was not significantly different from the CTB-covered area of the DREZ in vehicle-treated animals (0.9 ± 0.3% area covered). Using CTB to trace large diameter neurons, rhGall-Ox-treated animals did not have a significantly greater amount of CTB staining in the D R E Z (0.002 ± 0.001% of DREZ) than vehicle treated animals (0.001 ± 0.0002%). Scale bar = 100 microns.  73 Figure 3.11 rhGall-Ox promotes growth of CGRP and WGA labeled fibers rhGall-Ox promotes limited growth of small diameter fibers across the DREZ 14 days after rhizotomy. In triple-labeled images of G F A P (to delineate the DREZ), CGRP (peptidergic fibers) and W G A (neuronal tracer), 0.016 ± 0.001% of the DREZ area (delineated by GFAP, see methods) was covered by CGRP-IR after rhGall-Ox infusion: this was significantly greater than the CGRP-covered area of the DREZ in vehicle-treated animals (0.005 ± 0.0007%). Using W G A to trace small diameter neurons, rhGall-Ox treated animals had a significantly greater amount of W G A staining (0.006 ± 0.001% of DREZ) than vehicle- treated animals (0.002 ± 0.0002%). Arrows indicate fibers double-stained for both CGRP and WGA. Scale bar = 100 microns, * indicates p<0.05.  75 Discussion Gall expression in the naive DRG In the adult cervical DRG, 56.8 ± 5.6% of the sensory nuclei had an ISH signal for Gal l mRNA 5 times greater than background levels. The majority of these profiles were small diameter neurons. This is the first report to quantify the size distribution of Gal l mRNA expression in the DRG, and these results are consistent with previous reports that show small diameter sensory neurons with high Gal l mRNA expression (Hynes et al., 1990; Sango et al., 2004). I show 47.5 ± 1.1% of the nuclei had some Gall-IR which is similar to a previous finding of 63% of all DRGs having some Gall-IR with 46% having strong Gall-IR (Regan et al., 1986). In lumbar DRGs most cells also have Gall-IR but only 20-26% of these somata were intensely Gall-IR (Imbe et al., 2003; Sango et al., 2004). These differences between cervical and thoracic Gall-IR cellular proportion are not surprising. Significant variations in the rostro-caudal distribution of sensory phenotypes exist in DRGs. For example, a greater overall proportion and size distribution of P2X 3-IR somata occurs in cervical DRGs when compared to lumbar DRGs (Ramer et al., 2001a). The majority of Gall-IR neurons were either IB4-binding (33%) or CGRP-IR (28%) small diameter neurons within cervical DRGs. In the lumbar D R G , Gall-IR cells co- localized mainly to c-Ret mRNA (94%) expressing somata and also to a limited number of TrkA mRNA (6.8%) expressing neurons (Imbe et al., 2003). In these lumbar DRGs, only 63% of the c-Ret mRNA expressing neurons bind IB4 (Molliver et al., 1997) and approximately 50% of the IB4-binding neurons also have CGRP-IR (Wang et al., 1994; Bergman et al., 1999). Accordingly, the binding of IB4 to both CGRP-IR and c-Ret mRNA expressing neurons is consistent with both the report of Gall-IR occurring mainly in c-Ret mRNA expressing lumbar neurons and Gall-IR occurring in both IB4-binding and CGRP-IR cervical neurons (this report). Gall expression after injury Using two different injury paradigms, peripheral axotomy and rhizotomy, two different injury responses were elecited within the D R G and spinal cord. These results are summarized in Table 3. After a peripheral nerve lesion, which normally elicits a strong cell body response, I observed a significant increase of both Gal 1 mRNA and -IR, mainly within the large diameter NF200-IR sensory neurons. Within the spinal cord, increased Gall-IR 76 occurred within the deeper layers of the dorsal horn. This is consistent with previous findings in both an L4/L5 spared nerve injury model (SNL) of neuropathic pain and sciatic nerve transection increased Gall-IR within deeper layers of the dorsal horn (Imbe et al., 2003). Following rhizotomy, Ga l l mRNA or - IR within the D R G was not significantly different than control animals but there was a complete absence of Gall-IR within the dorsal horn, demonstrating that G a l l protein is normally transported to the nerve terminals in uninjured sensory neurons. Gal l mRNA expression and not Gall-IR was observed within the degenerating fiber tracts within the spinal cord; however, Ga l l mRNA was more readily detected in the D R G than Ga l l protein. A semi-quantitative polymerase chain reaction for Ga l l mRNA on rhizotomized spinal cord would further demonstrate these increases. Reactive astrocytes could be expressing Gal l mRNA in the deafferented spinal cord since astrocytomas are the only known Gal l expressing glial cells within the CNS (Camby et al., 2001). However, since astrocytes become reactive throughout the dorsal horn following rhizotomy, and since silver grain density was present mainly along the paths of large diameter (myelinated) afferents in the medial dorsal horn, it may also be that activated microglia or oligodendrocytes upregulated Gal l mRNA. Using antibodies to either G F A P (astrocytes) or Rip (oligodendrocyte cell bodies) combined with a G a l l ISH may aid in resolving which cells express Gal l mRNA after rhizotomy in the dorsal horn. On the PNS side of the DREZ there was a large increase in Gal l mRNA expression. Schwann cells were most likely expressing G a l l mRNA here since both sensory neurons and Schwann but not macrophages express Gal l in the PNS (Sango et al., 2004). 77 changes in Gall expression location rhizotomy axotomy D R G t DREZ t (PNS side) dorsal horn I (IR) t cuneate fasciculus t (mRNA) Table 3. A summary of changes in Gall expression in sensory neurons following injury. 78 Redox state affects Gall functions When secreted from cells, some of the Ga l l changes from a reduced (Gall-Red) to an oxidized state (Gall-Ox), transforming Gal l from a lectin to a trophic molecule (Tracey et al., 1992; Horie and Kadoya, 2000). Within neurons, Gall-Red has been identified as a component of the Survival of Motor Neuron complex which is involved in neuronal growth and survival (Park et al., 2001; Rossoll et al., 2003). Once released into the extracellular space, Gall-Red facilitates axonal growth by increasing adhesion (Mahanthappa et al., 1994). Exogenous application of Gall-Ox but not Gall-Red increases axonal outgrowth by acting as a growth factor and/or a cytokine to stimulate DRG outgrowth in vitro, and increases the rate of sensory and motor growth into acellular nerve grafts and tubes in vivo (Horie et al., 1999; Horie and Kadoya, 2000; Fukaya et al., 2003). This occurs by increasing Schwann cell migration and eliciting the release of a yet unidentified growth-promoting factor from macrophages (Horie et al., 1999; Horie and Kadoya, 2000; Fukaya et al., 2003; Horie et al., 2004). These data suggest that both endogenous Ga l l redox states increase neuronal outgrowth, but only the exogenous application of Gall-Ox increases the rate of peripheral nerve regeneration. Accordingly, I attempted to promote sensory neuron axonal regeneration after a rhizotomy by exogenous Gall-Ox application. Significant numbers of large diameter fibers were not observed regenerating into the spinal cord. This suggests that Gal l does not promote large diameter neuronal growth into the CNS or a reduction in Gal l ' s efficacy. Future experiments should also repeat Horie et al., (1999) tube ligation study to confirm Gall-Ox activity. However Gall-Ox did promote some regeneration of small diameter nociceptive C G R P and/or W G A labeled fibers suggesting that some Gall-Ox was active. Unlike the long distance and robust growth of rhizotomized sensory neuronal growth observed after neurotrophin delivery (Ramer et al., 2000; Ramer et al., 2002), Gall-Ox induced only a very limited amount of nociceptive fiber growth into the CNS. The G a l l - induced growth in the DREZ was more reminiscent of a localized sprouting response than that of robust regeneration. The precise role of G a l l expression in the uninjured nervous system remains uncertain. Over half of the uninjured DRGs express Gal l (Figure 3.2) (Regan et al., 1986; Imbe et al., 2003; Sango et al., 2004). Once expressed, Gal l is released from central nerve 79 terminals where it can bind to astrocytes which then release B D N F (Sango et al., 2004; Sasaki et al., 2004). B D N F has been implicated in such diverse roles as synaptic plasticity to nociception modulation (Thoenen, 2000; Pezet et al., 2002a). Interestingly, sensory neurons of Ga l l -/- mice do not make appropriate central connections during development. For example, olfactory neurons in Gal l -/- mice that would normally express Gal l grow towards their targets but do not make the final appropriate connections (Puche et al., 1996). Small- diameter primary sensory neurons of Gal l -/- mice also grow to the dorsal horn but terminate within deeper laminae, an observation which correlates with reduced nocifensive responses to noxious thermal stimuli compared to Gal l wt mice (Chapter 4). These data suggest that the intrinsic expression of G a l l is not critical for neuronal outgrowth in the CNS during development but may rather be involved in making or maintaining appropriate connections. 80 CHAPTER 4: INCREASED THERMAL-PAIN THRESHOLDS IN GALECTIN-1 NULL MUTANT MICE: CORRELATION WITH ALTERED NOCICEPTIVE PRIMARY AFFERENT NEURONAL NUMBER AND SPINAL TERMINAL FIELDS 81 Small diameter D R G afferents transmit nociceptive information from the periphery to the CNS. These thinly-myelinated and unmyelinated small diameter neurons comprise approximately 70% of all cells within the DRG (Snider and McMahon, 1998). The small- caliber group can be further subdivided based on neurochemistry and termination pattern within the spinal cord: neurons expressing the neuropeptide C G R P terminate in laminae I and II outer (Ho) (Averill et al., 1995); and, neurons that express the A T P receptor P2X 3 (non-peptidergic neurons) and bind IB4 terminate in lamina II inner (Hi) of the spinal cord (Chen et al., 1995; Molliver et al., 1997; Bradbury et al., 1998). Large-caliber axons carrying proprio- and mechanoceptive information terminate in deeper laminae (III-X) are identifiable within the D R G by their expression of the large molecular weight neurofilament NF200 (Lawson etal., 1984). During rodent development, different neurotrophic factors regulate distinct functional classes of sensory neurons. For example, the NGF specific receptor TrkA localizes to 70- 80% of all DRG neurons early in development (Molliver et al., 1995; Molliver and Snider, 1997) and is required for survival (Crowley et al., 1994; Smeyne et al., 1994; Silos-Santiago et al., 1995). However, as development proceeds, half of the NGF-dependent neurons lose their TrkA expression and begin to express Ret, the signaling receptor for the GDNF family of neurotrophic factors (Molliver and Snider, 1997; Molliver et al., 1997) leading to an adult D R G population comprised of 40% TrkA and CGRP and 30% Ret-expressing and IB4- binding neurons. In the rat, during development and in the adult, these small diameter neurons also express the carbohydrate-binding protein Gal l (Regan et al., 1986). Following loose ligation of the rat sciatic nerve, which causes peripheral neuropathy and thermal allodynia, Gal l immunoreactivity increases in laminas I and II of the spinal cord (Cameron et al., 1997). Decreasing this activity using intrathecal application of Ga l l functional blocking antibodies leads to reduced mechanical allodynia (Imbe et al., 2003). Furthermore, exogenously applied Gal l protein promotes DRG axonal growth in vitro and in vivo (Horie et al., 1999; Horie and Kadoya, 2000; Horie et al., 2004). These data strongly suggest that Gal l plays a role in nociceptive sensory neuronal outgrowth and maintenance. However, to date no studies have attempted to examine the role of Gal l in sensory neuronal development. 82 The Ga l l -/- null mutant mouse is viable without obvious phenotypic abnormalities (Poirier and Robertson, 1993). Interestingly, in these mice, a neuronal subpopulation within the olfactory bulb that normally expresses Gal l does not reach appropriate targets in olfactory glomeruli (Puche et al., 1996). These data suggest that Ga l l may be required for axonal growth or pathfinding. Here I attempted to further elucidate the potential role of Gal l by examining Gal l expression in the DRG and spinal cord of G a l l wt mice, changes in neuronal populations and primary afferent terminations in Ga l l null-mutants, and correlate these changes in nociceptive behaviour. 83 Materials and Methods Animals A total of 22 adult age-matched 129P3/J (wild-type, Jackson Labs, Maine), 22 adult 129P3/J galectin-1 homozygous null mutant mice (Gall -/-)(Poirier and Robertson, 1993) and 7 CD-I mice (University of British Columbia's animal care facility) were used for these experiments. The generation of the Gal l -/- mice has been described (Poirier and Robertson, 1993). A l l experiments were performed in accordance with the Canadian Council for Animal Care and approved by the University of British Columbia Animal Care Committee. Behavioral testing A total of 7 CD-I mice, 7 Gal l wt, 7 Gal l -/- mice were used for the behavioral testing. Two tests (punctate pressure and either radiant heat or cold plate) were carried out three times per day with at least two hours between each trial. To avoid sensitization, the tests were repeated on four different days with three days between each testing day. Student's t-test was used to determine differences between groups with statistical significance attained when p<0.05. Progressive plantar punctate force test: A mouse was placed on a metal grate and allowed to adjust to the surroundings for 10 minutes. A dynamic plantar aesthesiometer (model # 37400 UGO Basile Biological Research, Comerio V A ) with a dull metal wire was maneuvered under a paw. The force was set at 20g increasing with ramp set to 7 seconds. Upon nocifensive withdrawal (involving some or all of: sustained elevation, biting, licking or shaking the paw), the instantaneous force applied to the plantar surface eliciting the withdrawal was recorded automatically. Random (non-nocifensive) paw movements were not recorded. Radiant heat: A mouse was placed on a Plexiglas surface and allowed to habituate to the surroundings for 20-30 minutes. When the mouse was still, a U V laser connected to a timer was turned on underneath a mouse paw (model #7371 UGO Basile Biological Research, Comerio V A ) . Latency to withdrawal was recorded. Only nocifensive movements were recorded. 84 Cold plate test: A mouse was placed on a 1°C cold plate (model # 0134-0021 Columbus Instruments, OH) and a timer was immediately started. The latency to the initial nocifensive response was recorded. c-fos activation Fos protein expression is used as a marker for neuronal activation in the spinal cord (Hunt et al., 1987). A total of 8 G a l l wt and 8 Gal l -/- mice were used to determine if there are differences between Ga l l -/- or wt mice in second order neuron activation after noxious temperature stimulation. Under light anesthesia, the mouse's left front paw was carefully submerged in a water bath (1 or 52°C) three times for 10 seconds with a 30 second delay between each submersion. Animals were killed 2 hours after thermal stimulation when c-fos activation in the dorsal horn has peaked (Hunt et al., 1987; Dai et al., 2001). Perfusion I cryosectioning Perfusion was carried out as described in Chapter 2, Material and Methods Perfusion. Immunohistochemistry Standard immunohistochemical techniques and controls for indirect-immunofluorescence were used in order to visualize specific antigens on cryosectioned tissue. Slides were washed in 0.1m PBS for 20 minutes then blocked for 20 minutes in 10 % normal goat serum and then either goat anti mouse-Gall (1:500 R & D systems, MN), rabbit anti-NeuN (1:100, Chemicon, Temecula CA) , rabbit anti-CGRP (1:2000, Sigma, Oakville, ON), followed by biotin- conjugated IB4 (1/50, Vector Labs), p i l l tubulin (1:500, Sigma) or mouse anti c-fos (1:5000, Oncogene, Cambridge, M A ) , in 0.1M PBS (in 0.2% Triton X-100, and 0.1% sodium azide) was applied to the slides overnight. After washing, secondary antibodies raised in donkey and conjugated to either Cy3, Alexa 488, A M C A (1/300, Jaskson Immunological Research, West Grove, PA) or extravidin conjugated Cy3 or FITC (1/500, Sigma) was applied for 1 hour at room temperature. After a final wash, slides were coverslipped with a 3:1 solution of glycerohPBS. A fluorescent microscope (Carl Zeiss, Axioskop, Toronto, ON) was used to visualize chromophore labeled tissue and then greyscale images were captured using a digital camera (Carl Zeiss, Axioskop, Toronto, ON) in combination with Northern Eclipse software (Empix Inc, Mississauga ON). A l l images for an individual antigen were taken at the same time and under the same light intensities. 85 DRG - IR quantification The same D R G quantification technique was used as described in Chapter 3, Materials and Methods: DRG quantification. Dorsal horn - IR quantification Dorsal horn quantification for CGRP and IB4 was carried out as described by Ramer et al. (2001a) and Chapter 3 Materials and Methods, dorsal horn quantification. c-fos-IR quantification Three c-fos images were captured for each cervical section of the left dorsal horn for each animal. These images were imported into SigmaScan Pro 5.0 (SPSS Inc., Chicago, IL), a threshold was applied to remove any background staining and then c-fos positive cells were automatically counted. NeuN-IR quantification For each animal, three images of NeuN-IR cells were captured for each cervical section (C7- C8) of the left dorsal horn. These images were then imported into Photoshop (Adobe Systems, San Jose CA) and only lamina I -II was selected. Using the Image Processing toolkit 3.0 (Reindeer Graphics, Asheville NC) a threshold was used to separate the individual NeuN positive cells. The cells were then automatically counted. In situ hybridization The mouse GAL1 probe was a 51-mer oligonucleotide complementary to the 3'-untranslated sequence of G A L 1 and 5'-TCA C T C A A A G G C C A C G C A CTT A A T CTT G A A GTC TCC A T C C G C C G C C A T G T A -3' (GenBank accession number BC002063). The GAL1 probe was complementary to bases 424-474. The mouse probes were end-labeled with 33P- dATP (Perkin-Elmer, Woodbridge, On) by using deoxynucleotide terminal transferase according to a standard protocol (Kobayashi et al., 1996). Perfusion-fixed sections were hybridized to 1.2 x 106 cpm of probe for 16-18 h at 44°C. The slides were dipped in Kodak NTB-2 emulsion and exposed for 3 days. Slides were then dehydrated in a series of alcohols and stored at room temperature. Spinal cord sections were later re-hydrated in dH 20 for 1 hour and then the fluorescent nissl stain; Neurotrace (1:200, Molecular Probes Inc. Eugene, 86 OR) was added to the slides. Slides were then dehydrated in a series of alcohols and coverslipped with Entallen (Fisher Scientific, Nepean, ON). Image analysis and statistics A l l images were imported into Photoshop (7.0, Adobe, Ottawa, ON) and adjustments were made to brightness and contrast to the whole image. Some images were false coloured in Photoshop to provide clarity. Quantification was performed blind with respect to the treatment groups. A l l results were analyzed using SigmaStat 3.0 (SPSS Inc., Chicago, IL) and the criterion for significance was p<0.05. Unless otherwise stated, a Student's t-test was used to determine significance. A l l results are stated as mean ± standard error of the mean (SEM). 87 Results Functional differences between Gall null mutant and wild type mice The responses to both noxious and non-noxious stimuli of Gal l -/-were compared to that of the inbred Gal l wt mice. In addition, the responses of an outbred line of mice were examined (CD-I) to ensure that the particular line of 129P3/J wt mice responds similarly to thermal stimulation. When placed on a 1°C cold plate, null mutant mice displayed significantly longer latencies before displaying a nociceptive withdrawal response (involving some or all of: sustained elevation, biting, licking or shaking the paw) when compared to either Gal l wt or CD-I mice (75.4 ± 6.8 for Gal l -/-, mean ± S E M , compared to 18.4 ±1 .3 seconds for Gal l wt, or 21.1± 1.5 seconds for C D - I ; A N O V A , p<0.001, Figure 4.1). The CD-I mice were significantly different from Gal l -/- mice (ANOVA, p<0.001) but not from Gal l wt mice. Gal l -/-mice had increased withdrawal latency from radiant heat (8.3 ± 0.3 seconds, front paw; 8.7 ± 2.6 seconds, hind paw) compared to Gal l wt mice (5.3 ± 0.3 seconds, front paw; 5.4 ± 2.6 seconds, hind paw, A N O V A , p<0.012) and CD-I mice (6.1 ± 0.6 seconds, front paw; 6.2 ± 0.7 seconds, hind paw p<0.025). A dynamic plantar punctate pressure test was used to assess the amount of force (grams) at which a mouse would withdraw. Here no significant difference was observed between the Gal l - / - (front: 6.7 ± 0.6 hind paw: 7.5 ± 2.6 grams of force) compared to Gal l wt groups (front: 5.7 ±1.3 hind paw: 6.7 ± 0.6 grams of force, Figure 4.1). Gall expression in Gall wt mice Small diameter nociceptive afferents are known to express the A T P receptor P2X 3 , terminate in lamina II inner of the dorsal horn in the spinal cord, and require the neurotrophin GDNF for neuronal development (McMahon and Moore, 1988; Chen et al., 1995; Molliver et al., 1997). Ga l l -/- mice displayed reduced thermal nocifensive responses when compared to either Gal l wt or CD-I mice. Using immunohistochemistry, Ga l l protein expression occurs in 68 ± 8.3% of all somata within the C7 and C8 DRGs and that this expression is not limited to any particular size class (Figure 4.2, histogram). Peptidergic neurons, as indicated by CGRP immunoreactivity, showed 32 ± 3.8% staining overlap with G a l l (Figure 4.2, histogram inset). 88 Figure 4.1 Gall -/- and wt sensory tests Gall -/- mice have reduced nocifensive thermal response compared to Gal l wt mice and CD- 1 mice (n=7 for each group). Nocifensive withdrawal involves some or all of: sustained elevation, biting, licking or shaking the paw during stimulation. Cold Plate, top graph: G a l l -/- mice (grey bar) could remain on a 1°C cold plate for a significantly longer period of time when compared to Gal l wt and CD-I mice. Gal l -/- mice remain on the cold plate for 75.4 ± 6.8 seconds (mean ± SEM) before exhibiting a nociceptive cold response as compared to Gal l wt mice which lasted for 18.4 ±1.3 (p<0.001) seconds and CD-I mice (white bar) which lasted 21.1± 1.5 seconds (p<0.001). No significant difference was found between the Gal l wt and CD-I mice strains. The * indicates p<0.05 compared to either Gal l wt or CD-I mice. Radiant heat test, middle graph: Gal l -/- mice (grey bars) withstand radiant heat 8.3 ± 0.3 seconds for the front and 8.7 ± 2.6 seconds for the hind paw which is significantly longer than the time seen for either the Gal l wt mice (black bars) of 5.3 ± 0.3 seconds (p<0.012) for the front and 5.4 ± 0.3 seconds for the hind paw (p<0.025) or the CD-I mice which had a response of 6.1 ± 0.6 seconds (p<0.012) for the front arid 6.2 ± 0.7 seconds for the hind paw (p<0.025). There was no significant difference observed between Gal l wt or CD-I mice. The * indicates p<0.05 compared to either Gal l wt or CD-I mice. Progressive punctate force, bottom graph: There is no significant difference in the amount of non-noxious force required to illicit a response between Ga l l -/- mice (grey bars) and the Gal l wt mice (black bars). Gal l -/- mice required 6.8 ± 0.42 grams of force for the front paw and 7.5 ± 2.6 grams of force for the hind paw compared to the Gal l wt mice that required 5.7 ±1.3 grams of force for the front paw and 6.7 ± 0.6 grams of force for the hind paw. 100 coldplate CD-1 Gall wt Gall -/- radiant heat front paw hind paw progressive punctate force > CD-1 Gall wt Gall -/- front paw hind paw Gall wt Gall -/- 90 Figure 4.2 Gall-IR in the C 7 and C 8 DRGs of Gall wt mice Representative photomicrographs of a C8 mouse D R G triple labeled for Gal l (red), IB4 (green), C G R P (blue) and the merged picture of all three images (colour). G a l l immunohistochemistry (red) shows that 68 ± 8.3% of all somata express Gal l distributed evenly across somata size (grey bars, large histogram). Of this Gal l expressing population, 37 ± 2.4 % also bind the lectin IB4, 32 ± 3.8% express CGRP, 18 ± 0.6% bind both IB4 and express CGRP and 23 ± 4.1% do not bind IB4 nor express CGRP (histogram bottom inset). Scale bar = 50 microns. 91 92 Non-peptidergic neurons that bind IB4 also co-express Ga l l with a 37 ± 2.4% frequency (Figure 4.2, histogram inset). 18 ± 0.6% of DRG neurons bind both IB4 and express CGRP and 23 ± 4.1% do not bind IB4 nor express CGRP (Figure 4.2, histogram inset). Anatomical differences Using radioactive in situ hybridization for Gal l mRNA followed by autoradiography Gal l mRNA was observed in the DRG somata (Figure 4.3) and the silver grains predominately co- localized to neurons within the DRG (Figure 4.3). Silver grain density was at background levels in Gal l -/- tissue sections, even when exposed for 2 days longer than Gal l wt sections, confirming that Gal l mRNA expression was undetectable in the Ga l l -/- mice (Figure 4.3 middle panel). Differences in peptidergic (CGRP expressing) and non-peptidergic (IB4 binding) nociceptive neurons within the D R G between -/- and G a l l wt mice were examined. C G R P immunohistochemistry indicated a non-significant difference of 55 ± 5% CGRP-positive somata in Ga l l wt mice and 49 ± 4 % CGRP positive somata in -/- mice (Figure 4.4). Interestingly, there was a significant reduction in the proportion of cells binding IB4 from 59 ± 2% in Gal l wt mice to 38 ± 4 % in Gal l -/- (p<0.05, Figure 4.4). The co-localization of both IB4-binding and CGRP expressing neurons was also significantly decreased from 37 ± 3 % in Gal l wt mice to 20 ± 2 % in -/- mice (p<0.05, Figure 4.4). Changes in sensory neuronal distribution and spinal termination in Gall null mutant mice Interestingly, when IB4 staining was examined in the dorsal horn, Ga l l - / - mice showed a significant increase in the depth of IB4 binding within the dorsal horn (middle panel Figure 4.5). The difference between the distances of the red arrows illustrate that the binding of IB4 has not only shifted to deeper level, but expanded in depth in Ga l l -/- mice. Compared to Gal l -/- mice, the peak IB4 binding density occurred between 70 - 80 microns deep to lamina I, whereas in Gal 1 wt mice, the maximum binding intensity occurred 30-40 microns deep to lamina I (Figure 4.5 bottom right graph). C G R P immunohistochemistry also showed a significant difference of the depth of CGRP staining within the dorsal horn between -/- and wt mice (Figure 4.5, bottom left graph). Here, maximum CGRP immunoreactivity was also observed to be significantly deeper in Gal l -/- mice when compared to Gal l wt mice. When 93 the CGRP (green image) and IB4 (red image) are superimposed (Figure 4.5, colour picture), the white arrowheads illustrate the increased depth of IB4 binding within the dorsal horn. Using c-fos expression as a marker for neuronal primary afferent-elicited activity, in second-order neurons in the dorsal horn, I tested whether the null mutant and wild type mice differed in their expression of c-fos after thermal stimulation (Hunt et al., 1987; Dai et al., 2001). Two hours after exposure of the front paw to noxious thermal stimuli, the dorsal horn of spinal segments C7 and C8 were examined. These two segments were examined since these two spinal levels receive the majority of the sensory input from the forepaw. After exposure to 1°C water (Figure 6, left panel) Gal l -/- mice had 14.7 ± 0.9 c-fos positive cells at per section C7 and 13.1 ±0 .6 c-fos positive nuclei at C8 compared to Gal l wt mice, which had 21.1 ± 1.9 c-fos positive nuclei per section at C7 and 24.1 ± 2.7 c-fos positive cells per section at C8 (p<0.05 compared at the same level between Ga l l -/- and Gal l wt animals, Figure 4.6). After exposure to 52°C water, Ga l l -/- mice had 18.7 ± 2 . 1 c-fos positive nuclei per section at C7 and 28.0 ±1 .9 c-fos positive cells per section at C8 compared to Gal l wt mice, which had 27.5 ± 2.9 c-fos positive nuclei at per section C7 and 34.5 ± 1 . 5 c-fos positive nuclei per section at C8 (*p<0.05 compared at the same level between Ga l l -/- and wt animals, Figure 4.6). The neuronal specific antibody NeuN, as previously described by McPhail and colleagues (McPhail et al., 2004), was used to determine if the differences in c- fos expression can be attributed to differences in the number of second order neurons. There was no significant difference in the number of neuronal cell bodies at either the C7 or C8 spinal levels between Gal l -/- or wt mice (figure 4.7). 9 4 Figure 4.3 Gall ISH signal in the DRG Gal l in situ hybridization signal (red) can clearly be seen in Ga l l wild-type (wt) mice (middle left panel) and that this localizes to neuronal somata (blue arrows, top right panel). Sections have been counter stained with the fluorescent nissl stain (green) within the mouse D R G in mice (top panels). Ga l l -/- (middle panel) do not show any ISH signal within the DRG or specific cell somata (vertical blue arrows) confirming that Ga l l -/- do not express Gal l mRNA. Scale bar = 100 microns. 95 4-> IS 7 96 Figure 4.4 CGRP and IB4 in Gall -/- and wt mice Distribution of CGRP (green) and IB4-binding (red) neurons in the D R G between Gal l -/- and Gal wild type (wt) mice (n=4 for both groups). I observed approximately 55 ± 5% of cells expressing CGRP in cell bodies within the C7 and C8 dorsal D R G within Gal l wt (top left panel) and 49 ± 4 % of cells in -/- mice (top right panel) and there was no significant difference between these two groups (bottom graph). There was 59 ± 2% of cells binding IB4 in cell bodies within the C7 and C8 dorsal D R G within G a l l wt (middle left panel) which was significantly different to the 38 ± 4 % of cells binding IB4 in the -/-mice as indicated in the bottom graph. A significant decrease in the proportion of cells expressing both CGRP and binding IB4 decreased from 37 ± 3 % in Gal l wt to 20 ± 2 % in Gal l -/- neurons. * indicates p< 0.05, Scale bar = 100 microns 97 98 Figure 4.5 CGRP and IB4 in the dorsal horn of Gall -/- and wt mice Differences in distribution of nociceptive fiber terminals within the dorsal horn of Gal l -/- and Gal l wt mice (n=4 for both groups). CGRP immunohistochemistry of -/- mice (top right panel) within the cervical dorsal horn reveals a significant increase of C G R P immunoreactivity within the deeper lamina of the dorsal horn when compared to wt mice (top right panel). This is clearly observed when the CGRP staining intensity of Ga l l wt (black line) and -/- (grey line) Gal l mice are plotted as a function of depth (bottom left graph). IB4 binding neurons within the dorsal horn appear in deeper lamina in the -/- mice (middle right panel) when compared to the Gal l wt mice (middle left panel). Red arrows (middle panel) illustrate the increase in depth of IB4 binding in Gal l -/- mice when compared to wild-type mice. When IB4 binding was plotted against depth (bottom right graph), there was a significantly shift of IB4 binding intensity within deeper lamina of the spinal cord in -/- mice as compared to Gal l wt mice. This was clearly demonstrated when IB4-binding intensity is plotted as a function of depth. Ga l l -/- (grey line) had increased staining in deeper lamina compared to wild-type (black line) mice. When the CGRP and IB4 images were overlapped (right panel), white arrowheads indicate the increased depth of IB4 binding (red) comparing Gal l wt (bottom left) and -/- (bottom right) animals. * indicates p<0.05, scale bar = 100 microns, error bars of graphs represent SEM. 99 100 Figure 4.6 c-fos-IR in the mouse dorsal horn after noxious temperature Two hours after thermal nociception, fewer c-fos positive cells in the dorsal horn of Gal l -/- mice is observed when compared to Gal l wt mice. After exposure to 1°C water (left panel) Gal l -/- mice (grey bar, bottom left) had significantly less 14.7 ± 0.9 c-fos positive cells per section at C7 compared to 21.1 ± 1.9 c-fos positive cells per section in Gal l wt (p =0.02). At C8 there were 13.1 ± 0.6 c-fos positive cells per section in G a l l -/- mice that was significantly less than G a l l wt mice (black bar, bottom left), which had 24.1 ± 2.7 c-fos positive cells per section (p=0.007). * indicates p<0.05 compared at the same level between Gal l -/- and Gal l wt animals, n=4 for both -/- and Gal l wt mice. After exposure to 52°C water (right panel) Ga l l -/- mice (grey bar, bottom left) had significantly less 18.7 ± 2 . 1 c-fos positive cells per section at C7 compared to 27.5 ± 2.9 c- fos positive cells per section in Gal l wt (p<0.05). At C8 there were 28.0 ±1 .9 c-fos positive cells per section in Gal l -/- mice which was significantly less than Gal l wt mice (black bar, bottom left), which had 34.5 ± 1 . 5 c-fos positive cells per section (p<0.04). * indicates p<0.05 compared at the same level between Gal l -/- and Gal l wt animals, n=4 for both -/- and Gal 1 wt mice. 101 Figure 4.6 c-fos in the mouse dorsal horn 102 Figure 4.7 NeuN-IR in the mouse dorsal horn The reduced number of c-fos positive neurons in Gal l -/- is not a result of a difference in the number of second order neurons. NeuN immunohistochemistry was performed on Gal l -/- (top panel) and wild type (middle panel) spinal cords at C7 and C8 to quantify the number of neuronal cell bodies within the dorsal horn (n=4 for both groups). The graph (bottom panel) shows that there is no significant difference between the Gal l -/- and Gal l wt mice at either C7 or C8 levels. Ga l l -/- mice had 248 ± 15 NeuN positive cells at C7 and 299 ± 17 NeuN positive cells at C8 compared to wild-type mice (black bar, bottom right), which had 278 ± 20 NeuN positive cells at C7 and 333 ± 19 NeuN positive cells at C8. Scale bar = 100 microns. 103 104 Discussion Neurodevelopment of nociceptive fibers Nociception is a basic requirement for the avoidance of actual or potential tissue damage. Not surprisingly, the majority of primary afferent fibers transmit a variety of nociceptive information ranging from thermal and chemical to mechanical sensitivity. These diverse nociceptive inputs are transmitted along two major nociceptive pathways terminating in laminas I and II (Snider and McMahon, 1998). The segregation of these small diameter afferent fiber projections into a laminar specific topology is a key event during neurodevelopment and suggests different functional roles. Different classes of sensory neurons enter the spinal cord in sequence. In the thoracic cord, large diameter sensory fibers enter the spinal cord at E14.5 followed by the small diameter fibers at E15.5 (Ozaki and Snider, 1997). Interestingly, in the rat Gal l expression was first seen in the DRG at E14, just as small diameter fibers grew towards the spinal cord (Regan et al., 1986). At PO in the superficial dorsal horn, Gal l expression was seen in laminae I and II with the highest staining intensity seen from P0-P7. This correlates with the time at which appropriate connections are established in the dorsal horn of the spinal cord (Regan et al., 1986). These observations led to the speculation that Gal l was involved in either axonal outgrowth or synaptic stability of nociceptive fibers during neurodevelopment (Dodd and Jessell, 1986). Once connections are made, these fibers continue to express G a l l at lower levels. After a peripheral neuropathy in the rat, where increased nociception occurs, Gal l mRNA increases in the DRG as well as protein levels increase in lamina I and II (Cameron et al., 1997; Xiao et al., 2002). Furthermore, G a l l function blocking antibodies attenuate the mechanical allodynia associated with neuropathic pain (Imbe et al., 2003). Taken together, these data strongly suggest that Ga l l plays a significant role in both neurodevelopment and maintenance of small diameter sensory afferents. To compare with results in the rat, Gal 1 expression was examined in wt mice and then determined if the lack of Ga l l in G a l l -/- mice correlates with anatomical and functional differences in nociceptive behaviour. Gal l -/- mice displayed reduced nocifensive thermal responses compared to Gal l wt mice. These attenuated responses correlates with differences in primary afferent distribution and termination within the dorsal horn of Gal l -/- mice. Noxious thermal stimulation also 105 stimulated less second order neurons as indicated by c-fos activation. Taken together, these data suggest Gal l is involved connecting and/or maintaining adult sensory neurons in vivo. Gall distribution Approximately 68% of all neurons within the cervical DRGs are Gall-immunoreactivity (IR) in Gal l wt mice. Since this is the first report of Gal l expression in mice DRGs these results can only be compared to previous findings in rat DRGs. In adult rats, Regan et al (1986) report 63% of all DRGs examined (it is not stated from which spinal cord segment) are positive for Gall-IR with 46% of these DRGs having strong Gall-IR. Most DRG neurons from the 4 t h and 5 t h lumbar (L) levels in the rat have some Gall-IR, but only 20-26% are intensely Gall-IR positive (Imbe et al., 2003; Sango et al., 2004). The Gall-IR within Gal l wt mice is somewhat consistent with previous reports in rats given that there are observed differences in the portion of sensory neuron types between cervical and lumbar DRGs (Ramer etal., 2001a). Gall expressing neurons fail to make appropriate connections in Gall -I- mice Gal l -/- mice have neither gross morphological differences from G a l l wt mice, known compensatory changes in the expression of other galectins, nor any changes in immune cell numbers (Poirier and Robertson, 1993). During olfactory development, Ga l l is expressed by Dolichos biflorus agglutinin binding neurons as they are growing towards their target and this expression continues, albeit at lower levels, once connections have been made (Puche et al., 1996). Furthermore, when grown in vitro, Gal l increases olfactory neurite outgrowth (Puche et al., 1996). In the Gal l -/- mice, these Dolichos biflorus agg/urmm-binding neurons do not reach their appropriate target during development (Puche et al., 1996). Somewhat reminiscent of the olfactory system, analogous abnormalities of small-diameter primary afferents (which express G a l l , Figure 4.2) have a significantly different terminal distribution in the dorsal horn of Ga l l -/- mice compared to Gal l wt mice (Figure 4.5). The altered immunoreactivity of nociceptive markers in the dorsal horn (Figure 4.2) and the decrease in c-fos activation after noxious thermal stimulation in mice lacking Gal l (Figure 4.6), suggest that the significant increase in noxious pain threshold that is observed (Figure 4.1) is in part due to the inappropriate connections of the small diameter fibers in lamina Hi (Figure 4.5), and not due to any potential differences in the number of second order neurons as indicated 106 by NeuN staining (Figure 4.7). There are many potential mechanism which are discussed below leading to the reduced proportion of IB4-binding cell bodies in the DRG, and the binding of IB4 and CGRP immunoreactivity within deeper layers of the dorsal horn. Neuronal Galectn-1 interactions There are many putative mechanisms by which Gal l can maintain sensory neuronal function. Gal l has been shown to act as a growth factor and/or a cytokine by stimulating DRG outgrowth in vitro and increasing the rate of sensory and motor regeneration in vivo by increasing Schwann cell migration and possibly eliciting the release of a yet unidentified factor from macrophages (Horie et al., 1999; Horie and Kadoya, 2000; Fukaya et al., 2003; Horie et al., 2004). A neuronal Ga l l receptor has not been identified but under normal circumstances Gal l is secreted, via the non classical pathway, (Cooper and Barondes, 1990) by Schwann cells and dorsal root ganglion neurons (Sango et al., 2004). This secreted protein may then act in an autocrine/paracrine fashion on sensory neurons and/or glial cells within the D R G during development. Therefore, the lack of Ga l l in the null mutant mouse may lead to a reduction of trophic support within the D R G , resulting in a decreased proportion of IB4-binding neurons. Gall alters ECM binding Gal l also promotes axonal growth by altering adhesion properties of the extracellular matrix molecules (ECM). For example, Gal l mediates self-aggregation of primary sensory olfactory neurons through the cross linking of carbohydrate ligands, and facilitates D R G fasciculation in vitro (Outenreath and Jones, 1992; Mahanthappa et al., 1994). This inappropriate neuronal targeting was previously reported in the olfactory system (Puche et al., 1996) and now in the primary sensory afferent neurons terminating on the dorsal horn of the spinal cord. The absence of Ga l l in the null mutant mice may then lead to inappropriate targeting by either hindering axonal fasciculation or altering cellular adhesion during small diameter sensory afferent growth into the spinal cord. IB4 neurons in thermal nociception Both peptidergic and non-peptidergic (IB4-binding) small diameter sensory neurons are implicated in thermal nociception although their precise role remains somewhat elusive (Snider and McMahon, 1998). These two neuronal populations are functionally distinct in 107 their response to heat, suggesting that they may respond to distinct aspects of noxious stimulation (Stucky and Lewin, 1999). Heterogeneity in thermal responses exists within the non-peptidergic neurons. Only half of the IB4-binding nociceptors are sensitive to noxious heat (Stucky and Lewin, 1999). This is most likely due to the differences in receptor expression on these neurons. Of all IB4-binding neurons approximately 78% express the capsaicin and thermal sensitive vanilloid receptor (VR1/TRPV1) and 67% also express the ATP-gated receptor P2X 3 (Bradbury et al., 1998; Guo et al., 1999). Application of a P2X 3 or VR1 antagonist to rats with inflammation leads to reductions in thermal hyperalgesia (Garcia-Martinez et al., 2002; Jarvis et al., 2002). IB4-binding neurons also express the neurturin co-receptor GFRa2 (Bennett et al., 1998). In GFRa2 -/- mice, a decrease in heat- evoked currents is observed when compared to GFRa2 +/+ but there were no observed changes in the number of IB4-postive cells (Stucky et al., 2002). Chemically killing non- peptidergic neurons via a single injection of IB4 conjugated to the toxin saporin leads to a temporary decrease in thermal nocifensive behavior (Vulchanova et al., 2001). These data indicate that IB4-binding neurons do play a role in thermal nociception. The reduction in the proportions of IB4-binding cells observed in this report as well as the alterations in laminar termination in these neurons correlate with the observed attenuated thermal nocifensive responses in Gal l -/- mice. Although Gal l expression within the CNS was first reported in 1984 (Dodd et al.), we are only now beginning to understand its role in neurodevelopment, axonal injury and regeneration. In this chapter I show that Gal 11 null mutant mice have anatomical and sensory deficits compared to Gal l wt mice, and that these differences correlate with changes in behavioral responses to noxious stimuli. The precise role of G a l l during neurodevelopment remains elusive, and more research is required to determine not only its role in potential inter- and intracellular signaling, but also the specific receptors involved in this signaling process. 1 0 8 CHAPTER 5: ENDOGENOUS MOTONEURONAL GALECTIN-1 INCREASES AFTER AXOTOMY AND PROMOTES FUNCTIONAL RECOVERY AFTER FACIAL NERVE INJURY 109 Introduction Neuronal Gal l expression occurs within primary sensory neurons and motoneurons (Regan et al., 1986). During the period of motor axonal outgrowth in embryonic rats, Ga l l mRNA expression increases within somata of spinal motoneurons until target muscles are reached, following which, expression is maintained at a lower level throughout adult life (Hynes et al., 1990). Although the precise functions of this protein remain uncertain, Puche (1996) demonstrated that recombinant Gal l modifies cellular adhesion of mouse olfactory neurons in vitro. Although Gal l -/- mice are viable and do not display any overt phenotype, they also demonstrated that specific olfactory neurons that normally express G a l l grew to inappropriate targets in the absence of the protein in vivo (Puche et al., 1996). RhGall-Ox, but not the reduced Gal l form, promotes axonal growth both in vitro and in vivo (Horie et al., 1999; Horie and Kadoya, 2000; Fukaya et al., 2003; Horie et al., 2004). Specifically, after a sciatic nerve injury exogenously applied Gall-Ox increased the rate and success of spinal motor and sensory axonal growth as indicated by either neurofilament staining or retrograde tracer application (Horie et al., 1999; Fukaya et al., 2003). This effect was reversed using Gal l function-blocking antibodies that significantly reduced axonal regrowth. In both of these reports, the promotion of axonal growth by Gal l was also associated with increased Schwann cell migration into an acellular environment. The neuronal expression of Gall-Ox acts as a cytokine by stimulating macrophages to release an unidentified factor to promote both neuronal outgrowth and Schwann cell proliferation and migration in vitro (reviewed in Horie and Kadoya, 2000; Horie et al., 2004). Although exogenous application of recombinant Gal l facilitates motor axon regrowth and G a l l mRNA expression occurs within naive motoneurons, there is a paucity of information regarding changes in the endogenous expression of G a l l mRNA following axonal injury. Previous studies used injured sciatic nerves as a model for assessing axonal regeneration. This nerve contains sensory, motor and sympathetic neurons and therefore does not allow for a clear assessment of functional regeneration within specific neuronal populations. Here I have used a facial motoneuron model of axonal injury. The facial nerve carries almost entirely motor fibers, and functional recovery can be assessed by movement of vibrissae (Paxinos, 1985; Isokawa-Akesson and Komisaruk, 1987). After a facial nerve 110 crush, whisker movement ceases until the injured axons begin to re-innervate their targets (Gilad et al., 1996; Ferri et al., 1998; Serpe et al., 2002; Kami jo et al., 2003). The amount and character of whisking movement, (amplitude and frequency) is directly proportional to the success of axonal regeneration (Tomov et al., 2002). In the present study, I examined the endogenous Gal l mRNA expression in the adult mouse facial nucleus after axonal injury. I used both a nerve resection and crush injury to examine Gal l mRNA changes within the motoneuron cell bodies to ascertain whether Gal l expression correlates with the regenerative state of the axons. To determine the significance of endogenous Gal l for axonal regeneration, the recovery of whisking movement of Gal l -/- to Gal l wt mice was compared after facial nerve crush. I l l Materials and Methods Surgery For anesthetic procedures and information on Ga l l wt, -/- and CD-I mice see Chapter 4 Materials and Methods. Facial Nerve Lesion Facial nerve lesions were performed as described previously (McPhail et al., 2004). Briefly, under anesthesia, the facial nerve was exposed at its exit from the stylomastoid foramen. The buccal branch of the facial nerve was either transected and a 2-3 mm nerve segment was removed to prevent nerve regeneration, or the nerve was crushed twice for a period of 5 seconds with #5 forceps (Fine Science Tools, North Vancouver, BC) and the wound was closed with sutures. Facial Nerve Injections Facial nerves were exposed as described above. Using a Hamilton syringe and a pulled glass micropipette, 1 pi of saline solution, 50 yM colchicine (Sigma, Oakville, ON) or 2 pig/pil of G D N F (gift from Regeneron Pharmaceuticals, Tarrytown, N Y ) was injected into the uninjured nerve at the same site where the nerve was crushed in previous experiments. The skin was sutured closed and the animals were allowed to survive for three days. Perfusion I cryosectioning See Chapter 2 Materials and Methods for perfusion and cryosectioning. For mouse facial tissue all sections were cut at 14 microns. In situ hybridization See Chapter 4 Materials and Methods for Gal l ISH methods. Note for mouse facial tissue autoradiographic signal was exposed for 3 days. Galectin-1 ISH analysis At least three sections per animal were analyzed, and to prevent a neuron being analyzed twice each tissue section was a least 100 pim apart. Nissl and darkfield (silver grain) images were taken of both the injured and the contralateral (uninjured) side using a digital camera attached to a fluorescent microscope (Carl Zeiss, Axioskop) in combination with Northern Eclipse software (Empix Inc, Mississauga ON). A l l images were analyzed with SigmaScan 112 Pro 5 software (SPSS Inc., Chicago, IL). The percent area occupied by silver grains was determined. This was accomplished by outlining the individual neuronal cell bodies using the nissl image and applying the resulting layer to the darkfield image. Background autoradiographic signal was then subtracted to obtain the corrected area occupied by silver grains. For each animal, the percentage area occupied by ISH signal per soma was determined for both the axotomized and contralateral (uninjured) side. The data were expressed as percentage of the mean ISH signal per soma on the contralateral uninjured side (as described previously by Fernandes et al., 1999). Mouse whisker movement analysis The Gal l -/- (n=4) and wt (n=4) mice used in this study were born within 24 hours of each other and were 4 weeks old when surgery was performed. Prior to surgery and under light anesthesia, all but two whiskers in the caudal C-row were trimmed from the whisker pad as previously described by Tomov et al. (2002) and the wound was sutured closed. To analyze changes in whisker movement over time as an indicator of regeneration of the crushed facial nerve, before surgery and after the first 3 days following a facial nerve crush, 2-5 minutes of whisker movement was recorded using a digital video camera (Cannon, XR50MC) as described by (Tomov et al., 2002). Digital images were then transferred to a Macintosh computer (Apple Computer, Cupertino CA) and individual frames were obtained using iMovie 3.03 (Apple Computer, Cupertino CA). Individual frames of the maximal protraction were obtained (Figure 5.6A). Using Image J (1.30p, NIH, Bethesda, M L ) a straight line was drawn between the tear ducts of the right and left orbits of the eyes (Figure 5.6B). This line represented the 0° angle. An angle was then measured and recorded between this line and the maximum forward sweep of the vibrissae (Figure 5.6C). Frequency was measured by counting the number of vibrissae sweeps per second. For each animal on each day until 14 days after surgery, 4 separate vibrissae movements were measured and averaged. Statistics Quantification was performed blind with respect to the treatment groups. A l l data are represented as mean ± standard error of the mean (SEM), and all tested were carried out using SigmaStat 3.0 (SPSS Inc, Chicago, IL). A 1-way A N O V A with Holm-Sidak post hoc test was used to determine significance between groups. Significance was assigned at p<0.05. 113 Results Gall mRNA expression in the facial nucleus In the facial nucleus of Ga l l wt (129P3/J) mice, in situ hybridization (ISH) for Gal l mRNA followed by autoradiography was used to determine motoneuronal Gal l mRNA expression (Figure 5.1). Silver grain density was at background levels in Ga l l -/- tissue sections, even when exposed for 2 days longer than G a l l wt sections, confirming that G a l l mRNA expression was undetectable in the Gal l -/- mice (Figure 5.1). Ga l l wt mice are an inbred line and were used for comparison. Motoneurons in these mice show moderate amounts of silver grains that leveled around 7.23 ± 0.89 times background. In addition, Gal l expression was examined in an outbred line of mice (CD-I) to ensure that this particular line of 129P3/J wt mice has similar levels of Gal l mRNA expression and responds similarly to axonal injury. The baseline levels of Ga l l ISH signal in the uninjured facial motoneurons of these CD1 mice were comparable to the 129P3/J line (data not shown). Control experiments with a sense probe to Gal l revealed only background levels of silver grains on the tissue sections in either the Gal l wt, Ga l l -/- or CD-I facial motor nuclei. Gall expression after crush or resection of the facial nerve After a facial nerve crush in G a l l wt mice, ISH signals for G a l l mRNA significantly increased in the axotomized motoneurons (Fig. 2). The silver grain density after 3 days was 307.3 ± 46.6% of contralateral and 360.9 ± 57.9% after 7 days. Statistically, these increased mRNA levels were significantly different from the levels found in uninjured motoneurons (p<0.001, p<0.005 respectively; Figure 5.2). By 14 days after a crush injury the nerves have successfully regenerated back to their targets as indicated by complete behavioral recovery (Figure 5.6). At this time, silver grain density decreased to 147.8 ± 8.1% of contralateral. This level of expression was no longer significantly different from the uninjured control animals (p>0.05, Figure 5.2 histogram). 114 Figure 5.1 Gall is expressed in uninjured facial motoneurons Photomicrographs of Gal l in situ hybridization signal (white middle panel, red bottom panel) that have been counter stained with the fluorescent nissl stain (green) within the mouse facial motor nucleus (FMN). Angled arrows on the G a l l wt photomicrographs (left panels) indicate G a l l mRNA is neuronally expressed. In G a l l -/- mice (right panels) the photographic emulsion was left on 2 days longer than the Ga l l wt mice sections ensured the absences of mRNA signal was not due to a reduction in signal. Vertical arrows indicate no silver grains over G a l l -/- neuronal cell bodies and that G a l l mRNA does not show any greater ISH signal than background levels (right panels). Scale bar = 50 microns.  116 Figure 5.2 Gall ISH in Gall wt mice after a nerve crush At 3 days post lesion (top panel), the silver grain density over facial motoneurons after a crush was 307.3 ± 46.6% (mean ± SEM) of the contralateral and 360.9 ± 57.9% after 7days (middle panel). These increased mRNA levels were significantly different from the levels found in uninjured motoneurons (p<0.001, p<0.005 respectively). By 14 days after a crush (bottom panel), there was an increase of 147.8 ± 8.1% of contralateral which was not significantly different to uninjured animals (p>0.05, bottom histogram). * indicates p<0.05 compared to uninjured animals, scale bar = 50 microns.  118 Numerous studies have demonstrated strain specific responses in mice to a variety of neurological insults (reviewed in Steward et al., 1999). In the present study, in addition to the inbred 129P3/J mice, Ga l l expression was examined in an outbred strain of mice (CD-I) to ensure that the axonal injury induced increase in Gal mRNA was not a strain specific response. I observed a similar Gal l mRNA expression after axonal injury in both mouse strains; compare the Gal-1 wt mouse in Figure 5.2 to the CD-I mouse in Figure 5.3. After a facial nerve crush in CD-I mice, an increase in grain density to 254.8 ± 27.9% of the contralateral side after 3 days and 346.6 ± 30.4% after 7 days was observed. When compared to the Gal-1 expression in uninjured motoneurons these increases were significant (p<0.001, p<0.005 respectively (Figure 5.3). At 14 days after crush a decrease in grain density to 191.1 ± 46.6% was observed. Similar to the Gal-1 wt strain this level of expression was no longer significantly different to the uninjured control animal (p=0.05, Figure 5.3 histogram). After a facial nerve resection, an injury preventing axonal reconnection with the targets, a significant and rapid increase of Gal l mRNA occurred within 3 days and persisted until at least 14 days after the injury (Figure 5.4), the longest time point examined. Quantification of these results at 3 and 7 days post injury revealed a increase in silver grain density to 257.3 ± 26.6% and 299.6 ± 29.1% of contralateral, (Figure 5.4). Compared to the levels in uninjured motoneurons these ISH signals were significantly different with p values smaller than p<0.01. At 14 days after a resection injury the Gal l mRNA expression remained significantly elevated (309.8 ± 83.2% of contralateral) which contrasts with the reduced Gal l expression at 14 days after a nerve crush (Figure 5.2 and 5.3). 119 Figure 5.3 Gall ISH in CD-I mice after a nerve crush Gall mRNA is transiently upregulated by facial nerve crush in outbred CD1 mice. At 3 days post lesion (top panel), the silver grain density over facial motoneurons after a crush was 254.8 ± 27.9% (mean ± SEM) of contralateral and 346.6 ± 30.4% after 7 days (middle panel). When compared to the Gal-1 expression in uninjured motoneurons these increases were significant (p<0.001, p<0.005 respectively). 14 days post axotomy (bottom panel), silver grain density was 191.1 ± 46.6% of contralateral (bottom histogram) and was not significantly different from the uninjured control animals (p=0.05, bottom histogram). * indicates p<0.05 when compared to uninjured animals, scale bar = 50 microns. 120 400 H ra 300 4 c o o 200 4 100 4 * 1 uninjured 3 7 14 days post injury 121 Figure 5 .4 Gall ISH in CD-I mice after a nerve resection Gal l mRNA is persistently upregulated by facial nerve transection in outbred CD1 mice. At 3 days post lesion (top panel), the average area occupied by silver grains over axotomized facial motoneurons was 257.3 ± 26.6% (mean ± SEM) of the contralateral side and 299.6 ± 29.1% at 7 days (middle panel). Compared to the levels in uninjured motoneurons these ISH signals were significantly different with p<0.01. At 14 days following axotomy (bottom panel), there was an increase ISH signal of 309.8 ± 83.2% of contralateral which was significantly greater than uninjured control animals (p<0.015, bottom histogram). * indicates p<0.05 compared to uninjured animals, scale bar = 50 microns. uninjured 3 7 14 days post injury 123 Signals increasing Gall mRNA expression Since the experiments above suggested a regulation of G a l l by target-derived factors I investigated the potential role of retrograde transport on G a l l mRNA regulation. The cessation of axonal transport can be achieved through the application of the plant alkaloid colchicine, which halts axonal transport by disassembling neuronal microtubules. Colchicine or vehicle solution (n=3 per group) was injected into the facial nerve of CD-I mice. Colchicine increased the Ga l l ISH signal to 243.9 ± 45.2% of contralateral by 3 days, rendering it significantly different from uninjured (p<0.01) or saline (p<0.02) injected motoneurons (Figure 5.5). Saline injection alone increased the average ISH signal to 147.3 ± 7.0% of the untreated contralateral side (Figure 5.5) but this level of expression was not significantly different from untreated animals. The non-significant increased Gal l mRNA observed with saline injection is likely due to the neuronal damage caused by the nerve manipulation and injury induced by the injection. In addition to the depletion of target derived factors playing a role in regulating gene expression after axonal injury, the release of trophic factors and cytokines at the site of injury may serve as a positive signal driving the neuronal gene expression response (Fu and Gordon, 1997). Activated peri-lesion Schwann cells increase the expression of glial cell line- derived neurotrophic factor (GDNF) (Hoke et al., 2000) and here G D N F was injected into the intact nerve of CD-I mice (n=3 per group). Like colchicine GDNF significantly increased the ISH signal to 253.5 ± 36.8% of the contralateral side, a level significantly higher than the level in uninjured (p<0.01) or saline injected (p<0.02) motoneurons (Figure 5.5). 124 Figure 5 . 5 Positive and negative signals regulate Gall mRNA Saline treatment (top right panel) increases the average area occupied by silver grains on the treated side to 147.3 ± 7.0% (mean ± SEM) of the untreated contralateral side. This level of expression was not significantly increased above the untreated group alone (p>0.05, top left panel and bottom histogram). Colchicine injection (middle right panel) results in a 243.9 ± 45.2% increase (middle left panel) that was significantly greater than the uninjured animal (p<0.01) or vehicle injection (p<0.02). GDNF injection (bottom right panel) also leads to a 253.5 ± 36.8% increase (bottom left panel and bottom histogram) compared to the uninjured animal (p<0.01) or saline injected animal (p<0.02). * indicates p<0.05 compared to uninjured and saline treated animals, scale bar = 50 microns. 125 contralateral treatment 126 Functional analysis ofFMN injury Previous reports have demonstrated that exogenously applied recombinant Gall-Ox increases the rate of transected sensory and motor axonal regeneration (Horie et al., 1999; Horie and Kadoya, 2000; Fukaya et al., 2003). Conversely, function-blocking Gal l antibodies applied to the transection site reduce the rate of axonal regrowth (Horie et al., 1999; Horie and Kadoya, 2000; Fukaya et al., 2003). Here I wanted to examine the whether endogenous expression affects functional recovery. To accomplish this, the facial nerve was crushed in Gal l wt and -/- and then assessed functional recovery by recording and measuring whisking movements. Except for two whiskers in the C-row of the whisker pad all whiskers were trimmed close to the skin (Figure 5.6A). Using video analysis (see materials and methods) the maximum angle and frequency of whisker movement was measured before and after a nerve crush. When compared to the maximum angle achieved before injury, Gal l -/- mice required 12 days to return to pre-injury levels (Figure 5.6D, grey +) as compared to the 11 days required for the Gal l wt mice (Figure 5.6D, black +). The duration of 11 days in the wt mice is very similar to other mouse strains (Ferri et al., 1998; Serpe et al., 2002; Kamijo et al., 2003). Also, Ga l l wt had achieved a significantly larger maximum angle when compared to Gal l -/- mice at 9 and 10 days post injury (Figure 5.6D, p<0.05). When the frequency of movement was measured, there was no difference between Gal 1 -/- or wt animals prior to or after a nerve crush except at 10 days post- lesion (Figure 5.6E). At this time point Gal l wt mice had a significantly higher whisking frequency than Gal l -/- mice. These data show that both Gal l -/- and wt motor axons successfully regenerate to their targets after a nerve crush but that the rate of full functional recovery is slower in Gal l -/- mice. 127 Figure 5.6 Behavioural recovery following a facial nerve crush in Gall wt and -/- mice Using video analysis, mouse whisker movement (whisking) was recorded. A horizontal line was drawn between the inner orbits of each eye (6B). This line became the 0 degree angle from which the maximum whisking angle and frequency were measured (Figure 5.6C). Seven days after a nerve crush, whisking movements were observed. There was a significant difference in the total angle moved on day 9 and 10 between Ga l l -/- and wt animals (* indicates p<0.05, A N O V A F=138.5). The maximum angle of movement returns to uninjured levels one day later in Ga l l -/- mice (+ indicates when maximum angle movement is not significant different prior to crush lesion). No significant difference in the frequency of movement was observed between Gal l -/- or wt animal prior to or after a nerve crush except 10 days after the lesion (* indicates p<0.05, 6E, A N O V A F=40.9). fj 70 -1 4 5 6 7 8 9 10 11 12 13 14 -1 4 5 6 7 8 9 10 11 12 13 14 days post lesion I G a l 1 w t r- Gall -/- 12? Discussion Both crush and resection of the mouse facial nerve resulted in a significant increase in Gal l mRNA expression by 7 days post injury. Furthermore, I demonstrated that both interneural injections of the axonal transport inhibitor colchicine or the trophic factor GDNF induced increased G a l l mRNA expression in uninjured facial motoneurons. These results are summarized in Table 4. The absence of Gall in Gal -/- mice attenuated the rate of recovery of whisker movement after a facial nerve crush. Gall mRNA expression is regulated by target-derived as well as injury-derived signals In the crush model, Ga l l mRNA expression was not significantly different from control levels by 14 days after injury. At this time complete functional whisker movement was restored indicating target reinnervation. This observation indicates that the increase of Gal l mRNA after axonal injury is in part due to the loss of target-derived factor(s). A number of putative signals exist to regulate gene expression after axotomy, but to date they are poorly understood. These are postulated to be either positive signals, such factors originating at the injury site to initiate neuronal changes, or negative signals, such as the interruption of target- derived factors (Cragg, 1970; Fernandes and Tetzlaff, 2000; McGraw et al., 2002). Colchicine application to the uninjured nerve halts axonal transport through microtubule disassembly, resulting in the loss of retrograde transport. The loss of a retrograde signal derived from a target has long been thought of as one of the injury-signaling mechanisms (Cragg, 1970). For example, in the uninjured nerve, when transport is interrupted through colchicine or cold block, gene expression increases significantly for specific genes such as T a i tubulin and GAP-43 (Woolf et al., 1990; Wu et al., 1993; Bormann et al., 1998). In the present study, the increased G a l l expression observed after axonal transport inhibition suggests that G a l l expression is partly suppressed by (a) target derived factor(s). After axotomy or transport blockade, the putative repressor(s) would be absent, thus increasing Gal l mRNA expression. 130 galectin -1 mRNA changes treatment 3da\s 7 d«ij s 14 days nerve resection t t . t nerve crush t t saline injection colchicine injection t B D N F injection t Table 4. A summary of Gall mRNA changes in Facial motoneurons. Changes of G a l l mRNA expression in facial motoneurons as observed in Chapter 5 following facial nerve treatment in CD-I or Gal l wt mice. 131 Neuronal gene expression changes also occur due to positive injury signals produced and/or released at the injury site. For example, the neurotrophin G D N F has potent effects on motoneuronal regeneration (Henderson et al., 1994; Blesch and Tuszynski, 2001; Boyd and Gordon, 2003). After a nerve injury, GDNF expression increases in Schwann cells distal to the lesion site where it is taken up by injured axons and retrogradely transported to promote axonal regrowth (Yan et al., 1995; Naveilhan et al., 1997; Burazin and Gundlach, 1998; Hoke et al., 2000; Blesch and Tuszynski, 2001). Intrathecal G D N F application also results in an increase of neuronal proteins such as calcitonin gene-related peptide in uninjured motoneurons (Ramer et al., 2003). Here, I report that intraneural G D N F injection increases Gal l mRNA expression within uninjured motoneurons and illustrates its potential role as a positive regulator of Ga l l . Taken together, these data indicate that both injury site- and target-derived factors regulate Gal l mRNA expression after axotomy. Endogenous Gall facilitates functional recovery Through the use of Ga l l -/- mice I have attempted to ascertain the role of Ga l l in neuronal injury and repair. This null mutant mouse does not have any known compensatory changes in the expression of other galectins or in immune cell numbers (Poirier and Robertson, 1993). To determine differences in regenerative responses between animals a facial crush was performed on both Gal l wt and Gal l -/- mice. In the adult mouse, a facial nerve crush results in regeneration and functionally complete whisking behavior within 11 days in both CD-I and ICR mice which is comparable to the behavioural recovery time of Gal wt mice (Ferri et al., 1998; Serpe et al., 2002). Using video analysis, I quantified the rate of facial nerve functional recovery by recording the angle and frequency of vibrissae movement (Guntinas- Lichius et al., 2002). In agreement with other whisker movement data (Guntinas-Lichius et al., 2002), I observed that the measured average maximum angle was 49.9 ± 3.6 with a frequency of 7.2 ± 0.6 Hz (Figure 5.6). After a facial nerve crush, the motoneuronal regenerative response, as indicated by whisker movement, proceeded through three distinct phases. The first stage, occurring 0-7 days post injury, resulted in a complete paralysis of moment (Figure 5.6). During this time high levels of Gal l mRNA were expressed within the injured facial somata (Figures 5.3 and 5.4). The second stage, characterized as minimal 132 whisker movement, occurs from 8-10 days post injury (Figure 5.6). Here Ga l l mRNA expression presumably remained elevated. The third stage is characterized by complete functional recovery of whisker movement, which is correlated with a decrease in Gal l mRNA expression in the injured cell bodies (Figure 5.2). The behavioural recovery usually occurs after the 10 th day post injury. Although little difference in the frequency movement of whiskers was detected, whisking returned to its pre-injury state one day earlier in the wt mice than in the Gal l -/- mice. Since recovery of whisking involves both axonal regeneration rate and synaptogenesis, (which have not been separated in this analysis), it is unclear as to which of these processes involves Ga l l . However, both gain and loss of function experiments have ascribed Gal l with a role in both regeneration rate and synaptogenesis (see below). Gall's role in axonal repair The data presented here supports other studies demonstrating that Gal l increases the rate of regeneration through its involvement in the initiation of the regeneration process (Horie et al., 1999; Horie and Kadoya, 2000; Fukaya et al., 2003). Exogenous Gal l infusion into an acellular bridge at the site of a sciatic nerve injury increased both the rate of regeneration and Schwann cell migration (Horie et al., 1999; Fukaya et al., 2003). Conversely, in this model, application of Ga l l function-blocking antibodies significantly reduced the rate of axonal regeneration compared to vehicle-treated animals (Horie et al., 1999; Fukaya et al., 2003). The endogenous neuronal expression of Ga l l has been reported in neuroblastoma cells (Avellana-Adalid et al., 1994), sensory neurons (Imbe et al., 2003; Sango et al., 2004) and motoneurons (Hynes et al., 1990; Fukaya et al., 2003). G a l l immunoreactivity is also detected within distal axons of motor and sensory neurons (Horie et al., 1999; Fukaya et al., 2003). Externalization of the G a l l protein can occur across the growth cone plasma membrane via the non-classical pathway, despite lacking the recognizable acetylated N terminus secretion sequence (Cooper and Barondes, 1990; Inagaki et al., 2000; Schafer et al., 2003; Sango et al., 2004). Once in the extracellular space, Gal l becomes oxidized, changes conformation and exhibits neuronal growth-promoting abilities (Horie and Kadoya, 2000; Horie et al., 2004). In the oxidized form, Gal l acts as a cytokine by binding to and activating macrophages, which then release an unidentified growth-promoting factor that is larger than 10 kDa (Horie et al., 2004). Interestingly, the macrophage-stimulating factor zymosan also causes the release of a 14 kDa factor from macrophages that increases neuronal regrowth of 133 axotomized retinal ganglion cells (Yin et al., 2003). Therefore after injury the neuronal- expressed Gal l is likely to be secreted into the extracellular space at the injury site thereby inducing macrophages to release both growth-promoting and Schwann cell-activating factors (Cooper and Barondes, 1990; Horie et al., 1999; Fukaya et al., 2003; Horie et al., 2004; Sango et al., 2004). Both macrophages and Schwann cells are known to promote successful regeneration through a variety of mechanisms such as removing myelin debris and providing trophic support (reviewed in Fu and Gordon, 1997). Experiments that alter the responses of macrophages and Schwann cells demonstrate their importance in the regenerative process. For example, acellular nerve grafts do not support peripheral nerve growth when Schwann cell migration or proliferation is inhibited (Chong et al., 1994a; Enver and Hall, 1994). In addition, inhibiting the macrophage response also reduces regenerative success (Calcutt et al., 1994; Dailey et al., 1998). In the C57BL/Wld s mutant mouse, delayed Wallerian degeneration occurs due to mutation in an ubiquitination factor (Conforti et al., 2000). In these mutant mice, functional recovery after a facial nerve crush is also delayed due to the failure of macrophages to clear axonal and myelin debris and inhibitory proteins (Perry et al., 1990; Chen and Bisby, 1993; Glass et al., 1993). Conversely, activating macrophages promotes sensory neuronal outgrowth (Lu and Richardson, 1991; Luk et al., 2003). In the present study the absence of Gal l in mutant mice may have acted to delay the initial degenerative response following a facial nerve crush, thus reducing the rate of functional recovery. The results presented in this study demonstrate that endogenous neuronal Gal l mRNA expression increases after a nerve injury. This elevated expression may be a result of both neuronal target loss as well as expression of factors at the injury site. Furthermore this data suggests that endogenous Ga l l expression may contribute to the rate of functional recovery after a nerve crush. 134 CHAPTER 6: GENERAL DISCUSSION Summary of Results This thesis set out to address the hypothesis that the endogenous neuronal Gall expression influences the growth potential of axotomized neurons. The results presented in the previous chapters and summarized in Table 5 support this hypothesis. In chapters 2 and 5,1 demonstrated increased Ga l l expression within regenerating axotomized motoneurons and reduced Ga l l expression in non-regenerating axotomized rubrospinal neurons. After the infusion of the growth-promoting trophic factor B D N F into the vicinity of the red nucleus, the axotomy-induced reduction of Ga l l was reversed, resulting in an increase in Ga l l expression. In the facial nucleus (Chapter 5), Ga l l expression remained elevated until the axotomized nerve re-innervated its target. Further evidence that Gal l plays an important role in functional recovery was observed in the Gal l -/- mouse. The absence of Gal l in Gal -/- mice attenuated the rate of whisker movement recovery after a facial nerve crush (Chapter 5). These data suggest that G a l l contributes to the regenerative success of the injured motoneurons. Sensory neurons, which are able to regenerate successfully after a peripheral injury, were also examined for Gal l expression. In Chapter 3, Gal l expression within the DRG was examined after both a peripheral axotomy and a rhizotomy. Only after a peripheral axotomy and not rhizotomy was Ga l l expression significantly increased within the sensory neurons. Intrathecal rhGall-Ox application to the rhizotomized DRG did promote limited regeneration of small diameter fibers into the CNS. These data further support the observation in Chapters 2 and 5 that Gal l expression correlates with regenerative ability and that Gall-Ox promotes limited growth into the CNS. 135 neuronal population change in Gall expression following a\otoim spinal motoneurons t red nucleus i red nucleus + vehicle red nucleus + BDNF t D R G - central axotomy DRG - peripheral axotomy t facial nucleus T Table 5. A summary of changes observed in Gall expression following injury. Summary of chapters 2-5 of the changes observed in Gal l expression in neurons following an axotomy. 136 The sensory behavior and anatomy of both Gal l wt and Ga l l -/- mice was examined to investigate potential importance of Ga l l in sensory function. Unexpectedly, normal nociception was impaired in Ga l l -/- mice when compared to G a l l wt mice. The data presented in Chapter 4 demonstrate that the attenuated nocifensive responses correspond to differences in primary afferent distribution and termination within the dorsal horn of Gal l -/- mice. These data suggest that endogenous Gal l expression may contribute to normal sensory function. Overall, the four data-containing chapters of this thesis examined a variety of neuronal populations and their Ga l l responses to injury. In every neuronal system examined, neuronal Gal l expression was found to correlate with the regenerative propensity of injured neurons. Furthermore, the absence of Gal l affected both normal sensory functions and functional recovery following injury. Limitations and future remedies In Chapters 2 through 5 of this thesis I examined the intrinsic expression of Ga l l . Previous work demonstrated that Gal l exists in two conformations, each of which causes a different effect (see Introduction, Ga l l : 2 redox forms). The inability to distinguish between Gall-Ox and Gall-Red within (or outside) of neurons is a major limitation of the experiments within this thesis. To overcome this issue, Dr. Horie is attempting to create monoclonal antibodies specific to Gall-Ox or Gall-Red (Dr. Horie, personal communication). Once created, these Gal l redox specific antibodies are expected to give further insight into the location of G a l l - Ox activity. Although Gal l protein has been observed in Schwann cells as well as neurons (Horie et al., 1999; Sango et al., 2004) most of the results presented in this thesis focuses on the neuronal expression of Ga l l . . Chapter 3, Figure 3.9 shows an increase in Gal l mRNA expression within the peripheral side of the DREZ after a rhizotomy. This increased Ga l l mRNA is associated with an increased number of Nissl stained cells. In vitro, both Schwann cells and neurons but not macrophages express G a l l , therefore the cells central to the rhizotomy site are most likely to be Schwann cells (Sango et al., 2004). Unfortunately for 137 many of the experiments in this thesis the properties of radioactive decay and photographic emulsion do not allow for fine spatial resolution of silver grains to small glial cells. Although previous studies have demonstrated the increase in Ga l l expression at the peripheral nerve lesion site, in Chapters 2, 3 or 5 the lesion site was not examined for Gal l mRNA or protein expression. Furthermore, Ga l l protein is transported from the neuronal cell body to the axon terminals (Chapter 3, Figure 7 and Sango et al., 2004). The amount of Gal l transport to the terminals remains uncertain. In light of this, Ga l l expression at the injury site, as well as the potential Gal l transport to the injury site should examined. The peripheral expression of Gal l protein is not only limited to neurons and possibly Schwann cells; smooth and skeletal muscle cells also express G a l l (Gu et al., 1994; Moiseeva et al., 2000). In muscle cells, Gall-Red is involved in proliferation, differentiation and adhesion (Gu et al., 1994; Moiseeva et al., 1999; Moiseeva et al., 2000). Since expression also occurs in the muscle and possibly Schwann cells, differences in skin and muscle cytology should be examined between Gal l wt and -/- mice. In particular, differences in primary sensory endings within the periphery and their distribution relative to sensory transducers or specific receptor channels should be considered. Temperature-sensitive channels called temperature-activated transient receptor potential ion channels (thermo TRPs) are expressed in DRGs and the spinal cord (reviewed in Patapoutian et al., 2003). In Chapter 4, thermal nociceptive tests revealed increased nocifensive response times in Gal l -/- mice when compared to Ga l l wt. Changes were observed in both sensory fiber termination within the dorsal horn and an alteration in the proportion of sensory neuronal phenotype that may have led to difference of nocifensive responses between wt and mutant mice. Differences in sensory transducer expression between these mice may also lead to the observed behavioral differences. Specifically the expression of thermoTRPs associated with high heat (>42 °C) and low cold (<12 °C) temperatures, called Trpvl and Anktml respectively, should be examined in both the Gal l -/- and wt mice (Tominaga et al., 1998; Patapoutian et al., 2003; Story et al., 2003). A family of two-pore K + channel controls some of these thermoTRPs (Fink et al., 1998; Maingret et al., 2000). Gall-Ox is suggested to bind an unspecified K + channel (Dr. Horie, personal communication). The peripheral distribution 138 of receptors and thermo TRPs should be examined considering Gal 1 expression may also occur in Schwann and muscle cells in the periphery. In Chapter 5, the absence of Ga l l in Gal l -/- mice attenuated the restoration of function. A number of different factors that were not examined could lead to these observed changes. Since Gall-Ox increases the rate of neuronal regeneration in rats it seems likely that the regenerative rate would be decreased in Gal l -/- mice compared to Gal l wt mice. However examination of the rate of growth within the axotomized nerve was not undertaken. The differences in functional recovery could also be attributed to alterations in restoring functional synaptic connections. In uninjured adult Ga l l -/- mice both olfactory and DRG neurons are in the correct vicinity of their appropriate targets but they appear to have made inappropriate terminations. This suggests Gal l ' s involvement in either target finding or maintenance of the axon. In the periphery, muscles cells also express Ga l l (Watt et al., 2004). In vitro, Ga l l promotes olfactory neuron growth through cell adhesion mechanisms (Mahanthappa et al., 1994). It is unknown whether Ga l l expressed in target muscle of growing axons can facilitate axonal growth via attractant or adhesive mechanisms. Accordingly I can only demonstrate that functional recovery is attenuated in Gal l -/- mice but the underlying mechanism by which this happens remains unanswered. In Chapters 4 and 5, the 129P3/J Ga l l -/- was compared to the 129P3/J Gal l wt mouse strain for both functional and anatomical assessments. Gal 1 wt strain was obtained from a commercial supplier and used for both behavioral and anatomical comparisons whereas the Gal l -/- was obtained from Dr. Poirier. Even though the Ga l l -/- and wt mice are both inbred mice strains that reduce genetic variability, a limited amount of generational genetic change could occur. These possible genetic changes could lead to differences, other than those attributed to the Ga l l null mutation, between the G a l l -/- and Gal l wt mouse strains. However, the heritability of nociceptive response of inbred mouse strains is considered high compared to those of outbred mouse strains (Mogil et al., 1999b). In particular, thermal nociceptive traits of 129 mice have little variation (Mogil and Adhikari, 1999; Mogil et al., 1999a, b). When testing thermal nociception in mice, there is a greater variability between experimenters than between differing inbred and outbred mouse strains (Chesler et al., 2002a, b). Great care was taken to minimize testing variability by age 139 matching mice and having the same experimenter perform the behavioral tests (see methods Chapter 4 and 5). How does Gall promote axonal growth? This thesis examined neuronal Gal l expression, its putative involvement in regeneration and the effect of the absence of Gal l expression. These experiments do not address the putative growth-promoting mechanisms of Ga l l . Evidence from both in vitro and in vivo research suggests that Gal l can promote axonal growth through both extracellular and intracellular mechanisms. In light of the results presented in this thesis, the mechanisms by which this might occur are discussed below. Redox State Redox modulation by covalent modification of sulfhydryl groups on cysteine residues can regulate protein function (Lipton, 1999). Gal l is an example of the biological activity being dependant on the redox state. As previously stated, direct application of Gall-Ox promotes axonal growth in vitro and in vivo whereas Gall-Red does not promote neurite outgrowth (Horie et al., 1999). In the extracellular space of neurons or Schwann cells, Ga l l exists in both the reduced and oxidized forms but within the cell Ga l l is believed to exist mainly in the reduced form (Sango et al., 2004). The protein's redox environment is determined by equilibrium between oxidants, such as reactive oxygen species, and antioxidant mechanisms. Contributing to an oxidizing environment are reactive nitrogen intermediates produced by nitric oxide synthase (NOS). Modification of critical cysteine residues by reactive nitrogen intermediates has been shown to regulate various ion channels, G-proteins, growth factors, enzymes and transcription factors (reviewed in Stamler et al., 1997). NOS may also regulate Gall 's redox state. The expression of NOS increases after a peripheral nerve injury site and mediates thermal hypersensitivity (Zochodne et al., 1999; Levy et al., 2000). Gal l binding to macrophages regulates nitric oxide production by NOS and the nitric oxide may also promote neuronal hyperexcitability in neuropathic pain models of injury (Wiesenfeld-Hallin et al., 1993; Michaelis et al., 1995; Correa et al., 2003). Interestingly, Ga l l -/- mice have decreased responses to thermal noxious stimuli that may be, in part, related to changes in nitric oxide levels. The mechanisms regulating Gal l ' s redox state are not understood. We are gaining a greater insight into roles of both Gall-Ox and Gall-Red within and outside the cell. 140 Extracellular actions T H E EXTRA CELLULAR M A T R I X (ECM) The E C M plays an important role in successful axonal regeneration of sensory neurons in both peripheral and central nervous systems. Condroitin proteoglycan (CSPG), an E C M molecule, has specifically been shown to prevent D R G growth in the central nervous system (McKeon et al., 1991; Davies et al., 1997) and also limits successful regrowth after a peripheral axotomy (Zuo et al., 2002). The ability of axotomized CNS or PNS to successfully regrow increases when CSPGs are enzymatically reduced with chondroitinase A B C at the lesion site (McKeon et al., 1991; Moon et al., 2001; Bradbury et al., 2002; Morgenstern et al., 2002; Zuo et al., 2002; Yick et al., 2003). Recent in vitro evidence suggests that Ga l l red binds to CSPG, thus preventing its incorporation into the E C M (Moiseeva et al., 2003a). This implies that high G a l l expression reduces CSPG incorporation, creating an environment more permissive to growth. MACROPHAGES Macrophages are an important component of successful peripheral nerve regeneration. By removing myelin debris, modulating the Schwann cell proteases, promoting Schwann cell proliferation and providing trophic support of neurons the macrophages create a more permissive environment for peripheral regeneration (Fu and Gordon, 1997). For example, during the peripheral regeneration process macrophages secret the anti-inflammatory cytokine IL-10 and the trophic factor transforming growth factor - 6 serving to limit secondary damage while providing a permissive growth environment. After a CNS injury, peripheral macrophages enter they injury site where they can exacerbate the injury (Popovich and Hickey, 2001). This is demonstrated when peripheral macrophages are depleted, reducing the number that would normally enter the CNS, and thus reducing CNS tissue damage (Popovich et al., 1999). Simulating macrophages and/or microglia with either Zymosan or lysophophatidylcholine within the CNS leads to further tissue damage (Fitch and Silver, 1997b; Ousman and David, 2000; Popovich et al., 2002). These results suggest that peripheral activated macrophages support axonal peripheral regeneration whereas i f activation occurs within the CNS these cells inhibit repair and exacerbate the injury. This notion is further demonstrated using macrophage transplant techniques. Macrophages can reduce secondary tissue damage and promote limited axonal growth within the injured CNS 141 if these macrophages are cultured on degenerating peripheral myelin first and then transplanted into the injured CNS (Lazarov-Spiegler et al., 1996; Rapalino et al., 1998). This notion was illustrated when macrophages that were stimulated with either Zymosan injected directly into the D R G (Steinmetz et al., 2003) or by an intraperitoneal injection of Corynebacterium parvum (Lu and Richardson, 1991) led to increase neuronal growth. To find any putative factors that these peripherally activated macrophages might release, Yin et al (2003) injected Zymosan into the vitrius of the eye to simulate peripheral macrophages. Not only did they observe increased growth of injured CNS rentinal ganglion cells compared to saline injections but also they discovered that Zymosan stimulated macrophages in vitro led to the release of an unidentified 14-kDa protein that increased axonal growth. These results suggest that peripherally-primed macrophages release a specific factor that promotes axonal regrowth. Recently, Gallox has been shown to specifically bind peripheral macrophages in vitro (Horie et al., 2004). Although the macrophage receptor remains unidentified, this binding led to both kinase phosphorylation and the release of an unidentified factor that was larger than 10 kDa (Horie et al., 2004). The Gall/macrophage-conditioned media stimulated both axonal growth and Schwann cell migration. Both Zymosan and Gal l stimulate peripheral macrophages to promote axonal growth in vitro and in vivo (Horie et al., 1999; Yin et al., 2003; Horie et al., 2004). It would be interesting to know if the 14 kDa molecule released from Zymosan stimulated macrophages is the same (or similar) molecule released from Gal l stimulated macrophages. Also peripherally derived macrophages could be stimulated with Gal l and then transplanted into the injured CNS which may reduce secondary damage and/or induce regeneration. Another approach would be through the use of the well-established peripheral nerve graft paradigm. Specifically, degenerated peripheral nerve grafts from either Gal l -/- or wt mice could be transplanted into either CNS tissue (rubrospinal tract) or onto another peripheral nerve of Gal l wt mice to assess the growth permissiveness of Gal l - /- tissue. Currently, the search for the Ga l l macrophage receptor and the unidentified compound(s) released from Gall-stimulated macrophages is currently underway (Dr. Horie, personal communication). Once these two pieces of information are revealed, further 142 investigation into the mechanisms into Ga l l macrophage activation can be explored and exploited for possible CNS repair. M I C R O G L I A The full complement of cells that bind Gal l is unknown. Specifically, no in vitro Ga l l binding assays have been undertaken with CNS cells. Astrocytes and olfactory neurons are the only known cell within the CNS to bind Gall-Red (Sasaki et al., 2004). Once bound to astrocytes, Ga l l causes B D N F release and astrocyte differentiation (Sasaki et al., 2004). In chapters 2 and 5 of thesis, I demonstrate the increased mRNA expression in the axotomized facial nucleus and the decrease Gal l expression in the axotomized rubrospinal nucleus. The glial response is also quite different in these two injury paradigms: after a facial nerve axotomy, there is an increase both astrocyte and microglial reactivity around the neuronal cell bodies (Graeber et al., 1988; Tetzlaff et al., 1988; Barron et al., 1990; Tseng et al., 1996). After a rubrospinal tract injury, a muted or non-existent glial response occurs around the red nucleus (Tseng et al., 1996; Liu et al., 2003). Although the gliotic response following injury creates an inhibitory barrier to growing axons, these inflammatory reactive cells also can produce trophic factors that support growth (Streit et al., 1998; Fitch and Silver, 1999). For example, a significant glial response occurs around the facial nucleus following a facial nerve axotomy. These reactive glial cells are thought to be growth-promoting since they are able to release trophic factors around the cell body (Ridet et al., 1997; Batchelor et al., 2002). Since Gal l can be released from cell bodies (Sango et al., 2004) and I demonstrated increased Gal l expression in both facial and spinal motoneuronal cell bodies following axotomy, it seems plausible that Gal l released from neurons may beneficially stimulate glial cells around the neuronal cell bodies. Therefore the decease in Gal 1 expression observed in the red nucleus after axotomy may contribute to the observed regenerative failure by not initiating a glial response. Ga l l infusions into the red nucleus after a rubrospinal tract axotomy might further aid in determining whether this postulated hypothesis is accurate. Intracellular actions The early reports that Ga l l promoted axonal growth in vitro and in vivo focused on the exogenous application of this lectin. In chapters 2-5 of this thesis, I have reported either mRNA or protein expression within the neuronal nucleus or cytoplasm. These observations 143 of the intrinsic Gal l expression are not new. The first paper describing Gal l expression in the DRG demonstrated nuclear and/or cytoplasmic Gal 1 immunoreactivity in the nervous system was in 1986 (Dodd and Jessell, 1986). In recent reports, only application of Gall-Ox at the injury site was examined even though neurons express G a l l . Although the exact role this lectin plays in the uninjured neuron remains unclear, there are indications that it serves important basic neuronal functions. A greater understanding of the intracellular processes in which Gal l is involved will lead to some indication of its overall role both uninjured and axotomized neurons. S U R V I V A L OF M O T O R N E U R O N BINDING Gal l has been identified as a component of the Survival of Motor Neuron complex (Park et al.,.2001). More specifically, Gall-Red binds to Gemin4 within this 12-protein structure. This Survival of Motor Neuron complex, found within both the nuclear and cytoplasmic compartments of motoneurons, associates indirectly or directly with RNA, RNA polymerase II and zinc-finger binding proteins and is required for cell viability (Gangwani et al., 2001; Pellizzoni et al., 2001; Frugier et al., 2002). CVe-mediated deletion of the S M A gene (which disrupts the Survival of Motor Neuron complex) instigated within a Cre-LoxP SMA mutant mouse leads to death of the mouse four weeks later due to severe motoneuronal deficits. Specifically, there was a 73% reduction of motor axons and a 23% reduction in cell body size with neurofilaments accumulating in synaptic terminals (Frugier et al., 2000; Cifuentes-Diaz et al., 2001). Conversely, over-expression of S M N within motoneurons in vitro enhances neuronal outgrowth (Rossoll et al., 2003). Gal l binding to the S M N complex is not required for mouse survival since the Gal l -/- mice are completely viable. This may be in part due to the ability of galectin-3 to also bind Gemin4 (Park et al., 2001). The interaction of Gal l with neuronal survival and growth proteins within the neuron may contribute to neuronal maintenance and growth. In the fatal neurodegenerative disease familial amyotrophic lateral sclerosis (ALS), abnormal accumulations of neurofilaments within axons and perikarya of motor neurons occur. These neurofilamentous lesions have a high accumulation of Gal l (Kato et al., 2001). The mechanisms and potential significance of the close association between the 144 neurofilaments and G a l l remain uncertain, but evidence from in vitro and pathological conditions offers some insight. SIGNALING C A S C A D E S Within the neuron, the regenerative machinery required to maintain the active extension of the axon and axonal growth cone (e.g. cytoskeletal proteins), as well as factors which are involved in maintaining neuronal phenotype (such as neurotrophins and their receptors) are required for successful regeneration. Included in the first category is GAP-43, which acts as a link between membrane rafts and the actin cytoskeleton, and mediates growth cone motility (Frey et al., 2000; Laux et al., 2000). GAP-43 is massively and persistently upregulated in spinal motoneurons, but is less robustly and only transiently upregulated in cervically axotomized RSNs (Fernandes et al., 1999). In the second category are neurotrophins and their receptors such as B D N F and trkB, respectively. Receptor binding by the appropriate neurotrophin leads to various signal cascades. Depending upon whether the Raf - l /MEK/ERK or PI3K signaling cascade is activated the outcome can effect neuronal growth and/or survival respectively (Kaplan and Miller, 2000; Patapoutian and Reichardt, 2001). Within cells, Ga l l may act in an intermediate role by altering second messenger cascades of this neurotrophin signaling. Gall-Red recently has been shown to stabilize the GTPase H-ras to non-raft microdomains resulting in stabilization of the signaling domains of the second messenger system (Prior et al., 2003). This stabilization by Gall-Red increases the potential for the R a f - l / M E K / E R K activation, and not the PI3K signaling cascade (Paz et al., 2001; Elad-Sfadia et al., 2002). The R a f - l / M E K / E R K signaling pathway is involved in axon extension (Markus et al., 2002). Thus Gal l may facilitate intracellular signaling cascades and alter the growth capacity of neurons. A C T I N C Y T O S K E L E T O N Cellular motility and growth require the cytoskeletal rearrangement of actin. In astrocytes, motility is dependant on F-actin (Abd-el-Basset et al., 1991). In astrocytic tumours (glioblastoma), for example, malignancy is a direct result of the cell's ability to move and diffusely infiltrate normal brain tissue. In these glioblastomas, malignancy is directly associated with Ga l l expression (Kopitz et al., 1998; Yamaoka et al., 2000; Camby et al., 2001). In vitro, Ga l l antisense injuected into gliomas results in arrested growth (Yamaoka et 145 al., 2000). After G a l l application to glioblastoma cells in vitro, at relatively low concentrations (0.1 ng/ml), there is a 30% increase in cell motility and F-actin polymerization, and up to a 75% increase in RhoA expression. Unfortunately, the authors of this study did not report whether Gal 1 induced changes in RhoA activation. The regulation of actin polymerization/depolymerizafion involves the small GTPases of the Rho family (Maekawa et al., 1999). In growing neurons, F-actin polymerization is required for neurite extension and outgrowth (Mackay et al., 1995). Changes in RhoA activation leads to changes in a neuron's outgrowth ability in vitro and in vivo (Lehmann et al., 1999; Borisoff et al., 2003). Although no published study exists demonstrating Gall-induced changes in neuronal RhoA expression, many of the original studies examining signaling to the cytoskeleton were first carried out in fibroblasts and later repeated in cultured neurons. Since G a l l promotes neurite outgrowth and Schwann cell migration in vitro and in vivo (Mahanthappa et al., 1994; Horie et al., 1999; Fukaya et al., 2003; Horie et al., 2003), and high expression is associated with growing neurons (Chapters 2, 3 and 5) it might be fruitful to examine whether G a l l also has direct effects on either RhoA expression or activation within neurons. Schwann cells have a highly regulated mechanism to establish myelin sheaths around axons in either development or regeneration. These cells express laminin receptors, one of which, a 6p\ integrin, is directly linked to F-actin. Function blocking p\ integrin receptor antibodies prevents both proper Schwann cell myelination and basal laminal formation. The p\ integrin null mutant mouse is embryonically non-viable but the conditional mutant mouse demonstrates that this receptor is required for proper myelination. In vitro, Gal l binds to the integrin of smooth muscle cells and increases cell proliferation and migration (Moiseeva et al., 2000; Moiseeva et al., 2003b). In Schwann cells, (3, integrin activation causes dimerization of the receptor that then leads to the autophosphorylation of the focal adhesion kinase (FAK), promoting myelination and migration (Siciliano et al., 1996; Chen et al., 2000; Taylor et al., 2003). In both astrocytes and Schwann cells, Gal l promotes changes in F-actin to increase migration. In the CNS, astrocyte migration or movement is usually associated with malignancy whereas in the PNS, Schwann cell migration is viewed as beneficial for peripheral axonal repair. 146 Consequences of an enhanced neuronal growth mode Axonal Regeneration As outlined in the General Introduction, there are many obstacles the damaged neuron must overcome to achieve successful regeneration in both peripheral can central nervous systems. After peripheral nerve injury, Ga l l may increase regenerative success by facilitating some of these repair mechanisms. After injury, neurons have a reduced ability to regulate membrane permeability leading to in an increased Gal l release into the extracellular space (Povlishock and Pettus, 1996). Once in the extracellular space, Gal l promotes Schwann cell migration and macrophage release of neuronal growth-inducing factors to promote axonal repair and functional recovery (Horie et al., 1999; Horie et al., 2004; Sango et al., 2004). Within the injured neuron, G a l l expression increases possibly leading to more G a l l release and increased G a l l within the cell body. Neuronal G a l l is involved in signal cascade modulation, neuronal survival protein binding and possibly actin cytoskeleton interactions, all of which contribute to an increased capability of injured neurons to survive and grow both extra and intracellular processes. Obviously these cellular processes are not dependant on G a l l since injured neurons in Ga l l -/- mice (Chapter 5) are able to make functional connections (albeit at a slower rate). This illustrates that Gal l is involved but is not essential for successful neuronal repair. Neuropathic pain Partial nerve injury is associated with hyperalgesia (increased pain sensitivity), allodynia (pain from non-noxious stimuli), spontaneous and general ongoing pain. These conditions are due in part to axotomy-induced changes within the peripheral neuron. Large-diameter axotomized afferents begin to discharge spontaneously and increase their expression of BDNF (Michael et al., 1999; Boucher et al., 2000; Liu et al., 2000a; Liu et al., 2000b). This increase in spontaneous activity is thought to sensitize spared fibers by reducing the dorsal horn neurons threshold for activation in a process called central sensitization (Woolf, 2000). Furthermore, the spared fibers have greater access to target-derived trophic factors, such as NGF, due to the loss of axonal transport in injured axons. NGF has been shown to regulate behavioural sensitivity to pain (Koltzenburg et al., 1999). These injury-induced changes increase the expression of the excitatory neuropeptide substance P as well as B D N F 147 (Noguchi et al., 1995; Michael et al., 1997; Pezet et al., 2002a). In the dorsal horn, BDNF increases hyperalgesia, whereas reduction in BDNF attenuates hyperalgesia in a neuropathic pain model (Groth and Aanonsen, 2002; Pezet et al., 2002b). In Chapter 3 of this thesis, I demonstrated that large-diameter fibers also increase their Ga l l expression in the DRG and dorsal horn after axotomy. In a neuropathic pain rat model, a similar change in Gal l protein distribution is observed within the dorsal horn (Imbe et al., 2003). At these sensory neuron terminals, Ga l l release can then occur (Sango et al., 2004). Once released, Gal l can bind to astrocytes causing these glial cells to release B D N F into the extracellular environment, thereby further increasing hyperalgesia (Sasaki et al., 2004). Interestingly, the infusion of Gal l function blocking antibodies to the intrathecal space around the spinal cord in a neuropathic pain model reduces mechanical allodynia as well as substance P receptor expression (NK-1) in the dorsal horn (Imbe et al., 2003). In Gal -/- mice, I demonstrated reduced thermal nociceptive responses when compared to either G a l l wt or CD-I mice strains. The increased expression of Ga l l in neurons that have increased activity may contribute to axotomy-induced pain. Further experiments to determine Gal l ' s role in neuropathic pain seem warranted. Using established mouse models for neuropathic pain, differences in thermal allodynia and hyperalgesia between G a l l wt and -/- mice should be examined. It would be useful to test whether exogenous G a l l application in either Ga l l wt or -/- mice increases thermal nociception in either naive mice or in mice with neuropathic pain. Furthermore, differences in the expression of substance P, NK-1 and BDNF within the dorsal horn in these mice (Gall wt and -/-) in the uninjured and neuropathic pain model should be investigated. Axotomy induced sprouting and plasticity In the E C M , there are other molecules that limit axonal plasticity. For example, the E C M molecule that inhibits axonal and neurite outgrowth, CSPG, also inhibits plasticity in the spinal cord and ocular dominance columns (McKeon et al., 1991; Davies et al., 1997; Bradbury et al., 2002; Pizzorusso et al., 2002). When the CSPGs are enzymatically cleaved using chondroitinase A B C , plasticity terminal sprouting increases in the ocular dominance columns (Pizzorusso et al., 2002). After injury to ascending sensory fibers in the dorsal column, chondroitinase A B C increases plasticity and not growth, to increase some sensory 148 function (Bradbury et al., 2002). Recent in vitro evidence suggests that Gall-Red binds to CSPG, thus preventing its incorporation into the E C M (Moiseeva et al., 2003a). It is interesting to speculate whether this process occurs within the nervous system as well. If this does occur after injury then high G a l l expression could potentially attenuate CSPG incorporation in the E C M , creating an environment more permissive to growth. The maintenance and refinement of the hypersensitivity associated with acute and chronic pain is due in part to neuronal plasticity. In particular nociceptive stimuli instigate a variety of intracellular signaling cascades including the E R K 1/2 pathway (Aley et al., 2001; Dai et al., 2002). ERKs have been implicated in neuronal plasticity associated with learning and memory as well as plasticity associated with nociceptor sensitization (Bailey et al., 1997; Martin et al., 1997; Aley et al., 2001). The hyperalgesia associated with ERK1/2 activation depends on cytoskeletal changes (Dina et al., 2003). Accordingly, modifications to cytoskeletal signaling may modify nociceptive neuronal activity and contribute to chronic pain. After partial peripheral nerve injuries, activation of injury nociceptive pathways leads to central sensitization of intact pathways in turn, leading to long-lasting changes in a sensory neuron's receptive field (expansion or contraction). These changes may also be elicited in the absence of injury by the increased activity of nociceptive afferents (McMahon and Wall, 1984; Cook et al., 1987). This synaptic re-organization requires molecular links to the neuronal cytoskeleton (Dina et al., 2003). Intracellular Gal l interacts with both signaling and cytoskeletal elements. As previously described (see SIGNALING CASCADES), Ga l l increases E R K activation over the PI3K cascades (Paz et al., 2001; Elad-Sfadia et al., 2002) and Gal l expression can lead to cytoskeletal re-arrangement (Maekawa et al., 1999). This may occur with Rho GTPases that also have a prominent role in affecting axonal and dendritic structure (reviewed in Hall and Nobes, 2000). Gal l has been shown to effect F-actin polymerization in astrocytes and has been associated with neurofilaments in the lesions of A L S patients. Furthermore, the involvement of Ga l l in R a f - l / M E K / E R K signaling cascades suggests Gal l ' s involvement in cytoskeletal re-arrangements and outgrowth. In neuropathic pain, in which synaptic remodeling occurs, function blocking antibodies to Gal l reduces mechanical hyperalgesia (Imbe et al., 2003). Also, sensory neurons, specifically D R G and olfactory 149 system as well as spinal motoneurons, are all neuronal systems with activity-dependent induced synaptic plasticity (Wong and Ghosh, 2002). In these systems, we observe inappropriate neuronal termination in Gal l -/- mice when compared to wt mice (Chapter 4)(Puche et al., 1996). It is uncertain whether these changes are a result of inappropriate outgrowth during development or changes in neuronal maintenance in the adult. After a facial nerve injury there is a delay in restoring functional recovery in Ga l l -/- mice compared to Gal l wt mice. This delay could be due to a reduced initiation rate as suggested by Gal l function blocking antibodies in the rat (Horie et al., 1999; Fukaya et al., 2003), or by a change in the rate or success of synapse formation at the target. Furthermore, examination of Gall-Ox-induced growth of axotomized sciatic nerves shows that Gal l not only increases growth but also the branching of injured neurons (Horie et al., 1999; Fukaya et al., 2003). In Chapter 5, Gal l -Ox application to rhizotomized DRGs neurons did not stimulate robust regeneration into the CNS but promoted a limited growth both in the number and distance of injured neurons (Chapter 3). These Gall-Ox effects on both the limited CNS growth after rhizotomy and increased branching after peripheral nerve lesion could be attributed to an increase sprouting response. A l l of this circumstantial evidence suggests that endogenously expressed G a l l is involved in the neuronal maintenance as well as the remodeling of functional connections in pathological conditions. However, more direct evidence clearly is required to substantiate this hypothesis. Further assessment of the differences between the Gal 1 -/- and wt mice will provide insight into Gal l ' s involvement in axonal sprouting and/or growth. In particular, interactions between Gal l and the cytoskeleton could be examined using cultured cells from Gal l -/- and wt animals. Changes in CSPG incorporation in the E C M , of both Gal l -/- and wt mice should be examined, both before and after injury. Other Future directions Galectin-3 and 8 This thesis focused exclusively on Gal l but there are other galectins in the CNS that bear further investigation. These are Galectin-3 and 8. As discussed in the Chapter 1 of this thesis, each of these galectins belongs to a separate subfamily based on structural similarities. Of this group, GaI3 is the second most studied galectin due to its early discovery and immune 150 system involvement. In the CNS, Gal3 immunoreactivity occurs in a subset of D R G small diameter neurons that slightly overlaps with Gal l immunoreactivity (Regan et al., 1986). Gal3 appears to be expressed and regulated by TrkA expressing sensory neurons since NGF maintains Gal3 expression within D R G in vitro and Gal3 expression is observed in laminae I and II (Cameron et al., 1997; Pesheva et al., 2000). In vitro, Gal3 also promotes adhesion and growth of D R G and cerebellar neurons (Pesheva et al., 1998a; Mahoney et al., 2000), and after a partial peripheral nerve lesion Gal3 immunoreactivity increases in the dorsal horn (Cameron et al., 1997). Within the CNS, Gal3 expression occurs in cultured microglia (Pesheva et al., 1998b). After a facial nerve axotomy, Gal3 expression appears to increase within the motoneurons but not in microglia around the somata (Walther et al., 2000) suggesting that like G a l l , Gal3 also may be involved in neuronal maintenance and possibly repair. These injury-induced changes of Gal3 protein in motoneurons remains to be properly assessed. Interestingly, the Gal3 -/- and the double null mutant G a l l - / - Gal3-/- mice are viable suggesting that neither of these lectins are essential for development but they would be useful for examining both neuronal injury and neuropathic pain (Colnot et al., 1998). The recently discovered galectin, Gal8, is also expressed in CNS tissue, but the precise location of such expression is not known (Hadari et al., 1995). Although Gal8 is not in the same family as G a l l , the two share quite a few similarities. Gal8 has four cysteine residues that could possibly alter redox states, as has been observed in Gal l (Hadari et al., 1995). Furthermore, elevated levels of Gal8 expression also occur in malignant astrocytomas (Colnot et al., 1998). Gal8 affects the E R K 1/2 signal cascade that then promotes cellular motility via F-actin (Levy et al., 2003). These data suggest that like G a l l , Gal8 may also be involved in signaling and outgrowth mechanisms. Only recently has the significance of G a l l expression within the nervous system begun to be understood. With evidence now indicating that both Gal3 and 8 also may serve important roles in neuronal development and in neuropathological states, further investigation appears to be warranted. 151 Overall Significance Although neuronal Galectin (Gal)-l protein expression has been known for a considerable amount of time, only recently has the role of this protein in both sensory and spinal motor nerve regeneration been investigated. To date, only the developmental and adult expression of Ga l l mRNA and protein has been reported in cranial nerves, primary sensory afferents and spinal motor nerves and that exogenous G a l l ox application increases neuronal regeneration. The studies in this thesis demonstrate four findings. First I show increases in Gal l mRNA and protein expression in DRGs and spinal motor neurons after injury. Second, this is the first report of Gal l expression in the red nucleus, and in contrast to injured spinal motor neurons, this expression decreases after injury. Third, this is the first report of a neurotrophic factor (BDNF), effecting Ga l l mRNA expression within any neuronal population. Fourth, the absence of Gal l in Gal l -/- mice leads to functional and anatomical nociceptive deficits. Collectively, these findings further demonstrate the importance of Gal l within the nervous system and as a result pose many more questions with each finding. Concluding remarks We are only beginning to elucidate galectin neurobiology. The potential involvement of galectin proteins in such wide-ranging conditions such as A L S , neuropathic pain, axonal injury and possibly synaptic plasticity further underscores the wide-ranging role galectins may have within the nervous system. 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