Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Minocycline as a neuroprotective agent following spinal cord injury Stirling, David Paul 2005

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

Item Metadata

Download

Media
831-ubc_2005-105755.pdf [ 15.77MB ]
Metadata
JSON: 831-1.0092356.json
JSON-LD: 831-1.0092356-ld.json
RDF/XML (Pretty): 831-1.0092356-rdf.xml
RDF/JSON: 831-1.0092356-rdf.json
Turtle: 831-1.0092356-turtle.txt
N-Triples: 831-1.0092356-rdf-ntriples.txt
Original Record: 831-1.0092356-source.json
Full Text
831-1.0092356-fulltext.txt
Citation
831-1.0092356.ris

Full Text

MINOCYCLINE AS A NEUROPROTECTIVE AGENT FOLLOWING SPINAL CORD INJURY by DAVID P A U L STIRLING B.Sc, The University of British Columbia, 2000 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY In THE F A C U L T Y OF G R A D U A T E STUDIES (ZOOLOGY) THE UNIVERSITY OF BRITISH C O L U M B I A August 2005 © David PaulStirling, 2005 ABSTRACT The mechanical impact of a spinal cord injury (SCI) triggers a cascade of secondary damage that progressively destroys an increasing amount of tissue adjacent to the primary lesion over a period of hours to weeks. The pathophysiological changes lead to necrotic, as wel l as apoptotic death, inflammation, demyelination, and axonal damage. In their wake, a fluid-filled cavity often forms leaving a small r im of spared white matter. Whi le the initial mechanically destructive events cannot be reversed, the cellular inflammatory reactions occur over several hours to weeks, a timeframe during which therapeutic intervention may be achieved. This thesis examines the efficacy of minocycline, a 2nd generation tetracycline-derivative, in attenuating secondary degeneration after SCI. The results of the first series of experiments indicate that the peak of apoptosis within the ascending sensory tracts (AST) following dorsal column transection in rats occurs at one to two weeks after injury. Numerous apoptotic profiles are located within both the proximal and distal segments of the A S T after injury. Although oligodendrocytes undergo apoptosis as wel l as microglia, the latter are the main cell type to undergo apoptosis in this model. Importantly, minocycline administration delayed up to 30 minutes after injury, significantly reduces apoptosis, prevents corticospinal tract axonal dieback, diminishes lesion size and promotes functional recovery following a dorsal column transection. The results from the second series of experiments show that minocycline treatment reduces levels of m R N A and activation of p38 mitogen-activated protein kinase ( M A P K ) after SCI. In addition, p38 M A P K increases early after SCI and colocalizes with neutrophils, and microglia/macrophages. The final series of experiments assessed the efficacy of minocycline or the p38 M A P K inhibitor SB203580 in promoting neurological recovery uti l izing a contusion model of SCI in rats to more closely mimic the injury seen in humans. The results from these i i experiments indicate that delayed minocycline treatment (1 hour after injury) administered intravenously promotes tissue preservation and functional recovery after SCI. Collectively, these findings suggest that several aspects of the secondary degeneration that occurs after SCI can be prevented by minocycline application, and supports its use as a potential neuroprotective treatment following human SCI. i i i T A B L E O F C O N T E N T S A B S T R A C T i i T A B L E OF C O N T E N T S iv L IST OF T A B L E S x L IST OF F I G U R E S x i L IST OF A B B R E V I A T I O N S x i i i A C K N O W L E D G E M E N T S xv i C O - A U T H O R S H I P S T A T E M E N T xv i i C H A P T E R 1. G E N E R A L INTRODUCTION 1 Rationale 2 Pathophysiology of SCI 3 Primary injury 3 Secondary injury 3 Vascular disturbances, ischemia and reperfusion 4 Free radicals and l ipid peroxidation 6 Excitotoxicity 7 Inflammatory response to SCI 7 Apoptosis 10 Apoptosis after SCI 11 Neuroprotective strategies for mitigating secondary damage after acute SCI 14 Corticosteroids 14 Gangliosides 15 Opiate antagonists 16 iv Glutamate receptor, calcium, and sodium channel antagonists 17 Potential pharmacological agents for acute SCI 18 Minocycl ine 19 Minocycl ine improves outcome of SCI 22 Mechanisms of minocycline's anti-inflammatory actions 23 Mechanisms of minocycline's survival promoting actions 28 P38 M A P K inhibition after minocycline treatment 30 Chapter overviews and hypotheses 30 C H A P T E R 2. T E M P O R A L AND SPATIAL EXPRESSION O F APOPTOSIS F O L L O W I N G C E R V I C A L SPINAL C O R D INJURY AND R E D U C T I O N F O L L O W I N G M I N O C Y C L I N E T R E A T M E N T 34 S U M M A R Y 35 I N T R O D U C T I O N :. 36 M A T E R I A L S A N D M E T H O D S 39 Cervical spinal cord dorsal column transection and corticospinal tract tracing 39 Minocycl ine administration 40 Preparation of tissue 40 Immunohistochemistry 42 Ce l l counts... 43 Statistics 43 R E S U L T S 43 Spatial and temporal expression of active caspase-3-positive profiles within both proximal and degenerating white matter after C7 dorsal column transection 43 v Active caspase-3-mediated apoptosis of oligodendrocytes and microglia within both proximal and degenerating white matter after C7 dorsal column transection 45 Effect of minocycline treatment on active caspase-3-mediated oligodendrocyte death after SCI 49 D I S C U S S I O N 50 C H A P T E R 3. M I N O C Y C L I N E T R E A T M E N T R E D U C E S M I C R O G L I A L / M A C R O P H A G E A C T I V A T I O N , CORTICOSPINAL A X O N A L D I E B A C K , L E S I O N SIZE, AND IMPROVES F U N C T I O N A L O U T C O M E A F T E R SCI 55 S U M M A R Y 56 I N T R O D U C T I O N 57 M A T E R I A L S A N D M E T H O D S 59 Spinal cord dorsal column transection and corticospinal tract tracing 59 Minocycl ine administration 60 Preparation of tissue 60 Immunohi stochemistry 61 Microgl ia l / macrophage activation/ recruitment 61 C S T dieback 62 Lesion area 62 Footprint analysis 63 Statistics 64 R E S U L T S ...64 Effect of minocycline treatment on E D I density seven days after SCI 64 Effect of minocycline treatment on C S T dieback after SCI 65 vi Effect of minocycline treatment on lesion size after SCI 67 Improved functional outcome following minocycline treatment 67 D I S C U S S I O N 69 C H A P T E R 4. M I N O C Y C L I N E T R E A T M E N T R E D U C E S P38 M A P K EXPRESSION AND M I C R O G L I A L / M A C R O P H A G E A C T I V A T I O N F O L L O W I N G SCI 75 S U M M A R Y 76 I N T R O D U C T I O N 78 M A T E R I A L S A N D M E T H O D S 82 Spinal cord dorsal column transection 82 Contusion model 83 Minocycl ine administration 84 Preparation of tissue 84 R T - P C R and Microarray 84 Western blot 87 Immunohistochemistry 88 R E S U L T S 88 Minocycl ine treatment reduces p38 M A P K m R N A expression 14 days after SCI 88 Minocycl ine treatment reduces p38 M A P K expression following SCI 89 Spatial and temporal expression of active p38 M A P K following contusion SCI 92 Minocycl ine reduces active p38 M A P K and CD1 l b in microglia/macrophages following dorsal column transection 94 D I S C U S S I O N 94 v i i C H A P T E R 5. INTRAVENOUS A P P L I C A T I O N O F M I N O C Y C L I N E IS N E U R O P R O T E C T I V E F O L L O W I N G SPINAL C O R D CONTUSION IN A D U L T RATS 105 S U M M A R Y 106 I N T R O D U C T I O N 107 M A T E R I A L S A N D M E T H O D S 110 Animal care 110 Contusion model 110 Intravenous drug delivery system I l l Minocycl ine dosage and delivery I l l Intrathecal administration of SB203580-HCL 112 Preparation of tissue 112 Residual white and grey matter 113 Behavioral analysis 113 Statistics 114 R E S U L T S 114 Delayed (1 hour) intravenous delivery of minocycline improves hindlimb function.... 114 Delayed (1 hour) intravenous delivery of minocycline improves fine aspects of locomotion following contusion SCI 118 Delayed intravenous application of minocycline increases residual sparing of both white and grey matter fol lowing SCI 122 D I S C U S S I O N 122 v i i i C H A P T E R 6. G E N E R A L DISCUSSION AND F U T U R E DIRECTIONS 129 Introduction 130 Gl ia l apoptosis following SCI 131 Extracellular mechanisms of oligodendrocyte apoptosis after SCI 133 Relevance of oligodendrocyte apoptosis 135 Minocycl ine as an anti-apoptotic agent 136 Microglia/macrophage response to SCI 137 Minocycl ine's anti-inflammatory properties 140 P38 M A P K , a putative target of minocycline's mode of action 143 Minocycl ine treatment of SCI 145 Conclusions 146 R E F E R E N C E S 148 i x L I S T O F T A B L E S Table 5.1 Mean force and displacement for all groups of animals 115 Table 5.2 Basso, Beattie, and Bresnahan (BBB) Locomotor Rating Scale 117 Table 5.3 B B B Subscoring Scale 120 x LIST OF FIGURES Figure 1.1 Pathophysiology of spinal cord injury 5 Figure 1.2 Inflammation after SCI 9 Figure 1.3 Apoptotic cell death pathways ,. 12 Figure 1.4 Structure o f tetracyclines 20 Figure 2.1 A schematic diagram of the dorsal column transection (DCT) model 41 Figure 2.2 Act ive caspase-3 positive profiles within both proximal and distal segments of the C S T and A S T after SCI 46 Figure 2.3 Spatial and temporal expression of active caspase-3 positive profiles following a C7 dorsal column transection 47 Figure 2.4 Cellular localization of active caspase-3 profiles after SCI 48 Figure 2.5 Minocycl ine treatment inhibits dorsal column transection-induced glial cell death within the distal and proximal A S T 51 Figure 3.1 Minocycl ine treatment reduces ED1(+) (microglial/macrophage) density 7 days postinjury 66 Figure 3.2 Minocycl ine treatment reduces C S T dieback and lesion size at both 7 & 14 days post-lesion 68 Figure 3.3 Minocycl ine treatment improves inter-limb coordination and reduces hindlimb angle of rotation after SCI 70 Figure 3.4 Minocycl ine treatment reduces hindlimb toe spread at 7 days post-injury 71 Figure 4.1 Minocycl ine reduces p38 M A P K m R N A after SCI 90 Figure 4.2 Western blot analysis of active p38 M A P K and total p38 M A P K protein isolated from rat spinal cord 91 Figure 4.3 Colocalization of active p38 M A P K and microglia/macrophages following x i contusion injury to the spinal cord 93 Figure 4.4 Differential staining of active p38 M A P K within immune cells at the lesion site at 3hr, 24hr and 15 days following a moderate contusion injury 95 Figure 4.5 Co-localization of active p38 M A P K within microglia/macrophages following dorsal column transection 96 Figure 4.6 Minocycl ine treatment reduces both CD1 l b and active p38 M A P K (low magnification images) 97 Figure 4.7 Minocycl ine treatment reduces both CD1 l b and active p38 M A P K (high magnification confocal images) 98 Figure 5.1 Examples of individual rat force curves derived from impact data 116 Figure 5.2 Delayed intravenous application of minocycline improves hindlimb function after SCI 119 Figure 5.3 Delayed intravenous application of minocycline improves fine aspects of hindlimb function after SCI 121 Figure 5.4 Delayed intravenous application of minocycline promotes preservation of both white and grey matter fol lowing contusion SCI 123 Figure 6.1 Apoptotic cell death pathways and intervention after minocycline application 138 Figure 6.2 SCI-induced inflammatory response and intervention following minocycline treatment 142 x i i ABBREVIATIONS 33P-dCTP Phosphorus isotope 33 labeled deoxycytosine-triphosphate A IF apoptosis inducing factor A L S amyotrophic lateral sclerosis A M P A alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid A P C adenomatous polyposis coli Apaf-1 apoptotic protease activating factor A S I A American spinal injury association A S T ascending sensory tracts A T F - 2 activating transcription factor-2 A T P adenosine triphosphate B B B Basso, Beattie, and Bresnahan locomotor rating scale Bc l -2 B-cel l CLL/ lymphoma 2 B D A biotin dextran amine B D N F brain derived-neurotrophic factor C A D caspase-activated deoxyribonuclease C C R 6 chemokine receptor six C D 11 b cluster of differentiation-11 -beta C N S central nervous system C O X cyclooxygenase C S T corticospinal tract C X C R 3 chemokine ( C - X - C motif) receptor 3 D I A B L O direct inhibitor of apoptosis-binding protein with low pi D N A deoxyribonucleic acid x i i i DNase deoxyribonuclease E A E experimental allergic encephalomyelitis G F A P glial fibrillary associated protein G M - 1 ganglioside mono-1 H I V human deficiency virus LAP inhibitor o f apoptosis protein I C A D inhibitor of caspase-activated deoxyribonuclease I C E interleukin-converting enzyme LFNy interferon-gamma IL interleukin iNOS inducible nitric oxide synthase LP intraperitoneal FV intravenous L P S lipopolysaccharide M A P K mitogen-activated protein kinase M A P K A P K 2 mitogen-activated protein kinase-activated protein kinase 2 M L N O minocycline MLP-1 a macrophage inhibitory protein-1 M M P matrix metalloproteinases M P T P 1-methyl-4-phenyl-l ,2,3,6-tetrahydropyridine N A S C I S national acute spinal cord injury study N G F nerve growth factor N M D A N-methyl-D-aspartate P2X7 purinergic receptor 2X7 xiv P B S phosphate-buffered saline P C R polymerase chain reaction P E A 15 Phosphoprotein enriched in astrocytes P G E prostaglandin P L A phospholipase A P N S peripheral nervous system R A N T E S regulated upon activation, normal thymus cell expressed and presumably secreted R N A ribonucleic acid R T - P C R reverse-transcriptase polymerase chain reaction SCI spinal cord injury S D S - P A G E sodium dodecyl sulphate polyacrylamide gel electrophoresis Smac second mitochondria-derived activator of caspases T G F transforming growth factor Th l /2 helper thymus cell-1/2 T N F tumor necrosis factor T T B S tris-buffered saline with Tween-20 T T X tetrodotoxin T U N E L terminal deoxynucleotidyl transferase dUTP nick-end labeling X I A P X- l inked inhibitor of apoptosis xv A C K N O W L E D G E M E N T S I would like to thank my parents for their inspiration throughout my life, their unconditional love, and support as I strive to achieve my goals. I thank my supervisor, Dr. Wolfram Tetzlaff, for his excellent guidance and support throughout this journey. I would also like to thank the members of my supervisory committee, Dr. Matt Ramer, Dr. John Steeves, Dr. Lynn Raymond and Dr. Jane Roskams for their continued support and helpful suggestions. I am also indepted to Dr. Chris McBr ide for his advice, training and for always finding the time to review and edit my work. I am also very grateful for the help, support and friendship so generously offered from my labmates: Carmen Chan, Kourosh Khodarahmi, Br ian Kwon , Clarrie Lam, Rene Lane, Jie L iu , Lowel l McPha i l , Loren Oschipok, Ward Plunet, Egidio Spinell i and Darren Sutherland. I would also like to thank the members of Dr. Steeves' and Dr. Ramer's lab for their encouragement and friendship. Most ly I dedicate this thesis to my darling wife Tiffany for all her patience and love through the "ups and downs" of this journey and to my precious daughter M i a . I thank both of you for showing me what is truly important in life. xv i C O - A U T H O R S H I P S T A T E M E N T This thesis contains work that has been previously published or submitted for publication. Stirling D P , Koochesfahani, K . M . , Steeves, J.D., Tetzlaff, W . (2005) Minocycl ine as a neuroprotective agent. Neuroscientist 11:308-322. Stirling D P , Khodarahmi K, L i u J , McPha i l L T , McBr ide C B , Steeves JD , Ramer M S , Tetzlaff W (2004) Minocycl ine treatment reduces delayed oligodendrocyte death, attenuates axonal dieback, and improves functional outcome after spinal cord injury. J Neurosci 24:2182-2190. Stirling D, Plunet, W , Khodarahmi, K, L iu , J , Becker, K G , Dyer, J K, McBr ide, C B , Steeves, JD , Tetzlaff, W . (2003) Minocycl ine treatment reduces p38 MAPK inase expression within microglia/macrophages and improves functional outcome after spinal cord injury. In: Program No. 76.9. 2003 Abstract Viewer/Itinerary Planner. Washington, D C : Society for Neuroscience. Online. The thesis author David Stirling was the primary researcher for all the results presented in the above articles. Technical expertise with gene array experiments was provided by W. Plunet and surgical assistance by J . L iu . Both D. Sutherland and K. Khodaramhi assisted with behavioral analysis. xv i i CHAPTER 1 GENERAL INTRODUCTION 1 Rationale Spinal cord injury (SCI) is an important contributor to morbidity and mortality, particularly in young males. In North America alone, approximately 11,000 people per year sustain an injury to the spinal cord and over 200,000 people live with serious and permanent disabilities as a result of these injuries (Dumont et al., 2001; Nashmi and Fehlings, 2001). These often devastating events leave in their wake profound emotional and economic burdens to individuals, families and society as a whole. Even though the life expectancy has greatly increased for SCI patients, the inability of the spinal cord to repair itself (regeneration failure) and lack of efficacious treatments offers little hope to persons who have suffered from this infliction. Although the damage to the spinal cord by the mechanical trauma is immediate, and therefore not amenable to treatment, secondary degeneration of the cord occurs more slowly over hours to weeks. As many SCI 's are initially incomplete, this indirect destruction of neural tissue can exacerbate neurological deficits, making it an important target for therapeutic intervention. In support of this notion, results from the Second National Acute Spinal Cord Injury Study (NASCIS) , showed some therapeutic potential for the anti-inflammatory steroid methylprednisolone in reducing the secondary damage when administered in high doses within 8 hours after SCI (Bracken et al., 1990; Hal l and Springer, 2004). However, the modest benefits and detrimental side-effects associated with high-dose steroid therapy has prompted the search for more efficacious neuroprotective strategies. To this end, the work performed in this thesis investigates minocycline, a second-generation tetracycline derivative, as a potential neuroprotective agent in the SCI setting. 2 Pathophysiology of SCI Primary injury The primary injury refers to the mechanical damage to the spinal cord inflicted by the insult or trauma. There are four main types of injury observed in the clinical setting which include impact with continued or transient compression, distraction, or laceration/transection, for review see (Dumont et al., 2001). The most common type of SCI in humans results from compression or contusion of the cord from fractured vertebrae and/or discs extending into the lesion site, whereas complete lacerations/transections are rarely observed (Schwab and Bartholdi, 1996; Dumont etal . , 2001). Early after the primary insult, hemorrhages are evident within the center of the cord due to disruption and shearing of neuronal and endothelial membranes. The propensity for damage of the central grey matter damage, is l ikely due to its exceedingly vascular nature, soft consistency and increased metabolic requirements compared to the overlying white matter (Tator, 1995). In addition, models that mimic spinal cord tissue movement dynamics following contusion injury to the cord show a predominantly rostral and caudal displacement, and thus opposing forces are experienced at the center of the cord in the highly vascular grey matter (Blight, 1988). The lower force experienced at the cord periphery may explain why axons located near the pia mater are often spared after injury and form a subpial axonal r im, whereas centrally located axons are often severely damaged after SCI (Schwab and Bartholdi, 1996; Young, 2002). Secondary injury The mechanical impact of a SCI triggers a cascade of secondary damage that progressively destroys an increasing amount of tissue adjacent to the primary lesion over a 3 period of hours to weeks. These secondary mechanisms include vascular disturbances including ischemia and reperfusion, metabolic failure, ionic dysregulation, excitotoxicity, free radical formation, l ipid peroxidation, cytokine/chemokine release, inflammation and cell death, see Figure 1.1, (Schwab and Bartholdi, 1996; Hausmann, 2003). Vascular disturbances, ischemia and reperfusion Although, the exact mechanisms which cause ischemia are unknown, microangiographic and electron microscopic studies have shown a reduction in both microcirculation and spinal cord blood flow after SCI (Koyanagi et al., 1993a, b; Koyanagi et al., 1993c). Loss of blood flow is thought to be a key mechanism of secondary injury, as a substantial reduction in oxygen wi l l lead to a depletion of cellular energy stores. Once cellular A T P is depleted, cells are incapable of maintaining ionic homeostasis, swell and eventually rupture. Thus, decreased blood flow propagates necrosis in C N S tissues, leading to a necrotic wave of cell death originating from the primary injury site (Young, 2002). O f particular interest, the period of ischemia may be followed by a period of hyperemia or reperfusion that may worsen injury through production of highly reactive and destructive oxygen-derived free radicals (Tator and Fehlings, 1991; Lukacova et al., 1996). Neurogenic or spinal shock typically results from a change in systemic vascular resistance mediated by trauma to the spinal cord (Dumont et al., 2001). Neurogenic shock may be caused by paralysis of the nerves that mediate vasomotor input that control the size of the blood vessels, leading to widespread dilation and inadequate tissue perfusion (Dumont et al., 2001). Bradycardia, hypotension, decreased peripheral resistance and blood flow are serious consequences of neurogenic shock and i f left untreated may exacerbate tissue damage (Guha and Tator, 1988). 4 Figure 1.1 Pathophysiology of spinal cord injury. A summary diagram of the major vascular, biochemical and cellular events that contribute to the overall pathology after spinal cord injury are shown as well as when these events occur relative to the primary injury. 5 Free radicals and lipid peroxidation Free radicals are unstable and highly reactive molecules that have unpaired electrons and can cause damage to biological membranes, proteins and D N A . Several studies have demonstrated the appearance of free radical oxidation products following SCI (Braughler and Hal l , 1992; Barut et al., 1993; Ha l l and Braughler, 1993; Springer et al., 1997). The significance of these products is suggested by the fact that: (i) they increase after SCI ; (ii) they decrease following treatment with a steroidal antioxidant; (iii) application of high doses of steroidal antioxidants preserve tissue and promote functional restoration (Hall and Braughler, 1986; Ha l l and Springer, 2004). The role of pathologic free radical reactions is also inferred from the loss of C N S antioxidants following SCI (Braughler and Ha l l , 1992; Barut et al., 1993; Hal l and Braughler, 1993; Springer et al., 1997). In addition to reperfusion injury, mitochondrial dysfunction, cyclooxgenase, lipoxygenase and nitric oxide synthase activity can all lead to the formation of free radicals (Anderson et al., 1985; Hal l and Braughler, 1986; Ha l l and Springer, 2004). The peroxynitrite anion, a potent free radical, can cause damage to cell membranes and organelles through a process termed l ipid peroxidation (Anderson et al., 1985; Hal l and Braughler, 1986). In addition, hemorrhage and glycolytically derived lactate provides a source of iron to catalyze and propagate oxygen radical and l ipid peroxidation reactions making the SCI site unfavorable for cell survival (Hall and Braughler, 1986). Indeed, the benefit afforded by antioxidant compounds including methylprednisolone and tirilazad mesylate, supports the concept that free radical-mediated l ipid peroxidation is an important therapeutic target for acute SCI (Hall , 1993). 6 Excitotoxicity It is wel l documented that excitatory amino acids rapidly accumulate to neurotoxic levels after SCI (Demediuk et al., 1989; Panter et al., 1990; L i u et al., 1991; Park et al., 2004). Excitotoxicity refers to the ability of excitatory amino acids, in particular glutamate, to mediate the death of neurons usually as a result of excessive activation of glutamate receptors (Choi, 1992). Exposure of glutamate or agonists of glutamate receptors dose-dependently induce cell death in neuronal cultures (Regan and Choi , 1991), and following injections into the naive spinal cord (L iu et al., 1999). In contrast, treatments that antagonize ionic glutamate receptors promote tissue preservation and functional recovery after SCI (Faden et al., 1988; Wrathall et al., 1992; Agrawal and Fehlings, 1997; Kanellopoulos et al., 2000). Although most studies have focused on grey matter toxicity due to activation of N M D A glutamate receptors and subsequent C a 2 + overload, white matter toxicity due to glutamate accumulation is increasingly documented. In support of this notion, antagonism of A M P A receptors is neuroprotective after SCI (Wrathall et al., 1992; Wrathall et al., 1996; Agrawal and Fehlings, 1997; Kanellopoulos et al., 2000). Although A M P A receptors are expressed on astrocytes and oligodendrocytes (Agrawal and Fehlings, 1997), the latter are thought to be highly vulnerable to glutamate-mediated excitotoxicity and their death may lead to demyelination and functional deficits after SCI (Yoshioka et al., 1995; McDonald et al., 1998; Sanchez-Gomez and Matute, 1999). Inflammatory response to SCI Local resident microglia, the immuno-competent cells of the C N S , are activated early after SCI and initially represent the primary source of pro-inflammatory cytokines, tumor necrosis factor (TNF-a), interleukin-lbeta (IL-1B), and IL 6 (Figure 1.2) (Dusart and Schwab, 7 1994; Bartholdi and Schwab, 1997; Streit et al., 1998). This early microglial response is followed by neutrophil invasion which peaks at one day, but is still present up to three days after injury (Dusart and Schwab, 1994; Streit et al., 1998). Neutrophils produce and release many potential cytotoxic factors, including proteases, reactive oxygen species, and nitric oxide, and contribute to l ipid peroxidation and protein nitration (Taoka and Okajima, 2000). The damaging effects of neutrophils have been illustrated in several loss-of-function experiments. For example, inhibition of neutrophil invasion with an antibody to the CD1 Id subunit of the leukocyte integrin diminished myeloperoxidase levels, macrophage invasion, l ipid peroxidation, protein nitration and improved neurological recovery after SCI (Bao et al., 2004). Therefore, strategies to prevent the detrimental effects of neutrophil invasion and toxicity are thought to be beneficial after SCI. Fol lowing the peak of neutrophil activation, and lasting for several weeks after injury, hematogenous macrophages are the next immune cells to infiltrate the lesion epicenter (Blight, 1985; Dusart and Schwab, 1994). The effects of macrophage recruitment following SCI have been described as both detrimental as wel l as beneficial. Whi le the controlled activation of macrophages and immune cells may be of benefit at some stage of SCI (Rapalino et al., 1998; David, 2002), other studies have shown that activated macrophages (CNS resident microglia and/or blood-derived macrophages), and cytotoxic products associated with their activation, contribute to the secondary damage after SCI (Popovich, 2000). Depletion of peripheral monocytes/macrophages reduced the loss of myelinated axons in a guinea-pig model of SCI (Blight, 1994), and similarly in rats, hematogeneous macrophage depletion reduced activated macrophages in the injured cord, improved hindlimb recovery, increased sparing of myelinated axons and reduced cavitation after SCI (Popovich et al., 1999). In agreement, zymosan, a potent activator of microglia/macrophages caused cavitation, demyelination and permanent axonal 8 Figure 1.2 Inflammation after SCI. A. normal spinal cord. B. After the mechanical injury to the spinal cord a progressive secondary wave o f degeneration occurs that involves a robust inflammatory response. Microg l ia , the resident immuno-competent cells o f the C N S become activated and release several inflammatory mediators. Neutrophils invade the lesion site followed closely by macrophages and lymphocytes (not shown) amplifying the inflammatory response. C. Detrimental components o f the inflammatory response may contribute to the death o f oligodendrocytes, demyelination with subsequent axonal conduction failure, axonal degeneration and functional loss after SCI . Ongoing phagocytosis, axonal retraction/dieback and the formation o f a glial scar further contribute to lack o f regeneration o f severed axons after spinal cord trauma. injury when injected into the spinal cord (Popovich et al., 2002). Furthermore, fibroblasts expressing a potent version of the inflammatory cytokine IL-6 (Hyper-IL-6), transplanted into the spinal cord injury site, dramatically augmented inflammatory cell invasion, increased lesion size and reduced axonal sprouting after SCI (Lacroix et al., 2002). Collectively these studies provide compelling evidence of the deleterious potential associated with the activation of microglia, infiltration of neutrophils and invasion of hematogenous macrophages fol lowing SCI. In summary, the role of inflammation after SCI is an ongoing matter of intense study and the inflammatory response appears to be both detrimental in the early stages and perhaps beneficial at later phases. It w i l l be of great importance to determine the destructive components of the inflammatory response and the optimal time to intervene without disturbing any possible beneficial aspects associated with tissue repair. Apoptosis Apoptosis is an active and highly regulated form of cell death characterized morphologically by cytoplasmic shrinkage, chromatin condensation and fragmentation, membrane blebbing, and the disintegration of the cell into membrane-bound intra-cellular inclusion bodies. The apoptotic cell is subsequently removed by phagocytosis, an event, which in contrast to necrosis, does not induce an inflammatory response. Many of the morphological hallmarks of apoptosis can be attributed to the specific cleavage of cellular proteins mediated by a family of cysteine proteases known as caspases. Mammalian caspases are synthesized as inactive procaspases, which require cleavage for activation, and are often grouped according to function. Initiator caspases (caspase-2, 8, 9, 10) function to activate effector caspases (caspase-3, 6, 7), which cleave several apoptotic substrates involved in the orderly breakdown of the cell (Nicholson and Thornberry, 1997). 10 Two main apoptotic pathways, the death receptor (extrinsic) pathway and the mitochondrial (intrinsic) pathway, have been described (see Figure 1.3). The death receptor pathway involves binding of a death receptor (e.g. Fas/CD95, T N F a receptor) with its ligand, recruitment and activation of initiator caspase-8, which in turn activates the effector caspases (e.g. caspase-3). The mitochondrial pathway involves activation of upstream pro-apoptotic factors that cause mitochondrial release of cytochrome-c and Smac /D IABLO, formation of an apoptosome, and sequestering of the inhibitor of apoptosis proteins (IAP's) respectively. These processes lead to the activation of initiator caspase-9, which in turn activates effector caspase-3. In addition, caspase-independent cell death pathways involve the release of apoptosis inducing factor (AIF) from the mitochondria into the cytosol, where it translocates to the nucleus and contributes to D N A condensation and fragmentation (Cregan et al., 2004). Once caspase-3 is activated, it cleaves many substrates including I C A D (inhibitor of caspase-activated deoxyribonuclease (CAD)) allowing for the liberation of C A D , an endonuclease that mediates the apoptotic internucleosomal cleavage of D N A (Kaufmann and Hengartner, 2001). Apoptosis after S C I . Apoptosis of both neurons and glia occurs in a variety of neurodegenerative diseases and following C N S trauma (Ekshyyan and A w , 2004). Apoptosis has also been demonstrated to occur in animal models of SCI and after human SCI, and contributes to secondary cell death, reviewed in (Beattie et al., 2002a). Caspases are activated within neurons and oligodendrocytes, suggesting caspase-dependent cell death plays an important role in the execution of these cells following injury (Springer et al., 1999; McBr ide et al., 2003). Oligodendrocyte apoptosis after SCI is biphasic, with an early response (within 24hr) occurring at the injury site, and a delayed response (days to weeks) remote to the lesion site within degenerating and non-degenerating 11 Death receptor pathway Mitochondrial pathway Figure 1.3 Apoptotic cell death pathways. The death receptor pathway involves activation o f procaspase-8 and subsequent activation o f effector caspases such as pro-caspase-3. The mitochondrial pathway involves cytochrome-c release and formation o f an apoptosome leading to activation o f procaspase-9 and subsequent activation o f procaspase-3. Active caspase-3 cleaves several cytosolic proteins and liberates a nuclease ( C A D ) involved in D N A condensation and fragmentation. Inhibitor o f apoptosis protein (IAPs) and members o f the Bcl-2 family o f anti-apoptotic proteins oppose cell death by inhibiting the activation o f caspases and preventing the release o f cytochrome-c, respectively. 12 tracts (Beattie et al., 2002a). Both the late and early phases of oligodendrocyte apoptosis may contribute to demyelination of intact axons spared from the mechanical injury. Since oligodendrocytes myelinate several internodes on several axons, the loss of these cells fol lowing trauma may have profound consequences to the functional integrity of the C N S (see Figure 1.2). Beattie and colleagues reported approximately 50% of oligodendrocytes are lost at 8 days post-SCI with significant reductions existing to 42 days post-injury (latest time point examined (Beattie et al., 2002b). Since oligodendrocyte apoptosis after SCI is temporally and spatially correlated with the actual loss of oligodendrocytes, apoptosis might be an important mechanism leading to the death of these cells after SCI (Beattie et al., 2002b). Potential cytotoxic products released from degenerating axons or activated microglia/macrophages fol lowing injury may be responsible for inducing oligodendrocyte death. For example, oligodendrocytes are sensitive to glutamate excitotoxicity via AMPA/Ka ina te receptors and antagonists to AMPA/Ka ina te receptors on oligodendrocytes reduced Kainate-induced excitotoxic death (McDonald et al., 1998). Microglia/macrophages are thought to produce large amounts of T N F - a , and transgenic mice that over-express T N F - a within the C N S , spontaneously develop a chronic demyelinating disease (Prober! et al., 1995). In addition, T N F -a injected into the spinal cord augments glutamate-mediated neuronal cell death (Hermann et al., 2001). Furthermore, Beattie and colleagues showed that pro-nerve growth factor (pNGF) -mediated activation of p75 caused oligodendrocyte apoptosis following SCI (Beattie et al., 2002b). Since activated microglia/macrophages produce T N F - a , N G F and glutamate after C N S trauma, they may contribute to the remote oligodendrocyte death observed after SCI (Shuman et al., 1997; Bethea, 2000). This "bystander effect" leading to the death of oligodendrocytes may explain the chronic demyelination observed in areas where axons are left intact after SCI. In 13 addition to oligodendrocytes, the apoptotic death of neurons has also been reported after SCI (Liu et al., 1997b) however, the triggers of their death are less clear. Microgl ia also undergo apoptosis after SCI, although the functional significance of their death is unknown (Shuman et al., 1997; Stirling et al., 2004). The majority of reports suggest that astrocytes rarely undergo apoptosis after SCI, implicating their resistance to apoptosis promoting factors released after SCI. Neuroprotective strategies for mitigating secondary damage after acute SCI A n emerging understanding of the pathophysiology associated with SCI has prompted much interest in developing therapeutic approaches to target key elements of secondary injury. The following few paragraphs w i l l summarize some of the more wel l known pharmacological strategies to illustrate the prior successes and failures for treating SCI and the complexity of the secondary injury response. Corticosteroids Corticosteroids, including methylprednisolone have had a long history of use in the SCI setting (Ducker and Zeidman, 1994). Although the protective mechanisms are not completely understood, animal models of SCI have shown that administration of methylprednisolone reduced lesion size and promoted functional recovery, reduced edema by restoring the blood-brain barrier, augmented spinal cord blood flow, stabilized membranes, altered electrolyte concentrations, attenuated the inflammatory response, and prevented damage mediated by oxygen derived free radicals following C N S trauma (Ducker and Hamit, 1969; Means et al., 1981; Anderson et al., 1982; Braughler and Ha l l , 1982; Hal l and Braughler, 1982; Young and Flamm, 1982; Young et al., 1988; Bracken et al., 1990; Saville et al., 2004; Y u et a l , 2004; Fu and Saporta, 2005). It is of importance to note that not all studies have shown neurological 14 improvement with methylprednisolone therapy after SCI and many questions regarding its usage remain unresolved (Rabchevsky et al., 2002; Wells et al., 2003a). Nevertheless, based on some of the earlier studies showing beneficial effects of high-dose methylprednisolone, the Second National Acute Spinal Cord Injury Study (NASCIS II) was started and demonstrated that methylprednisolone given within 8 hr after injury, at a dosage of 30 mg/kg and maintained at 5.4 mg/kg/hr for 24 hr, improved neurological outcome after SCI in humans (Bracken et al., 1990). Thus the N A S C I S II study was important as it was the first clinical study to show that pharmacological manipulation after SCI could indeed promote functional recovery, established methylprednisolone's use in the clinic for treating SCI, and also substantiated the importance of the secondary injury concept. Although the N A S C I S II study propagated the widespread use of methylprednisolone, much criticism has been generated towards the methodology used in the trial and the risk of serious side effects with steroid treatment versus the modest neurological benefits afforded, for recent comprehensive reviews of the N A S C I S I, II and III trials see (Hurlbert, 2001; Bracken, 2002; Hal l and Springer, 2004; Kwon et a l , 2004; Lammertse, 2004). However, methylprednisolone remains the only widely used treatment available to persons suffering from a SCI. Cangliosides Gangliosides are plasma membrane components thought to play important roles in cell surface interactions, cell differentiation, and transmembrane signaling. The rationale to use gangliosides to promote neurological recovery stems from animal studies that provide evidence that they promote neurite outgrowth, sprouting, regeneration, and survival (Toffano et al., 1984; Bose et al., 1986; Skaper and Leon, 1992). 15 Based on these positive effects, clinical trials were conducted using G M - 1 ganglioside (Sygen) following SCI (Geisler et al., 2001). Although the primary outcome measure was negative the study did show there was a significant effect in all patients in the primary outcome variable (the percentage of marked recovery) at week 8, the end of the dosing period. In addition, there was a significant effect in all patients in the time at which marked recovery was first achieved. Furthermore, American Spinal Cord Injury Association (ASIA) motor, touch and pinprick scores, bowel and bladder function, sacral sensation and anal contraction showed a consistent trend favoring treatment. Opiate antagonists A significant release of endogenous opioid peptides occurs after SCI and contributes to the secondary injury cascade by reducing blood flow, increasing hypotension and augmenting ischemia (Faden and Holaday, 1981a, b; Faden et al., 1981a; Faden et al., 1981b; Holaday and Faden, 1981; Naftchi, 1982; Holaday, 1984). Several studies in animal models have shown that treatment with antagonists of opioids including naloxone, increased blood flow, reduced neurologic shock and improved neurological recovery (Faden and Holaday, 1981a, b; Faden et al., 1981a; Faden et al., 1981b; Holaday and Faden, 1981; Holaday, 1984). The positive effects of naloxone treatment demonstrated in animal models provided the rationale to assess naloxone in the N A S C I S II study, but the results of the trial were negative (Bracken et al., 1990). However, subsequent reanalysis of the data from the trial suggested that naloxone did improve functional outcome in incomplete patients that received naloxone within 8 hours of injury (Bracken and Holford, 1993). Furthermore, it was also suggested that the dosage used in the N A S C I S II trial was subtherapeutic and additional studies are needed (Hall and 16 Springer, 2004). Thus much work remains to be done to validate the use of opiate antagonists in the SCI setting including determining proper dosage and timing of administration. Glutamate receptor, calcium, and sodium channel antagonists Excitotoxicity due to excessive activation of glutamate receptors has prompted the search for effective glutamate receptor antagonists in the SCI setting. Towards this end, studies have shown that antagonists to N M D A and A M P A receptors prevented excitotoxicity, reduced lesion size and promoted functional recovery after SCI (Faden et al., 1988; Agrawal and Fehlings, 1997; L i u et al., 1997a; Gavir ia et al., 2000; Kanellopoulos et al., 2000; Colak et al., 2003). O f these antagonists GK11 was tested in human trials but the results from the study have yet to be published (Kwon et al., 2004). The calcium channel antagonist nimodipine have been demonstrated to exert neuroprotective effects after SCI by improving blood flow and motor and somatosensory evoked potentials (Guha et al., 1987; Fehlings et al., 1989; Guha et al., 1989; Winkler et al., 2003). However, it is important to note that other studies in animal models have failed to show any benefit (Faden et al., 1984; Ford and Ma lm, 1985; Haghighi et al., 1988). In addition, a small cl inical study conducted in France testing the effects of methylprednisolone or nimodipine alone, methylprednisolone and nimodipine combined, or placebo, failed to show a significant improvement in motor or sensory scores with either of the treatment groups (Pointillart et al., 2000). Thus, at least with the results from the nimodipine studies, the future of this calcium channel antagonist in SCI therapy is unlikely to develop further in the near future. Several animal studies have shown that treatment with sodium channel antagonists such as tetrodotoxin (TTX) , ri luzole, and phenytoin, is neuroprotective after SCI by reducing acute white matter pathology and increasing both axon density and hindlimb function after SCI 17 (Fehlings and Agrawal, 1995; Agrawal and Fehlings, 1996; Teng and Wrathall, 1997; Rosenberg et al., 1999; Schwartz and Fehlings, 2001; Hains et a l , 2004; Rosenberg et al., 2005). However, studies uti l izing sodium channel blockers for treatment of human SCI have not yet materialized although animal models have shown promising results. Potential pharmacological agents for acute SCI Several other agents have been tested in animal models of SCI and have been shown to improve functional outcome after SCI. Some of the promising approaches include treatment with antioxidants (vitamin E, vitamin A , vitamin C, selenium, and coenzyme Q) (Saunders et al., 1987; Anderson et al., 1988; Iwasa et al., 1989; Katoh et a l , 1996a; Kaptanoglu et al., 2002; Hi l lard et al., 2004; L i u et al., 2004) , anti apoptosis promoting agents (caspase inhibitors, calpain inhibitors, neutraling antibodies to fas ligand, dexamethasone, erythropoietin, B D N F , P2X7 receptor inhibition) (L i et al., 2000; Brandoli et al., 2001; Cel ik et al., 2002; Koda et al., 2002; McBr ide et al., 2003; Demjen et al., 2004; Yune et al., 2004; Colak et al., 2005), immune modulators (FK506, cyclosporine, neutralizing antibodies to T N F - a and Fas ligand, nitric oxide sythase inhibitors, chemokine receptor antagonists, I L - i p receptor antagonists, statins ) (Lee et al., 2000b; Satake et al., 2000; Springer et al., 2000; Ghirnikar et al., 2001; Nesic et al., 2001; Zhang et al., 2003c; Pannu et al., 2005), and hormones (estrogen, progesterone) (Thomas et al., 1999; Sribnick et al., 2003; Yune et al., 2004). However, many of these agents have been applied prior to injury and require further investigation to validate their cl inical applicability. In addition, many of these studies have yet to be replicated. These hurdles w i l l l ikely have to be overcome in animal models before large scale human trials w i l l be initiated. Furthermore, given the complexity of SCI , it is l ikely that therapies w i l l need to be combined or include agents that 18 target several aspects of the secondary cascade, to promote tissue preservation and functional recovery after SCI. Minocyc l ine Since their discovery in the 1940's, tetracyclines have undergone a variety of molecular modifications to enhance their antibacterial activity, improve their tissue absorption, and prolong their half-life. Towards this end, minocycline, a second-generation semi-synthetic tetracycline, was derived with improved tissue absorption into the cerebrospinal fluid and the C N S with a longer half-life compared to 1st generation tetracyclines, see Figure 1.4 (Klein and Cunha, 1995). In addition to its antimicrobial actions, minocycline has been shown to possess anti apoptotic and anti-inflammatory properties. Given that neurodegenerative diseases, traumatic or ischemic C N S insults, often involve inflammation and apoptotic cell death, and the lack of treatment options available for these conditions, has led to an explosion of studies to re-examine established drugs for other purposes. The following briefly summarizes the successful preclinical applications of minocycline as wel l as discussing what is known about the anti-inflammatory, immunosuppressive, and anti-apoptotic mechanisms of action for minocycline. In addition, the p38 M A P K pathway is discussed as a potential target that may account for some of these multiple effects. Minocycl ine has been successfully applied to animal models of focal and global ischemia (Yrjanheikki et al., 1998; Yrjanheikki et al., 1999; A rv in et al., 2002; Wang et al., 2003a; X u et al., 2004b), Parkinson's disease (Du et al., 2001; He et al., 2001; Thomas and Le, 2004), Huntington's disease (Chen et al., 2000; Hersch et al., 2003; Thomas et al., 2003; Wang et al., 2003b), amyotrophic lateral sclerosis (ALS) (Kr iz et al., 2002; Van Den Bosch et al., 2002; Zhu et al., 2002; Zhang et al., 2003b), Alzheimer's disease (Ryu et al., 2004), multiple sclerosis 19 O H O O H Conserved structure o R1 R2 R3 R4 tetracycline H CH3 OH H doxycycline H CH3 H OH minocycline N(CH3)2 H H H Figure 1.4 Structure of tetracyclines. Tetracycline and tetracycline derivatives share a common four fused six-membered ring structure. The semi-synthetic derivatives consist of modifications made at one or more of four sites (RI to R 4 ) on the conserved structure. 20 (Brundula et al., 2002; Popovic et al., 2002; Yong , 2004), traumatic brain injury (Sanchez Mej ia et al., 2001), and spinal cord injury (Lee et al., 2003; Wells et al., 2003a; Stirling et al., 2004; Teng et al., 2004). Pioneering studies of minocycline revealed that following global brain ischemia in gerbils, minocycline increased the survival of C A 1 pyramidal neurons by 77% and 71%, when administered 12 hours prior to, as wel l as 30 minutes after the insult, respectively (Yrjanheikki et al., 1998). Treatment with minocycline also decreased infarct volume (by 63%) when given 4 hours after the onset of focal ischemia (Yrjanheikki et al., 1999). Importantly, these neuroprotective effects were associated with a marked reduction in microglia activation, suggesting a deleterious role of these cells fol lowing C N S insult that may be overcome by minocycline treatment. In addition, m R N A levels for caspase-1 (also known as interleukin-1 beta converting enzyme (ICE)), a key enzyme with dual roles in inflammation and cell death, is markedly reduced following application of minocycline (Yrjanheikki et al., 1998). These landmark studies provided important initial clues for a dual role of minocycline as an anti-inflammatory and anti-apoptotic agent. Subsequent to these initial stroke studies Friedlander's group reported beneficial effects of minocycline treatment in the R6/2 mouse model of Huntington's disease. Minocycl ine extended the survival of these mice by 14% and delayed the characteristic decline in Rotorod performance (Chen et al., 2000). Other studies have shown that minocycline treatment reduced lesion volume and neurological deficits fol lowing traumatic brain injury (Sanchez Mej ia et al., 2001), protected nigral cells after 6-hydroxydopamine injection into mouse striatum (He et al., 2001), attenuated nigrostriatal dopaminergic neurodegeneration in the l-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson's disease (Du et al., 2001; W u et al., 21 2002), suppressed disease progression in experimental allergic encephalomyelitis (EAE) models of multiple sclerosis (Brundula et al., 2002; Popovic et al., 2002), delayed the onset of disease and extended the survival in a mouse model of A L S (Zhu et al., 2002) and promoted the survival and myelination of transplanted oligodendroglial progenitors (Zhang et al., 2003a). A long with a proven and safe clinical track record as an antibiotic and anti-inflammatory for acne and arthritis (Alarcon, 2000; Feldman et al., 2004), it is not surprising that minocycline has become an attractive therapeutic drug candidate and that several "off-label use" clinical trials are under way for a variety of neurodegenerative diseases ( A L S , Huntington's disease, multiple sclerosis), stroke, and SCI. However, minocycline failed to be of benefit in some models of Huntington's disease, Parkinson's disease, and hypoxia-ischemia for review see (Diguet et al., 2004a), stressing the importance of a thorough understanding of minocycline's modes of action, its pharmacokinetics, and the targeted cellular pathology mechanisms. Minocycline improves outcome of SCI Several independent studies, including my experiments detailed in this thesis, have demonstrated that minocycline treatment is neuroprotective following SCI in rodents. During my PhD, the first publication assessing minocycline in SCI was published, and unfortunately preceeded my study by 6 months. In this important study, the authors used a murine clip compression model and administered minocycline one hour after thoracic (T)3/4 SCI. The results showed that minocycline effectively reduced lesion size, increased axonal sparing of rubrospinal axons and improved hindlimb function (Wells et al., 2003a). Importantly, minocycline was more effective in improving functional outcomes when compared with methylprednisolone, the only current treatment option to patients with SCI (Wells et al., 2003a). Ut i l iz ing the dorsal column transection model in rats , I have observed that minocycline 22 treatment, given 30 minutes after dorsal column injury of the spinal cord, reduced neural cell death (Chapter 2), inflammation, axonal dieback, and improved functional recovery (Chapter 3) (Stirling et al., 2004). After spinal cord contusion in rats, minocycline treatment decreased pro-inflammatory cytokine expression, increased anti-inflammatory cytokine expression (Lee et al., 2003), reduced lesion size, and promoted the survival of motoneurons (Teng et al., 2004). Importantly, these studies reported enhanced functional outcomes with minocycline in both open-field locomotion and inclined-plane tests. Taken together, minocycline's neuroprotective effect after experimental SCI , and in neurodegenerative disease models, appears to involve modulation of both immune responses and cell death. Thus, minocycline may have therapeutic benefits across a broad spectrum of C N S insults. However, the molecular targets of minocycline remain somewhat unclear. Mechanisms of minocycline's anti-inflammatory actions Minocycl ine has been suggested to exert its anti-inflammatory effects by modulating microglia, immune cell activation and subsequent release of cytokines, chemokines, l ipid mediators of inflammation, metalloproteases (MMPs) , and nitric oxide release. Cel l cultures of monocytes, microglia, and macrophages stimulated with activating compounds, in the presence or absence of minocycline, combined with in vivo studies have been useful in addressing some of the potential cellular signaling pathways affected by the treatment. Cytokines are small, secreted proteins produced and released by many cell types de novo and mediate and regulate inflammation, immunity, and hematopoiesis. Pro-inflammatory cytokines, such as T N F a , I L - l p , and IL-6 are produced by microglial cells, astrocytes, neutrophils, macrophages, and augment both inflammation and subsequent immune responses. Common to many of the minocycline studies, is the consistent reduction in caspase-1-mediated 23 liberation of I L - i p (Yrjanheikki et al., 1998; Yrjanheikki et al., 1999; Chen et a l , 2000; Sanchez Mej ia et al., 2001). Similarly, minocycline treatment diminished T N F - a m R N A levels after SCI, and prevented the lipopolysaccharide (LPS)-induced production of T N F - a in primary glial cell cultures (Lee et al., 2003; Lee et al., 2004). In addition, the anti-inflammatory cytokine IL-10, was up-regulated following minocycline application immediately after SCI (Lee et al., 2003). Therefore, modulation of pro-inflammatory cytokines may contribute to minocycline's neuroprotective effects. In agreement, other treatment paradigms that lessen I L - l p and T N F - a levels have been shown to be neuroprotective in several animal models of C N S disease or injury, underscoring the detrimental role associated with the release of these potent pro-inflammatory cytokines (Viviani et al., 2004). Chemokines act as chemoattractants guiding microglia, astrocytes and peripheral immune cells to sites of infection, trauma or during disease progression. Microgl ia l cells, astrocytes, and tissue-infiltrating immune cells are all sources of chemokines. Minocycl ine may be neuroprotective by decreasing resident and immune cell migration to sites of inflammation by altering levels of chemokines and/or chemokine receptor expression. For example, minocycline suppressed LPS-induced production of chemokines ( M l P - l a , R A N T E S ) and diminished the chemokine receptor C X C R 3 expression in microglia-like B V - 2 cells (Kremlev et al., 2004). O f particular interest, the chemokine receptor C X C R 3 is largely expressed on T h i (pro-inflammatory CD4+ T-cells), that are implicated in the development of autoimmune C N S inflammation, microglia/macrophage activation and demyelination (Ambrosini and A lo is i , 2004). Thus down-regulation of C X C R 3 by minocycline may act to reduce the damaging aspects of T h i recruitment and activation after injury. In addition, M l P - l a and R A N T E S are chemotaxic for monocytes, microglia, astrocytes, and biases T-cells to T h i commitment (Karpus 24 and Kennedy, 1997). Furthermore, an t i -MLF- la treatment prevented the onset of acute E A E , and therefore may prevent the damaging aspects of inflammation after C N S insult (Karpus and Kennedy, 1997). Minocycl ine can also modify l ipid mediators of inflammation. Phospholipases, such as phospholipase A 2 , hydrolyze cellular membrane phospholipids releasing the short-lived arachidonic acid. Arachidonic acid is metabolized by cyclooxygenases ( C O X ) to produce prostaglandins or metabolized by lipoxygenases to produce lipoxins or leukotrienes. Prostaglandins, e.g. P G E 2 , are a family of soluble, hormone-like molecules produced by several cell types such as monocytes, macrophages and neutrophils, and contribute to pyrogenicity, vasodilation and vascular permeability. Minocycl ine treatment inhibited both secreted and non-secreted forms of phospholipase A 2 (sPLA2) activity in vitro (Pruzanski et al., 1992). In addition, minocycline treatment inhibited P G E 2 production and C O X - 2 expression in (LPS)-challenged B V - 2 murine microglial cells (K im et al., 2004). In a rat model of focal ischemia, pretreatment with minocycline almost completely reduced C O X - 2 protein levels, and reduced prostaglandin (PGE2) levels by 55% (Yrjanheikki et al., 1999). Interestingly, minocycline treatment abolished 5-lipoxygenease translocation to the nuclear membrane in P C 12 cells subjected to oxygen-glucose deprivation (Song et al., 2004). Thus, lipid-mediated inflammatory signaling may be attenuated at several steps following minocycline treatment. Although originally thought to be mediators of extracellular matrix degradation, an important role for matrix metalloproteinases (MMPs) in inflammation is now wel l established, reviewed in (Parks et al., 2004). For example, some M M P s have been shown to enhance the effects of pro-inflammatory cytokines such as TNF-ct, and IL-lf3, and regulate chemokine activity either directly through cleavage or indirectly, enhancing or sequestering their response. 25 In addition, M M P ' s have been shown to activate defensins, important antimicrobials, and facilitate neutrophil accumulation and activation through local chemokine gradients. Both in vitro and in vivo studies have shown that minocycline treatment may reduce inflammation by inhibiting M M P ' s . For example, daily injections of minocycline reduced M M P - 2 expression in a chronic relapsing remitting model of multiple sclerosis (Popovic et al., 2002). In vitro, high doses of minocycline inhibited gelatinase ( M M P - 2 & -9) activity in a zymography assay, reduced M M P - 9 protein expression in cultured T-cells, and attenuated T-cel l migration across a fibronectin barrier (Brundula et al., 2002; Power et al., 2003). Therefore, minocycline's observed effects on M M P s may act to reduce inflammation by diminishing cell infiltration and migration. Several M M P s , especially M M P - 1 2 , increase after SCI (Wells et al., 2003b) and following brain hemorrhage (Power et al., 2003), and minocycline application down-regulated M M P - 1 2 (Power et al., 2003). Many studies have shown that minocycline may be neuroprotective by regulating nitric oxide production. Microglia/macrophage release of nitric oxide is thought to mediate neurotoxicity in several neurodegenerative diseases (Dawson and Dawson, 1998). In cultured microglial cells, minocycline reduced hypoxia- and excitotoxin-induced nitric oxide production (Tikka et al., 2001; Suk, 2004). Similarly, in murine macrophage cells stimulated with either L P S or IFN-y, as wel l as in osteoarthritis (OA)-affected cartilage cells stimulated with I L - l p , minocycline inhibited i N O S activity indirectly through diminishing iNOS m R N A expression and suppressed subsequent protein levels (Amin et al., 1996). Since supplemental C a 2 + did not have a significant influence on minocycline-dependent N O S inhibition, these effects are most l ikely independent of minocycline's C a 2 + chelating abilities. 26 In a gerbil model of global ischemia, minocycline treatment reduced m R N A levels of i N O S by 30% compared to saline treated controls, suggesting minocycline is effective at reducing iNOS expression in vivo (Yrjanheikki et al., 1998). In agreement, daily injections of minocycline inhibited i N O S activity by 72% in a transgenic mouse model of Huntington's disease (Chen et al., 2000). Furthermore, minocycline has been shown to reduce macrophage production of nitric oxide (Amin et al., 1997), a molecule that may be involved in axonal retraction (He et al., 2002). These studies provide compelling evidence that minocycline may reduce inflammatory-induced production of nitric oxide and subsequent generation of peroxynitrite through attenuating iNOS expression and preventing peroxynitrite formation. In addition to its effects on microglia/macrophage activation, minocycline has also been shown to lessen the astrocytic response after SCI (McPhai l et al., 2004; Teng et al., 2004), and following P-amyloid injection into the hippocampus (Ryu et al., 2004). Thus, reducing the astrocytic response to injury may reduce astrocyte-mediated release of pro-inflammatory mediators, and lessen scar formation. However, in other models minocycline did not appear to affect astrocyte activation (Yrjanheikki et al., 1999; D u et al., 2001). Further studies are needed to determine minocycline's effects on the astrocyte response after C N S trauma or disease. Collectively, minocycline acts on several aspects of the inflammatory response during neurodegenerative diseases or fol lowing C N S trauma. Since neurons and oligodendrocytes are vulnerable to many of the inflammatory factors discussed in the previous sections, minocycline's effects on attenuating several aspects of the inflammatory response may reduce 'bystander death" of these cells during disease progression or following trauma. In addition to an indirect effect on cell death through dampening inflammatory reactions, minocycline has been shown to directly prevent apoptotic cell death. 27 Mechanisms of minocycline's survival promoting actions Associated with minocycline's neuroprotective properties is the consistent finding that several caspases are inhibited by minocycline after treatment in animal models of C N S trauma and disease. We and others have shown that minocycline treatment reduced caspase-3 activation (see Chapter 2) (Stirling et al., 2004) and activity (Lee et al., 2003) following SCI , traumatic brain injury (Sanchez Mej ia et al., 2001), and Huntington's disease (Chen et al., 2000). However, a direct effect of minocycline on caspase-1 and -3 activity was ruled out using cell-free extracts, suggesting minocycline may act upstream of caspase activation (Chen et al., 2000). In support of these findings, reconstitution experiments, by adding cytochrome-c to isolated cytosol extracts of kidney cells, confirmed that minocycline acts at the level of the mitochondria, and not downstream of cytochrome-c release (see Figure 1.3)(Wang et al., 2004a). Since cytochrome c release from the mitochondria plays a crucial role in activating downstream caspases, Zhu et al (Zhu et al., 2002) tested the effects of minocycline on the release of cytochrome c from cell-free mitochondria isolated from rodent liver and brain. Minocycl ine prevented both C a 2 + and Bid-induced cytochrome c release, decreased mitochondrial depolarization, and swelling in purified mitochondria (Zhu et al., 2002). Furthermore, minocycline treatment also reduced cytochrome c release from mitochondria isolated from spinal cords of A L S mice and from ischemic mouse brains (Zhu et al., 2002). Moreover, minocycline prevented cytoplasmic accumulation of cytochrome-c, Smac /D IABLO and A IF (Wang et al., 2003b) and, in nuclear fractions minocycline prevented nuclear translocation of A IF . Thus, minocycline-mediated protection targets both caspase-dependent (cytochrome-c, Smac/Diablo) and caspase-independent (AIF) forms of cell death. 28 Minocycl ine may also prevent apoptosis by increasing levels of anti-apoptotic factors potentially upstream of cytochrome-c release. Recent in vitro studies have shown that minocycline treatment protected kidney epithelial cells against apoptosis induced by hypoxia, azide, cisplatin, and staurosporine by selectively increasing the anti-apoptotic protein Bcl-2 (both m R N A and protein) (Wang et al., 2004a). In addition, Bcl-2 down-regulation using a specific anti-sense oligonucleotide, reversed the rescue effects of minocycline-pretreatment suggesting, minocycline is neuroprotective in part by up-regulating Bcl-2 expression (Wang et al., 2004a). Minocycl ine may also be neuroprotective by increasing the levels of inhibitor of apoptosis proteins (IAPs) such as X I A P and thus preventing caspase activation and subsequent cell death. In support of this view, it was demonstrated that in vivo pretreatment with minocycline before ischemia/reperfusion injury of isolated rat hearts, reduced both protein and m R N A expression of several initiator and effector caspases, diminished infarct volume, and lessened apoptotic cell death (Scarabelli et al., 2004). Concomitantly, minocycline treatment reduced caspase activity as wel l as cytosol levels of cytochrome-c and Smac /D IABLO, and increased X I A P expression. In addition, treatment with minocycline alone without ischemia/reperfusion, also reduced caspases-1, 3, 7, 8, 9, 12 expression below basal levels (Scarabelli et al., 2004). Collectively, the results suggest that minocycline has a direct effect in inhibiting cell death by priming a "survival mode" rendering a cell less vulnerable to apoptotic stimuli. This may be due to up-regulation of anti-apoptotic proteins such as members of the Bcl-2 and/or LAP family that have been shown to antagonize the pro-apoptotic members of the Bcl -2 family and reduce caspase activation (Figure 1.3). In addition, minocycline may target the mitochondria directly and prevent the liberation of pro-apoptotic molecules such as cytochrome-c, 29 Smac /D IABLO and A IF . Minocycl ine's anti-apoptotic combined with its anti-inflammatory properties protect cells from several death inducing stimuli and l ikely explain minocycline's neuroprotective effects in C N S trauma and disease. P38 M A P K inhibition after minocycline treatment Tikka and coworkers showed that minocycline treatment increased neuronal survival in mixed spinal cord cultures treated with glutamate, kainate or N-methyl-D aspartate, presumably by reducing microglia activation through a p38 mitogen-activated protein kinase ( M A P K ) -dependent mechanism (Tikka et al., 2001; T ikka and Koistinaho, 2001). P38 M A P K ' s are serine threonine kinases that are activated by several upstream kinases in response to a wide variety of stressors and cytokines (Dong et al., 2002; Koistinaho and Koistinaho, 2002; Kumar et al., 2003) and play an important role in inflammatory signal transduction and cell death (Dong et al., 2002; Koistinaho and Koistinaho, 2002; Kumar et al., 2003; Wada and Penninger, 2004). Further studies revealed that minocycline reduced nitric oxide-induced death in rat cerebellar granule neurons which correlated with a reduction in p38 M A P K (Lin et al., 2001), and reduced p38 M A P K activation in a simian immunodeficiency virus (SIV) model of HIV-associated C N S disease in pigtailed macaques (Zink et al., 2005). Collectively, these studies provide evidence that minocycline may be neuroprotective by inhibiting p38 MAPK-dependent microglial-induced neurotoxicity and preventing p38 MAPK-dependent neuronal cell death. Chapter overviews and hypotheses Oligodendrocytes have been shown to undergo apoptosis following SCI and as a result may contribute to demyelination and subsequent functional deficits. In Chapter 2 of this thesis, I hypothesized that minocycline treatment would prevent oligodendrocyte apoptosis after SCI. To test this hypothesis, I performed a cervical dorsal column transection in adult rats and 30 assessed the spatial and temporal expression of apoptotic profiles within the corticospinal tract (CST) and ascending sensory tract (AST) . To ensure the apoptotic profiles were localized to the A S T or C S T I prelabelled the C S T with B D A to visualize the proximal and distal areas of the tract. Ut i l iz ing immunohistochemistry with antibodies specific for active caspase-3, cell specific markers, and Hoechst dye to assess condensed chromatin, I compared the amount of cell death between saline and minocycline treated animals. The effects of macrophage recruitment following SCI have been described as both detrimental as wel l as beneficial. Whi le the controlled activation of macrophages and immune cells may be of benefit at some stage of SCI (Rapalino et al., 1998; David, 2002), other studies have shown that activated macrophages (CNS resident microglia and/or blood-derived macrophages), and cytotoxic products associated with their activation, contribute to the secondary damage after SCI (Blight, 1994; Popovich et al., 1999; Popovich, 2000; Popovich et al., 2002). Recent studies have shown that minocycline treatment reduced microglia activation following ischemia and thus provided the impetus to examine these effects in the SCI setting (Yrjanheikki et al., 1998; Yrjanheikki et al., 1999). In chapter 3 I hypothesized that minocycline is neuroprotective after SCI by inhibiting microglia/macrophages and thus dampening the inflammatory response would reduce lesion size and promote functional recovery. Whi le examining the microglial/macrophage response within the lesion site, I noticed in some animals labeled corticospinal axons did not dieback from the center of the lesion and many of these animals had smaller lesions. I therefore assessed C S T axonal dieback and measured lesion size and compared the outcomes in minocycline or saline treated rats (chapter 3). To assess functional outcome between minocycline and saline treated animals their fore and hindpaws were dipped in ink and they were trained to run across a narrow platform. The tracks 31 were digitized and fine aspects of locomotion were measured and compared between treatment groups. Based on the beneficial effects exerted following minocycline application in the SCI setting, I then sought after the possible molecular targets of minocycline's mode of action. In chapter 4,1 hypothesized that minocycline treatment would decrease pro-inflammatory mediators following SCI. In collaboration with Ward Plunet, gene array was util ized to examine changes in m R N A expression between saline and minocycline treated rats following SCI. The results of the gene array experiments were subsequently confirmed by reverse-transcriptase Polymerase chain reaction (RT-PCR) . I then examined p38 M A P K protein expression (western blot with specific antibodies to the active, dual phosphorylated form) in saline vs. minocycline treated animals based on the results from the gene expression studies. I hypothesized that minocycline treatment would decrease p38 M A P K expression in microglia/macrophages. In addition, I determined the spatial and temporal expression of active p38 M A P K and determined what cell types expressed active p38 M A P K . Previously, minocycline has been demonstrated to be neuroprotective after spinal cord injury (SCI). However, the mechanisms and optimal treatment paradigms remain poorly defined. In chapter 5,1 examined minocycline's neuroprotective effects following a contusion injury to the spinal cord that more closely mimics human SCI. I also tested different routes of delivery of minocycline and hypothesized that intravenous application of minocycline would exert greater neuroprotective effects than intraperitoneal application. This hypothesis is based on results from a previous study that showed that peak plasma levels of minocycline was achieved immediately after intravenous injection, and less variability was detected between animals compared to intraperitoneal delivery (Fagan et al., 2004). In collaboration with Dr. Jie 32 L iu , intravenous assessable rodent access ports were transplanted underneath the skin and a catheter (connected to the port) was inserted into the jugular vein to facilitate intravenous delivery of minocycline or saline as a control. Outcome measures compared residual tissue sparing (% area of white or grey matter) and behavioral analysis ( B B B locomotor rating scale and subscores, see methods chapter 5). Since minocycline's neuroprotective effects were associated with a reduction in p38 M A P K expression, I further hypothesized that p38 M A P K inhibition would reduce inflammation and improve functional outcome after SCI. To test this hypothesis, I applied a p38 M A P K inhibitor, SB203580 via osmotic mini-pump using two doses and assessed behavioral outcome using the B B B and subscores. 33 CHAPTER 2 TEMPORAL AND SPATIAL EXPRESSION OF APOPTOSIS FOLLOWING CERVICAL SPINAL CORD INJURY AND REDUCTION FOLLOWING MINOCYCLINE TREATMENT 34 SUMMARY Oligodendrocytes have been demonstrated to undergo apoptosis following SCI. The death of these cells may contribute to the demyelination and subsequent loss of function observed following human SCI. Our group and others have previously shown that oligodendrocytes are highly vulnerable to axotomy-induced apoptosis and can be rescued following caspase inhibition. Minocycl ine has been demonstrated to be neuroprotective after spinal cord injury (SCI). However, the cellular consequences of minocycline treatment on the secondary injury response are poorly understood. In this chapter I examined minocycline's effects on oligodendrocyte apoptosis after SCI. Adult rats were subjected to a C7 dorsal column transection and the presence of apoptotic oligodendrocytes was assessed within the ascending sensory tract (AST) and descending corticospinal tract (CST) in segments (3-7 mm) both proximal and distal to the injury site. To detect apoptotic profiles immunostaining for active caspase-3 and Hoechst 33258 staining in combination with CC1 or 0 X 4 2 was utilized. Surprisingly, the number of dying oligodendrocytes in the proximal and distal segments was comparable, suggesting more than the lack of axon-cell body contiguity played a role in their demise. Minocycl ine or vehicle control was injected into the intraperitoneal cavity 30 minutes and 8 hours after SCI and thereafter twice daily for 2 days. The findings are summarized as follows: The peak of apoptosis within the A S T occurs within one to two weeks after injury. Microglia/macrophages and not oligodendrocytes are the predominant cell type to undergo apoptosis in this model. Importantly, SCI-induced apoptosis of oligodendrocytes and microglia was reduced following minocycline treatment. 35 INTRODUCTION In the wake of spinal trauma, the degenerative response spreads from the site of impact causing destruction of neuronal tissue that survived the initial insult, but succumbed to secondary cell death. Although originally thought to occur via necrosis (Dusart and Schwab, 1994), several reports provide evidence that apoptosis, an active and highly regulated type of cell death, contributes to secondary cell death fol lowing SCI (Katoh et al., 1996b; L i et al., 1996; Crowe et al., 1997; L i u et al., 1997b; Emery et al., 1998; Lou et al., 1998; Yong et al., 1998; Beattie et al., 2002a). Oligodendrocytes, the myelin-forming cells of the C N S , have been reported to undergo apoptosis not only in animal models (L i et al., 1996; Crowe et al., 1997; L i u et al., 1997b; Shuman et al., 1997; Yong et al., 1998; Abe et al., 1999; L i et al., 1999a; L i et al., 1999b; Springer et al., 1999; L i et al., 2000; L u et al., 2000; Springer et al., 2000; Casha et al., 2001; Warden et al., 2001; Beattie et al., 2002b; Koda et al., 2002; Dong et al., 2003; McBr ide et al., 2003; Abe et al., 2004; Stirling et a l , 2004; Wang et al., 2004b), but also following human SCI (Emery et al., 1998). The apoptotic death of oligodendrocytes is thought to contribute to the chronic demyelination of spared axons and the consequential conduction block may cause further functional loss after SCI (Gledhil l et al., 1973; Blight, 1983; Blight and Decrescito, 1986; Blight, 1992; Waxman, 1992; Bunge et al., 1993). Recently, Beattie and colleagues reported approximately 50% of oligodendrocytes are lost at 8 days post-SCI in the rat with significant reductions existing to 42 days post-injury (latest time point examined) (Beattie et al., 2002b). Since oligodendrocyte apoptosis after SCI is temporally and spatially correlated with the actual loss of oligodendrocytes, the authors concluded that apoptosis must be an important mechanism leading to the death of these cells after SCI (Beattie et al., 2002b). 36 Although, the exact cause of oligodendrocyte apoptosis after SCI remains unknown, recent studies have suggested that the loss of axonal-derived trophic support induces their death after injury (Crowe et al., 1997; Warden et al., 2001). Although oligodendrocytes require axon-derived signals for survival during development (Barres et al., 1992) it is less clear for adult oligodendrocytes. Transection of the optic nerve in adult rats resulted in less oligodendrocyte death within areas of complete axonal degeneration compared to the developing optic nerve and argues against the loss of axonal trophic-support as a mechanism that induces oligodendrocyte apoptosis following injury (Ludwin, 1990). In addition, many oligodendrocytes survive for up to 6 weeks (longest survival period examined) within the degenerating white matter tracts following a cervical (C7) dorsal column transection in adult rat (personal observations). In addition to loss of axonal trophic support, microglia/ macrophage activation and subsequent release of cytotoxic factors, may induce their death (Shuman et al., 1997). For example, mature oligodendrocytes are sensitive to glutamate excitotoxicity via AMPA/Ka ina te receptors (Yoshioka et al., 1996; L i and Stys, 2000; Beattie et al., 2002a; Park et al., 2004; X u et al., 2004a) and antagonists to AMPA/Ka ina te receptors reduced Kainate-induced excitotoxic death in mature rats (Park et al., 2004). Microglia/macrophages produce glutamate following injury and treatment with N B Q X , an antagonist of Ka ina te /AMPA channels, reduced oligodendrocyte death after experimental autoimmune encephalitis (EAE) , an experimental model of multiple sclerosis (Werner et al., 2000). Microglia/macrophages are thought to produce large amounts of T N F - a , and transgenic mice that overexpress T N F - a within the C N S , spontaneously develop a chronic demyelinating disease (Probert et al., 1995). Recent studies provide evidence that T N F - a injected into the spinal cord potentiates glutamate-mediated neuronal cell death (Hermann et al., 2001). In addition, Beattie and colleagues show pro-nerve 37 growth factor (pNGF) -mediated activation of p75 causes oligodendrocyte apoptosis following SCI (Beattie et al., 2002b). Since activated microglia/macrophages produce TNF-oc, N G F and glutamate after C N S trauma, they may cause the remote oligodendrocyte cell death observed after SCI (Bethea, 2000). This "bystander effect" leading to the death of oligodendrocytes may explain the chronic demyelination observed in areas where axons are left intact after SCI (Blight, 1985, 1992). Minocycl ine, a 2nd generation tetracycline, may be an attractive candidate in the treatment of many neurodegenerative and trauma-induced C N S injuries due to both anti-inflammatory and neuroprotective properties. Collectively, minocycline's biological effects include inhibition of microglial activation, reduction of m R N A of both I L - i p and inducible nitric oxide synthase ( iNOS) (Yrjanheikki et al., 1998), cyclooxygenase-2 expression and prostaglandin E2 production (Yrjanheikki et al., 1999). Minocycl ine has also been shown to attenuate production of matrix metalloproteinases (MMPs) and decrease T lymphocyte transmigration (Brundula et al., 2002; Power et al., 2003). In addition, minocycline has been shown to inhibit caspase expression (Chen et al., 2000), cytochrome-c release (Zhu et al., 2002) and caspase-dependent and caspase-independent cell death (Wang et al., 2003). Recently, minocycline has been shown to improve functional outcome, reduce lesion size, reduce cell death, and alter cytokine expression following SCI (Wells et al., 2003; Lee et al., 2003). Since minocycline inhibits apoptosis through caspase-dependent mechanisms, I hypothesized that minocycline treatment would prevent the "bystander death" of oligodendrocytes after SCI. Specifically, I util ized a dorsal column transection model at the cervical (C) 7 level of the spinal cord to examine the effects of minocycline treatment on delayed oligodendrocyte death within the distal degenerating and proximal dorsal columns. The results 38 indicate that SCI induces a massive apoptotic response within both the proximal and distal regions of the dorsal columns at both 7 and 14 days post-injury, microglia are the predominant cell type to undergo apoptosis, and minocycline treatment effectively attenuates apoptosis even when delayed up to 30 minutes post-injury. MATERIALS AND METHODS Cervical spinal cord dorsal column transection and corticospinal tract tracing A l l experiments were conducted in accordance with the University of Brit ish Columbia Animal Care ethics committee adhering to guidelines of the Canadian Counci l on Animal Care. Adult Wistar rats were anesthetized with an intraperitoneal injection of a mixture of ketamine hydrochloride (72 mg/kg; B imeda-MTC, Cambridge, ON) and xylazine hydrochloride (9 mg/kg; Bayer Inc., Etobicoke, ON) . The animals were placed in a stereotaxic frame and a laminectomy was performed at the 7th cervical vertebra. A n adjustable wire knife model 120 (David K o p f Instruments, Tujunga, C A ) was util ized to minimize the variability in depth of lesion. The knife was lowered to the dura mater 1 mm lateral to midline on the animal's right side. Fol lowing a pre-puncture of the dura mater with a fine needle, the wire knife was lowered 1.1 mm into the dorsal horn between the dorsal roots of C7/C8. The wire knife blade (3 mm curvature length) was extended to a further depth of 0.5 mm (total 1.6 mm) and horizontally to a total diameter of 1.9 to 2 mm. The wire knife was drawn up while gently pushing the dorsal columns down with a cotton swab. This severs most dorsal column axons, but it does not sever the dura except at its point of entry and its extreme distal end that points upwards. The wire knife was removed and a pair of microscissors was introduced into the two small holes produced by the wire knife in order to cut the dura and dorsal vein and to ensure complete transection of the dorsal columns and central canal. Eff icacy of the lesion was confirmed with histology at the end of the study. 39 Two weeks prior to the dorsal column transection, rats were injected with the anterograde tracer biotin dextran amine ( B D A , Molecular Probes, Eugene, OR) in order to visualize the corticospinal tract (CST) both proximal and distal to lesion (see Figure 2.1). A burr hole overlying the right side of the cortex was made to access the cell bodies of the C S T . To effectively label the C S T , 200 nl of (25%) B D A was injected into each of 8 sites spanning the rat sensorimotor cortex using a glass micropipette fitted to a Hamilton syringe. Minocycline administration To test the effects of minocycline treatment on reducing apoptosis after SCI , additional rats were randomly assigned into several treatment groups and were treated twice per day for two days post-injury (six injections in total) with intraperitoneal injections of either saline or 50 mg/kg minocycline (Apotex Inc., Weston, ON) in saline (non buffered), beginning 30 minutes post-injury. There was a minor grooming response at the site of injection in some animals lasting a few seconds. Otherwise these treatments were wel l tolerated. Preparation of tissue At either 1, 7, 14, 28, and 35 days post-injury, animals were anesthetized with a lethal dose of chloral hydrate ( B D H Inc., Toronto, ON) , perfused with phosphate buffered saline (PBS), followed by perfusion fixation with a solution of 4% paraformaldehyde in P B S . The spinal cords (brain-stem to midthoracic region) were removed and post-fixed in 4% paraformaldehyde overnight and subsequently cryoprotected in 30% sucrose. The spinal cords were then cut into 3 blocks, frozen and stored at -80°C until sectioned. For the spatial and temporal expression studies, a 10 mm lesion block, centered at the lesion site, was used to assess cell death within the lesion area. A rostral block (+15 mm to +5 mm) and caudal block (-5 mm to -15 mm) was used to assess remote apoptotic cell death. For the minocycline or saline treated 40 A B C7/8 AST CST +7 +3 distal AST proximal CST proximal AST Figure 2.1 A schematic diagram of the dorsal column transection (DCT) model. A, The dorsal columns consist of the descending corticospinal tract (CST) located ventral to the dorsal ascending sensory tracts (AST). Fourteen days prior to a C7 dorsal column transection, the left CST was labeled with the anterograde tracer BDA. B, Following transection of the dorsal columns, the distal segments of the AST and CST degenerate albwing for comparison of oligodendrocyte death within degenerating and proximal segments after injury and analysis of CST dieback. DRG, dorsal root ganglion. 41 animals, a rostral block (+7 mm to +3 mm) and caudal block (-3 mm to -7 mm) was used to assess remote apoptotic cell death based on results from the spatial and temporal studies that showed peak numbers of apoptotic cells within these regions at 7 and 14 days post-injury. Blocks were cryosectioned in the sagittal plane at a thickness of 10 urn and tissue sections were collected on Superfrost slides (Fisher Scientific, Houston, T X ) organized into five adjacent section series. Immunohistochemistry Fluorescent immunohistochemistry was performed on slides containing sections of 1, 7, 14, 28, or 35 day post-injured spinal cords. In general, slides were rinsed three times in P B S , and blocked in 10 % normal goat serum in P B S for 30 minutes at room temperature. To detect active-caspase-3 positive apoptotic cells within the remote distal and proximal dorsal A S T and C S T , slides were incubated with a rabbit monoclonal antibody for active caspase-3 (1:500, B D Pharmingen, San Diego, C A ) . Fol lowing three washes in P B S , slides were incubated for one hour at room temperature with the A lexa Fluor 488 goat anti-rabbit secondary antibody (1:200, Molecular Probes, Inc, Eugene, OR) and Cy3-conjugated Streptavidin (Jackson ImmunoResearch, West Grove, P A ) to visualize active caspase-3 and the BDA- labeled C S T , respectively. Slides were then washed three times in P B S and submerged in Hoechst 33258 (1 jxg/ml, Sigma Aldr ich Canada Ltd., Oakvil le, ON) to detect condensed nuclei indicative of apoptosis. To assess cell identity of apoptotic profiles, the following mouse primary antibodies were used: A P C (Ab-7; 1:200, Oncogene Research Products, Boston, M A ) for oligodendrocyte cell bodies, G F A P (1:1000, Sigma Aldrich) for astrocytes, 0 X 4 2 (1:500, Serotec, Oxford, U K ) for microglia/macrophages, and E D I (1:500, Serotec) for activated microglia/macrophages. A Cy3-conjugated donkey anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories 42 Inc, West Grove, P A ) or A lexa Fluor 488 goat anti-mouse secondary antibody (1:200, Molecular Probes) was used to visualize the mouse primary antibodies listed above. Cell counts To determine the number of apoptotic profiles within the distal degenerating and proximal segments remote from injury, active-caspase-3 positive cells with a condensed nucleus were counted. Tracing the C S T with B D A prior to lesion allowed us to discriminate the ascending sensory tracts (AST) from the CST. Ut i l iz ing the central canal as a reference point, 10 um thick sagittal sections (3 sections approximately 50 um apart) from either the rostral, central or caudal block were assessed and the amount of apoptosis was expressed as the mean number of active-caspase-3 positive profiles with condensed nuclei per 10 tim section. Ce l l counts of active-caspase-3 positive oligodendrocytes and microglia were expressed in a similar manner. Counts were performed blind with respect to treatment util izing a Zeiss Axioskop microscope. Three sections from 2-3 animals (spatial and temporal studies) or 4-6 animals (minocycline studies) in each group per time point were analyzed. Statistics Statistical analysis was performed using SigmaStat software (SPSS Inc.). Data were compared between groups using a Student's t-test. In the case of unequal variances, the Mann-Whitney Rank Sum test was used. Differences with a P value less than 0.05 were considered significant. R E S U L T S Spatial and temporal expression of active caspase-3-positive profiles within both proximal and degenerating white matter after C7 dorsal column transection. I chose a dorsal column transection model to compare cell death within both proximal and distal regions of two wel l defined systems, the descending dorsal corticospinal tract (CST) 43 and ascending sensory tracts (AST), namely the cuneate and gracile funiculi (see Figure 2.\A,B). Thus, the surgery selectively and completely transects all dorsal column tracts allowing for a comparative study between two defined systems and their response to injury. It also facilitates a comparison of the apoptotic response within proximal (closest to cell body) segment of the tract vs. the distal segment of the tract that undergoes complete degeneration. If the degeneration of cut axons and subsequent loss of axonal-derived trophic support triggers oligodendrocyte apoptosis, then oligodendrocyte apoptosis would not be expected to occur in the proximal segments after injury as the axons are severed but remain intact. Thus, the appearance of oligodendrocyte apoptosis within the proximal regions would indicate that other mechanisms may have caused their death. A previous study utilizing a similar injury model at the thoracic level reported numerous T U N E L positive glia within degenerating and proximal (to a lesser extent) segments of the A S T and C S T , and identified a subpopulation of the dying cells as oligodendrocytes (Warden et al., 2001). Based on results from this study, I examined death of oligodendrocytes within the proximal and distal regions of the A S T and C S T remote from a cervical lesion, a region not previously studied using this model in mature rodents. Pre-labeling of the right motor cortex with the anterograde tracer biotinylated-dextrine amine (BDA) allowed me to discriminate the proximal and distal segments of the C S T from the proximal and distal segments of the A S T following injury (see Figure 2.2A,G). A role for caspase-3-mediated oligodendrocyte apoptosis after SCI has been shown in rat, mouse, chick and humans (Emery et al., 1998; Springer et al., 1999; Beattie et al., 2002; McBride et al., 2003; Dong et al., 2003). Previously, our group has shown that large numbers of oligodendrocytes undergo apoptosis following transection of the chick spinal cord, were 44 immunopositive for active-caspase-3, and application of caspase inhibitors attenuated SCI-induced apoptosis (McBride et al., 2003). I therefore assessed active cell death utilizing immunohistochemistry for active caspase-3 and Hoechst 33258 stain to visualize nuclear condensation, a hallmark of apoptosis. Active-caspase-3 positive profiles with a condensed nucleus were rarely seen within the uninjured spinal cord, however, their number greatly increased overtime after SCI. As shown in Figure 2.2, active caspase-3-positive profiles with a condensed nucleus were located within the CST (Figure 2.2B-F) and AST (Figure 2.2H-L). The peak of apoptosis within the proximal and distal AST occurred within 7 to 14 days post-injury (Figure 2.3^4), whereas, fewer apoptotic profiles were present, and the peak of cell death was delayed within the CST at 28 days post-injury (Figure 2.35). Visually, under low magnification, active caspase-3 positive profiles were concentrated adjacent to the lesion site and gradually tapered off remote to injury. Therefore, based on the results of the spatial and temporal expression studies, I narrowed future analysis of cell death to the proximal and distal regions (3 mm to 7 mm remote to lesion) of the AST at the determined peak of apoptosis within these regions (7 & 14 days). Active caspase-3-mediated apoptosis of oligodendrocytes and microglia within both proximal and degenerating white matter after C7 dorsal column transection. Utilizing cell specific markers CC1 and 0X42,1 confirmed that the majority of these apoptotic profiles were oligodendrocytes (Figure 2AA-D and/) and microglia/macrophages (Figure 2 AE-H and J), respectively. No apoptotic astrocytes (GFAP) were observed (data not shown). To ensure microglia and oligodendrocytes were truly apoptotic and did not represent apoptotic bodies contained within a phagocyte which would possess a normal, nucleus as well, I used confocal microscopy to assess the nucleus within the entire boundaries of the cell. 45 Si -* Figure 2.2 Active caspase-3 positive profiles within both proximal and distal segments of the CST and AST after SCI. The dorsal funiculus 5 mm rostral (A-F) or caudal (G-L) to the injury site, 14 days post-lesion. The BDA labeled CST is easily distinguished from the adjacent ascending fibers allowing for analysis of apoptotic profiles within the CST or AST. B, Most CST fibers are intact, but some degenerate (arrowheads). C-F, Boxed area in (B), An active caspase-3(+) profile (D, arrow) with a condensed nucleus (E, arrow). F, Merged image of C-E. Caudal to the injury site, the CST completely degenerates (G). Apoptotic profiles are found within both the degenerating CST and within the proximal segment of the ascending fibers (boxed area in H). I-L, Boxed area in (H), showing an active caspase-3(+) profile (J) with a condensed nucleus (/T.arrow). L, Merged image ofl-K. Scale bars: A,G„ 100 um; B,H, 25 um; C-F, I-L, 10 um. 46 • rostral (15 mm to 5 mm) • epicenter (5 mm to -5 mm) • caudal (-5 mm to -15 mm) 28 d 35 d B • rostral (15 mm to 5 mm) • epicenter (5 mm to -5 mm) • caudal (-5 mm to -15 mm) control 1 d 7 d 14 d 28 d 35 d Figure 2.3 Spatial and temporal expression of active caspase-3 positive profiles following a C7 dorsal column transection. Quantification of apoptotic profiles (active caspase-3 positive with a condensed nucleus) over time within the AST (A) and the CST (B). Data represent the mean number of apoptotic profiles per 10 um section ± S E M , n=2-3 animals per time point. 47 C ' ' I'1 K IJ Figure 2.4 Cellular localization of active caspase-3 profiles after SCI. (A-D) CC1 immunohistochemistry (A) reveals several oligodendrocytes undergoing apoptosis (B active caspase-3, C Hoechst) within the degenerating AST, 7 days post-injury (D, triple filter image). {E-H), OX42 immunoreactivity (£) reveals apoptotic microglia/macrophages were also present within the degenerating AST, (F) active caspase-3, (G, Hoechst), (H, triple filter image). I,J, Representative merged confocal images of CC1(+) (/, arrow) or OX42(+) (J, arrow),active caspase-3(+) profiles with (/', J') or without a condensed nucleus (/, arrowhead). K-M, Numerous active caspase-3(+) profiles (green) are present within the proximal (AT) and distal (L) AST at 7 days post-lesion. Minocycline treatment greatly reduces the number of active caspase-3(+) profiles within both the proximal and distal AST (M, distal AST). Scale bars: A-D, 25 um ; E-H, 10 um; K - M , 100 \im Examples of apoptotic nuclei (Figure 2.4/') within the boundaries of an oligodendrocyte are shown in Figure 2.4/ and within a microglial/macrophage in Figure 2AJ,J' confirming their apoptotic phenotype. Approximately 10-15% of apoptotic profiles were oligodendrocytes (CC1 positive) whereas the majority of apoptotic profiles (-65-70%) were of microglial/macrophage origin (0X42 positive) (Figure 2.5,4). Effect of minocycline treatment on active caspase-3-mediated oligodendrocyte death. To test the efficacy of minocycline treatment in attenuating the apoptotic response after SCI, apoptotic profiles were counted within both proximal and distal segments of the AST remote from a C7 dorsal column transection site and compared between saline treated versus minocycline treated groups (Figure 2.55). The experimental paradigm (above) was repeated except the amount of tissue analyzed was reduced to a rostral (+3 mm to +7 mm) and caudal (-3 mm to -7 mm) block as the majority of apoptotic profiles were located in these regions at the peak of SCI-induced apoptosis (7 to 14 days post-injury)(Figure 2AK,L). In the untreated controls there was significantly less apoptotic profiles within the proximal segment compared to the distal segment within the AST at both 7 and 14 days after injury (7d, P < 0.001; 14d, P < 0.01)(Figure 2.55 ). Minocycline treatment significantly reduced the number of apoptotic profiles within the proximal AST (32.7 ± 2.7; mean ± SEM) vs saline treated animals (56.1 ± 3.9; P < 0.001) at 7 days post-injury (Figure 2.4M and 2.5B). Within the degenerating (distal) AST, minocycline treatment also reduced the mean number of apoptotic profiles (47.9 ± 4.0) when compared to saline treatment (78.7 ± 3.5; P < 0.001)(Figures 2.4Mand 2.55). Minocycline treatment continued to prevent cell death at 14 days post-lesion within both proximal (minocycline, 19.8 ± 3.3 vs saline, 37.6 ± 3.8; P < 0.01) and distal (minocycline, 36.8 ±4 .1 vs. saline, 58.2 ± 5.0; P < 0.01) segments (Figure 2.55). These results suggest that minocycline 49 treatment continued for 2 days after injury is sufficient to reduce secondary cell death by approximately 39% (distal) and 42% (proximal) at 7 days and by 37% (distal) and 47% (proximal) at 14 days after injury. Utilizing triple labeling with CC1 as a marker for oligodendrocytes combined with the two apoptotic markers used above, we counted the number of apoptotic oligodendrocytes from minocycline or saline treated rats. Minocycline significantly reduced the mean number of apoptotic oligodendrocytes within both the proximal (minocycline, 3.9 ± 0.5 vs. saline, 6.2 ± 0.6; P < 0.01) and distal (minocycline, 4.8 ± 0.8 vs. saline, 7.9 ± 0.8; P < 0.01) segments of the AST at 7 days post-injury compared to saline treated animals (Figure 2.5Q. At 14 days post-lesion, only the proximal segment from minocycline treated rats (4.6 ± 0.7) had significantly less apoptotic oligodendrocytes compared to saline treated animals (7.4 ± 0.9; P < 0.05). Interestingly, in the untreated group, the number of oligodendrocytes in the distal segments at both time points was only marginally higher than their proximal counterparts, but did not reach significance (7d, 14d, P > 0.05)(Figure 2.5C). DISCUSSION My findings revealed large numbers of active caspase-3 positive apoptotic profiles located within both degenerating and proximal segments of the AST remote to lesion. Systemic minocycline treatment significantly attenuated the number of apoptotic profiles, including oligodendrocytes within both degenerating and proximal segments. These results are in agreement with a recent study that assessed the effects of minocycline treatment on cell death at the lesion site, following SCI (Lee et al., 2003). 50 A 90 .0X42 C 1 2 distai 1 proximal ' distal 1 proximal 7d 14d Figure 2.5 Minocycline treatment inhibits dorsal column transection-induced glial cell death within the distal and proximal AST. A, Percentage of apoptotic microglia/macrophages (OX42 (+)) or oligodendrocytes (CC1(+)) within the proximal and distal AST, 7 days post-lesion (n = 6/group). B, Minocycline treatment significantly reduced the mean number of apoptotic profiles per 10 um section ± SEM within distal and proximal segments of the AST at both 7 and 14 days post-injury compared to saline treated controls. (*P < 0.05, **P < 0.01, ***P < 0.001 ; 7d n = 6/group, 14d n = 4-5/group). C, Apoptotic oligodendrocytes (CC1(+)), active caspase-3(+) profiles with a condensed nucleus were significantly decreased in the minocycline treated group compared to saline controls within the proximal AST at both 7 and 14 days post-injury. However, only the proximal segment of the AST from minocycline treated animals contained significantly fewer apoptotic oligodendrocytes at 14 days post-injury. There was no difference between the mean number of apoptotic oligodendrocytes within the proximal vs. distal segments of the AST at both 7 and 14 days post-injury (data represent mean ± SEM, *P < 0.05 ; 7d n = 5/group, 14d n = 4-5/group). Quantification of the mean number of apoptotic profiles within the distal and proximal segments of the AST. 51 Surprisingly, microglia/macrophages were found to be the principle cell type to undergo apoptosis after dorsal column transection. At seven days post-injury, approximately 70% of apoptotic profiles within the proximal and degenerating AST were positive for 0X42, a cell specific marker of microglia/macrophages, whereas only -11 % were identified as oligodendrocytes (CC1 immunopositive). My results are in agreement with previous studies that found large numbers of apoptotic microglia are present after contusion SCI in rats (Shuman et al., 1997; Yong et al., 1998). However, other studies have reported that microglia/macrophages were not among the apoptotic profiles present after partial or complete transection injuries in rat (Abe et al., 1999; Warden et al., 2001). Differences in apoptotic detection methods (TUNEL vs. active caspase-3), microglial/macrophage markers (0X42 vs. BS1 or 0X6), or injury paradigms may partially account for the discrepancy between studies. I could not entirely rule out the possibility that phagocytes engulfing apoptotic cells may be included in the counts of apoptotic microglia/macrophages thereby inflating their number. In an attempt to limit this problem, confocal microscopy was used to visualize the entire boundary of the cell. Of the approximately twelve randomly selected cells analyzed in this manner, all displayed an apoptotic nucleus within an active caspase-3(+) profile, confirming their apoptotic phenotype. Previously, it has been shown that activation and proliferation of microglia within the ventral grey matter after sciatic nerve injury is followed by the elimination of these cells via apoptosis (Gehrmann and Banati, 1995). Since minocycline treatment reduces both microglial/macrophage activation and proliferation (Yrjanheikki et al., 1998; Tikka and Koistinaho, 2001; Tikka et al., 2001), a resultant decrease in microglial/macrophage apoptosis would be expected as well. In addition, minocycline has been shown to inhibit caspase-dependant cell death (Chen et al., 2000; Wang et al., 2003; Lee et al., 2003). Therefore, the 52 reduction in microglial/macrophage cell death following minocycline treatment in this study is probably due to a direct effect on microglial/macrophage activation as well as their death. Interestingly, apoptotic oligodendrocytes were present at the same levels in both proximal (beyond the level of axonal dieback) and degenerating segments within the transected dorsal columns, suggesting that lack of axonal-derived trophic support may not be the only mechanism to induce oligodendrocyte apoptosis after SCI. These results are in contrast to previous studies that showed apoptotic profiles are predominantly localized within white matter tracts undergoing Wallerian degeneration (Crowe et al., 1997; Shuman et al., 1997; Abe et al., 1999; Casha et al., 2001; Warden et al., 2001). However, these studies did not directly assess oligodendrocyte apoptosis within distinguished boundaries of degenerating and intact tracts within the injured dorsal columns. In the present study, I applied the anterograde tracer B D A to label the CST previous to injury. This approach facilitates a clear distinction between the boundaries of the AST and CST, and allowed the precise localization of apoptotic profiles, including oligodendrocytes, to these two tracts. We cannot rule out the possibility that some of the proximal fibers degenerate after injury and, therefore, may induce oligodendrocyte apoptosis. However, large numbers of ascending fibers and CST fibers are present within their proximal stumps, suggesting oligodendrocyte apoptosis within the proximal segments remote from injury is not due to pronounced axonal degeneration. If this were the case we would not expect the same number of apoptotic oligodendrocytes within both segments as the distal segment completely degenerates. Furthermore, oligodendrocyte apoptosis has been demonstrated to occur within areas of intact axons following SCI (Li et al., 1999a), and in demyelinated regions in MS (Vartanian et al., 1995; Akassoglou et al., 1998; Hisahara et a l , 2000). 53 Regardless of the precise stimuli/us that induces oligodendrocyte apoptosis, its prevention may prevent the chronic demyelination observed in areas where axons are left intact after SCI and promote functional improvement (Blight and Decrescito, 1986; Blight, 1991; Bunge et al., 1993; Waxman, 1993; Werner et al., 2000). 54 CHAPTER 3 MINOCYCLINE TREATMENT REDUCES MICROGLIAL/MACROPHAGE ACTIVATION, LESION SIZE, CORTICOSPINAL AXONAL DIEBACK AND IMPROVES FUNCTIONAL OUTCOME FOLLOWING SPINAL CORD INJURY 55 S U M M A R Y Minocycline has been demonstrated to be neuroprotective after spinal cord injury (SCI). However, the cellular consequences of minocycline treatment on the secondary injury response are poorly understood. I examined minocycline's ability to reduce microglial/macrophage activation, corticospinal tract (CST) dieback, lesion size and to improve functional outcome after SCI. Minocycline or vehicle control was injected into the intraperitoneal cavity 30 minutes and 8 hours after SCI and thereafter twice daily for 2 days. EDI positive microglial/macrophage density was reduced remote to the lesion as well as at the lesion site. Both CST dieback as well as lesion size was diminished following minocycline treatment. Footprint analysis revealed improved functional outcome after minocycline treatment. Thus, minocycline ameliorates multiple secondary events after SCI, rendering this clinically used drug an attractive candidate for SCI treatment trials. 56 I N T R O D U C T I O N The loss of function after spinal cord injury (SCI) results from both the primary mechanical insult and the subsequent, multi-faceted secondary degenerative response (Dumont et al., 2001; Sekhon and Fehlings, 2001; McDonald and Sadowsky, 2002). The secondary degenerative response spreads from the site of impact, causing demyelination, axonal injury, and destruction to neuronal tissue that survived the initial insult. The secondary degenerative response also involves glutamate excitotoxicity, ionic dysregulation, free radical generation, and ischemia (Balentine and Spector, 1977; Young and Flamm, 1982; Panter et al., 1990; Choi, 1992; Wrathall et al., 1994). A massive inflammatory response results from the release of cytokines and chemokines from resident and blood-derived inflammatory cells, which may further aggravate cellular and axonal damage (Popovich, 2000; Bethea, 2000; Hausmann, 2003). While the controlled activation of macrophages and immune cells may be of benefit at some stage of SCI (Rapalino et al., 1998; David, 2002), other studies have shown that activated macrophages (CNS resident microglia and/or blood-derived macrophages), and cytotoxic products associated with their activation, contribute to the secondary damage after SCI (Popovich, 2000). Depletion of peripheral monocytes/macrophages reduced the loss of myelinated axons in a guinea-pig model of SCI (Blight, 1994), and similarly in rats, hematogeneous macrophage depletion reduced activated macrophages in the injured cord, improved hindlimb recovery, increased sparing of myelinated axons and reduced cavitation after SCI (Popovich et al., 1999). In agreement, zymosan, a potent activator of microglia/macrophages caused cavitation, demyelination and permanent axonal injury when injected into the spinal cord (Popovich et al., 2002). Furthermore, fibroblasts expressing a potent version of the inflammatory cytokine IL-6 (Hyper-IL-6), transplanted into the spinal cord injury site, 57 dramatically augmented inflammatory cell invasion, increased lesion size and reduced axonal sprouting after SCI (Lacroix et al., 2002). Collectively these studies provide compelling evidence of the deleterious potential associated with the activation of microglia and invasion of hematogenous macrophages following SCI. Minocycline, a 2nd generation tetracycline, has been shown to reduce microglia/ macrophage activation and release of potential cytotoxic factors (Yrjanheikki et al., 1998; Yrjanheikki et al., 1999). For example, minocycline treatment reduced mRNA of both IL- ip and inducible nitric oxide synthase (iNOS) (Yrjanheikki et al., 1998), cyclooxygenase-2 expression and prostaglandin E2 production (Yrjanheikki et al., 1999). Minocycline has also been shown to attenuate production of matrix metalloproteinases (MMPs) and decrease T lymphocyte transmigration (Brundula et al., 2002; Power et al., 2003). Since many of the above factors are toxic to oligodendrocytes and neurons, preventing microglial/macrophage activation may be neuroprotective after SCI. Recently, minocycline has been shown to improve functional outcome, reduce lesion size, reduce cell death, and alter cytokine expression following SCI (Lee et al., 2003; Wells et al., 2003a). In a murine model of SCI, minocycline treatment was superior to methylprednisolone, the only treatment available to SCI patients, in promoting functional improvement (Wells et al., 2003a). However, the cellular and molecular basis for minocycline's neuroprotective effects remains largely unknown. I therefore sought to elucidate the cellular consequences of minocycline treatment and how it leads to attenuation of the pathophysiological response after SCI. Specifically, I utilized a dorsal column transection model at the C7 level of the spinal cord to examine the effects of minocycline treatment on the microglial/macrophage response, CST dieback, lesion size and functional outcome following SCI. The results indicate 58 that minocycline influences several aspects of the secondary degenerative response, a characteristic that is likely responsible for its ability to promote improved functional outcome after SCI. M A T E R I A L S AND M E T H O D S Spinal cord dorsal column transection and corticospinal tract tracing A l l experiments were conducted in accordance with the University of British Columbia Animal Care ethics committee adhering to guidelines of the Canadian Council on Animal Care. Adult Wistar rats were anesthetized with an intraperitoneal injection of a mixture of ketamine hydrochloride (72 mg/kg; Bimeda-MTC, Cambridge, ON) and xylazine hydrochloride (9 mg/kg; Bayer Inc., Etobicoke, ON). The animals were placed in a stereotaxic frame and a laminectomy was performed at the 7th cervical vertebra. An adjustable wire knife model 120 (David Kopf Instruments, Tujunga, C A) was utilized to minimize the variability in depth of lesion. The knife was lowered to the dura mater 1 mm lateral to midline on the animal's right side. Following a pre-puncture of the dura mater with a fine needle, the wire knife was lowered 1.1 mm into the dorsal horn between the dorsal roots of C7/C8. The wire knife blade (3 mm curvature length) was extended to a further depth of 0.5 mm (total 1.6 mm) and horizontally to a total diameter of 1.9 to 2 mm. The wire knife was drawn up while gently pushing the dorsal columns down with a cotton swab. This severs most dorsal column axons, but it does not sever the dura except at its point of entry and its extreme distal end that points upwards. The wire knife was removed and a pair of microscissors was introduced into the two small holes produced by the wire knife in order to cut the dura and dorsal vein and to ensure complete transection of the dorsal columns and central canal. Efficacy of the lesion was confirmed with histology at the end of the study. Two weeks prior to the dorsal column transection, rats were injected with the anterograde tracer biotin 59 dextran amine (BDA, Molecular Probes, Eugene, OR) in order to visualize the corticospinal tract (CST) both proximal and distal to lesion and to assess CST dieback. A burr hole overlying the right side of the cortex was made to access the cell bodies of the CST. To effectively label the CST, 200 nl of (25%) B D A was injected into each of 8 sites spanning the rat sensorimotor cortex using a glass micropipette fitted to a Hamilton syringe. Minocycline administration Rats were randomly assigned into two treatment groups and were treated twice per day for two days with intraperitoneal injections of either saline or 50 mg/kg minocycline (Apotex Inc., Weston, ON) in 1.0 ml of saline (non buffered), beginning 30 minutes post-injury. There was a minor grooming response at the site of injection in some animals lasting a few seconds. Otherwise these treatments were well tolerated. Preparation of tissue At 7 and 14 days post-injury, animals were anesthetized with a lethal dose of chloral hydrate (BDH Inc., Toronto, ON), perfused with phosphate buffered saline (PBS), followed by perfusion fixation with a solution of 4% paraformaldehyde in PBS. The spinal cords (brain-stem to midthoracic region) were removed and post-fixed in 4% paraformaldehyde overnight and subsequently cryoprotected in 30% sucrose. The spinal cords were then cut into 3 blocks, frozen and stored at -80°C until sectioned. Specifically, a 6 mm lesion block, centered at the lesion site, was used to assess lesion size, CST dieback, and EDI density (% area). A rostral block (+7 mm to +3 mm) and caudal block (-3 mm to -7 mm) was used to assess EDI density (% area). Blocks were cryosectioned in the sagittal plane at a thickness of 14 um (lesion studies) and tissue sections were collected on Superfrost slides (Fisher Scientific, Houston, TX) organized into five adjacent section series. 60 Immunohistochemistry Fluorescent immunohistochemistry was performed on slides containing sections of 7 day or 14 day post-injured spinal cords. In general, slides were rinsed three times in PBS, and blocked in 10 % normal goat serum in PBS for 30 minutes at room temperature. Slides were then incubated with primary antibodies specific for either astrocytes (GFAP, 1:1000, Sigma Aldrich) or for activated microglia/macrophages (EDI, 1:500, Serotec, Oxford, UK) . Following three washes in PBS, slides were incubated for one hour at room temperature with the Alexa Fluor 488 goat anti-mouse secondary antibody (1:200, Molecular Probes, Inc, Eugene, OR) and Cy3-conjugated Streptavidin (Jackson ImmunoResearch, West Grove, PA) to visualize either astrocytes or macrophages and the BDA-labeled CST, respectively. Slides were then washed three times in PBS and submerged in Hoechst 33258 (1 Lig/ml, Sigma Aldrich Canada Ltd., Oakville, ON), rinsed and mounted. Microglial/macrophage activation/recruitment To assess microglial/macrophage activation/recruitment within the corticospinal or ascending sensory tracts 3 to 3.5 mm remote from lesion, an overlay of three rectangular boxes (dimensions of each box: 100 um width x 500 |im length) was placed onto digitally captured sagittal sections containing B D A labeled axons (red channel) using SigmaScan Pro software (SPSS, Chicago, IL). With regards to the placement of the rectangles, the dorsal most edge of the CST/AST interface is set at 0 um. The CST rectangle was placed so its top edge was aligned with the dorsal edge of the CST and captured an area (50,000 um2) within the CST. The ASTioo rectangle was positioned 100 um from the dorsal edge of the labeled CST and captured an area (50,000 um2) within the adjacent AST. The AST300 rectangle was positioned 100 um from the dorsal edge of the ASTioo rectangle and captured an area (50,000 um2) 300 \im dorsal to the 61 CST. The overlay of the rectangles was then copied onto thresholded images depicting the EDI positive cells (green channel) and the percentage of the area occupied by EDI signal was reported. One or two sections from at least four animals per group were assessed. Similarly, EDI density within the proximal CST stump was quantified using the same approach with a standardized overlay box (250 um width x 750 um length) positioned within the proximal CST stump. At least two sections from five animals per group were assessed. C S T dieback To quantify CST dieback, 14 um thick sections containing the lesion site were subjected to GFAP immunohistochemistry to delineate the lesion area and Cy3-conjugated Streptavidin (Jackson ImmunoResearch) to visualize the BDA-traced CST. Digital images were captured and CST dieback was measured using SigmaScan Pro software (SPSS). At least two sections from each animal (7d, six or seven animals per group; 14d, four or five animals per group) were assessed and the mean distance of leading intact CST fibers from the center of the lesion site per 14 um section was reported. To ensure equal sampling, I used the central canal as a reference point (midline) and sampled the midline section as well as the adjacent sections within approximately 100 to 200 um area lateral to the midline respectively. Thus, all sections run through the gracile funiculus containing the axons from the hindlimbs and do not contain unlesioned cervical sensory afferents. Lesion area The digital images captured and used for CST dieback analysis (see above) were used to quantify the lesion area (delineated by GFAP immunohistochemistry). Lesion area was measured using SigmaScan Pro software (SPSS Inc.). At least two sections from each animal 62 (Id, six or seven animals per group; 14d, four or five animals per group) were assessed and the mean lesion area (mm2) per 14 urn section was reported. Footpr int analysis Walking track footprint analysis was modified from de Medinaceli et al. (de Medinaceli et al., 1982). The animal's forepaws were dipped in green dye and the hindpaws dipped in red dye (non-toxic). The rats were trained to avoid a brightly illuminated starting box by walking across a narrow wooden board (1 meter long and 7 cm wide) leading to a darkened box containing their familiar housing mates. Two marks were placed 70 cm apart, centered on the platform, to ensure the rat footprints were not analyzed during the beginning (acceleration) or ending (deceleration) of the movement. The rats were timed as they passed from mark 1 to mark 2. If the animal failed to complete the test within 1-2 seconds or paused while traversing the track, the trial was excluded from analysis, and the test repeated. Each animal (observer was blind to treatment conditions) performed one training session without paws being dipped, followed by two separate baseline sessions taken before injury and one session on 3, 5, 7, and 14 days post-injury. Each session consisted of three separate traverses of the track. The footprints were scanned and digitized images were measured using Adobe Photoshop Version 6.0 (Adobe Systems Canada, Ottawa, ON). Toe spread was measured as the distance between the first and fifth toe in the hindpaw. Inter-limb coordination was measured as the distance between the ipsilateral forepaw (center of pad) and hindpaw (center of pad). Angle of rotation was measured as the angle made by the third toe, center of the footpad, and a line parallel to the direction of travel. To assess toe spread, the maximal toe spread index (TSI) was calculated according to the Brown TSI formula (Brown et al., 1989). TSI = (ETS-NTS)/NTS 63 ETS - experimental toe spread. NTS- normal toe spread. Similar to toe spread, an index was used to calculate both inter-limb coordination and angle of rotation. At least nine footprints per side from three sessions were measured per animal per group (n=8, 7 days; n=4, 14 days) to determine the mean values of each parameter assessed. Statistics Statistical analysis was performed using SigmaStat software (SPSS Inc.). Data were compared between groups using a Student's t-test. In the case of unequal variances, the Mann-Whitney Rank Sum test was used. Differences with a P value less than 0.05 were considered significant. R E S U L T S Effect of minocycline treatment on EDI density seven days after SCI. Previously, it has been shown that the microglial/macrophage response peaks at 7 days post-SCI and is associated with demyelination and axonal degeneration (Blight, 1985; Giulian and Robertson, 1990; Blight, 1992; Popovich et al., 1997; Popovich et al., 1999). In addition, Shuman and colleagues had previously hypothesized that activated microglia may induce oligodendrocyte apoptosis within degenerating tracts after SCI (Shuman et al., 1997). I therefore assessed whether minocycline.treatment would inhibit microglial/macrophage activation in areas of oligodendrocyte apoptosis, i.e., the segments several millimeters distant to the lesion site (see chapter 2) as well as within the lesion site itself. Untreated injured animals had large numbers of EDI positive microglia/macrophages located within the degenerating distal segments of the AST and CST (Figure 2>AA,B,E,F). Treatment with minocycline significantly reduced EDI density within the distal (rostral to lesion site) degenerating segments of the AST measured from 100 um (ASTioo, minocycline, 1.6% ± 0.5; mean ± SEM, vs. saline, 6.0% ± 1.1; P < 0.01) and 300 um 64 dorsal to the CST (AST 3 0 0 , minocycline, 2.0% ± 0.3, vs. saline, 4.7% ± 0.7; P < 0.01)(Figure 3AC,G,M). Within the distal (caudal to lesion site) degenerating segment of the CST, minocycline treatment also significantly reduced EDI density (minocycline, 5.1% ± 2.1 vs. saline, 11.8% ± 1.4; P < 0.05)(Figure 3.\D,H,M). Although there was a trend towards a reduction of EDI density within the proximal segment of the AST (caudal to lesion) closest to the CST (compare Figure 3.15,Fwith 3.\D,H) and within the proximal CST (rostral to lesion) in minocycline treated rats, this failed to reach significance (Figure 3 AM). At the injury site, large numbers of microglia/macrophages were found within the proximal stump of the anterogradely traced CST, i.e. immediately adjacent to the lesion, as well as in areas of cavitation in saline treated rats (Figure 3.1I,J). This EDI density was reduced by minocycline treatment at 7 days post-injury (Figure 3.\K,L). Quantification of EDI density within the proximal CST stump confirmed the visual impression given in Figure 3.1, revealing a significant reduction in EDI density in minocycline treated animals (8.2% ± 1.6; mean ± SEM) compared to saline treated controls (19.4% ± 3.8; P < 0.01)(Figure 3.17V). Effect of minocycline treatment on CST dieback after SCI. Previously, it has been shown that following transection of the adult rat CST, the proximal axon stumps degenerate during the first few weeks after injury (Pallini et al., 1988). This extensive axonal dieback may have implications for regeneration and sprouting after injury. We measured the distance between the closest, intact, anterogradely traced CST fibers and the center of the lesion site to test i f minocycline treatment would prevent proximal CST degeneration after SCI. In agreement with earlier findings (Pallini et al., 1988), the proximal stump of the lesioned CST axons showed pronounced axonal dieback (Figure 3.2A,C,E). 65 A saline B saline C minocycline D minocycline rostral caudal rostral caudal E ED1 F G H I J f »*' — __' K L * 10. 1C« I M Rostral Caudal Figure 3.1 Minocycline treatment reduces ED1(+) (microglial/macrophage) density 7 days post-injury. A-H, ED1(+) profiles 3 mm rostral (A.C.E.G) and caudal (B,D,F,H) to injury. Minocycl ine significantly reduced E D I density within both rostral A S T , (100 and 300 um dorsal to C S T ) ( C , G , M ) , and within the caudal C S T (D,H,M) compared to saline treated animals (A,B,E,F,M). Triple immunofluorescence images of the lesion site from saline (I,J) and minocycline (K,L) treated animals. The proximal C S T , ED1(+) microglia/macrophages, Hoechst 33258. Note reduced ED1(+) signal in (AT) compared with (/). J,L, Higher magnification o f boxed area in (1,K). Less ED1(+) signal is evident within the proximal C S T o f minocycline treated animals compared to saline treated animals. N, Quantification o f the density of ED1(+) signal within the proximal C S T . Data represent mean percentage ± S E M (n=4,5/group). * P < 0.05; * * P < 0.01. Scale bars: A - H , j , L 100 um; 1,K, 500 um. 66 Following treatment with minocycline, CST dieback was significantly attenuated at both 7 and 14 days post-injury compared to saline treated animals (7d, P < 0.001; 14d, P < 0.001) (Figure 3.2B,D,E). At 7 days post-lesion, the mean distance between the proximal CST axons to the centre of the lesion site was 598.4 ± 48.6 um (mean ± SEM) in minocycline treated vs. 877.1 ± 47.7 um in saline treated animals. At 14 days post-injury, CST dieback reached 751.2 um ± 58.0 in minocycline treated animals compared to 1120.2 urn ± 80.5 saline treated controls. On average minocycline treatment reduced the distance of the leading CST axons from the injury site by -32% and 33% at 7d and 14d, respectively. Effect of minocycline treatment on lesion size after SCI. Recently, Wells and colleagues reported minocycline treatment significantly reduced lesion area after SCI in mice (Wells et al., 2003a). However, the sequelae of secondary damage following SCI in the mouse vary from that in humans (Guth et al., 1999; Inman et al., 2002). Therefore, we assessed whether minocycline treatment would reduce lesion size in our rat model of SCI. The region demarcated by activated astrocytes (determined by GFAP immunofluorescence) was used to delineate the boundaries of the lesion. Lesion area was greatly enhanced in saline (Figure 3.2A,C,F) versus minocycline treated animals (Figure 3.2B,D,F). Quantification of these results showed minocycline treatment significantly reduced lesion area at both 7 and 14 days post-injury compared to saline treated animals (7d, p < 0.01; 14d, p < 0.001)(Figure 3.2F). Improved functional outcome following minocycline treatment. To assess functional outcome after SCI, rat footprints were collected and assessed for perturbations in limb coordination, angle of rotation and toe spread. Footprints from animals during pre-surgical training revealed highly coordinated forelimb and hindlimb foot placements 67 A saline c <g> A i 1 wf^ f ... -jfT'? .'7***' • « B minocycl ine D • ^ A -f'+z^:. <- mm A • ** 0 e *** 3 e 0 i » ! 8 i 0 1 ) § i I 0 o ' M S m s 7d 14d Figure 3.2 Minocycline treatment reduces CST dieback and lesion size at both 7 & 14 days post-lesion. A,B, Images of the dorsal columns revealing the lesion site at 7 days post-injury from saline [S] (A) vs. minocycline [M] treated (B) rats. Note the lesion area (delineated by GFAP staining) and CST dieback is clearly reduced in minocycline vs. saline treated animals. C, Boxed areas in (A), (D) boxed area in (B) showing a higher magnification of the proximal BDA-traced CST axons. E, Quantification of CST die-back. Overall minocycline treatment significantly reduced CST axonal dieback by -32% and -33% at 7d and 14d respectively. F, Quantification of lesion area. Minocycline treatment significantly reduced lesion area by -34% and -43% at 7d and 14d. Data represent mean ± S E M (n=4-7/group). **P < 0.01; **P < 0.001. Scale bars: AJB 500 um; C,D 100 um. 68 (Figure 3.3,4). Following a cervical dorsal column transection, forelimb/hindlimb coordination was compromised (saline treated animals) whereas minocycline treatment significantly improved forelimb/hindlimb co-ordination at both 7 days and 14 days post-injury (P < 0.05)(Figure 3.35). Minocycline treatment also reduced the injury-induced hindlimb angle of rotation compared to saline treated animals (P < 0.05)(Figure 3.3Q. In addition, minocycline treatment significantly attenuated the SCI-induced increase in hindlimb toe spread compared to saline treated animals at 7 days after injury (toe spread index; mean ± SEM, minocycline, 0.07 ± 0.02 vs. saline, 0.13 ± 0.01; P < 0.01)(Figure 3.4). However, at 14 days there was no significant difference between treatment groups, suggesting minocycline reduced the time of the recovery process in this paradigm (Figure 3.4). DISCUSSION Here, I report that following a cervical dorsal column transection, minocycline treatment attenuated microglial/macrophage activation/recruitment, prevented corticospinal axonal dieback and reduced lesion size. In addition, minocycline treatment improved inter-limb coordination, and normalized hindpaw angle of rotation and toe spread after SCI. Several studies provide strong evidence that activated mononuclear phagocytes (CNS resident microglia and/or blood-derived macrophages) and products associated with their activation contribute to the secondary damage after SCI, including demyelination and/or axonal degeneration of previously spared axons (Blight, 1985; Giulian and Robertson, 1990; Blight, 1992; Blight, 1994; Popovich et al., 1999; Popovich et al., 2002). Following SCI in cats, demyelination of intact axons within the outer rim of the spinal cord is temporally correlated with an invasion of the CNS parenchyma by phagocytes (Blight, 1985). Treatments designed to inhibit the macrophage response after SCI, have promoted survival of motoneurons, reduced the 69 A Training 7d Minocycline »••# 7d Saline to« spread ' < t ' * ^ ^ - » anglo of rotation •« ' ft ' > » » ^ ' coordination B • saline • minocycline 3d 5d 7d 14d Figure 3.3 Minocycline treatment improves inter-limb coordination and reduces hindlimb angle of rotation after SCI. A, Representative footprints collected from saline and minocycline treated animals. Minocycline treatment improved limb coordination (B), and reduced hindlimb angle of rotation (Q at both 7 and 14 days post-injury. Indexes were calculated as (experimental value - baseline value/ baseline value). Data represent mean ± SEM (n=8/group 7d; n=4/group 14d). *P < 0.05. 70 0.18 0.16 £ 0.14 I 0.12 -o 0.10 £ 0.08 % 0.06 | 0.04 0.02 0.00 B 0.18 0.16 £ 0.14 1 0.12-1 -o 0.10 CO 2 0.08 0.06 g 0.04 0.02 0.00 • saline • minocycline Figure 3.4 Minocycline treatment reduces hindlimb toe spread at 7 days post-injury. Minocycline treatment significantly reduces left (A) and right (B) hindfoot toe spread at 7 days post injury compared to saline treated controls. However, at 14 days post-injury, there was no significant difference between treatment groups, suggesting minocycline reduced the time of the recovery process in this paradigm. 71 loss of myelinated axons, reduced cavitation and improved function after SCI (Giulian and Robertson, 1990; Blight, 1994; Popovich et al., 1999). Here, I show that minocycline treatment effectively reduced microglial/macrophage activation at the lesion site within the proximal CST stump and remote from injury within the distal segments of the dorsal column tracts. In addition, inhibition of the microglial/macrophage response was associated with reduced lesion size, reduced axonal dieback and improved functional outcome. In agreement with previous studies, minocycline treatment reduced lesion size following SCI (Lee et al., 2003; Wels et al., 2003a). Although the mechanism is unknown, lesion size is often decreased following attenuation of the inflammatory response (Bethea et al., 1999; Oudega et al., 1999; Popovich et al., 1999). Indeed treatment with the anti-inflammatory cytokine IL-10 decreases SCI-induced and monocyte/macrophage production of the pro-inflammatory cytokine TNF-a, with a concurrent decrease in lesion size (Bethea et al., 1999; Takami et al., 2002). Interestingly, minocycline treatment increased mRNA levels of the anti-inflammatory cytokine IL-10, with a concomitant decrease in the pro-inflammatory cytokine TNF-a, mirroring the neuroprotective effects of IL-10 treatment (Lee et al., 2003). However, despite a large reduction in macrophages following methylprednisolone treatment, the loss of spinal tissue and macrophage number was not correlated (Oudega et al., 1999). Further studies are needed to elucidate the mechanism/s underlying spinal tissue loss after SCI. A possible limitation to successful regeneration/plasticity of severed axons after SCI is the progressive dieback of the proximal stump that may cause additional loss of function. The mechanisms underlying axonal dieback are currently unknown. Our CST dieback data from saline treated animals are consistent with Pallini and colleagues who showed CST dieback occurs at a rate of approximately 40-90 urn per day over the first two weeks following 72 midthoracic spinal cord transection (Pallini et al., 1988). The results of the present study suggest that minocycline treatment prevents CST axonal dieback compared to saline treated animals at both 7 and 14 days post-injury. Similarly, we have also shown that minocycline treatment was effective in reducing AST dieback and microglia/macrophage density within the proximal region of the AST (McPhail et al., 2004). Interestingly, methylprednisolone treatment failed to reduce corticospinal tract dieback (Oudega et al., 1999), suggesting minocycline may be more effective in preventing proximal corticospinal tract axonal degeneration after SCI. In this study I have demonstrated reduced EDI density within the proximal CST stump in minocycline treated animals. Inhibition of the microglial/macrophage response may have prevented axonal dieback by preventing phagocytosis of axonal ends or reducing the liberation of cytotoxic or axonal repellant molecules associated with their activation. Furthermore, minocycline has been shown to reduce macrophage production of nitric oxide (Amin et al., 1997), a molecule that may be involved in axonal dieback (He et al., 2002). However, we cannot rule out a direct effect of the drug on axonal dieback. Minocycline has been shown to be a strong chelator and may sequester excess C a 2 + released after injury (Chopra and Roberts, 2001). Reduced axonal C a 2 + levels may prevent activation of calpains and preserve axonal integrity (Balentine and Spector, 1977; Ray and Banik, 2003). Future mechanistic studies are needed to elucidate the role of minocycline treatment in preventing axonal degeneration. Previously, it has been shown that minocycline treatment improved functional outcome following SCI (Lee et al., 2003; Wells et al., 2003a). Furthermore, the murine study showed that minocycline was more effective in improving functional outcome when compared to methylprednisolone, the current treatment option to patients suffering SCI (Wells et al., 2003a). We extend these results to show that minocycline improved inter-limb coordination, and reduced 73 both hindpaw angle of rotation and toe spread after SCI. From the results of the present study, I conclude that minocycline's positive effects on the improved functional outcome following SCI result from its multifaceted impact on the secondary degenerative response. Minocycline's neuroprotective properties combined with its proven safety record in humans and animals suggests its efficacy as an effective treatment option following human SCI and justifies further examination. 74 CHAPTER 4 MINOCYCLINE TREATMENT REDUCES P38 MAPK EXPRESSION AND MICROGLIAL/ MACROPHAGE ACTIVATION FOLLOWING SCI 75 S U M M A R Y Minocycline is neuroprotective after SCI, however the mechanisms remain elusive. To further our understanding of the potential mechanism/s of minocycline's neuroprotective properties, male adult Wistar rats were subjected to a C7 DCT to completely sever the dorsal ascending fibers and the underlying corticospinal tract. Gene microarray experiments were conducted on mRNA isolated from the lesion site of minocycline (single bolus 90 mg/kg immediately after injury) or saline treated animals. The results from the gene array studies revealed a 5-fold reduction in p38 M A P K in minocycline vs. saline treated animals at 14 days post-injury. Subsequent analysis with primers specific for rat p38 M A P K confirmed the microarray data. In addition, western blot analysis with dual-phosphorylation site specific (active) p38 M A P K antibodies showed that delayed minocycline treatment reduced active p38 M A P K levels within the lesion site compared to saline treated controls. Utilizing immunohistochemistry for active-p38 M A P K I demonstrated that active p38 M A P K was localized to microglia/macrophages within the degenerating AST 7 days post-injury, and is reduced after minocycline treatment. I also examined active p38 M A P K expression acutely following a moderate contusion injury (OSU Impactor, 1.2 mm displacement) to more closely mimic the type of injury seen in human SCI. The results from these experiments revealed that active p38 M A P K is primarily localized to resting microglia within the non-injured spinal cord. However, three hours following a contusion injury, a few neutrophils (lobular nuclei) and reactive microglia (0X42+) within the lesion site were immunopositive for active p38 M A P K . At 24 hours post-injury, a marked increase in active p38 M A P K was seen in large numbers of neutrophils and activated microglia. At 15 days post-injury, active p38 M A P K was localized to macrophages (ED1+) that dominated the lesion site and presumably astrocytes 76 delineating the lesion cavity. Collectively, my results demonstrate that active p38 M A P K increased within resident and invading immune cells after DCT and contusion injury, and therefore, may be an important target to regulate the inflammatory cascade after SCI. 77 I N T R O D U C T I O N Minocycline has been demonstrated to be neuroprotective in several different models of CNS disease and trauma (Yong et al., 2004). However, the mechanism behind minocycline's neuroprotective properties within the SCI setting is largely unknown. Chapter 4 of this thesis investigates the potential pathways regulated by minocycline application after SCI. Previous studies have suggested that minocycline's neuroprotective effects include modulating the inflammatory response and preventing apoptosis. Minocycline has been suggested to exert its anti-inflammatory effects by modulating microglia, immune cell activation and subsequent release of cytokines, chemokines, lipid mediators of inflammation, matrix metalloproteases (MMPs), and nitric oxide release. Specifically, minocycline treatment reduced liberation of IL-ip (Yrjanheikki et al., 1998; Yrjanheikki et al., 1999; Chen et al., 2000; Sanchez Mejia et al., 2001), diminished TNF-a mRNA levels after SCI, and prevented the lipopolysaccharide (LPS)-induced production of TNF-a (Lee et al., 2003; Lee et al., 2004). In addition, the anti-inflammatory cytokine IL-10, was also up-regulated following minocycline application immediately after SCI (Lee et al., 2003). Chemokines are important chemoattractants guiding microglia, astrocytes and peripheral immune cells to sites of infection, trauma and recent studies have demonstrated that minocycline suppressed LPS-induced production of M l f l a and RANTES, and diminished the chemokine receptor CXCR3 expression in microglia-like BV-2 cells (Kremlev et al., 2004). Therefore, modulation of cytokines and chemokines following CNS trauma may contribute to minocycline's neuroprotective effects. Minocycline has also been shown to modify lipid mediators of inflammation. Minocycline treatment inhibited phospholipase A2 (sPLA2) activity (Pruzanski et al., 1992), 78 PGE2 production and COX-2 expression (Kim et al., 2004). Similarly, in a rat model of focal ischemia, pretreatment with minocycline reduced COX-2 and PGE2 levels (Yrjanheikki et al., 1999). Interestingly, minocycline treatment abolished 5-lipoxygenease translocation to the nuclear membrane in PC12 cells subjected to oxygen-glucose deprivation (Song et al., 2004). Thus, lipid-mediated inflammatory signaling may be attenuated at several steps following minocycline treatment. Minocycline may also exert anti-inflammatory effects by inhibiting matrix metalloproteinases (MMPs). Minocycline treatment reduced MMP-2 expression (Popovic et al., 2002) , inhibited gelatinase (MMP-2 & -9) activity, down-regulated MMP-12 (Power et al., 2003) , reduced MMP-9 protein expression in cultured T-cells, and attenuated T-cell migration across a fibronectin barrier (Brundula et al., 2002; Power et al., 2003). Therefore, minocycline's observed effects on MMPs may act to reduce inflammation by diminishing cell infiltration and migration. A role for minocycline in inhibiting nitric oxide release has also been suggested. For example, minocycline reduced hypoxia- and excitotoxin-induced nitric oxide production (Tikka et a l , 2001; Suk, 2004). Similarly, in murine macrophages stimulated with either LPS or IFN-y, as well as in osteoarthritis (OA)-affected cartilage cells stimulated with IL- lp , minocycline inhibited iNOS activity indirectly through diminishing iNOS mRNA expression and suppressed subsequent protein levels (Amin et al., 1996). Since supplemental C a 2 + did not have a significant influence on minocycline-dependent NOS inhibition, these effects are most likely independent of minocycline's C a 2 + chelating abilities. In a gerbil model of global ischemia, minocycline treatment reduced mRNA levels of iNOS by 30% suggesting minocycline is effective at reducing iNOS expression in vivo (Yrjanheikki et al., 1998). In agreement, daily injections of 79 minocycline inhibited iNOS activity by 72% in a transgenic mouse model of Huntington's disease (Chen et al., 2000). These studies provide compelling evidence that minocycline may reduce inflammatory-induced production of nitric oxide and subsequent generation of peroxynitrite through attenuating iNOS expression and preventing peroxynitrite formation. Minocycline may also directly inhibit apoptosis following CNS trauma or during disease progresssion. We and others have shown that minocycline treatment reduced caspase-3 activation (Stirling et al., 2004) and activity (Lee et al., 2003) following SCI, traumatic brain injury (Sanchez Mejia et al., 2001), and Huntington's disease (Chen et al., 2000). In addition, minocycline treatment prevented the release of pro-apoptotic factors (cytochrome c, Smac/DIABLO and AIF) from the mitochondria (Zhu et al., 2002; Wang et al., 2004a) and therefore, minocycline may be neuroprotective in part, by targeting both caspase-dependent (cytochrome-c, Smac/Diablo) and caspase-independent (AIF) forms of cell death. Minocycline may also prevent apoptosis by increasing levels of anti-apoptotic factors potentially upstream of cytochrome-c release. Recent in vitro studies have shown that minocycline treatment protected kidney epithelial cells against apoptosis induced by hypoxia, azide, cisplatin, and staurosporine by selectively increasing the anti-apoptotic protein Bcl-2 (both mRNA and protein) (Wang et al., 2004a). In addition, Bcl-2 down-regulation using a specific anti-sense oligonucleotide, reversed the rescue effects of minocycline-pretreatment suggesting, minocycline is neuroprotective in part by up-regulating Bcl-2 expression (Wang et al., 2004a). Minocycline may also be neuroprotective by increasing the levels of inhibitor of apoptosis proteins (IAPs) such as XIAP and thus preventing caspase activation and subsequent cell death. In support of this view, it was demonstrated that pretreatment with minocycline before ischemia/reperfusion injury of isolated rat hearts, reduced both protein and mRNA expression of 80 several initiator and effector caspases, diminished infarct volume, and lessened apoptotic cell death (Scarabelli et al., 2004). Concomitantly, minocycline treatment reduced caspase activity as well as cytosol levels of cytochrome-c and Smac/Diablo, and increased XIAP expression. Collectively, the results suggest that minocycline has a direct effect in inhibiting cell death by priming a "survival mode" rendering a cell less vulnerable to apoptotic stimuli. The p38 mitogen-activated protein kinase (MAPK) pathway has been demonstrated to be an important pathway for mitigating inflammatory responses in immune cells and regulating survival/cell death, and thus has become an important target for several inflammatory and degenerative diseases, for review see (Barone et a l , 2001; Saklatvala, 2004; Wada and Penninger, 2004). Tikka and coworkers showed that minocycline treatment increased neuronal survival in mixed spinal cord cultures treated with glutamate, kainite or N-methyl-D aspartate, presumably by reducing microglia activation through a p38 MAPK-dependent mechanism (Tikka et al., 2001; Tikka and Koistinaho, 2001). Further studies revealed that minocycline reduced nitric oxide-induced death in rat cerebellar granule neurons which correlated with a reduction in p38 M A P K (Lin et al., 2001). Recently, Pi and colleaques were able to demonstrate that minocycline directly inhibited p38 M A P K activity (Pi et al., 2004).These studies provide evidence that minocycline may be neuroprotective by inhibiting p38 MAPK-dependent microglial-induced neurotoxicity and preventing p38 MAPK-dependent neuronal cell death. Minocycline's anti-apoptotic combined with its anti-inflammatory properties protects cells from several death inducing stimuli and likely explains part of minocycline's neuroprotective effects in CNS trauma and disease. I therefore sought after common signaling pathways that may account for minocycline's dual effects on cell death and inflammation. Utilizing microarray technology in collaboration with Ward Plunet, we demonstrated that p38 81 M A P K increased after SCI, and minocycline treatment reduced mRNA levels approximately 5-fold compared to saline-treated controls. In addition, active p38 M A P K increased acutely after contusion SCI and was expressed in neutrophils and microglia/macrophages and remained elevated within the latter for several weeks after injury. Furthermore, minocycline treatment reduced both CD1 lb and active p38 M A P K levels predominantly in microglia/macrophages. Thus minocycline may be neuroprotective in part by reducing the microglia/macrophage response and p38 M A P K expression after SCI. M A T E R I A L S AND M E T H O D S Spinal cord dorsal column transection A l l experiments were conducted in accordance with the University of British Columbia Animal Care ethics committee adhering to guidelines of the Canadian Council on Animal Care. Adult Wistar rats were anesthetized with an intraperitoneal injection of a mixture of ketamine hydrochloride (72 mg/kg; Bimeda-MTC, Cambridge, ON) and xylazine hydrochloride (9 mg/kg; Bayer Inc., Etobicoke, ON). The animals were placed in a stereotaxic frame and a laminectomy was performed at the 7th cervical vertebra. An adjustable wire knife model 120 (David Kopf Instruments, Tujunga, CA) was utilized to minimize the variability in depth of lesion. The knife was lowered to the dura mater 1 mm lateral to midline on the animal's right side. Following a pre-puncture of the dura mater with a fine needle, the wire knife was lowered 1.1 mm into the dorsal horn between the dorsal roots of C7/C8. The wire knife blade (3 mm curvature length) was extended to a further depth of 0.5 mm (total 1.6 mm) and horizontally to a total diameter of 1.9 to 2 mm. The wire knife was drawn up while gently pushing the dorsal columns down with a cotton swab. This severs most dorsal column axons, but it does not sever the dura except at its point of entry and its extreme distal end that points upwards. The wire knife was removed and a 82 pair of microscissors was introduced into the two small holes produced by the wire knife in order to cut the dura and dorsal vein and to ensure complete transection of the dorsal columns and central canal. Contusion model Adult Wistar rats were anesthetized with an intraperitoneal injection of ketamine hydrochloride (72 mg/kg; Bimeda-MTC, Cambridge, ON) and xylazine hydrochloride (9 mg/kg; Bayer Inc., Etobicoke, ON) diluted in 20 m M PBS. While anesthetized, rats received a spinal contusion injury utilizing the Ohio State impactor device (Ohio State University, OH, USA). The injury model has been well characterized and produces a moderate lesion in a reproducible manner (Stokes et al., 1992; Popovich et al., 1997). Prior to injury, the animal's backs were shaved (l"x 2" area), disinfected with Betadine and a midline incision was made. Utilizing both blunt and sharp dissection, the musculature was separated from the dorsal vertebral bone (T-8 to T-10). A small (~3 mm round diameter) laminectomy was made in the dorsal bone to visualize the spinal cord and allow access of the impactor tip. Following the laminectomy, the spine was stabilized by clamping the dorsal processes one segment above and below the proposed site of lesion (T9/10). A scale and adjustable stage was placed underneath the animal to support 50% of the animal's weight. The impact probe was lowered onto the dura to a force of 1.5 kilodynes and then the surface of the cord was rapidly displaced 1.2 mm to produce a moderate contusion. Following injury the rats were maintained at 37 °C and received 10-15 ml Ringers-Lactate solution (to compensate for loss of blood and dehydration). Bladders were manually expressed 2-3 times daily until spontaneous voiding returned. 83 Minocycline administration To test potential mechanisms of minocycline's mode of actions within the injured spinal cord setting, rats were randomly assigned into several treatment groups and were treated with a single bolus of minocycline (90 mg/kg) (Apotex Inc., Weston, ON) immediately after injury (immediate group) or twice per day for two days (post-injury) with intraperitoneal injections of either saline or 50 mg/kg minocycline (Apotex Inc., Weston, ON) in saline (non buffered), beginning 30 minutes post-injury (delayed group). There was a minor grooming response at the site of injection in some animals lasting a few seconds. Otherwise these treatments were well tolerated. Preparation of tissue Animals were anesthetized with a lethal dose of chloral hydrate (BDH Inc., Toronto, ON). For the fresh kil l animals, one centimeter of spinal cord centered at the lesion site (or equivalent location in the uninjured spinal cord from control animals) was rapidly dissected and immediately frozen on a metal surface placed on top of dry-ice and stored at -80°C until further use. Additional animals were perfused with phosphate buffered saline (PBS), followed by perfusion fixation with a solution of 4% paraformaldehyde in PBS. The spinal cords (brain-stem to midthoracic region) were removed and post-fixed in 4% paraformaldehyde overnight and subsequently cryoprotected in 30% sucrose. The spinal cords were then cut into 3 blocks, frozen and stored at -80°C until sectioned. R T - P C R and Microarray We obtained our immunomicroarray's from the National Institute on Aging (NLA) and followed the manufacturer's instructions (Mayne et al., 2001). The immunomicroarrays contain a defined set of 1,152 immune response-related cDNAs encoding immune response-specific cell 84 differentiation antigens; cytokines and their receptors, chemokines; structural and cytoskeletal genes; and signal transduction genes, transcription factors, growth factors, and other cellular and metabolic molecules (Mayne et al., 2001). A n entire list of the genes printed on the immunomicroarray can be found at www.Rrc.nia.nih.gov/branches/rrb/dna/dna.htm. Briefly, total R N A was extracted from fresh killed spinal cords of minocycline or saline treated animals using TRIzol reagent (Life Technologies, Gaithersburg, MD). Each sample was then DNase treated to remove contaminating D N A and quality and quantity of R N A assessed using a spectrometer. 10 pg of total R N A was used for each microarray experiment. The R N A was reverse transcribed with RT Superscript (GIBCO/BRL, Gaithersburg, MD) and labeled with 33P dCTP. A total count (CPM) for the radioactive labeled probes was at least one million. The microarrays were placed in a 50 ml Falcon tube and pre-hybridized for 4 hours at 50 °C. The labeled R N A of each sample was split equally between two Falcon tubes after the pre-hyb was finished. Hybridization occurred for 12-18 hours at 50 °C. At the end of this time period the microarrays were washed several times then exposed to a phosphorimager screen for 2 days. The phosphorimager screens were scanned in a Molecular Dynamics Storm Phosphorimager at 50 mm resolution and the Molecular Dynamics ImageQuant program (Amersham Biosciences Corp, NJ, USA) was used to crop, straighten the scanned images, and quantify the results. A grid was placed on the scanned image and adjusted to fit each spot into a separate box in the grid. A l l pixels within each box of the grid were given a score of 0-255 greyscale index. The raw intensity scores were transformed by the use of log 10 to reduce the variance due to extreme values, as per the suppliers instructions (Cheadle et al., 2003). The mean gene expression of each independent array is set at 0 and a standard deviation of 1 by calculating a distribution of z-85 scores using the following formula: Observed Gene z-score = (Observed Gene log 10 intensity -Mean microarray Pool log 10 intensity) / (Standard deviation microarray Pool log 10 intensity). To compare between arrays (experimental vs. control) the average of 4 measurements of each gene in one group is subtracted from the other. These z-scores differences are translated into Z-ratios using: z-score difference gene 1/ standard deviation of the z differences distribution. A l l of the calculated Z ratios for a given comparison can be rank ordered on this basis. Z ratios were determined for all of the individual.genes in the appropriate comparisons; for example, a Z ratio for IL-1 would be calculated by determining the ratio of the IL-1 Z scores obtained from minocycline and saline treated animals. Three independent sets of arrays (experimental and control) were performed and only those genes which displayed differential regulation in all three comparisons with a z-ratio of +/- 1.0 or more were reported. A z-ratio of 1 signifies approximately a 3 fold change whereas a z-ratio of 1.5 corresponds to an approximately 5 fold change (Mayne et al., 2001). Samples of minocycline -treated or saline-treated reverse transcribed R N A from above were amplified by polymerase chain reaction (RT-PCR) using the following primers specific for rat p38 M A P K : p38 M A P K right, 5 ' -ATG T C A GAT GGC A A G GGT TC-3' ; p38 M A P K left, 5 '-ATC T C A GTT GCT GGC GTT CT-3' . Amplication of p38 M A P K cDNA was performed using 30 cycles (45 sec 94°C, 1 min at 54 °C, 1.5 min at 72 °C). PCR for S12 was also performed as a control to ensure that an equal amount of input cDNA was analyzed. PCR products were visualized on 1.5% agrose gels using ethidium bromide and photographed under U V light using a polariod camera. 86 Western Blot Tissue from dissected spinal cords were placed into ice-cold homogenizing buffer (1 g tissue / 4 ml buffer) containing protease and phosphatase inhibitors (20 m M Tris, pH 7.0, 2 m M EGTA, 5 m M EDTA, 30 m M sodium fluoride, 40 m M glycerophosphate, pH 7.2, 20 m M sodium pyrophosphate, 1 m M sodium orthovanadate, 1 m M phenylmethylsulfonylfluoride, 3 m M benzamidine, 5 uM pepstatin A , 10 uM leupeptin and 0.5% Triton X-100). The final pH of the homogenizing buffer was adjusted to 7.2. The homogenate was centrifuged at a temperature of 4 °C and the supernatant was collected, and protein concentration was determined for each sample. A total of 20 p.g of protein was loaded into each well of a 15% SDS-PAGE gel and subjected to electrophoresis. The protein was then transferred to an Immobilon-P membrane (Millipore Corporation, M A , USA) overnight at 4°C. Membranes were then briefly soaked in ddH 2 0, blocked for one hour at room temperature in l x Tris-buffered saline/ 0.1 % Tween 20 (TTBS) containing 5% bovine serum albumin (BSA) (Sigma -Aldrich Canada Ltd., Oakville, ON). Membranes were probed with a rabbit polyclonal antibody specific for the active (dual phosphorylated form) p38 M A P K (Sigma-Aldrich) at 0.5 pg/ml or total p38 M A P K (Chemicon International, Inc,. CA, USA) at 2 pg/ml in TTBS containing 0.5% B S A for 1 hour at room temperature or or overnight at 4°C, washed four times with TTBS, then incubated with goat anti rabbit IgG conjugated to horseradish peroxidase at 1:10,000 in TTBS/5% goat serum. Membranes were washed four times with TTBS and developed using enhanced chemiluminescence (ECL) reagents (Amersham Biosciences, NJ, USA). The membranes were then exposed to autoradiographic film (Kodak Biomax, Eastman Kodak Company, Rochester, NY) . Membranes were stripped, blocked and re-probed with an antibody for actin (ICN Biomedicals, Costa Mesa, CA) to assess equal loading of samples. 87 Immunohistochemistry Fluorescent immunohistochemistry was performed on slides containing sections of injured spinal cords. In general, slides were rinsed three times in PBS, and blocked in 10 % normal goat serum in PBS for 30 minutes at room temperature. To detect active p38 M A P K positive profiles within the distal AST, or within the lesion area, slides were incubated with a rabbit polyclonal antibody specific for the active (dual phosphorylated form) p38 M A P K (1:500, Sigma Aldrich Canada Ltd., Oakville, ON). To assess cell identity of p38 MAPK-positive profiles, the following mouse primary antibodies were used: APC (Ab-7; 1:200, Oncogene Research Products, Boston, M A ) for oligodendrocyte cell bodies, GFAP (1:1000, Sigma Aldrich) for astrocytes, 0X42 (1:500, Serotec, Oxford, UK) for microglia/macrophages. Following three washes in PBS, slides were incubated for one hour at room temperature with the Alexa Fluor 488 goat anti-rabbit secondary antibody (1:200, Molecular Probes, Inc, Eugene, OR) or a Cy3-conjugated donkey anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories Inc, West Grove, PA) to visualize active p38 M A P K and the mouse primary antibodies listed above. Slides were then washed three times in PBS and submerged in Hoechst 33258 (1 pg/ml, Sigma Aldrich Canada Ltd.). R E S U L T S Minocycline treatment reduces p38 M A P K mRNA expression 14 days after SCI. To determine possible signaling pathways modulated by minocycline application following injury to the spinal cord, I utilized microarray analysis of mRNA isolated from spinal cord tissue containing the lesion area. Microarray analysis has been used successfully in the past to rapidly screen for drug targets and to test drug efficacy in a wide range of applications. To ensure the results were not due to chance, we ran three independent sets of arrays (experimental 88 and control) and only report those genes which displayed differential regulation in all three experiments with a z-ratio of ± 1.0 or more. It is important to note that a z-ratio of 1.0 corresponds to an approximate three-fold change, whereas a z-ratio of 1.5 represents approximately a five-fold change in gene expression (see methods). As shown in Figure 4.1,4, minocycline treatment (90 mg/kg single bolus immediately after injury) markedly down-regulated p38 M A P K , phosphoprotein enriched in astrocytes (PEA 15), and transferrin mRNA at 14 days post-lesion compared to saline treated controls. Concurrently, minocycline treatment up-regulated messages for chemokine receptor 6 (CCR6), and CD59 antigen pi8-20 (CD59). Since p38 M A P K has previously been shown to be inhibited following minocycline treatment in microglia cultures in vitro (Tikka et al., 2001; Tikka and Koistinaho, 2001), and plays a central role in the inflammatory response (Saklatvala, 2004), I utilized reverse transcriptase polymerase chain reaction (RT-PCR) with specific primers to rat p38 M A P K to verify the results from the gene array experiments. A representative gel from the RT-PCR experiments is shown in Figure 4.15 demonstrating a significant (P < 0.05) reduction in p38 M A P K mRNA following minocycline treatment vs. saline treated controls. Minocycline treatment reduces active p38 M A P K expression following SCI. To determine if minocycline treatment reduces activation of p38 M A P K , I isolated protein from the injured spinal cords of saline or minocycline treated animals and assessed p38 M A P K protein expression. Utilizing antibodies specific to the dual phosphorylated (active form) or total p38 M A P K , revealed a significant reduction of phospho-p38 M A P K in the minocycline treated groups vs. saline treated animals, 7 days post-injury (P < 0.05) (Figure 4.2). In contrast, a strong trend towards a reduction (P= 0.074) of total p38 M A P K levels was revealed from minocycline treated animals compared to saline treated animals. 89 A GENES EXP. 1. EXP. 2. EXP. 3. Up-regulated CCR6 1.68 1.55 1.66 CD59 1.50 2.40 1.49 Down-regulated p38 MAPK -3.15 -4.09 -1.83 PEA 15 -1.46 -1.57 -1.25 Transferrrin -2.55 -6.08 -3.20 B M M S S + - p38 MAPK -S12 1.* * iQirino ! i • satire i 1.2 rT | ,J a. £ 0 9 i S 0.6 1 0 4 f 0 2 |  0 1 Figure 4.1 Minocycline reduces p38 M A P K mRNA after SCI. A, Microarray analysis o f m R N A from saline vs. minocycline-treated rats 14 days after a dorsal column transection. c D N A spots with Z-ratio scores greater than 1.0 or less than -1 .0 in all three experiments. (>1 ~ 3 fold; >1.5 ~ 5 fold change). B, R T - P C R utilizing primers specific for rat p38 M A P K confirm the gene array results shown in A. Messenger R N A was isolated from saline (S) or minocycline (M) treated rats, 14 days after a dorsal column transection. The p38 M A P K c D N A bands are shown above the S12 c D N A bands. Quantification o f these results revealed a significant (P < 0.05) reduction in the ratio o f p38 M A P K and S12 intensity in minocycline treated animals (0.013 ± 0.005184) vs. saline treated (0.903 ± 0.298) animals (data represent mean ± S E M , *P < 0.05 ; 14d n = 2/group). Minocycl ine, mino; cortex, positive control, +; d H 2 0 , negative control, -; 100 bp ladder, L. 90 A Lam. DCT Saline DCT Mino. DCT Saline !p-p38 •^ ppwir actin D B DCT DCT Saline Mino. f t f I %~ - « total p38 MAPK actin Figure 4.2 Western blot analysis of active p38 MAPK and total p38 MAPK protein isolated from rat spinal cord. Western blot analysis for active p38 M A P K (A) and total p38 M A P K (5) from minocycline or saline treated rats 7 days after a dorsal column transection. Quantification of the ratio of active p38 M A P K ( Q , or total p38 M A P K (D) and actin intensity from saline treated vs. minocycline treated animals reveals a significant reduction in active p38 M A P K (data represent mean ± S E M , student's Mest, *P < 0.05 ; n = 3/group). Lam, laminectomy only control; D C T , dorsal column transection; Mino, minocycline. 91 Spatial and temporal expression of active p38 M A P K following contusion SCI. Since very little information is available pertaining to p38 M A P K expression in the injured spinal cord setting, I examined the spatial and temporal expression of active p38 M A P K following two types of spinal cord injuries, a partial-transection and contusion injury. Our group purchased the Ohio State impactor device (Ohio State University, OH, USA) during my partial-transection studies because the contusion model more closely mimics the injury seen in humans. Therefore the model is more appropriate for testing the efficacy of neuroprotective treatments than transection based models (Kwon et al., 2002), and thus I complimented my studies with this model. The OSU impactor device has been well characterized and produces a mild or moderate lesion in a reproducible manner (Stokes et al., 1992; Popovich et al., 1997). Using immunohistochemistry for activated p38 M A P K in combination with cell specific markers for microglia/macrophages (0X42), I examined uninjured and contused spinal cords at the lesion site at 3 hours, 1 day and 2 weeks after SCI. In uninjured animals, resting microglia (OX42-positive) were abundant and distributed evenly throughout the white and grey matter. Many of these profiles expressed low levels of active p38 M A P K (Figure 4.3). Three hours after contusion injury, microglia were reactive as demonstrated by increased levels of the complement receptor CD l i b (0X42 immunoreactivity) as previously shown (Popovich et al., 1997). Consistent with the peak invasion of the injured spinal cord by neutrophils (Carlson et al., 1998), many p38 M A P K positive profiles were evident at 24 hours. By 15 days post-lesion p38 M A P K immunoreactivity was highly expressed within microglia/macrophages at the lesion site and presumably within astrocytes surrounding the lesion. High resolution images are shown in 92 A B C D merged control E F *>** 3 hrs » 1 K L r» •„.*'i* 24 hrs M N 0 1 • P 15d Figure 4.3 Colocalization of active p38 M A P K and microglia/macrophages following contusion injury to the spinal cord. (A-D), In noninjured spinal cords microglia ( O X 4 2 , A) appear in a resting state with basal levels o f active p38 M A P K (B) (D, merge o f A,B,C). The complement receptor CD1 l b greatly increases 3 hours after contusion injury in reactive microglia/macrophages and invading immune cells ( 0 X 4 2 , E)(H, merge o f E,F,G,). B y 24 hours post-injury, several immune cells and microglia are immunopositive for active p38 M A P K (I-L). Act ive p38 M A P K (AO remains increased at 15 days post-lesion within microglia/macrophages (M) and presumably astrocytes delineating the lesion cavity. Note the massive increase in cell numbers over time following contusion injury (revealed by the bizbenzamide stain Hoechst 33258, (C,G,K,0)). Scale bar: 200 um. 93 Figure 4.4 confirming that p38 M A P K co-localizes with neutrophils (polymorphic nuclei) and microglia/macrophages early after injury and within microglia/macrophages at 15 days post-lesion. Minocycline reduces active p38 M A P K and C D l l b in microglia/macrophages following dorsal column transection. Remote from injury within the AST, p38 M A P K was localized to resting microglia within the uninjured spinal cord (Figure 4.5-4.7) and did not colocalize with oligodendrocytes from both non-injured or injured spinal cords (Figure 4.55,C). In contrast, both CD1 lb (0X42) and active p38 M A P K greatly increased within microglia/macrophages in saline treated animals at 7 days post-injury (Figure 4.6C and Figure 4.7I-L). However, following minocycline treatment signals for both CD1 lb and p38 M A P K were greatly reduced (figure 4.65 and 4.7E-H). These results suggest that minocycline is effective at reducing p38 M A P K activation and microglia/macrophage activation after SCI. DISCUSSION To determine the potential targets of minocycline's mode of action in the injured spinal cord setting, I isolated part of the spinal cord containing the lesion site from minocycline-treated and saline-treated rats, and utilized gene microarrays to unveil changes in gene expression, 14 days post-injury. Importantly, application of minocycline greatly reduced (~ 5 fold) p38 M A P K mRNA levels compared to saline treated animals (Stirling, 2003). Minocycline treatment also down-regulated mRNA expression of PEA-15 (phosphoprotein enriched in astrocytes), and transferrin and up-regulated CCR6, and CD59. PEA 15 is widely expressed in different tissues (including brain and spinal cord) and has been detected in several types of cells including astrocytes, neurons, microglia and oligodendrocytes. PEA 15 is implicated in cell death as 94 A 3 hrs B . • *• c 1 ^ D merge E 24 hrs F G H -. #* • 15 d K L yO ML 0 Figure 4.4 Differential staining of active p38 M A P K within immune cells at the lesion site at 3hr, 24hr and 15 days following a moderate T9 spinal contusion injury. (A-D) A t three hours post-injury (top panels), neutrophils (arrows) are immunopositive for active p38 M A P K (green) (Hoechst -blue to assess neutrophils characteristic multi-lobular nuclei, higher magnification, see insert C) . Some OX42-posi t ive microglia are reactive and express active p38 M A P K at this early time-point. (E-H) A t 24 hours post-injury (middle panels), many active p38 M A P K positive microglia and neutrophils (arrows) are present within the lesion site. (I-L) A t 15 days post-injury (bottom panels), activated macrophages (both O X 4 2 and active p38 M A P K positive) dominate the lesion cavity. Note the formation o f macrophage "rafts" consisting o f many macrophages connected by microvillae. Scale bar: 50 um. 95 B non injured non inured- injured Figure 4.5 Co-localization of active p38 MAPK within microglia/macrophages following DCT. A, Merged confocal image o f a microglial cell within the rostral dorsal columns from a non-injured rat ( O X 4 2 ; active p38 (dual-phosphorylated); Hoechst). B, Oligodendrocytes (CC1(+),Z?,C) within the rostral dorsal columns from injured (B) or non-injured ( Q do not colocalize with active p38 M A P K . However, cells with the morphological appearance o f microglia are positive for active p38 M A P K (arrows, B). 96 NON INJURED INJURED - minocycline INJURED-sal ine Figure 4.6 Minocycline treatment reduces both C D l l b and active p38 M A P K (low magnification images). L o w magnification merged images o f the rostral dorsal columns from uninjured (A), injured-minocycline treated (B), and injured-saline treated rats (C). L o w levels o f C D 1 l b and active p38 M A P K is evident in non-injured rat spinal cord. B, Minocycline-treated animals show reduced signal for C D 1 l b and active p38 M A P K within activated microglia/macrophages, compared to saline treated animals ( Q . Scale bar: 50 um. 97 A non injured B C f m e r g e d I E i... injured-minocycline F mm, \ G H * 1 -"-•Hr injured-saline J i ' K L Figure 4.7 Minocycline treatment reduces both C D l l b and active p38 M A P K (high magnification images). Confocal images o f the rostral dorsal columns from non-injured (A-D), injured-minocycline treated (E-H), and injured-saline treated rats (I-L). L o w levels o f C D 1 l b (A) and active p38 M A P K (B) are evident in non-injured rat spinal cord (D, merge, A-C). Minocycl ine treated animals show reduced signal for C D 1 l b and active p38 M A P K (middle panels) within activated microglia/macrophages, compared to saline treated animals (bottom panels). Scale bar: D.H.L, 10 um. C D 1 l b (A.E.l); active p38 M A P K (B,F,J); Hoechst (C.G.K) merged (D,H,L). 98 PEA15 expression enhanced TNF-a-induced apoptosis (Estelles et al., 1999). Thus, down-regulation of PEA15 is consistent with minocycline's anti-apoptotic properties. Of particular interest was the finding that transferrin, an iron transporting protein, was down-regulated following minocycline application. Transferrin mRNA is up-regulated in chronic multiple sclerosis plaques and is thought to play a protective role by reducing iron deposition and oxidative stress (Tajouri et al., 2003; Levine and Chakrabarty, 2004). The finding that transferrin message is down-regulated by minocycline treatment is consistent with the proposed chelating abilities of minocycline and may reflect less oxidative damage in treated animals (Chopra and Roberts, 2001). Alternatively, a reduction in iron usage may also result from a reduced microglia/macrophage response. Future studies will likely clarify the role of transferrin in CNS trauma and disease. Up-regulation of CD59, a complement regulatory protein, is also consistent with minocycline's anti-inflammatory effects as gene deletion of CD59 increased disease severity in a murine model of rheumatoid arthritis (Williams et al., 2004). Chemokine receptor 6 (CCR6) mRNA levels were increased (~3-fold), 14 days following SCI, in minocycline-treated compared to saline-treated rats. CCR6, and its sole known chemokine ligand, macrophage inflammatory protein-3a (MIP-3a), is involved in chemoattraction of immature dendritic cells, effector/memory T-cells and B-cells (Schutyser et al., 2003). However, whether up-regulation of CCR6 is detrimental or promotes tissue repair in the injured spinal cord setting is currently unknown. Based on the previously reported in vitro data implicating p38 M A P K as a potential target of minocycline's mode of action (Tikka et al., 2001), combined with my gene array data, and western blot data, I further explored p38 M A P K ' s role in the injured spinal cord using 99 phosphorylation site-specific antibodies for active p38 M A P K and found a marked reduction in active p38 M A P K in minocycline-treated animals after SCI. P38 M A P K s are serine threonine kinases that are activated by several upstream kinases in response to a wide variety of stressors and cytokines (Dong et al., 2002; Koistinaho and Koistinaho, 2002; Kumar et al., 2003) and play an important role in inflammatory signal transduction (Dong et al., 2002; Koistinaho and Koistinaho, 2002; Kumar et al., 2003). Several lines of evidence suggest p38 M A P K regulates the neutrophil response during an inflammatory reaction. P38 M A P K has been observed to be activated following TNF-a stimulation of neutrophils and inhibition of p38 M A P K abolished the production of IL-8, superoxide generation and neutrophil chemotaxis (Zu et al., 1998). Stimulation of neutrophils with TNF-a or GM-CSF (granulocyte-macrophage colony-stimulating factor) specifically activated upstream kinases of the p38 M A P K pathway (MAPKK3, M A P K K 6 ) and p38 M A P K (Suzuki et al., 1999). In a rodent model of acute renal inflammation, active p38 MAPK-positive neutrophils increased 8-fold within the inflamed kidney and p38 M A P K inhibition reduced neutrophil infiltration by 58% and greatly improved renal function (Stambe et al., 2003). In addition, p38 M A P K can promote survival by phosphorylation of caspase-8 and caspase-3 in human neutrophils (Alvarado-Kristensson et al., 2004). Since neutrophil invasion into the SCI lesion coincides with secondary cell damage (Taoka et al., 1998; Taoka and Okajima, 2000; Bao et al., 2004), inhibition of the release of cytotoxic factors from neutrophils or the induction of their death via p38 M A P K inhibition may reduce secondary degeneration after SCI. A role for p38 M A P K in cytokine production (TNF-a, IL-12, IL-6, IL-lp\ and NO release) by macrophages is also well established (Bhat et al., 1998; Dong et al., 2002). The downstream substrates of p38 M A P K ' s include other kinases, cytosolic proteins as well as 100 transcription factors. Notably, the downstream target M A P kinase-activated protein kinase-2 ( M A P K A P K2), is involved in the regulation of IL-ip, IL-6 and TNF-a production (Dong et al., 2002; Kumar et al., 2003). Mice lacking this kinase have low levels of these cytokines and pharmacological p38 inhibitors have been shown to decrease the production of these cytokines (Kumar and others 2003). In microglial cells, inhibition of the endotoxin-induced production of NO and the expression of iNOS and TNF-a was greatly decreased by the p38 inhibitor SB203580 (Bhat et al., 1998; Lee et al., 2000a). In addition to anti-inflammatory effects on monocytes/macrophages, p38 M A P K inhibition modifies the activation of T-cell responses. Inhibition of p38 M A P K with pharmacological inhibitors or in dominant negative p38 M A P K mice resulted in a reduced production of the pro-inflammatory IFN-y by Thi cells, but did not affect the anti-inflammatory IL-4 production in Th2 cells (Rincon and Pedraza-Alva, 2003). Hence, p38 M A P K seems to play a role in Thl/Th2 differentiation and thus possibly effector function (Rincon et al., 1998). The switch from a Thi response to a Th2 response has been proposed to be involved in the beneficial aspects of inflammation after SCI(David, 2002). P38 M A P K also plays a role in apoptosis of neurons and glia, however, the regulation of apoptosis by p38 M A P K s is complex with some studies supporting an anti-apoptotic effect whereas others report a pro-apoptotic effect (Wada and Penninger, 2004). Of interest is the demonstration that oligodendrocytes and neurons exposed to various apoptotic stimuli could be rescued by application of p38 M A P K inhibitors, e.g. (Hida et al., 1999; Kikuchi et al., 2000). Equally intriguing, is the demonstration that neutrophils can be induced to undergo apoptosis by p38 M A P K inhibition (Alvarado-Kristensson et al., 2004). Thus, differential effects of p38 M A P K inhibition may preserve neurons and oligodendrocytes, but induce cell death in immune 101 cells; all of which may have beneficial net effects to tissue preservation and functional outcomes after CNS damage. Combined with their known inhibitory effects on the inflammatory response, p38 M A P K inhibition may be of significant benefit for preserving neuronal function after CNS trauma or disease. Indeed, application of the p38 MAPK-inhibitor SB203580 into the ventricle of the brain was protective after transient focal ischemia where it reduced infarct volumes by 77% and improved functional deficits even when administered 12 hours after the event (Piao et al., 2003). These neuroprotective effects from inhibition of p38 M A P K , were associated with a reduction in iNOS, TNF-a, IL-1 (3 and COX-2 expression (Piao et al., 2003). Thus, the central role of p38 M A P K in inflammation and cell death has been widely established and it appears to be a major contributor to secondary cell damage in CNS trauma, and disease. Since p38 M A P K plays such an important role in inflammatory signal transduction (Dong et al., 2002; Koistinaho and Koistinaho, 2002; Kumar et al., 2003) and very little is known of p38 M A P K following SCI, I conducted a series of experiments to examine the spatial and temporal expression of p38 M A P K following a DCT and contusion SCI. Active p38 M A P K was primarily localized to resting microglia within the non-injured spinal cord. However, three hours following a contusion injury, a few neutrophils (lobular nuclei) and reactive microglia (0X42+) within the lesion site were immunopositive for active p38 M A P K . At 24 hours post-injury, a marked increase in active p38 M A P K was seen in large numbers of neutrophils and activated microglia. At 15 days post-injury, active p38 M A P K was localized to macrophages (ED1+) that dominated the lesion site and astrocytes (GFAP+) delineating the lesion cavity. Remarkably, active p38 M A P K was still highly expressed in microglia/macrophages at 6 weeks after injury (data not shown). In contrast both active p38 M A P K and EDI immunoreactivity was decreased in minocycline treated animals. Collectively, these results demonstrate that active p38 102 M A P K is increased within resident and invading immune cells after contusion injury and, therefore, may be an important target to regulate the inflammatory cascade after SCI. In agreement with my findings, other studies have shown that active p38 M A P K increases in microglia/macrophages after peripheral nerve injury (Kim et al., 2002; Tsuda et al., 2004) and ischemia (Lennmyr et al., 2002). Importantly, the intrathecal infusion of SB203580, a p38 M A P K inhibitor reduced hindlimb deficits after mild prolonged spinal cord compression, consistent with a detrimental role of p38 M A P K activation after SCI (Horiuchi et al., 2003). Of particular interest is the finding that the protective effects mediated by p38 M A P K inhibition are strikingly similar to the known effects asserted by minocycline application and fuel the possibility that p38 M A P K may be an important target of minocycline's mode of action. Tikka and coworkers showed that minocycline treatment increased neuronal survival in mixed spinal cord cultures treated with glutamate, kainite or N-methyl-D aspartate, presumably by reducing microglia activation through a p38 MAPK-dependent mechanism (Tikka et al., 2001; Tikka and Koistinaho, 2001). In addition, minocycline reduced nitric oxide-induced death or glutamate-induced death in rat cerebellar granule neurons correlated with a reduction in p38 M A P K (Lin et al., 2001; Pi et al., 2004). Further studies have shown that minocycline treatment reduced active p38 M A P K levels in hypoxia-induced microglia (Suk, 2004), gentamicin-induced hair cell damage (Wei et al., 2005), and HIV-associated CNS disease (Zink et al., 2005). Furthermore, Pi and colleaques were able to demonstrate that minocycline directly inhibited p38 M A P K activity (Pi et al., 2004). These studies provide evidence that minocycline may be neuroprotective by inhibiting p38 MAPK-dependent microglial-induced neurotoxicity and preventing p38 MAPK-dependent neuronal cell death. However, it is important to note that I was unable to demonstrate that 103 minocycline treatment directly inhibited p38 M A P K expression in this study as a reduced microglia/macrophage response would also result in decreased levels of active p38 M A P K (both protein levels as well as phosphorylation state). Further studies are required to unequivocally demonstrate that p38 M A P K is inhibited directly by minocyline following SCI. 104 CHAPTER 5 DELAYED INTRAVENOUS APPLICATION OF MINOCYCLINE IS NEUROPROTECTIVE FOLLOWING SPINAL CORD CONTUSION IN ADULT RATS 105 S U M M A R Y Previously, our group and others have demonstrated that minocycline, a 2nd-generation tetracycline, is neuroprotective after SCI. However, the mechanisms of minocycline's mode of action and optimal treatment paradigms remain poorly defined. I have recently shown that minocycline treatment reduced p38 M A P K expression following SCI. Since p38 M A P K is an important regulator of immune cell function, I hypothesized that p38 M A P K inhibition would reduce inflammation and improve functional outcome after SCI. Here I used an intravenous (I.V.) approach to achieve optimal delivery of minocycline to the contused spinal cord or administered the p38 M A P K inhibitor, SB203580 via osmotic mini-pump, and assessed lesion size and functional outcome (open-field locomotion) following a T-10 moderate contusion injury in adult rats (OSU device, 1.2 mm displacement). Delayed (1 hour) minocycline treatment (I.V.)( 20 mg/kg initial bolus followed by 10 mg/kg injections twice per day for 4 days post-injury) significantly improved hindlimb function compared to saline controls (P < 0.01) from one week to six weeks post-injury. Lesion site analysis revealed a significant increase in residual white and grey matter in minocycline compared to saline treated controls. In contrast to these results, intraperitoneal application of minocycline or application of a p38 M A P K inhibitor failed to improve functional outcome after SCI. 106 I N T R O D U C T I O N Following spinal cord injury (SCI) the lesion expands overtime due to a wave of secondary degenerative processes which include vascular disturbances, metabolic failure, ionic dysregulation, excitotoxicity, free radical formation, lipid peroxidation, cytokine/chemokine release, inflammation, and cell death, for review see (Dumont et al., 2001; Hausmann, 2003; Kwon et al., 2004). These processes exacerbate the primary mechanical injury to the cord and cause additional tissue destruction and functional deficits (Profyris et al., 2004). Therefore, treatment strategies designed to target several aspects of the secondary injury will likely be of value in the treatment of human SCI. Minocycline, a second-generation, semi-synthetic tetracycline, has an improved tissue absorption into the brain and cerebrospinal fluid and a longer elimination half-life compared to 1st generation tetracyclines (Klein and Cunha, 1995). In addition to its antimicrobial actions, minocycline has been shown to exert many neuroprotective and anti-inflammatory properties. Minocycline treatment has been successfully applied to animal models of focal and global ischemia (Yrjanheikki et al., 1998; Yrjanheikki et al., 1999; Arvin et al., 2002; Wang et al., 2003a), Parkinson's disease(Du et al., 2001; Wu et al., 2002; Lin et al., 2003; Thomas et al., 2003; Diguet et al., 2004b; Thomas and Le, 2004), Huntington's disease (Chen et al., 2000; Hersch et al., 2003; Thomas et al., 2003; Wang et al., 2003b), amyotrophic lateral sclerosis (Kriz et al., 2002; Van Den Bosch et al., 2002; Zhu et al., 2002; Zhang et al., 2003b), multiple sclerosis (Brundula et al., 2002; Popovic et al., 2002; Yong, 2004), and spinal cord injury (Lee et al., 2003; Wells et al., 2003a; Stirling et al., 2004; Teng et al., 2004). Our laboratory and others have shown that minocycline treatment is neuroprotective following SCI. In a clip compression murine model of SCI, Yong and colleagues reported that 107 minocycline treatment given one hour after thoracic (T)3/4 SCI, effectively reduced lesion size, increased axonal sparing of rubrospinal axons and improved hindlimb function (Wells et al., 2003a). Importantly, their study showed that minocycline was more effective in improving functional outcome when compared with methylprednisolone, the only treatment option to patients with SCI (Wells et al., 2003a). Minocycline treatment also promoted tissue preservation and enhanced functional recovery following SCI in rat (Lee et al., 2003; Stirling et al., 2004; Teng et al., 2004). M y previous studies have shown that minocycline treatment given 30 minutes after injury reduced oligodendrocyte cell death, microglia/macrophage activation and corticospinal axonal dieback (Stirling et al., 2004). In addition, Oh and colleagues (Lee et al., 2003) reported that minocycline treatment decreased TNF-a mRNA with a concomitant increase in the anti-inflammatory cytokine IL-10 (Bethea et al., 1999). Furthermore, minocycline has been shown to inhibit pro-apoptotic cytochrome-c release from mitochondria, a mechanism that may account for its reduction of cell death and caspase activation following SCI (Teng et al., 2004). Taken together, these studies show that minocycline's neuroprotective effects most likely involve modulating the immune response after injury and decreasing injury-induced cell death. These studies also provide the rationale to further explore the positive effects of minocycline on functional outcome after SCI and its multifaceted impact on the secondary degenerative response. Minocycline's biological effects include inhibition of microglial activation and reduction of the pro-inflammatory cytokines and cytotoxic products associated with their activation. For example, minocycline treatment reduced mRNA of both IL- ip and inducible nitric oxide synthase (iNOS) (Yrjanheikki et al., 1998), cyclooxygenase-2 expression and prostaglandin E2 production (Yrjanheikki et al., 1999). Moreover, minocycline inhibited the expression and 108 activity of several metalloproteases (Sadowski and Steinmeyer, 2001; Brundula et al., 2002; Power et al., 2003). In addition, minocycline has recently been shown to inhibit caspase expression and cytochrome-c release (Chen et al., 2000; Zhu et al., 2002; Wang et al., 2003b; Stirling et al., 2004; Teng et al., 2004). Although the exact mechanism remains elusive, in vitro data from spinal cord mixed cultures and isolated microglia cultures treated with glutamate or kainate has provided evidence that minocycline inhibits microglia-induced neurotoxicity by targeting the p38 M A P K pathway (Tikka et al., 2001). Our group has shown that minocycline inhibits p38 M A P K expression and activation within inflammatory cells and microglia after SCI (Stirling et al., 2003). Since p38 M A P K is an important regulator of immune cell function, I hypothesized that p38 M A P K inhibition would reduce inflammation and improve functional outcome after SCI. Here I used an intravenous approach to achieve optimal delivery of minocycline to the contused spinal cord or administered the p38 M A P K inhibitor, SB203580 via osmotic mini-pump, and assessed lesion size and functional outcome (open-field locomotion) following a T-10 moderate contusion injury in adult rats (OSU device, 1.2 mm displacement). Delayed (1 hour) minocycline treatment (I.V. 20 mg/kg initial bolus followed by 10 mg/kg injections twice per day for 4 days post-injury) significantly improved hindlimb function compared to saline controls (P < 0.01) from one week to six weeks post-injury. Lesion site analysis revealed a significant increase in residual white and grey matter tissue compared to saline treated controls. In contrast to these results, intraperitoneal application of minocycline, or applicatioin of a p38 M A P K inhibitor failed to improve functional outcome after SCI. These findings support minocycline treatment as a neuroprotective agent after SCI, however, the most efficacious route of administration and 109 optimal dosage paradigm after SCI needs further evaluation before translation into clinical setting. M A T E R I A L S A N D M E T H O D S A n i m a l care A l l experiments were conducted in accordance with the University of British Columbia Animal Care ethics committee adhering to guidelines of the Canadian Council on Animal Care. Adult Wistar rats were obtained from the University of British Columbia's Animal Care Center, Vancouver, BC. The rats were housed in groups of four, maintained in a 12hr light/dark cycle and provided rodent chow and water ad libitum. Contusion model Adult Wistar rats were anesthetized with an intraperitoneal injection of ketamine hydrochloride (72 mg/kg; Bimeda-MTC, Cambridge, ON) and xylazine hydrochloride (9 mg/kg; Bayer Inc., Etobicoke, ON) diluted in 20 m M PBS. While anesthetized, rats received a spinal contusion injury utilizing the Ohio State impactor device (Ohio State University, OH, USA). The injury model has been well characterized and produces a moderate lesion in a reproducible manner (Stokes et al., 1992; Popovich et al., 1997). Prior to injury, the animal's backs were shaved (l"x 2" area), disinfected with Betadine and a midline incision was made. Utilizing both blunt and sharp dissection, the musculature was separated from the dorsal vertebral bone (T-8 to T-10). A small (~3mm round diameter) laminectomy was made in the dorsal bone to visualize the spinal cord and allow access of the impactor tip. Following the laminectomy, the spine was stabilized by clamping the dorsal processes one segment above and below the proposed site of lesion (T9). A scale and adjustable stage was placed underneath the animal to support 50% of the animal's weight. The impact probe was lowered onto the dura to a force of 1.5 kilodynes and 110 then the surface of the cord was rapidly displaced 1.2 mm to produce a moderate contusion. Following injury the rats were maintained at 37°C and received 10-15 ml Ringers-Lactate solution (to compensate for loss of blood and dehydration). Bladders were manually expressed 2-3 times daily until spontaneous voiding returned (~2 weeks). Intravenous drug delivery system To facilitate intravenous delivery of drugs, rodent vascular access ports were used. One to three days prior to contusion injury, rats were anesthetized (as above), the area of skin overlying the left jugular vein was shaved, disinfected with Betadine, and a midline incision was made to expose the vein. A small diameter (3F) catheter coated with artificial heparin to prevent clotting, (Harvard Apparatus, Canada) was inserted into the jugular vein and secured with two sutures. The rodent vascular access port (Harvard Apparatus) attached to the catheter was placed under the skin to allow for easy access. Minocycline dosage and delivery To test the effects of minocycline treatment as a neuroprotective agent following contusion SCI, and to optimize drug delivery, rats were randomly assigned into several treatment groups, and received either intraperitoneal (LP.) or intravenous (I.V.) injections of minocycline (Apotex Inc., Weston, ON) or saline as a control. Rats in the LP. groups were treated twice per day for two days (post-injury) with LP. injections of 50 mg/kg minocycline in saline, immediately after injury. Rats in the I.V. groups were treated twice per day, beginning one hour after injury, and continued for 4 days thereafter. Intravenous injections consisted of an initial bolus of minocycline (20 mg/kg), followed by 10 mg/kg, for subsequent injections. I l l Intrathecal administration of SB203580-HCL To test p38 M A P K inhibition as a neuroprotective therapy for contusion SCI, rats were randomly assigned into three groups. The p38 M A P K inhibitor, SB203580 hydrochloride (Calbiochem San Diego, CA, USA) was applied intrathecally using an osmotic 7d mini-pump (Alzet no. 2001, 1 ul/h; Alzet, Palo Alto, CA) connected to a thin tube (0.3 mm outside diameter) inserted into the subarachnoid space at the level of the 4-5 lumbar vertebrae. These pumps have been well characterized and are designed to release substances lineally for 1 week when inserted into living tissue. Animals in the treatment groups were given either a low dose (3pg/ per day) or a high dose (7 ug/ per day) of inhibitor dissolved in a vehicle solution consisting of 20 m M PBS and 100 units of penicillin/streptomysin. Animals in the control group received vehicle alone. The dosage and delivery of SB203580 used in this study was based on the successful outcome after SCI compression as reported previously (Horiuchi et al., 2003). Preparation of tissue Animals were anesthetized with a lethal dose of chloral hydrate (BDH Inc., Toronto, ON), perfused with phosphate buffered saline (PBS), followed by perfusion fixation with a solution of 4% paraformaldehyde in PBS. The spinal cords (containing the lesion site and 3 segments rostral and caudal) were isolated and post-fixed in 4% paraformaldehyde overnight and subsequently cryoprotected in 30% sucrose (one to two days), frozen in dry ice cooled isopentane, and stored at -80°C until sectioned. The spinal cords were cryosectioned in the transverse plane at a thickness of 20 um and tissue sections were collected on Superfrost Plus slides (Fisher Scientific, Houston, TX) organized into five adjacent section series. 112 Residual white and grey matter To assess sparing of white and grey matter, transverse sections were stained with a modified eriochrome cyanine (EC) and cresyl violet protocol as previously described (Rabchevsky et al., 2001). Briefly, air-dried sections were cleared and hydrated before being placed for 10 min into a solution consisting of 2 mL of 10% FeCf? and 40 mL of 0.2% EC (Sigma) in 0.5% aqueous H2SO4 brought to a final volume of 50 mL with dH 2 0 . This was followed by washes in water and differentiation for 2 min in 0.5% aqueous NH4OH. The reaction was terminated with rinses in water before slides were immersed in cresyl violet solution for 10 minutes. Slides were then washed in dH 20, differentiated in cresyl violet differentiator (30 sees), dehydrated and mounted. Images were captured with a Zeiss Axioplan 2 microscope (2.5x magnification) and spared white matter and grey matter area was measured using Sigma Scan Pro software (SPSS, Chicago, IL). The cross- section with the smallest amount of spared tissue was defined as the lesion epicenter. Every 20th section (400um) was measured and recorded up to 3600 um rostral and caudal to the injury epicenter. Mean residual white or grey matter (mm2) per section was reported. Behavioral analysis To assess functional recovery of hindlimbs following SCI, rats were tested using the open-field Basso, Beattie, Bresnahan locomotor rating scale (BBB)(Basso et al., 1995, 1996). Animals were tested previous to injury to ensure no deficits in hindlimb function were evident and to expose the animals to an open-field testing environment. Following injury, animals were tested on the first or second day, fifth day and once per week thereafter until the end of the study. The mean scores per treatment group were plotted as a function of time post-injury. 113 Statistics Statistical analysis was performed using SigmaStat software (SPSS Inc.). For behavior analysis, differences among the treatment groups were tested using a repeated measures analysis of variance (ANOVA) and the Tukey-Kramer post-hoc analysis when warranted. For residual tissue analysis, data were compared between groups using a paired Student's t-test. In the case of unequal variances, the Mann-Whitney Rank Sum test was used. Differences with a P value less than 0.05 were considered significant. R E S U L T S Delayed (1 hour) intravenous delivery of minocycline improves hindlimb function. I chose a spinal contusion model to study the potential neuroprotective effects of minocycline treatment given that contusion models mimic many aspects of SCI in humans and provide a more clinically relevant scenario compared to transection-based models (Metz et al., 2000; Kwon et al., 2002). In addition, the Ohio State impactor model has been well characterized and produces graded injuries that are correlated to tissue sparing and functional outcome (Behrmann et al., 1992; Stokes et al., 1992). To ensure that all rats underwent a similar biomechanical injury to their spinal cords, criteria were developed to eliminate any rat from the study that showed evidence of severe "slippage" during injury and did not receive a final injury to the spinal cord of at least a magnitude of 175 kilodynes. Based on the computer-assisted analysis of force and displacement derived from the impact data, spinal cord contusions were comparable between all groups of rats used in the study (Table 5.1)(see also Figure 5.1 for examples of force curves). To assess the efficacy of minocycline treatment to promote functional recovery following a contusion injury to the rat spinal cord, locomotor ability within 114 G R O U P D I S P L A C E M E N T (mm) F O R C E (kdvnes) Saline (LP.) 1.19 ±0.005 250.77 ± 32.02 Minocycline (LP.) 1.20 ±0.005 216.89 ± 2 0 Saline (I.V.) 1.26 ±0.010 259.23 ± 13.82 Minocycline (I.V.) 1.25 ±0.018 247.14 ± 14.53 SB203580 (low) 1.20 ±0.008 237.07 ±6.29 SB203580 (high) 1.29 ±0.027 210.08 ± 16.42 vehicle 1.20 ±0.05 253.49 ± 9.94 Table 5.1 Mean force and displacement for all groups of animals. Notes: LP., intraperitoneal; I.V., intravenous. Al l values are means ± standard error of the mean (SEM) 115 0 1 2 3 4 5 6 7 time (msecs) Figure 5.1 Examples of individual rat force curves derived from impact data. Force (kdynes, y-axis, left) and displacement (red line) (mm, y-axis, right) is plotted against time (msecs, x-axis). The baseline force is recorded from a "blank" hit o f the impactor rod into air until it hits the physical stop (large downward deflection o f the baseline force curve at approximately 6.9 seconds). From this deflection point, the peak force is calculated for al l individual rats in the experiment. Examples o f individual rat force curves are shown. Note that severe "slippage" is easily seen in the force curves (example rat 5) a l lowing the experimenter to remove the animal from the study. 116 Table 5.2 Basso, Beattie and Bresnahan Locomotor Rating Scale (BBB) 0 no observable hindlimb (HL) movement 1 slight movement of one or two joints, usually the hip and/or knee 2 Extensive movement of one joint or extensive movement of one joint and slight movement of one other joint 3 Extensive movement of two joints 4 slight movement of all three joints of the HL 5 Slight movement of two joints and extensive movement of the third 6 Extensive movement of two joints and slight movement of the third 7 Extensive movement of all three joints of the HL 8 Sweeping with no weight support or plantar placement of the paw with no weight support 9 Plantar placement of the paw with weight support in stance only (i.e. when stationary) or occasional, frequent, or consistent weight supported dorsal stepping and no plantar stepping 10 Occasional weight-supported plantar steps; no FL-HL coordination 11 Frequent to consistent weight-supported plantar steps and no FL-HL coordination 12 Frequent to consistent weight-supported plantar steps and occasional FL-HL coordination 13 Frequent to consistent weight-supported plantar steps and frequent FL-HL coordination 14 Consistent weight-supported plantar steps, consistent FL-HL coordination, and predominant paw position during locomotion is rotated (internally or externally) when it makes initial contact with the surface as well as just before it is lifted off at the end of stance; or frequent plantar stepping, consistent FL-HL coordination, and occasional dorsal stepping 15 Consistent plantar stepping and consistent FL-HL coordination and no toe clearance or occasional toe clearance during forward limb advancement; predominant paw position is parallel to the body at initial contact 16 Consistent plantar stepping and consistent FL-HL coordination during gait and toe clearance occurs frequently during forward limb advancement; predominant paw position is parallel at initial contact and rotated at lift off 17 Consistent plantar stepping and consistent FL-HL coordination during gait and toe clearance occurs frequently during forward limb advancement; predominant paw position is parallel at initial contact and lift off 18 Consistent plantar stepping and consistent FL-HL coordination during gait and toe clearance occurs consistently during forward limb advancement; predominant paw position is parallel at initial contact and rotated at lift off 19 Consistent plantar stepping and consistent FL-HL coordination during gait, toe clearance occurs consistently during forward limb advancement, predominant paw position is parallel at initial contact and lift off, and tail is down part or all of the time 20 Consistent plantar stepping and consistent coordinated gait, consistent toe clearance, predominant paw position is parallel at initial contact and lift off, and trunk instability; tail consistently up 21 Consistent plantar stepping and coordinated gait, consistent toe clearance, predominant paw position is parallel throughout stance, and consistent trunk stability; tail consistently up Note. Slight: Partial joint movement through less than half the range of joint motion. Extensive: Movement through more than half of the range of joint motion. Sweeping: Rhythmic movement of HL in which all three joints are extended and then fully flex and extend again; animal is usually side lying and plantar surface of paw may or may not contact the ground; no weight support across the HL is evident. No weight support: No contraction of the extensor muscles of the H L during plantar placement of the paw; or no elevation of the hindquarter. Weight support: Contraction of the extensor muscles of the HL during plantar placement of the paw; or, elevation of the hindquarter. Plantar stepping: The paw is in plantar contact with weight support and then the HL is advanced forward and plantar contact with weight support is reestablished. Dorsal stepping: Weight is supported through the dorsal surface of the paw at some point in the step cycle. FL-HL coordination: For every FL step a HL step is taken and the HLs alternate. Occasional: Less than or equal to half; =50%. Frequent: More than half but not always; 51-94%. Consistent: Nearly always or always; 95-100%. Truck instability: Lateral weight shifts which cause waddling from side to side or a partial collapse of the trunk. From (Basso, D.M., et al, 1996) 117 an open-field environment was scored utilizing the B B B scale (see Table 5.2) (Basso et al., 1995, 1996). A l l contused rats showed severe bilateral hindlimb paralysis immediately after surgery and regained substantial recovery over the following six weeks. In sham-operated rats, the mean B B B score remained at 21 (normal locomotion) throughout the entire study (Figure 5.2,4). Suprisingly, minocycline administered (LP.) failed to improve B B B scores compared to saline treated controls (Figure 5.2,4). I then turned to an intravenous (I.V.) approach to administer minocycline as this approach proved successful in a model of ischemia (Xu et al., 2004b). In addition, peak plasma levels are obtained immediately after injection and less variability in plasma levels are seen between animals (Fagan et al., 2004). As revealed in Figure 5.25, contused rats treated with minocycline (I.V.) delayed one hour after injury, significantly improved hindlimb function compared to saline treated animals from 1 week to 6 weeks post-injury. Importantly, some of the minocycline-treated (I.V.) animals demonstrated frequent to consistent weight-supported plantar steps, occasional or frequent coordination of the fore and hindlimbs and improvements in foot placement (see Table 5.2 for B B B scoring). In contrast to the 1 hour delay group, intrathecal administration of SB203580, a p38 M A P K inhibitor failed to improve hindlimb function compared to saline treated animals (Figure 5.2Q. Delayed (1 hour) intravenous delivery of minocycline improves fine aspects of locomotion following contusion SCI. To further assess the functional recovery seen in rats treated with minocycline or SB203580,1 utilized B B B subscoring that takes into account finer aspects of locomotor recovery including toe clearance and foot position during locomotion (see Table 5.3 for a description of the subscoring used here) (Popovich et al., 1999). As shown in Figure 5.3,4, there was no 118 -•-touch only (4) -*-salinelP(5) -tr mino IP (6) Od 2d 4d 7d 14d 21d 28d 35d 42d B -•-touch only (4) - * - saline IV (7) * mino IV (9) \ \ 1 * \ . * I - 4 - 4 \ y ^ r i t L \ \ YY \ fA — i — * — \ — i — i — i — i — i — i — i Od 2d 4d 7d 14d 21d 28d 35d 42d * • touch only (4) vehicle (6) SB203580 low (8) -*-SB203580high(4) ~i—*~h 1 1 1 1 r~ Od 2d 4d 7d 14d 21d 28d 35d 42d Figure 5.2 Delayed intravenous application of minocycline improves hindlimb function after SCI. Open field B B B scores for touch only (no contusion) and moderate contusion SCI injury groups over time. A, Minocycl ine (LP.) application fails to improve locomotion vs. saline treated animals. However, minocycline (I.V.) treatment significantly improved hindlimb function compared to saline treated controls (B). C, High or low dose intrathecal administration o f SB203580, a p38 M A P K inhibitor, failed to improve hindlimb function after SCI . Data are expressed as means ± S E M , * P < 0.05. 119 Table 5.3 BBB Subscoring Scale Paw position Toe clearance initial contact/lift off right left right left rotated / rotated 0 0 none 0 0 rotated / parallel 1 1 occasional 1 1 parallel / rotated 1 1 frequent 2 2 parallel / parallel .2 2 consistent 3 3 notes, paw position, rotated refers to both internal or external rotation relative to the body axis, for toe clearance, 0 = no clearance, 1 = occassional clearance ( = 50%), 2 = frequent clearance (51-95%), 3 = consistent clearance (>95%) a subscore, 0-5, was given to each hindlimb. Scores from individual hindlimbs were summed to yield a single score of 10 for each animal. (From Popovich et al, 1999). information derived from (Popovich et al, 1999) 120 10 o u 2 6 3 in xt | 4 c 0 B 10 = 8 O o U) XI 3 6 £ c 10 2 0 o 8 o 10 ^ 6 in Xt I 4 o -*— A A A "* *-7d 14d 21d 28d 35d 42d A A A 7d A. •+ 4- -4— 14d 21 d 28d 35d 42d 7 li 7 & *. saline IP (5) amino IP(6) * mean +/- SEM A salineiV(7) A mino IV (9) • mean +/- SEM vehicle (6). p38i low (8) p38i high (4) mean +/- SEM 7d 14d 21d 28d 35d 42d Figure 5.3 Delayed intravenous application of minocycline improves fine aspects of hindlimb function after SCI. A, Minocycline (LP.) application fails to improve subscores compared to saline treated animals. However, minocycline (I.V.) treatment significantly improved hindlimb function compared to saline treated controls. C, Intrathecal administration of SB203580, a p38 MAPK inhibitor, also failed to improve hindlimb subscores after SCI. Data are expressed as means ± SEM, * P < 0.05. 121 difference between minocycline (LP.) treated and saline treated animal subscores following SCI. In contrast, minocycline (I.V.) significantly improved hindlimb subscores compared to saline treated controls from 4 to 6 weeks (Figure 5.35). Rats in the minocycline (I.V.) group frequently showed improvements in foot position (more parallel to body) and demonstrated fewer toe drags compared to the saline treated animals. However, both regimens of SB203580, failed to improve hindlimb subscores compared to vehicle controls (Figure 5.3Q. Delayed intravenous application of minocycline increases residual sparing of both white and grey matter following SCI. To assess the effects of intravenous minocycline application on tissue sparing following contusion SCI, both residual white and grey matter area were measured and compared between minocycline and saline treated animals. As shown in Figure 5.4,4, delayed minocyline (I.V.) treatment promoted tissue sparing at the lesion site and both rostral and caudal to the injury epicenter in examples from a saline (top) and minocycline-treated rat (bottom). Quantification of residual white matter (Figure 5AB,D) and grey matter area (figure 5AC,E) reveals that minocycline significantly increased residual tissue sparing compared to saline-treated animals at six weeks following contusion injury. DISCUSSION In this study I examined the efficacy of minocycline or the p38 M A P K inhibitor, SB203580, on behavioural and histological outcome measures following a moderate contusion injury in rats. I hypothesized that minocycline treatment would improve functional outcome after contusion SCI and since p38 M A P K is a putative target of minocycline's neuroprotective effects, application of a p38 M A P K inhibitor would also improve functional outcome. The results of the study revealed that delayed intravenous (I.V.) application of minocycline 122 m It mm Umm Umm 1 • mm 12 mm B.l mm 9.1mm tpfcwwr 04 mm BJ mm 11 mm 11 mm l i n n 1.4 mm Figure 5.4 Delayed intravenous application of minocycline promotes preservation of both white and grey matter following contusion SCI. A, Serial transverse sections o f Eriochrome cyanine (EC) and cresyl violet stained sections throughout the entire lesion site reveal the extent o f tissue damage following a moderate contusion injury. Note the preservation o f both white and grey matter in the minocycline treated (best case scenerio, bottom) compared to a saline treated animal (top). Residual white matter (B) and grey matter (C) area as a function o f distance from the epicenter shows that minocycline increases residual tissue area compared to saline-treated animals at six weeks following contusion injury. Quantification o f total residual white matter area (D) and grey matter area ( £ ) reveal a significant increase in spared white and grey matter in minocycline compared to saline treated animals. Data represent mean ± S E M (n=5 to 6/group). *P < 0.05. 123 administered one hour after moderate contusion injury to the thoracic spinal cord in rats, improved open-field locomotion and preserved both white and grey matter tissue compared to saline-treated controls. In contrast, neither the intraperitoneal application of minocycline nor two dosage regimens of the p38 M A P K inhibitor, SB203580 improved functional outcome. The finding that minocycline administered interperitoneally failed to improve functional outcome is at odds with previous studies demonstrating that minocycline improved functional outcome following contusion SCI (Lee et al., 2003; Teng et al., 2004). The discrepancies between these studies may be due to the differences in the severity of injury, dose of minocycline, and the duration of treatment. Both the Lee study and Teng study used the N Y U contusion device to induce a mild contusion injury (lOg rod dropped from a height of 12.5 mm) at the T10 level (Lee et al., 2003; Teng et al., 2004). The authors of the Lee study reported that minocycline treatment significantly improved hindlimb function 24-38 days post injury compared to PBS treated controls, with mean B B B scores of 18 and 15 for the minocycline and saline-treated animals respectively (Lee et al., 2003). Similarly, Teng et al, reported improved B B B scores with a mean of 17 and 13, for minocycline and vehicle-treated animals, respectively (Teng et al., 2004). In contrast to these results, the mean B B B score in the present study was 11 for both I.P. groups. A rat with a B B B score of 11 demonstrates frequent to consistent weight-supported plantar steps but never achieves one-to-one forelimb-hindlimb coordination, a level extremely difficult to surpass following a moderate contusion injury, see table 5.2 (Basso et al., 1995, 1996; Popovich et al., 1999). Therefore, it is likely that the milder injury paradigm used in the Teng et al, and Lee et al, studies compared to the moderate injury reported here, partially explains the difference in outcome as the majority of the control animals in these studies demonstrated consistent forelimb-hindlimb coordination (Lee et al., 2003; Teng et al., 2004). 124 The dosage and duration of minocycline application in these reports may also partially explain the discrepancies between studies. The initial dosage used in the Lee study (90 mg/kg immediately after injury, followed by 2 doses of 45 mg/kg every 12 hours, total dose 180 mg/kg) was almost double the initial dosage used in the present study (50 mg/kg immediately after injury, followed by 50 mg/kg twice per day for 3 days, total dose: 300 mg/kg per rat) and may also contribute to the positive findings obtained in their study (Lee et al., 2003). Furthermore, the dosage given in the Teng et al study (90 mg/kg delayed one hour after injury, with subsequent doses of 45 mg/kg every 12 hours for 5 days, total dose: 495 mg/kg per rat) was delayed one hour and the initial bolus of minocycline would expectantly result in peak plasma levels of approximately 30 mg/L by two to three hours following injection (Fagan et al., 2004; Teng et al., 2004). The present dosage paradigm used in my studies would be expected to have peak plasma levels of 15 mg/L (assuming linearality) two to three hours after injection (Fagan et al., 2004). Since the half-life of minocycline in rodents following I.P. administration is approximately two to three hours (Popovic et al., 2002), the plasma levels in the Teng study would be expected to be approximately two-fold higher than the present study at three hours after injury, and may also contribute to the positive outcome in the their study (Teng et al., 2004). Based on the negative findings pertaining to the I.P. minocycline study, I investigated the efficacy of intravenous administration of minocycline as this approach has been proven successful after middle cerebral artery occlusion-reperfustion in rats (Xu et al., 2004b). The rationale for this approach is also supported by the demonstration that both uptake and absorption occurs immediately and variability between animals is greatly reduced (Fagan et al., 2004). Furthermore, due to the nature of the secondary injury after SCI, early intervention is 125 imperative and I.V. injection would be the preferred route of drug administration following human SCI (Tsai and Tator, 2005). In this study, I.V. administered minocycline delayed one hour after moderate contusion injury, significantly improved functional outcome and promoted tissue preservation compared to saline-treated controls. This confirms and extends previous studies that have demonstrated that minocycline is neuroprotective following milder forms of SCI in rat (Lee et al., 2003; Stirling et al., 2004; Teng et al., 2004) and severe clip compression injuries in mouse (Wells et al., 2003a). Since residual tissue sparing is positively correlated to functional outcome following contusion SCI (Basso et al., 1996), it is likely that the improved functional outcome reported here is a result of preserving descending ventral and lateral white matter tracts important in locomotion (Basso et al., 2002). The dosage used in the present study (initial bolus 20 mg/kg, followed by 10 mg/kg, twice per day for 4 days after injury, total dose: 110 mg) was chosen based on the positive results demonstrated in the previously reported LP. studies (90 mg/kg followed by 45 mg/kg twice per day) (Lee et al., 2003; Teng et al., 2004) and the equivalent dosage of I.V. minocycline required to reach the LP. peak plasma levels (peak plasma level of minocycline -32 mg/L obtained from 90 mg/kg LP. administered minocycline; peak plasma level of minocycline -28 mg/L obtained from 20 mg/kg I.V. administered minocycline) (Fagan et a l , 2004). Importantly, the dose used in the present study would by far exceed the 200 mg/kg per day dosage of minocycline used in humans for treatment of rheumatoid arthritis (Kloppenburg et al., 1994; Tilley et al., 1995). However, the half-live of minocycline in rodents is -2-3 hours vs. -12-18 hours in humans (Andes and Craig, 2002) and effective tissue levels may be comparable. Ungoing clinical trials of minocycline for Huntington's disease, amyotrophic lateral schlerosis, multiple sclerosis, 126 Parkinson's disease, stroke and SCI will likely provide important information regarding the safety, tolerability and efficacy of minocycline (Blum et al., 2004; Yong et al., 2004). Previously, I have demonstrated that p38 M A P K mRNA and activation is reduced following minocycline application, chapter 5 (Stirling, 2003). P38 M A P K may be an important therapeutic target for SCI as inhibitioin of p38 M A P K , utilizing SB203580, reduced neuropathic pain following L5 spinal nerve ligation (Jin et al., 2003) and improved hindlimb function after compression SCI (Horiuchi et al., 2003). To test whether direct p38 M A P K inhibition is neuroprotective after contusion SCI, I applied the p38 M A P K inhibitor, SB203580 intrathecally for seven days post-contusion injury. In contrast to previous results (Horiuchi et al., 2003), application of SB203580 failed to improve functional outcome after SCI. Horiuchi and colleagues reported that continuous intrathecal infusion of SB203580 (1 or 3 pg/day) immediately after mild compression (20g weight applied on the dural surface for 40 minutes) injury at the level of the 11th thoracic vertebrae, significantly improved frequency of standing compared to vehicle treated controls from two to three weeks post-injury (longest time-point examined) (Horiuchi et al., 2003). In addition, the authors stated that frequency of standing assessment was used instead of B B B as injured non-treated animals demonstrated B B B scores of 16-20 and therefore were not suitable in determining minor improvements in hindlimb function (Horiuchi et al., 2003). The discrepancy between their study and the current study may be due to the more severe contusion injury applied here as time of dosage and route of delivery were equivalent between studies. Of particular interest, the Horiuchi study revealed that high doses of SB203580 (10 pg/day) had no effect and the low dosage paradigm used in the current study (3 pg/day) was less efficacious than the 1 pg/day for 7 days regimen (Horiuchi et al., 2003). Therefore, the failure of both the 3 and 7 pg/day regimens in the current study to promote 127 functional recovery was probably a result of the dosing of the inhibitor. It is also likely that the weight placement model used in the Horiuchi study inflicts more of an ischemic lesion and less of a mechanical disruption that is seen after contusion injury, thus the pathophysiology between studies may also differ (Horiuchi et al., 2003). However, a diect effect of the p38 M A P K inhibitor on p38 M A P K activity was not measured and future studies should address this issue before final conclusions can be made. The results in the present study suggest that I.V. application of minocycline delayed up to one hour post-injury, promotes tissue preservation and functional recovery following contusion SCI. These results further support the use of minocycline for human SCI, however, the appropriate route of administration should be carefully considered. 128 CHAPTER 6 GENERAL DISCUSSION AND FUTURE DIRECTIONS 129 I N T R O D U C T I O N In this thesis I have used two established models of SCI, dorsal column transection and contusion, to further our understanding of the secondary injury and the neuroprotective effects of minocycline treatment in the SCI setting. Results of the studies in this thesis demonstrate (1) cervical (C7) dorsal column transection-induced apoptosis occurs within both distal and proximal regions of the AST and CST; (2) the peak of apoptosis occurs at one to two weeks post-injury within the AST, whereas, the peak of apoptosis within the CST occurs between two to four weeks post-injury; (3) although both microglia and oligodendrocytes undergo apoptosis, the former are the major cell type to undergo apoptosis in this model; (4) application of minocycline reduced oligodendrocyte apoptosis, diminished the microglial/macrophage response, prevented CST axonal dieback, lessened lesion size and improved functional outcome; (5) p38 M A P K mRNA and activation was increased after SCI and was attenuated following minocycline treatment; (6) active p38 M A P K colocalized with microglia and neutrophils acutely after SCI and remained elevated within microglia/macrophages at the lesion site and remote to lesion, up to six weeks post-injury (longest time point examined); (7) delayed intravenous application of minocycline increased both white and gray matter tissue sparing and improved open-field locomotion after T9/10 contusion SCI; (8) Intrathecal administration of the p38 M A P K inhibitor, SB203580, or intraperitoneal administered minocycline, failed to improve open-field locomotion following contusion SCI. Importantly, the work in this thesis has contributed to our understanding of the neuroprotective effects of minocycline treatment after SCI, however, further work in this area will be needed to elucidate the molecular targets of minocycline's mode of action in the SCI setting. 130 Glial apoptosis following SCI. The results of my studies on SCI-induced apoptosis (chapter 2) are consistent with previous studies that have shown large numbers of TUNEL positive and/or active caspase-3 positive apoptotic profiles contribute to SCI-induced cell death (Katoh et al., 1996b; L i et al., 1996; Crowe et al., 1997; Liu et al., 1997b; Shuman et al., 1997; Emery et al., 1998; Lou et al., 1998; Yong et al., 1998; Abe et al., 1999; L i et al., 1999a; Springer et al., 1999; Beattie et al., 2000; Citron et al., 2000; Lu et al., 2000; Casha et al., 2001; Grossman et al., 2001; Warden et al., 2001; Beattie et al., 2002b; Koda et al., 2002; Dong et al., 2003; McBride et al., 2003; Beattie, 2004; Stirling et al., 2004; Wang et al., 2004b; Yoshino et al., 2004; Yune et al., 2004). Oligodendrocyte apoptosis has been identified as a cellular constituent of the observed apoptotic response following SCI in rodents (Crowe et al., 1997; Yong et al., 1998; Abe et al., 1999; Beattie et al., 2000; Casha et al., 2001; Grossman et al., 2001; Warden et a l , 2001; Beattie et al., 2002b; Koda et al., 2002; Dong et al., 2003; Kim et al., 2003; Takagi et al., 2003; Stirling et al., 2004), chicks (McBride et al., 2003), monkeys (Crowe et al., 1997), and humans (Emery et al., 1998). Crowe and colleagues were first to report oligodendrocyte apoptosis after SCI (Crowe et al., 1997). They examined the temporal and spatial occurrence of apoptosis following a moderate contusion injury at the level of the 10th thoracic vertebrae in adult rats, utilizing a bisbenzimide dye, Hoechst 33342, to visualize clumped condensed chromatin, and TUNEL and D N A gel electrophoresis to assess oligonucleosomal D N A fragmentation, two hallmarks of apoptosis. Although apoptotic nuclei (both neurons and glia) were evident at the lesion site as early as 6 hr post-injury, a delayed response initiated several days to weeks later was reported within the white matter several segments rostral and caudal to the injury site. No apoptotic profiles were reported in non-injured spinal cords. 131 The apoptotic profiles within the degenerating white matter tracts were identified as oligodendrocytes utilizing monoclonal antibodies RIP and CC1, specific markers for these cells. Apoptotic profiles did not co-localize with markers for either microglia/macrophages (OX-42) or astrocytes (GFAP). The authors concluded that oligodendrocyte apoptosis in both SCI rat and monkey occurs in two phases: A n immediate phase localized to the lesion site and a delayed phase localized to degenerating white matter tracts that contribute to lesion-induced secondary cell death and subsequent neurological deficits (Crowe et al., 1997). Additional studies confirmed these results and reported the appearance of apoptotic microglia (Shuman et al., 1997). Whether astrocytes undergo SCI-induced apoptosis is highly debated, but at least one report provided evidence of astrocyte apoptosis within close proximity to the injury site (Yong et al., 1998). As discussed in chapter 2, my results are in agreement with previous studies that found large numbers of apoptotic microglia are present after contusion SCI in rats (Shuman et al., 1997; Yong et al., 1998). However, other studies have reported that microglia/macrophages were not among the apoptotic profiles present after partial or complete transection or contusion injuries in rats (Crowe et al., 1997; Abe et al., 1999; Warden et al., 2001). Differences in apoptotic detection methods (TUNEL vs. active caspase-3), microglial/macrophage markers (0X42 vs. BS1 or 0X6), or injury paradigms may partially account for the discrepancy between studies. As mentioned previously in chapter 2,1 was unable to rule out the possibility that phagocytes engulfing apoptotic cells may be included in the counts of apoptotic microglia/macrophages and thus may inflate their number. However, to try to limit this problem, I utilized confocal microscopy to visualize the entire boundary of the cell. This allowed me to definitively determine whether an apoptotic nucleus, normal nucleus, or both were present within the 132 boundaries of the cell. Of the approximately twelve randomly selected cells analyzed in this manner, all displayed an apoptotic nucleus within an active caspase-3 (+) profile, and colocalized to 0X42 (a marker for microglia/ macrophages) confirming that microglia were among the cells undergoing apoptosis following SCI. Extracellular mechanisms of oligodendrocyte apoptosis after SCI Even though the exact stimuli/us that cause oligodendrocyte apoptosis after SCI remains elusive, two hypotheses have been put forth; (1) Loss of axon-derived trophic support induces oligodendrocyte apoptosis (Crowe et al., 1997); or alternatively (2) activated microglia/macrophages or cytotoxic products associated with their activation may induce oligodendrocyte apoptosis (Shuman et al., 1997). Evidence to support the first hypothesis comes from studies that reported a spatial correlation between oligodendrocyte apoptosis and Wallerian degeneration after SCI (Crowe et al., 1997), and the dependence of oligodendrocytes on axon-derived trophic support during development, a mechanism that ensures oligodendrocyte number is precisely matched to the number and length of axons requiring myelination (Barres et al., 1993; Barres and Raff, 1994). Although oligodendrocytes require axon-derived signals for survival during development it is less clear for adult cells. Transection of the optic nerve in adult rats resulted in reduced oligodendrocyte death within areas of complete axonal degeneration and argues against the loss of axonal trophic-support as a mechanism that induces oligodendrocyte apoptosis following injury (Ludwin, 1990). In addition, many oligodendrocytes survived for up to 6 weeks (longest survival period examined) within the completely degenerating white matter tracts following a C7 dorsal column transection in adult rats (personal observations). Furthermore, L i and colleagues reported the appearance of apoptotic oligodendrocytes in proximal regions of the cord in which 133 axons were not damaged (Li et al., 1999a; L i et al., 1999b). The results in this thesis further support these findings as apoptotic oligodendrocytes were localized to the proximal areas of the AST and CST suggesting loss of axonal trophic support may not be the sole stimulus responsible for their death after SCI. Evidence to support the alternative hypothesis, that activated microglia/macrophages or cytotoxic products associated with their activation may induce oligodendrocyte apoptosis, is based on the observation that activated microglia/macrophages were found to be in close proximity to apoptotic oligodendrocytes after SCI (Shuman et al., 1997) and microglia/macrophages express and release death inducers (such as Fas ligand, TNF-a, and NGF) known to induce apoptosis in these cells (McLaurin et al., 1995; Casaccia-Bonnefil et al., 1996; D'Souza et al., 1996; Beattie et al., 2002b). The results in chapter 3 of this thesis are inconclusive regarding the role of microglia/macrophages in inducing oligodendrocyte death, as minocycline treatment may directly inhibit apoptosis as well as microglia/macrophage activation. In addition, microglia density within the proximal areas of oligodendrocyte apoptosis was not significantly reduced after minocycline administration (chapter 3). If microglia induced their death, it would be expected that the microglial response would be attenuated as well as oligodendrocyte apoptosis following minocycline treatment but no significant difference was detected in the microglia response compared to saline treated controls. Furthermore, activated microglia/macrophages may simply be responding to dying oligodendrocytes and be in the process of phagocytosis (Li et al., 1999b), an established mechanism to remove apoptotic cells. To further our understanding of the mechanism of SCI-induced oligodendrocyte apoptosis, future studies employing putative trophic molecules such as neuregulins, IGF-1, NT-3, and PDGF, could be used to determine the importance of survival factors in regulating 134 oligodendrocyte survival. Rescue of oligodendrocytes with such factors would indicate that the loss of trophic support after injury is an important death stimulus for these cells. Alternatively treatments to selectively inhibit the microglia/macrophage response after SCI would be expected to reduce oligodendrocyte apoptosis as well and would support a role for microglia/macrophages in oligodendrocyte cell death. Relevance of oligodendrocyte apoptosis Regardless of the precise cause of SCI-induced oligodendrocyte apoptosis, their death may contribute to the chronic demyelination of spared axons observed following human SCI (Bunge et al., 1993) and the resulting conduction block may cause further functional loss after SCI. Recently, Beattie and colleagues reported approximately 50% of oligodendrocytes are lost at one week after SCI with significant reductions existing to six weeks post-injury (Beattie et al., 2002b). Since oligodendrocyte apoptosis is temporally and spatially correlated with the actual loss of oligodendrocytes, the authors concluded that apoptosis must be an important mechanism leading to the death of these cells after SCI (Beattie et al., 2002b). Similarly, the apoptotic oligodendrocyte death in some forms and models of multiple sclerosis indicate a role in the progressive demyelination characterized by the disease (Dowling et al., 1997; Matute and Perez-Cerda, 2005). In addition, other pathological conditions also involve oligodendrocyte apoptosis, such as multiple system atrophy (Probst-Cousin et al., 1998), the childhood cerebral form of adrenoleukodystrophy (Feigenbaum et al., 2000), and cognitive and spastic deficits in premature infants (Noble and Mayer-Proschel, 1996). Thus the prevention of oligodendrocyte apoptosis is an important goal in arresting the progression of these conditions. 135 Minocycline as an anti-apoptotic agent The finding that minocycline treatment attenuated SCI-induced oligodendrocyte apoptosis (chapter 3) is consistent with other studies that have demonstrated that minocycline reduced apoptosis in a variety of cell types, after application of many different death-inducing factors, and in a wide assortment of experimental paradigms (Arvin et al., 2002; Matsuki et al., 2003; Power et al., 2003; Wang et al., 2003b; Baptiste et al., 2004; Corbacella et al., 2004; Hughes et al., 2004; Kelly et al., 2004; Kern et al., 2004; Lee et al., 2004; Pi et al., 2004; Scarabelli et al., 2004; Wang et al., 2004a; Richardson-Burns and Tyler, 2005; Vera et al., 2005). M y results are also in agreement with other studies that have shown that minocycline promoted the survival and myelination of oligodendroglial progenitors transplanted into the spinal cord of myelin mutant rats (Zhang et al., 2003a), enhanced the survival of oligodendrocytes following contusion SCI (Teng et al., 2004) and after LPS-induced white matter injury in neonatal rat brain (Fan et al., 2005). However, it is important to note that long-term studies assessing the viability of oligodendrocytes after CNS trauma or disease have not been conducted. Therefore it will be important to establish whether minocycline's effects on oligodendrocyte survival and apoptosis are permanent or whether these effects are transient. Nevertheless, much recent work has been directed in elucidating the mechanisms behind minocycline's anti-apoptotic effects and potential targets include; (1) inhibiting caspase activation (Chen et al., 2000); (2) preventing the mitochondrial release of pro-apoptotic factors including cytochrome c, Smac/DIABLO and AIF (Zhu et a l , 2002; Wang et al., 2003b); and (3) increasing the expression of anti-apoptotic factors including Bcl-2 and XIAP (Scarabelli et al., 2004; Wang et al., 2004a). Thus, minocycline-mediated protection likely targets both caspase-136 dependent (cytochrome-c, Smac/Diablo) and caspase-independent (AIF) forms of apoptosis (Figure 6.1). Although the exact mechanism behind minocycline's anti-apoptotic effects remains unknown, a direct effect of minocycline on caspase-1 and -3 activity was ruled out using cell-free extracts, suggesting minocycline may act upstream of caspase activation (Chen et al., 2000). In support of these findings, reconstitution experiments, by adding cytochrome-c to isolated cytosol extracts of kidney cells, confirmed that minocycline acts at the level of the mitochondria, and not downstream of cytochrome-c release (Wang et al., 2004a). Collectively, these results suggest that minocycline has a direct effect in inhibiting cell death by priming a "survival mode" rendering a cell less vulnerable to apoptotic stimuli and likely explains in part, minocycline's neuroprotective effects in CNS trauma and disease. Microglial/macrophage response to SCI In chapter 3 and 4,1 demonstrated that minocycline treatment reduced the microglia/macrophage response after SCI and these effects were associated with improved functional outcome. These results are in agreement with several previous studies that revealed activated mononuclear phagocytes (CNS resident microglia and/or blood-derived macrophages) and products associated with their activation contribute to the secondary damage after SCI including demyelination and/or axonal degeneration of previously spared axons (Blight, 1985; Giulian and Robertson, 1990; Blight, 1992, 1994; Bethea et al., 1999; Popovich et al., 1999; Popovich et al., 2002). Following SCI in cats, demyelination of intact axons within the outer rim of the spinal cord is delayed (2 days to 1 week post-injury) and is temporally correlated with an invasion of the CNS parenchyma by phagocytes (Blight, 1992). Choroquine/colchicine treatment significantly reduced the appearance of mononuclear phagocytes after spinal cord 137 Death receptor pathway ligand ^ | ^ ^ . . procaspake-3 Cleavage of cytosolic proteins Figure 6.1 Apoptotic cell death pathways and intervention following minocycline application. The death receptor pathway involves activation o f procaspase-8 and subsequent activation o f effector caspases such as pro-caspase-3. The mitochondrial pathway involves cytochrome-c release and formation o f an apoptosome leading to activation of procaspase-9 and subsequent activation o f procaspase-3. Act ive caspase-3 cleaves several cytosolic proteins and liberates a nuclease ( C A D ) involved in D N A condensation and fragmentation. Minocyc l ine may inhibit apoptosis by preventing the release o f pro-apoptotic molecules (cytochrome-c, S m a c / D I A B L O and A I F , red stop signs = inhibition) and up-regulating anti-apoptotic proteins including Bc l -2 and X I A P , green traffic light = stimulation. 138 ischemia in rabbits and resulted in improved recovery of hindlimb and bladder function, promoted survival of motoneurons, and preserved somatosensory evoked potentials (Giulian and Robertson, 1990). Silica-induced death of monocytes/macrophages following SCI significantly reduced the loss of myelinated axons in a guinea-pig model of SCI (Blight, 1994). In addition, Popovich and colleagues depleted hematogenous macrophages and reported reduced activated macrophages (EDI staining), improved hindlimb recovery, increased sparing of myelinated axons and reduced cavitation after SCI (Popovich et al., 1999). Furthermore, zymosan, a potent activator of microglia/macrophages caused cavitation, demyelination and permanent axonal injury when injected into the rat brain and spinal cord (Fitch et al., 1999; Popovich et al., 2002). Even though macrophages are essential in wound healing and repair in peripheral tissues (Fahey et al., 1990), many of the potential cytotoxic substances released during their response may cause "bystander death" to uninjured oligodendrocytes and neurons, furthering the neurological deficits after SCI (Bethea, 2000; Popovich, 2000). Collectively these studies provide compelling evidence of the deleterious potential associated with the activation of these cells following CNS trauma. Although microglia/macrophages may cause "bystander" cell death following SCI, others have suggested a beneficial role following injury. In support of this notion, transplantation of activated macrophages (pre-stimulated with peripheral nerve) into the injured spinal cord improved axonal regeneration and functional outcome (Rapalino et al., 1998; Schwartz et al., 1999). In addition, neurite outgrowth was enhanced following transplantation of cultured microglial cells into injured spinal cord (Prewitt et al., 1997; Rabchevsky and Streit, 1997). Furthermore, microglia/macrophages may augment remyelination following experimental 139 demyelination and inhibiting their response may hinder spontaneous remyelination attempts (Kotter et a l , 2005). It is unknown why these often conflicting results exist but they may be due to the manner in which the macrophages are activated and differences in the injury milieu that influences their response. For example, the benefits afforded by macrophage transplantation after SCI may result from promoting a more "PNS macrophage response" where pre-stimulating macrophages within peripheral myelin before transplantation aides removal of inhibitory molecules and facilitates axonal regeneration (Perry et al., 1993; Rapalino et al., 1998). Further studies are needed to resolve these important issues and to determine the potential damaging and/or regenerative capacity pertaining to the microglial/macrophage response following SCI and the appropriate time to intervene. Minocycline's anti-inflammatory properties The finding that minocycline treatment attenuated the microglia/macrophage response after SCI (chapter 3&4) is consistent with other studies that have demonstrated that minocycline reduced the microglial/macrophage response following a variety of experimental manipulations and animal models of CNS trauma and disease (Yrjanheikki et al., 1998; Yrjanheikki et al., 1999; He et al., 2001; Tikka et al., 2001; Tikka and Koistinaho, 2001; Wu et al., 2002; Ekdahl et al., 2003; Raghavendra et al., 2003; Zhang et al., 2003a; Kim et al., 2004; Kremlev et al., 2004; Stirling et al., 2004; Suk, 2004; Tomas-Camardiel et al., 2004; Fan et al., 2005; Krady et al., 2005; Ledeboer et al., 2005; Wang et al., 2005). Collectively, minocycline has been suggested to exert its anti-inflammatory effects by modulating microglia and peripheral immune cell activation/recruitment by: (1) preventing the release of cytokines and chemokines ; (2) reducing lipid mediators of inflammation; (3) 140 inhibiting metalloproteases (MMPs); (4) and limiting nitric oxide release, Figure 6.2 (Blum et al., 2004; Yong et al., 2004; Zemke and Majid, 2004). Since many of these substances are thought to contribute to the secondary injury after SCI (see Figure 1.1 and introduction), the finding that minocycline is neuroprotective after SCI is likely due in part to the inhibition of these inflammatory mediators. The results from my studies are also in agreement with other reports that have demonstrated that minocycline treatment diminished the immune response following SCI. In support of this notion, minocycline application reduced TNF-a mRNA levels after SCI, and prevented the lipopolysaccharide (LPS)-induced production of TNF-a in primary glial cell cultures (Lee et al., 2003; Lee et al., 2004). In addition, the anti-inflammatory cytokine IL-10, was also up-regulated following minocycline application immediately after SCI (Lee et al., 2003). Since administration of IL-10 improved functional outcome and reduced microglia/ macrophage-mediated production of TNF-a, modulating IL-10 expression may be an important component of minocycline's anti-inflammatory properties. Further studies are needed to define the role on IL-10 following SCI. Interestingly, minocycline has also been shown to reduce the astrocytic response after SCI (McPhail et al., 2004; Teng et al., 2004), and after p-amyloid injection into the hippocampus (Ryu et al., 2004). Since astrocytes contribute to axonal regeneration failure and proinflammatory cytokine production, inhibiting the astrocytic response to injury may be beneficial. However, minocycline did not appear to affect astrocyte activation in other models of neurodegeneration (Yrjanheikki et al., 1999; Du et al., 2001), providing the impetus to further explore whether minocycline is able to modulate the astrocyte response after injury or during disease progression. 141 Figure 6.2 SCI-induced inflammatory response and intervention following minocycline treatment. /. Resting microglial cell. 2. Microglia become activated after SCI and releases pro-inflammatory mediators that attract neutrophils (3) and macrophages to the site of injury. 4. The release of pro-inflammatory factors may cause additional axonal degeneration, oligodendrocyte death and demyelination. The p38 M A P K pathway (boxed region) regulates cytokine production, and release of both NO and PGE2. Minocycline (mino - red stop sign) is thought to reduce microglia activation, and levels of IL- ip , TNF-a , NO and PGE-2 release likely through inhibiting p38 M A P K activation, however the exact targets of minocycline are still unknown. Minocycline also reduces migration of immune cells through inhibition of matrix metalloproteinases (not shown). Abbreviations: TNF-a , tumor necrosis factor-alpha; IL, interleukin; M A P K , mitogen-activated protein kinase; M A P K K , mitogen-activated protein kinase, kinase; M A P K K K , mitogen-activate protein kinase, kinase, kinase; M A P K A P K2, mitogen-activated protein kinase-activated protein kinase 2; NO, nitric oxide; PGE2, prostaglandin E2; COX-2, cyclooxygenase-2 ; iNOS, .inducible nitric oxide synthase. 142 Taken together, minocycline acts on several aspects of the inflammatory response following CNS trauma and insult. Since neurons and oligodendrocytes are vulnerable to many of the inflammatory factors discussed in the previous sections, minocycline's effects on attenuating the inflammatory response may reduce 'bystander death" of these cells during disease progression or following trauma. However it is important to note that following facial nerve axotomy in rat, oral minocycline treatment failed to reduce microglial proliferation (Fendrick et al., 2005). It is likely that the lack of disruption of the blood brain barrier and differences between the microglia/macrophage response following CNS trauma and facial axotomy partially account for the lack of effect in the later study (Moran and Graeber, 2004). P38 M A P K , a putative target of minocycline's mode of action The results from chapter 4 in this thesis demonstrated that minocycline treatment was associated with a marked reduction in p38 M A P K expression and activation. The results also showed that p38 M A P K was localized to neutrophils and microglia/macrophages acutely after SCI. These findings led to the hypothesis that minocycline is neuroprotective during the early acute phase after SCI by inhibiting the microglial/macrophage response. I further hypothesized that minocycline's protective actions are mediated though inhibition of p38 M A P K ; therefore, p38 M A P K inhibitors are neuroprotective after acute SCI (chapter 5). Although minocycline-treated animals had reduced levels of p38 M A P K mRNA and activity state, I was not able to demonstrate that this was due to a direct effect of minocycline. Alternatively, minocycline application may have reduced the microglia/macrophage response and accordingly, p38 M A P K mRNA, total protein, and activation levels would be expected to be reduced as well. In addition, minocycline may also inhibit many other known (see above) or unknown mediators of the secondary damage after SCI, and as a result may also reduce tissue 143 damage and preserve axonal integrity. For example, minocycline may inhibit the neutrophil response after SCI and as a result reduce macrophage infiltration. Indeed treatments that are thought to directly target neutrophils after SCI also reduce macrophage infiltration as well (Saville et al., 2004). These unknown factors could also lead to the improved functional outcome demonstrated after minocycline application. However, as previously mentioned, several studies have demonstrated that minocycline directly inhibits microglia proliferation and activation in vitro and therefore it is probable that a direct effect on these cells occurs in vivo as well (Tikka et al., 2001; Tikka and Koistinaho, 2001). Minocycline treatment has been demonstrated to inhibit p38 M A P K activation in cultures of pure microglia stimulated by glutamate (Tikka et al., 2001) or N M D A (Tikka and Koistinaho, 2001) or under hypoxic conditions (Suk, 2004), in cultures of cerebellar granule neurons stimulated with nitric oxide (Du et al., 2001; Lin et al., 2001) or glutamate (Pi et al., 2004), and in hair cells treated with gentamicin (Wei et al., 2005). Since application of the p38 M A P K inhibitor, SB203580, mirrored the effects of minocycline treatment, whereas inhibitors of other M A P K family members did not, it was suggested that minocycline inhibits microglia activation and neuronal cell death through a p38 M A P K dependent mechanism (Du et al., 2001; Lin et al., 2001; Tikka et al., 2001; Tikka and Koistinaho, 2001; Pi et al., 2004; Suk, 2004; Wei et a l , 2005). Recently, Pi and colleagues were able to demonstrate that minocycline directly inhibited p38 M A P K activity (Pi et al., 2004). Utilizing specific kinase assays that isolate the dual phosphorylated (active) form of p38 M A P K , the authors measured the phosphorylation state of ATF-2, a well characterized, specific downstream target of p38 M A P K , and showed ATF-2 phosphorylation was significantly reduced following minocycline or SB203580 inhibition (Pi et 144 al., 2004). Collectively, these results provide strong evidence that minocycline directly inhibits p38 M A P K activation and activity. Based on the results from chapter 4, the established role p38 M A P K plays in regulation of immune cell function, and the previous in vitro minocycline studies above, the direct inhibition of p38 M A P K with the pharmacological inhibitor, SB203580 was expected to improve the outcome of SCI. However, contrary to my initial hypothesis, p38 M A P K inhibition failed to improve functional outcome after contusion SCI (chapter 5). P38 M A P K inhibitors are intensively pursued for their anti-inflammatory effects (Kumar et al., 2003). The prototypic p38 M A P K inhibitor SB203580 has been successfully used in a large number of in vivo studies, including stroke (Barone et al., 2001; Piao et al., 2003) and one study on mild spinal cord compression (non-contusive), where it had beneficial effects after intrathecal application (Horiuchi et al., 2003). It is not known why this approach failed in my hands, but severity of injury in the present study compared to the mild compression SCI used in the Horiuchi and coworker's report may partially account for the discrepancy between studies (Horiuchi et al., 2003). Minocycline treatment of SCI During my thesis studies, three other independent laboratories have also shown that minocycline treatment is neuroprotective following SCI. Given one hour after thoracic (T)3/4 clip compression in the mouse, minocycline treatment (LP.), effectively reduced lesion size, increased axonal sparing of rubrospinal axons and improved hindlimb function (Wells et al., 2003a). Importantly, minocycline was more effective in improving functional outcome when compared with methylprednisolone, the current treatment option for many patients with SCI (Wells et al., 2003a). Minocycline treatment, applied LP. immediately or at 1 hour post lesion, 145 promoted tissue preservation and enhanced functional recovery following mild contusion SCI in rats (Lee et al., 2003; Teng et al., 2004). However, treatment with LP. applied minocycline yielded variable results in my hands using a moderate contusion model. In chapter 5 of this thesis I utilized a novel delivery system to administer multiple injections of minocycline (delayed one hour) intravenously to overcome these problems and demonstrated that minocycline applied by this route promotes tissue preservation and functional recovery after a moderate contusion injury. However, several questions remain unanswered pertaining to the use of minocycline in the SCI setting. For example, further studies will be needed to determine the optimal dosage and most favorable time of minocycline application in order to reduce the damaging aspects of the secondary degenerative response. In addition, the type and severity of lesions that will benefit most from minocycline treatment remains unclear. Although the inflammatory response after SCI contributes to tissue destruction over a period of days to weeks after trauma (Blight, 1992; Popovich et al., 1999), microglia/ macrophages and other immune cells may also facilitate repair processes such as axonal sprouting and remyelination (Kotter et al., 2005; L i et al., 2005). Thus, further studies are needed to rule out possible adverse effects of late application and ensure the timing and duration of minocycline treatment does not impede any of the proposed benefits associated with an immune response or attempts of endogenous repair. Conclusions Based on the initial benefits of minocycline in animal models of stroke, an expansion of studies into several fields such as neurodegenerative diseases, spinal cord, and brain injury, has established minocycline, and possibly other tetracycline derivatives, as potential therapeutic agents. The findings of this thesis further support minocycline as a neuroprotective agent after 146 SCI. Although the exact molecular targets of minocycline's mode of action have yet to be characterized, several lines of evidence point to a convergent action in the apparent simultaneous suppression of both apoptosis and CNS inflammation. The p38 M A P K pathway may be a common signaling pathway for modulating both inflammation and apoptosis. Nevertheless, it is also possible that minocycline's benefit lies in its ability to target multiple pathways mediating different aspects of apoptosis and inflammation. Continued study in these areas will likely lead to additional answers, and unveil other targets for future neuroprotective strategies to promote functional improvement after human CNS trauma or disease. The emerging success of ongoing clinical trials raises optimism that minocycline treatment may soon be translated to clinical practice for neurodegeneration, stroke or trauma (Metz et al., 2004; Yong et al., 2004). 147 REFERENCES Abe Y , Yamamoto T, Sugiyama Y , Watanabe T, Saito N , Kayama H, Kumagai T (1999) Apoptotic cells associated with Wallerian degeneration after experimental spinal cord injury: a possible mechanism of oligodendroglial death. J Neurotrauma 16:945-952. Abe Y , Yamamoto T, Sugiyama Y , Watanabe T, Saito N , Kayama H , Kumagai T (2004) "Anoikis" of oligodendrocytes induced by Wallerian degeneration: ultrastructural observations. J Neurotrauma 21:119-124. Agrawal SK, Fehlings M G (1996) Mechanisms of secondary injury to spinal cord axons in vitro: role of Na+, Na(+)-K(+)-ATPase, the Na(+)-H+ exchanger, and the Na(+)-Ca2+ exchanger. J Neurosci 16:545-552. Agrawal SK, Fehlings M G (1997) Role of N M D A and non-NMDA ionotropic glutamate receptors in traumatic spinal cord axonal injury. J Neurosci 17:1055-1063. Akassoglou K, Bauer J, Kassiotis G, Pasparakis M , Lassmann H , Kollias G, Probert L (1998) Oligodendrocyte apoptosis and primary demyelination induced by local TNF/p55TNF receptor signaling in the central nervous system of transgenic mice: models for multiple sclerosis with primary oligodendrogliopathy. A m J Pathol 153:801-813. Alarcon GS (2000) Tetracyclines for the treatment of rheumatoid arthritis. Expert Opin Investig Drugs 9:1491-1498. 148 Alvarado-Kristensson M , Melander F, Leandersson K , Ronnstrand L, Wernstedt C, Andersson T (2004) p38-MAPK Signals Survival by Phosphorylation of Caspase-8 and Caspase-3 in Human Neutrophils. J Exp Med 199:449-458. Ambrosini E, Aloisi F (2004) Chemokines and glial cells: a complex network in the central nervous system. Neurochem Res 29:1017-1038. Amin AR, Patel RN, Thakker GD, Lowenstein CJ, Attur M G , Abramson SB (1997) Post-transcriptional regulation of inducible nitric oxide synthase mRNA in murine macrophages by doxycycline and chemically modified tetracyclines. FEBS Lett 410:259-264. Amin AR, Attur M G , Thakker GD, Patel PD, Vyas PR, Patel RN, Patel IR, Abramson SB (1996) A novel mechanism of action of tetracyclines: effects on nitric oxide synthases. Proc Natl Acad Sci U S A 93:14014-14019. Anderson DK, Waters TR, Means ED (1988) Pretreatment with alpha tocopherol enhances neurologic recovery after experimental spinal cord compression injury. J Neurotrauma 5:61-67. Anderson DK, Means ED, Waters TR, Green ES (1982) Microvascular perfusion and metabolism in injured spinal cord after methylprednisolone treatment. J Neurosurg 56:106-113. Anderson DK, Demediuk P, Saunders RD, Dugan L L , Means ED, Horrocks L A (1985) Spinal cord injury and protection. Ann Emerg Med 14:816-821. 149 Andes D, Craig W A (2002) Animal model pharmacokinetics and pharmacodynamics: a critical review. Int J Antimicrob Agents 19:261-268. Arvin K L , Han B H , Du Y , Lin SZ, Paul SM, Holtzman D M (2002) Minocycline markedly protects the neonatal brain against hypoxic-ischemic injury. Ann Neurol 52:54-61. Balentine JD, Spector M (1977) Calcification of axons in experimental spinal cord trauma. Ann Neurol 2:520-523. Bao F, Chen Y , Dekaban GA, Weaver L C (2004) Early anti-inflammatory treatment reduces lipid peroxidation and protein nitration after spinal cord injury in rats. J Neurochem:in press. Baptiste DC, Hartwick AT, Jollimore CA, Baldridge W H , Seigel G M , Kelly M E (2004) An investigation of the neuroprotective effects of tetracycline derivatives in experimental models of retinal cell death. Mol Pharmacol 66:1113-1122. Barone FC, Irving EA, Ray A M , Lee JC, Kassis S, Kumar S, Badger A M , Legos JJ, Erhardt JA, Ohlstein EH, Hunter A J , Harrison DC, Philpott K , Smith BR, Adams JL, Parsons A A (2001) Inhibition of p38 mitogen-activated protein kinase provides neuroprotection in cerebral focal ischemia. Med Res Rev 21:129-145. Barres B A , Raff M C (1994) Control of oligodendrocyte number in the developing rat optic nerve. Neuron 12:935-942. Barres B A , Jacobson M D , Schmid R, Sendtner M , Raff M C (1993) Does oligodendrocyte survival depend on axons? Curr Biol 3:489-497. 150 Barres B A , Hart IK, Coles HS, Burne JF, Voyvodic JT, Richardson WD, Raff M C (1992) Cell death and control of cell survival in the oligodendrocyte lineage. Cell 70:31-46. Bartholdi D, Schwab M E (1997) Expression of pro-inflammatory cytokine and chemokine mRNA upon experimental spinal cord injury in mouse: an in situ hybridization study. Eur J Neurosci 9:1422-1438. Barut S, Canbolat A , Bilge T, Aydin Y , Cokneseli B, Kaya U (1993) Lipid peroxidation in experimental spinal cord injury: time-level relationship. Neurosurg Rev 16:53-59. Basso D M , Beattie MS, Bresnahan JC (1995) A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma 12:1-21. Basso D M , Beattie MS, Bresnahan JC (1996) Graded histological and locomotor outcomes after spinal cord contusion using the N Y U weight-drop device versus transection. Exp Neurol 139:244-256. Basso D M , Beattie MS, Bresnahan JC (2002) Descending systems contributing to locomotor recovery after mild or moderate spinal cord injury in rats: experimental evidence and a review of literature. Restor Neurol Neurosci 20:189-218. Beattie MS (2004) Inflammation and apoptosis: linked therapeutic targets in spinal cord injury. Trends Mol Med 10:580-583. Beattie MS, Farooqui A A , Bresnahan JC (2000) Review of current evidence for apoptosis after spinal cord injury. J Neurotrauma 17:915-925. 151 Beattie MS, Hermann GE, Rogers RC, Bresnahan JC (2002a) Cell death in models of spinal cord injury. Prog Brain Res 137:37-47. Beattie MS, Harrington A W , Lee R, Kim JY, Boyce SL, Longo F M , Bresnahan JC, Hempstead B L , Yoon SO (2002b) ProNGF induces p75-mediated death of oligodendrocytes following spinal cord injury. Neuron 36:375-386. Behrmann DL, Bresnahan JC, Beattie MS, Shah BR (1992) Spinal cord injury produced by consistent mechanical displacement of the cord in rats: behavioral and histologic analysis. J Neurotrauma 9:197-217. Bethea JR (2000) Spinal cord injury-induced inflammation: a dual-edged sword. Prog Brain Res 128:33-42. Bethea JR, Nagashima H , Acosta M C , Briceno C, Gomez F, Marcillo A E , Loor K, Green J, Dietrich W D (1999) Systemically administered interleukin-10 reduces tumor necrosis factor-alpha production and significantly improves functional recovery following traumatic spinal cord injury in rats. J Neurotrauma 16:851-863. Bhat NR, Zhang P, Lee JC, Hogan E L (1998) Extracellular signal-regulated kinase and p38 subgroups of mitogen-activated protein kinases regulate inducible nitric oxide synthase and tumor necrosis factor-alpha gene expression in endotoxin-stimulated primary glial cultures. J Neurosci 18:1633-1641. Blight A (1988) Mechanical factors in experimental spinal cord injury. J A m Paraplegia Soc 11:26-34. 152 Blight A R (1983) Cellular morphology of chronic spinal cord injury in the cat: analysis of myelinated axons by line-sampling. Neuroscience 10:521-543. Blight A R (1985) Delayed demyelination and macrophage invasion: a candidate for secondary cell damage in spinal cord injury. Cent Nerv Syst Trauma 2:299-315. Blight A R (1991) Morphometric analysis of a model of spinal cord injury in guinea pigs, with behavioral evidence of delayed secondary pathology. J Neurol Sci 103:156-171. Blight A R (1992) Macrophages and inflammatory damage in spinal cord injury. J Neurotrauma 9 Suppl 1:S83-91. Blight A R (1994) Effects of silica on the outcome from experimental spinal cord injury: implication of macrophages in secondary tissue damage. Neuroscience 60:263-273. Blight AR, Decrescito V (1986) Morphometric analysis of experimental spinal cord injury in the cat: the relation of injury intensity to survival of myelinated axons. Neuroscience 19:321-341. Blum D, Chtarto A, Tenenbaum L, Brotchi J, Levivier M (2004) Clinical potential of minocycline for neurodegenerative disorders. Neurobiol Dis 17:359-366. Bose B, Osterholm JL, Kalia M (1986) Ganglioside-induced regeneration and reestablishment of axonal continuity in spinal cord-transected rats. Neurosci Lett 63:165-169. Bracken M B (2002) Steroids for acute spinal cord injury. Cochrane Database Syst Rev:CD001046. 153 Bracken M B , Holford TR (1993) Effects of timing of methylprednisolone or naloxone administration on recovery of segmental and long-tract neurological function in NASCIS 2. J Neurosurg 79:500-507. Bracken M B , Shepard MJ , Collins WF, Holford TR, Young W, Baskin DS, Eisenberg H M , Flamm E, Leo-Summers L , Maroon J, et al. (1990) A randomized, controlled trial of methylprednisolone or naloxone in the treatment of acute spinal-cord injury. Results of the Second National Acute Spinal Cord Injury Study. N Engl J Med 322:1405-1411. Brandoli C, Shi B, Pflug B, Andrews P, Wrathall JR, Mocchetti I (2001) Dexamethasone reduces the expression of p75 neurotrophin receptor and apoptosis in contused spinal cord. Brain Res Mol Brain Res 87:61-70. Braughler JM, Hall ED (1982) Correlation of methylprednisolone levels in cat spinal cord with its effects on (Na+ + K+)-ATPase, lipid peroxidation, and alpha motor neuron function. J Neurosurg 56:838-844. Braughler JM, Hall ED (1992) Involvement of lipid peroxidation in CNS injury. J Neurotrauma 9Suppl LS1-7. Brundula V , Rewcastle N B , Metz L M , Bernard CC, Yong V W (2002) Targeting leukocyte MMPs and transmigration: minocycline as a potential therapy for multiple sclerosis. Brain 125:1297-1308. Bunge RP, Puckett WR, Becerra JL, Marcillo A , Quencer R M (1993) Observations on the pathology of human spinal cord injury. A review and classification of 22 new cases with 154 details from a case of chronic cord compression with extensive focal demyelination. Adv Neurol 59:75-89. Carlson SL, Parrish M E , Springer JE, Doty K, Dossett L (1998) Acute inflammatory response in spinal cord following impact injury. Exp Neurol 151:77-88. Casaccia-Bonnefil P, Carter BD, Dobrowsky RT, Chao M V (1996) Death of oligodendrocytes mediated by the interaction of nerve growth factor with its receptor p75. Nature 383:716-719. Casha S, Y u WR, Fehlings M G (2001) Oligodendroglial apoptosis occurs along degenerating axons and is associated with FAS and p75 expression following spinal cord injury in the rat. Neuroscience 103:203-218. Celik M , Gokmen N , Erbayraktar S, Akhisaroglu M , Konakc S, Ulukus C, Gene S, Gene K, Sagiroglu E, Cerami A, Brines M (2002) Erythropoietin prevents motor neuron apoptosis and neurologic disability in experimental spinal cord ischemic injury. Proc Natl Acad Sci U S A 99:2258-2263. Cheadle C, Vawter MP, Freed WJ, Becker K G (2003) Analysis of microarray data using Z score transformation. J Mol Diagn 5:73-81. Chen M , Ona V O , L i M , Ferrante RJ, Fink K B , Zhu S, Bian J, Guo L, Farrell L A , Hersch SM, Hobbs W, Vonsattel JP, Cha JH, Friedlander R M (2000) Minocycline inhibits caspase-1 and caspase-3 expression and delays mortality in a transgenic mouse model of Huntington disease. Nat Med 6:797-801. 155 Choi D W (1992) Excitotoxic cell death. J Neurobiol 23:1261-1276. Chopra I, Roberts M (2001) Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol Mol Biol Rev 65:232-260 ; second page, table of contents. Citron B A , Arnold P M , Sebastian C, Qin F, Malladi S, Ameenuddin S, Landis M E , Festoff B W (2000) Rapid upregulation of caspase-3 in rat spinal cord after injury: mRNA, protein, and cellular localization correlates with apoptotic cell death. Exp Neurol 166:213-226. Colak A , Karaoglan A , Barut S, Kokturk S, Akyildiz AI, Tasyurekli M (2005) Neuroprotection and functional recovery after application of the caspase-9 inhibitor z-LEHD-fmk in a rat model of traumatic spinal cord injury. J Neurosurg Spine 2:327-334. Colak A , Soy O, Uzun H , Asian O, Barut S, Belce A , Akyildiz A , Tasyurekli M (2003) Neuroprotective effects of G Y K I 52466 on experimental spinal cord injury in rats. J Neurosurg 98:275-281. Corbacella E, Lanzoni I, Ding D, Previati M , Salvi R (2004) Minocycline attenuates gentamicin induced hair cell loss in neonatal cochlear cultures. Hear Res 197:11-18. Cregan SP, Dawson V L , Slack RS (2004) Role of AIF in caspase-dependent and caspase-independent cell death. Oncogene 23:2785-2796. Crowe M J , Bresnahan JC, Shuman SL, Masters JN, Beattie MS (1997) Apoptosis and delayed degeneration after spinal cord injury in rats and monkeys. Nat Med 3:73-76. 156 David S (2002) Recruiting the immune response to promote long distance axon regeneration after spinal cord injury. Prog Brain Res 137:407-414. Dawson V L , Dawson T M (1998) Nitric oxide in neurodegeneration. Prog Brain Res 118:215-229. Demediuk P, Daly MP, Faden AI (1989) Effect of impact trauma on neurotransmitter and nonneurotransmitter amino acids in rat spinal cord. J Neurochem 52:1529-1536. Demjen D, Klussmann S, Kleber S, Zuliani C, Stieltjes B, Metzger C, Hirt U A , Walczak H , Falk W, Essig M , Edler L , Krammer PH, Martin-Villalba A (2004) Neutralization of CD95 ligand promotes regeneration and functional recovery after spinal cord injury. Nat Med 10:389-395. Diguet E, Gross CE, Tison F, Bezard E (2004a) Rise and fall of minocycline in neuroprotection: need to promote publication of negative results. Exp Neurol 189:1-4. Diguet E, Gross CE, Bezard E, Tison F, Stefanova N , Wenning G K (2004b) Neuroprotective agents for clinical trials in Parkinson's disease: a systematic assessment. Neurology 62:158; author reply 158-159. Dong C, Davis RJ, Flavell R A (2002) M A P kinases in the immune response. Annu Rev Immunol 20:55-72. Dong H, Fazzaro A , Xiang C, Korsmeyer SJ, Jacquin MF, McDonald JW (2003) Enhanced oligodendrocyte survival after spinal cord injury in Bax-deficient mice and mice with delayed Wallerian degeneration. J Neurosci 23:8682-8691. 157 Dowling P, Husar W, Menonna J, Donnenfeld H, Cook S, Sidhu M (1997) Cell death and birth in multiple sclerosis brain. J Neurol Sci 149:1-11. D'Souza SD, Bonetti B, Balasingam V , Cashman NR, Barker PA, Troutt A B , Raine CS, Antel JP (1996) Multiple sclerosis: Fas signaling in oligodendrocyte cell death. J Exp Med 184:2361-2370. Du Y , Ma Z, Lin S, Dodel RC, Gao F, Bales KR, Triarhou L C , Chernet E, Perry K W , Nelson DL, Luecke S, Phebus L A , Bymaster FP, Paul S M (2001) Minocycline prevents nigrostriatal dopaminergic neurodegeneration in the MPTP model of Parkinson's disease. Proc Natl Acad Sci U S A 98:14669-14674. Ducker TB, Hamit HF (1969) Experimental treatments of acute spinal cord injury. J Neurosurg 30:693-697. Ducker TB, Zeidman S M (1994) Spinal cord injury. Role of steroid therapy. Spine 19:2281-2287. Dumont RJ, Okonkwo DO, Verma S, Hurlbert RJ, Boulos PT, Ellegala DB, Dumont AS (2001) Acute spinal cord injury, part I: pathophysiologic mechanisms. Clin Neuropharmacol 24:254-264. Dusart I, Schwab M E (1994) Secondary cell death and the inflammatory reaction after dorsal hemisection of the rat spinal cord. Eur J Neurosci 6:712-724. Ekdahl CT, Claasen JH, Bonde S, Kokaia Z, Lindvall O (2003) Inflammation is detrimental for neurogenesis in adult brain. Proc Natl Acad Sci U S A 100:13632-13637. 158 Ekshyyan O, Aw T Y (2004) Apoptosis in acute and chronic neurological disorders. Front Biosci 9:1567-1576. Emery E, Aldana P, Bunge M B , Puckett W, Srinivasan A , Keane RW, Bethea J, Levi A D (1998) Apoptosis after traumatic human spinal cord injury. J Neurosurg 89:911-920. Estelles A , Charlton CA, Blau H M (1999) The phosphoprotein protein PEA-15 inhibits Fas- but increases TNF-R1-mediated caspase-8 activity and apoptosis. Dev Biol 216:16-28. Faden AI, Holaday JW (1981a) A role for endorphins in the pathophysiology of spinal cord injury. Adv Biochem Psychopharmacol 28:435-446. Faden AI, Holaday JW (1981b) Endorphins in traumatic spinal injury. Pathophysiologic studies and clinical implications. Mod Probl Pharmacopsychiatry 17:158-174. Faden AI, Jacobs TP, Holaday JW (1981a) Opiate antagonist improves neurologic recovery after spinal injury. Science 211:493-494. Faden AI, Jacobs TP, Smith M T (1984) Evaluation of the calcium channel antagonist nimodipine in experimental spinal cord ischemia. J Neurosurg 60:796-799. Faden AI, Jacobs TP, Mougey E, Holaday JW (1981b) Endorphins in experimental spinal injury: therapeutic effect of naloxone. Ann Neurol 10:326-332. Faden AI, Lemke M , Simon RP, Noble LJ (1988) N-methyl-D-aspartate antagonist MK801 improves outcome following traumatic spinal cord injury in rats: behavioral, anatomic, and neurochemical studies. J Neurotrauma 5:33-45. 159 Fagan SC, Edwards DJ, Borlongan C V , X u L, Arora A , Feuerstein G, Hess DC (2004) Optimal delivery of minocycline to the brain: implication for human studies of acute neuroprotection. Exp Neurol 186:248-251. Fahey TJ, 3rd, Sherry B, Tracey KJ , van Deventer S, Jones W G , 2nd, Minei JP, Morgello S, Shires GT, Cerami A (1990) Cytokine production in a model of wound healing: the appearance of MIP-1, MIP-2, cachectin/TNF and IL-1. Cytokine 2:92-99. Fan LW, Pang Y , Lin S, Rhodes PG, Cai Z (2005) Minocycline attenuates lipopolysaccharide-induced white matter injury in the neonatal rat brain. Neuroscience 133:159-168. Fehlings M G , Agrawal S (1995) Role of sodium in the pathophysiology of secondary spinal cord injury. Spine 20:2187-2191. Fehlings M G , Tator C H , Linden RD (1989) The effect of nimodipine and dextran on axonal function and blood flow following experimental spinal cord injury. J Neurosurg 71:403-416. Feigenbaum V , Gelot A , Casanova P, Daumas-Duport C, Aubourg P, Dubois-Dalcq M (2000) Apoptosis in the central nervous system of cerebral adrenoleukodystrophy patients. Neurobiol Dis 7:600-612. Feldman S, Careccia RE, Barham K L , Hancox J (2004) Diagnosis and treatment of acne. A m Fam Physician 69:2123-2130. 160 Fendrick SE, Miller KR, Streit WJ (2005) Minocycline does not inhibit microglia proliferation or neuronal regeneration in the facial nucleus following crush injury. Neurosci Lett 385:220-223. Fitch MT, Doller C, Combs CK, Landreth GE, Silver J (1999) Cellular and molecular mechanisms of glial scarring and progressive cavitation: in vivo and in vitro analysis of inflammation-induced secondary injury after CNS trauma. J Neurosci 19:8182-8198. Ford RW, Malm D N (1985) Failure of nimodipine to reverse acute experimental spinal cord injury. Cent Nerv Syst Trauma 2:9-17. Fu ES, Saporta S (2005) Methylprednisolone Inhibits Production of Interleukin-lbeta and Interleukin-6 in the Spinal Cord Following Compression Injury in Rats. J Neurosurg Anesthesiol 17:82-85. Gaviria M , Privat A , d'Arbigny P, Kamenka J, Haton H , Ohanna F (2000) Neuroprotective effects of a novel N M D A antagonist, Gacyclidine, after experimental contusive spinal cord injury in adult rats. Brain Res 874:200-209. Gehrmann J, Banati RB (1995) Microglial turnover in the injured CNS: activated microglia undergo delayed D N A fragmentation following peripheral nerve injury. J Neuropathol Exp Neurol 54:680-688. Geisler FH, Coleman WP, Grieco G, Poonian D (2001) The Sygen multicenter acute spinal cord injury study. Spine 26:S87-98. 161 Ghirnikar RS, Lee Y L , Eng LF (2001) Chemokine antagonist infusion promotes axonal sparing after spinal cord contusion injury in rat. J Neurosci Res 64:582-589. Giulian D, Robertson C (1990) Inhibition of mononuclear phagocytes reduces ischemic injury in the spinal cord. Ann Neurol 27:33-42. Gledhill RF, Harrison B M , McDonald WI (1973) Demyelination and remyelination after acute spinal cord compression. Exp Neurol 38:472-487. Grossman SD, Rosenberg LJ , Wrathall JR (2001) Temporal-spatial pattern of acute neuronal and glial loss after spinal cord contusion. Exp Neurol 168:273-282. Guha A , Tator C H (1988) Acute cardiovascular effects of experimental spinal cord injury. J Trauma 28:481-490. Guha A , Tator C H , Piper I (1987) Effect of a calcium channel blocker on posttraumatic spinal cord blood flow. J Neurosurg 66:423-430. Guha A , Tator C H , Smith CR, Piper I (1989) Improvement in post-traumatic spinal cord blood flow with a combination of a calcium channel blocker and a vasopressor. J Trauma 29:1440-1447. Guth L, Zhang Z, Steward O (1999) The unique histopathological responses of the injured spinal cord. Implications for neuroprotective therapy. Ann N Y Acad Sci 890:366-384. Haghighi SS, Chehrazi B B , Wagner FC, Jr. (1988) Effect of nimodipine-associated hypotension on recovery from acute spinal cord injury in cats. Surg Neurol 29:293-297. 162 Hains BC, Saab C Y , Lo A C , Waxman SG (2004) Sodium channel blockade with phenytoin protects spinal cord axons, enhances axonal conduction, and improves functional motor recovery after contusion SCI. Exp Neurol 188:365-377. Hall ED (1993) Lipid antioxidants in acute central nervous system injury. Ann Emerg Med 22:1022-1027. Hall ED, Braughler J M (1982) Effects of intravenous methylprednisolone on spinal cord lipid peroxidation and Na+ + K+)-ATPase activity. Dose-response analysis during 1st hour after contusion injury in the cat. J Neurosurg 57:247-253. Hall ED, Braughler J M (1986) Role of lipid peroxidation in post-traumatic spinal cord degeneration: a review. Cent Nerv Syst Trauma 3:281-294. Hall ED, Braughler J M (1993) Free radicals in CNS injury. Res Publ Assoc Res Nerv Ment Dis 71:81-105. Hall ED, Springer JE (2004) Neuroprotection and Acute Spinal Cord Injury: A Reappraisal. Neurorx 1:80-100. Hausmann ON (2003) Post-traumatic inflammation following spinal cord injury. Spinal Cord 41:369-378. He Y , Appel S, Le W (2001) Minocycline inhibits microglial activation and protects nigral cells after 6-hydroxydopamine injection into mouse striatum. Brain Res 909:187-193. He Y , Y u W, Baas PW (2002) Microtubule reconfiguration during axonal retraction induced by nitric oxide. J Neurosci 22:5982-5991. 163 Hermann GE, Rogers RC, Bresnahan JC, Beattie MS (2001) Tumor necrosis factor-alpha induces cFOS and strongly potentiates glutamate-mediated cell death in the rat spinal cord. Neurobiol Dis 8:590-599. Hersch S, Fink K , Vonsattel JP, Friedlander R M (2003) Minocycline is protective in a mouse model of Huntington's disease. Ann Neurol 54:841; author reply 842-843. Hida H, Nagano S, Takeda M , Soliven B (1999) Regulation of mitogen-activated protein kinases by sphingolipid products in oligodendrocytes. J Neurosci 19:7458-7467. Hillard V H , Peng H, Zhang Y , Das K, Murali R, Etlinger JD, Zeman RJ (2004) Tempol, a nitroxide antioxidant, improves locomotor and histological outcomes after spinal cord contusion in rats. J Neurotrauma 21:1405-1414. Hisahara S, Araki T, Sugiyama F, Yagami K , Suzuki M , Abe K , Yamamura K , Miyazaki J, Momoi T, Saruta T, Bernard CC, Okano H, Miura M (2000) Targeted expression of baculovirus p35 caspase inhibitor in oligodendrocytes protects mice against autoimmune-mediated demyelination. Embo J 19:341-348. Holaday JW (1984) Opiate antagonists in shock and trauma. Am J Emerg Med 2:8-12. Holaday JW, Faden AI (1981) Naloxone reverses the pathophysiology of shock through an antagonism of endorphin systems. Adv Biochem Psychopharmacol 28:421-434. Horiuchi H, Ogata T, Morino T, Chuai M , Yamamoto H (2003) Continuous intrathecal infusion of SB203580, a selective inhibitor of p38 mitogen-activated protein kinase, reduces the 164 damage of hind-limb function after thoracic spinal cord injury in rat. Neurosci Res 47:209-217. Hughes EH, Schlichtenbrede FC, Murphy CC, Broderick C, van Rooijen N , A l i RR, Dick A D (2004) Minocycline delays photoreceptor death in the rds mouse through a microglia-independent mechanism. Exp Eye Res 78:1077-1084. Hurlbert RJ (2001) The role of steroids in acute spinal cord injury: an evidence-based analysis. Spine 26:S39-46. Inman D, Guth L , Steward O (2002) Genetic influences on secondary degeneration and wound healing following spinal cord injury in various strains of mice. J Comp Neurol 451:225-235. Iwasa K, Ikata T, Fukuzawa K (1989) Protective effect of vitamin E on spinal cord injury by compression and concurrent lipid peroxidation. Free Radic Biol Med 6:599-606. Jin SX, Zhuang Z Y , Woolf CJ, Ji RR (2003) p38 mitogen-activated protein kinase is activated after a spinal nerve ligation in spinal cord microglia and dorsal root ganglion neurons and contributes to the generation of neuropathic pain. J Neurosci 23:4017-4022. Kanellopoulos GK, X u X M , Hsu C Y , Lu X , Sundt T M , Kouchoukos NT (2000) White matter injury in spinal cord ischemia: protection by AMPA/kainate glutamate receptor antagonism. Stroke 31:1945-1952. 165 Kaptanoglu E, Sen S, Beskonakli E, Surucu HS, Tuncel M , Kilinc K , Taskin Y (2002) Antioxidant actions and early ultrastructural findings of thiopental and propofol in experimental spinal cord injury. J Neurosurg Anesthesiol 14:114-122. Karpus WJ, Kennedy K J (1997) MIP-1 alpha and MCP-1 differentially regulate acute and relapsing autoimmune encephalomyelitis as well as Thl/Th2 lymphocyte differentiation. J Leukoc Biol 62:681-687. Katoh D, Ikata T, Katoh S, Hamada Y , Fukuzawa K (1996a) Effect of dietary vitamin C on compression injury of the spinal cord in a rat mutant unable to synthesize ascorbic acid and its correlation with that of vitamin E. Spinal Cord 34:234-238. Katoh K, Ikata T, Katoh S, Hamada Y , Nakauchi K , Sano T, Niwa M (1996b) Induction and its spread of apoptosis in rat spinal cord after mechanical trauma. Neurosci Lett 216:9-12. Kaufmann SH, Hengartner M O (2001) Programmed cell death: alive and well in the new millennium. Trends Cell Biol 11:526-534. Kelly K J , Sutton TA, Weathered N , Ray N , Caldwell EJ, Plotkin Z, Dagher PC (2004) Minocycline inhibits apoptosis and inflammation in a rat model of ischemic renal injury. A m J Physiol Renal Physiol 287:F760-766. Kern RC, Conley DB, Haines GK, 3rd, Robinson A M (2004) Treatment of olfactory dysfunction, II: studies with minocycline. Laryngoscope 114:2200-2204. Kikuchi M , Tenneti L, Lipton SA (2000) Role of p38 mitogen-activated protein kinase in axotomy-induced apoptosis of rat retinal ganglion cells. J Neurosci 20:5037-5044. 166 Kim D H , Vaccaro AR, Henderson FC, Benzel EC (2003) Molecular biology of cervical myelopathy and spinal cord injury: role of oligodendrocyte apoptosis. Spine J 3:510-519. Kim SS, Kong PJ, K im BS, Sheen D H , Nam SY, Chun W (2004) Inhibitory action of minocycline on lipopolysaccharide-induced release of nitric oxide and prostaglandin E2 in BV2 microglial cells. Arch Pharm Res 27:314-318. Kim SY, Bae JC, K im JY, Lee HL, Lee K M , Kim DS, Cho HJ (2002) Activation of p38 M A P kinase in the rat dorsal root ganglia and spinal cord following peripheral inflammation and nerve injury. Neuroreport 13:2483-2486. Klein NC, Cunha B A (1995) Tetracyclines. Med Clin North A m 79:789-801. Kloppenburg M , Breedveld FC, Terwiel JP, Mallee C, Dijkmans B A (1994) Minocycline in active rheumatoid arthritis. A double-blind, placebo-controlled trial. Arthritis Rheum 37:629-636. Koda M , Murakami M , Ino H, Yoshinaga K, Ikeda O, Hashimoto M , Yamazaki M , Nakayama C, Moriya H (2002) Brain-derived neurotrophic factor suppresses delayed apoptosis of oligodendrocytes after spinal cord injury in rats. J Neurotrauma 19:777-785. Koistinaho M , Koistinaho J (2002) Role of p38 and p44/42 mitogen-activated protein kinases in microglia. Glia 40:175-183. Kotter MR, Zhao C, van Rooijen N , Franklin RJ (2005) Macrophage-depletion induced impairment of experimental CNS remyelination is associated with a reduced 167 oligodendrocyte progenitor cell response and altered growth factor expression. Neurobiol Dis 18:166-175. Koyanagi I, Tator C H , Lea PJ (1993 a) Three-dimensional analysis of the vascular system in the rat spinal cord with scanning electron microscopy of vascular corrosion casts. Part 1: Normal spinal cord. Neurosurgery 33:277-283; discussion 283-274. Koyanagi I, Tator C H , Lea PJ (1993b) Three-dimensional analysis of the vascular system in the rat spinal cord with scanning electron microscopy of vascular corrosion casts. Part 2: Acute spinal cord injury. Neurosurgery 33:285-291; discussion 292. Koyanagi I, Tator C H , Theriault E (1993 c) Silicone rubber microangiography of acute spinal cord injury in the rat. Neurosurgery 32:260-268; discussion 268. Krady JK, Basu A , Allen C M , X u Y , LaNoue K F , Gardner TW, Levison SW (2005) Minocycline reduces proinflammatory cytokine expression, microglial activation, and caspase-3 activation in a rodent model of diabetic retinopathy. Diabetes 54:1559-1565. Kremlev SG, Roberts RL, Palmer C (2004) Differential expression of chemokines and chemokine receptors during microglial activation and inhibition. J Neuroimmunol 149:1-9. Kriz J, Nguyen M D , Julien JP (2002) Minocycline slows disease progression in a mouse model of amyotrophic lateral sclerosis. Neurobiol Dis 10:268-278. Kumar S, Boehm J, Lee JC (2003) p38 M A P kinases: key signalling molecules as therapeutic targets for inflammatory diseases. Nat Rev Drug Discov 2:717-726. 168 Kwon B K , Oxland TR, Tetzlaff W (2002) Animal models used in spinal cord regeneration research. Spine 27:1504-1510. Kwon B K , Tetzlaff W, Grauer JN, Beiner J, Vaccaro A R (2004) Pathophysiology and pharmacologic treatment of acute spinal cord injury. Spine J 4:451-464. Lacroix S, Chang L, Rose-John S, Tuszynski M H (2002) Delivery of hyper-interleukin-6 to the injured spinal cord increases neutrophil and macrophage infiltration and inhibits axonal growth. J Comp Neurol 454:213-228. Lammertse DP (2004) Update on pharmaceutical trials in acute spinal cord injury. J Spinal Cord Med 27:319-325. Ledeboer A, Sloane E M , Milligan ED, Frank M G , Mahony JH, Maier SF, Watkins LR (2005) Minocycline attenuates mechanical allodynia and proinflammatory cytokine expression in rat models of pain facilitation. Pain 115:71-83. Lee SM, Yune TY, Kim SJ, Kim Y C , Oh Y J , Markelonis GJ, Oh T H (2004) Minocycline inhibits apoptotic cell death via attenuation of TNF-alpha expression following iNOS/NO induction by lipopolysaccharide in neuron/glia co-cultures. J Neurochem 91:568-578. Lee SM, Yune TY, Kim SJ, Park do W, Lee Y K , Kim Y C , Oh Y J , Markelonis GJ, Oh TH (2003) Minocycline reduces cell death and improves functional recovery after traumatic spinal cord injury in the rat. J Neurotrauma 20:1017-1027. Lee Y B , Schrader JW, Kim SU (2000a) p38 map kinase regulates TNF-alpha production in human astrocytes and microglia by multiple mechanisms. Cytokine 12:874-880. 169 Lee Y B , Yune TY, Baik SY, Shin Y H , Du S, Rhim H, Lee EB, K i m Y C , Shin M L , Markelonis GJ, Oh TH (2000b) Role of tumor necrosis factor-alpha in neuronal and glial apoptosis after spinal cord injury. Exp Neurol 166:190-195. Lennmyr F, Karlsson S, Gerwins P, Ata K A , Terent A (2002) Activation of mitogen-activated protein kinases in experimental cerebral ischemia. Acta Neurol Scand 106:333-340. Levine SM, Chakrabarty A (2004) The role of iron in the pathogenesis of experimental allergic encephalomyelitis and multiple sclerosis. A n n N Y Acad Sci 1012:252-266. L i GL, Farooque M , Holtz A , Olsson Y (1999a) Apoptosis of oligodendrocytes occurs for long distances away from the primary injury after compression trauma to rat spinal cord. Acta Neuropathol (Berl) 98:473-480. L i GL , Brodin G, Farooque M , Funa K , Holtz A , Wang W L , Olsson Y (1996) Apoptosis and expression of Bcl-2 after compression trauma to rat spinal cord. J Neuropathol Exp Neurol 55:280-289. L i M , Ona VO, Chen M , Kaul M , Tenneti L , Zhang X , Stieg PE, Lipton SA, Friedlander R M (2000) Functional role and therapeutic implications of neuronal caspase-1 and -3 in a mouse model of traumatic spinal cord injury. Neuroscience 99:333-342. L i S, Stys P K (2000) Mechanisms of ionotropic glutamate receptor-mediated excitotoxicity in isolated spinal cord white matter. J Neurosci 20:1190-1198. 170 L i WW, Setzu A , Zhao C, Franklin RJ (2005) Minocycline-mediated inhibition of microglia activation impairs oligodendrocyte progenitor cell responses and remyelination in a non-immune model of demyelination. J Neuroimmunol 158:58-66. L i Y , Field P M , Raisman G (1999b) Death of oligodendrocytes and microglial phagocytosis of myelin precede immigration of Schwann cells into the spinal cord. J Neurocytol 28:417-427. Lin S, Zhang Y , Dodel R, Farlow MR, Paul S M , Du Y (2001) Minocycline blocks nitric oxide-induced neurotoxicity by inhibition p38 M A P kinase in rat cerebellar granule neurons. Neurosci Lett 315:61-64. Lin S, Wei X , X u Y , Yan C, Dodel R, Zhang Y , Liu J, Klaunig JE, Farlow M , Du Y (2003) Minocycline blocks 6-hydroxydopamine-induced neurotoxicity and free radical production in rat cerebellar granule neurons. Life Sci 72:1635-1641. Liu D, Thangnipon W, McAdoo DJ (1991) Excitatory amino acids rise to toxic levels upon impact injury to the rat spinal cord. Brain Res 547:344-348. Liu D, X u G Y , Pan E, McAdoo DJ (1999) Neurotoxicity of glutamate at the concentration released upon spinal cord injury. Neuroscience 93:1383-1389. Liu JB, Tang TS, Yang HL, Xiao DS (2004) Antioxidation of melatonin against spinal cord injury in rats. Chin Med J (Engl) 117:571-575. Liu S, Ruenes GL, Yezierski RP (1997a) N M D A and non-NMDA receptor antagonists protect against excitotoxic injury in the rat spinal cord. Brain Res 756:160-167. 171 Liu X Z , X u X M , Hu R, Du C, Zhang SX, McDonald JW, Dong H X , Wu Y J , Fan GS, Jacquin M F , Hsu C Y , Choi DW (1997b) Neuronal and glial apoptosis after traumatic spinal cord injury. J Neurosci 17:5395-5406. Lou J, Lenke L G , Ludwig FJ, O'Brien M F (1998) Apoptosis as a mechanism of neuronal cell death following acute experimental spinal cord injury. Spinal Cord 36:683-690. Lu J, Ashwell K W , Waite P (2000) Advances in secondary spinal cord injury: role of apoptosis. Spine 25:1859-1866. Ludwin SK (1990) Oligodendrocyte survival in Wallerian degeneration. Acta Neuropathol (Berl) 80:184-191. Lukacova N , Halat G, Chavko M , Marsala J (1996) Ischemia-reperfusion injury in the spinal cord of rabbits strongly enhances lipid peroxidation and modifies phospholipid profiles. Neurochem Res 21:869-873. Matsuki S, Iuchi Y , Ikeda Y , Sasagawa I, Tomita Y , Fujii J (2003) Suppression of cytochrome c release and apoptosis in testes with heat stress by minocycline. Biochem Biophys Res Commun 312:843-849. Matute C, Perez-Cerda F (2005) Multiple sclerosis: novel perspectives on newly forming lesions. Trends Neurosci 28:173-175. Mayne M , Cheadle C, Soldan SS, Cermelli C, Yamano Y , Akhyani N , Nagel JE, Taub DD, Becker K G , Jacobson S (2001) Gene expression profile of herpesvirus-infected T cells 172 obtained using immunomicroarrays: induction of proinflammatory mechanisms. J Virol 75:11641-11650. McBride CB, McPhail LT, Vanderluit JL, Tetzlaff W, Steeves JD (2003) Caspase inhibition attenuates transection-induced oligodendrocyte apoptosis in the developing chick spinal cord. Mo l Cell Neurosci 23:383-397. McDonald JW, Sadowsky C (2002) Spinal-cord injury. Lancet 359:417-425. McDonald JW, Althomsons SP, Hyrc K L , Choi DW, Goldberg M P (1998) Oligodendrocytes from forebrain are highly vulnerable to AMPA/kainate receptor-mediated excitotoxicity. Nat Med 4:291-297. McLaurin J, D'Souza S, Stewart J, Blain M , Beaudet A, Nalbantoglu J, Antel JP (1995) Effect of tumor necrosis factor alpha and beta on human oligodendrocytes and neurons in culture. Int J Dev Neurosci 13:369-381. McPhail LT, Stirling DP, Tetzlaff W, Kwiecien JM, Ramer MS (2004) The contribution of activated phagocytes and myelin degeneration to axonal retraction/dieback following spinal cord injury. Eur J Neurosci 20:1984-1994. Means ED, Anderson DK, Waters TR, Kalaf L (1981) Effect of methylprednisolone in compression trauma to the feline spinal cord. J Neurosurg 55:200-208. Metz GA, Curt A , van de Meent H , Klusman I, Schwab M E , Dietz V (2000) Validation of the weight-drop contusion model in rats: a comparative study of human spinal cord injury. J Neurotrauma 17:1-17. 173 Metz L M , Zhang Y , Yeung M , Patry DG, Bell RB, Stoian C A , Yong V W , Patten SB, Duquette P, Antel JP, Mitchell JR (2004) Minocycline reduces gadolinium-enhancingnagnetic resonance imaging lesions in multiple sclerosis. Ann Neurol 55:756. Moran L B , Graeber M B (2004) The facial nerve axotomy model. Brain Res Brain Res Rev 44:154-178. Naftchi N E (1982) Prevention of damage in acute spinal cord injury by peptides and pharmacologic agents. Peptides 3:235-247. Nashmi R, Fehlings M G (2001) Mechanisms of axonal dysfunction after spinal cord injury: with an emphasis on the role of voltage-gated potassium channels. Brain Res Brain Res Rev 38:165-191. Nesic O, X u G Y , McAdoo D, High K W , Hulsebosch C, Perez-Pol R (2001) IL-1 receptor antagonist prevents apoptosis and caspase-3 activation after spinal cord injury. J Neurotrauma 18:947-956. Nicholson DW, Thornberry N A (1997) Caspases: killer proteases. Trends Biochem Sci 22:299-306. Noble M , Mayer-Proschel M (1996) On the track of cell survival pharmaceuticals in the oligodendrocyte type-2 astrocyte lineage. Perspect Dev Neurobiol 3:121-131. Oudega M , Vargas CG, Weber A B , Kleitman N , Bunge M B (1999) Long-term effects of methylprednisolone following transection of adult rat spinal cord. Eur J Neurosci 11:2453-2464. 174 Pallini R, Fernandez E, Sbriccoli A (1988) Retrograde degeneration of corticospinal axons following transection of the spinal cord in rats. A quantitative study with anterogradely transported horseradish peroxidase. J Neurosurg 68:124-128. Pannu R, Barbosa E, Singh A K , Singh I (2005) Attenuation of acute inflammatory response by atorvastatin after spinal cord injury in rats. J Neurosci Res 79:340-350. Panter SS, Yum SW, Faden AI (1990) Alteration in extracellular amino acids after traumatic spinal cord injury. Ann Neurol 27:96-99. Park E, Velumian A A , Fehlings M G (2004) The role of excitotoxicity in secondary mechanisms of spinal cord injury: a review with an emphasis on the implications for white matter degeneration. J Neurotrauma 21:754-774. Parks WC, Wilson CL, Lopez-Boado YS (2004) Matrix metalloproteinases as modulators of inflammation and innate immunity. Nat Rev Immunol 4:617-629. Perry V H , Brown M C , Andersson PB (1993) Macrophage responses to central and peripheral nerve injury. Adv Neurol 59:309-314. Pi R, L i W, Lee NT, Chan HH, Pu Y , Chan L N , Sucher NJ, Chang DC, L i M , Han Y (2004) Minocycline prevents glutamate-induced apoptosis of cerebellar granule neurons by differential regulation of p38 and Akt pathways. J Neurochem 91:1219-1230. Piao CS, Kim JB, Han PL, Lee JK (2003) Administration of the p38 M A P K inhibitor SB203580 affords brain protection with a wide therapeutic window against focal ischemic insult. J Neurosci Res 73:537-544. 175 Pointillart V , Petitjean M E , Wiart L , Vital JM, Lassie P, Thicoipe M , Dabadie P (2000) Pharmacological therapy of spinal cord injury during the acute phase. Spinal Cord 38:71-76. Popovic N , Schubart A , Goetz BD, Zhang SC, Linington C, Duncan ID (2002) Inhibition of autoimmune encephalomyelitis by a tetracycline. Ann Neurol 51:215-223. Popovich PG (2000) Immunological regulation of neuronal degeneration and regeneration in the injured spinal cord. Prog Brain Res 128:43-58. Popovich PG, Wei P, Stokes BT (1997) Cellular inflammatory response after spinal cord injury in Sprague-Dawley and Lewis rats. J Comp Neurol 377:443-464. Popovich PG, Guan Z, Wei P, Huitinga I, van Rooijen N , Stokes BT (1999) Depletion of hematogenous macrophages promotes partial hindlimb recovery and neuroanatomical repair after experimental spinal cord injury. Exp Neurol 158:351-365. Popovich PG, Guan Z, McGaughy V , Fisher L , Hickey WF, Basso D M (2002) The neuropathological and behavioral consequences of intraspinal microglial/macrophage activation. J Neuropathol Exp Neurol 61:623-633. Power C, Henry S, Del Bigio MR, Larsen PH, Corbett D, Imai Y , Yong V W , Peeling J (2003) Intracerebral hemorrhage induces macrophage activation and matrix metalloproteinases. Ann Neurol 53:731-742. 176 Prewitt C M , Niesman IR, Kane CJ, Houle JD (1997) Activated macrophage/microglial cells can promote the regeneration of sensory axons into the injured spinal cord. Exp Neurol 148:433-443. Probert L, Akassoglou K , Pasparakis M , Kontogeorgos G, Kollias G (1995) Spontaneous inflammatory demyelinating disease in transgenic mice showing central nervous system-specific expression of tumor necrosis factor alpha. Proc Natl Acad Sci U S A 92:11294-11298. Probst-Cousin S, Rickert C H , Schmid K W , Gullotta F (1998) Cell death mechanisms in multiple system atrophy. J Neuropathol Exp Neurol 57:814-821. Profyris C, Cheema SS, Zang D, Azari M F , Boyle K , Petratos S (2004) Degenerative and regenerative mechanisms governing spinal cord injury. Neurobiol Dis 15:415-436. Pruzanski W, Greenwald RA, Street IP, Laliberte F, Stefanski E, Vadas P (1992) Inhibition of enzymatic activity of phospholipases A2 by minocycline and doxycycline. Biochem Pharmacol 44:1165-1170. Rabchevsky A G , Streit WJ (1997) Grafting of cultured microglial cells into the lesioned spinal cord of adult rats enhances neurite outgrowth. J Neurosci Res 47:34-48. Rabchevsky A G , Fugaccia I, Sullivan PG, Scheff SW (2001) Cyclosporin A treatment following spinal cord injury to the rat: behavioral effects and stereological assessment of tissue sparing. J Neurotrauma 18:513-522. 177 Rabchevsky A G , Fugaccia I, Sullivan PG, Blades DA, Scheff SW (2002) Efficacy of methylprednisolone therapy for the injured rat spinal cord. J Neurosci Res 68:7-18. Raghavendra V , Tanga F, DeLeo JA (2003) Inhibition of microglial activation attenuates the development but not existing hypersensitivity in a rat model of neuropathy. J Pharmacol Exp Ther 306:624-630. Rapalino O, Lazarov-Spiegler O, Agranov E, Velan GJ, Yoles E, Fraidakis M , Solomon A , Gepstein R, Katz A , Belkin M , Hadani M , Schwartz M (1998) Implantation of stimulated homologous macrophages results in partial recovery of paraplegic rats. Nat Med 4:814-821. Ray SK, Banik N L (2003) Calpain and its involvement in the pathophysiology of CNS injuries and diseases: therapeutic potential of calpain inhibitors for prevention of neurodegeneration. Curr Drug Targets CNS Neurol Disord 2:173-189. Regan RF, Choi DW (1991) Glutamate neurotoxicity in spinal cord cell culture. Neuroscience 43:585-591. Richardson-Burns SM, Tyler K L (2005) Minocycline delays disease onset and mortality in reovirus encephalitis. Exp Neurol 192:331-339. Rincon M , Pedraza-Alva G (2003) JNK and p38 M A P kinases in CD4+ and CD8+ T cells. Immunol Rev 192:131-142. 178 Rincon M , Enslen H, Raingeaud J, Recht M , Zapton T, Su MS, Penix L A , Davis RJ, Flavell R A (1998) Interferon-gamma expression by Thi effector T cells mediated by the p38 M A P kinase signaling pathway. Embo J 17:2817-2829. Rosenberg LJ , Teng Y D , Wrathall JR (1999) Effects of the sodium channel blocker tetrodotoxin on acute white matter pathology after experimental contusive spinal cord injury. J Neurosci 19:6122-6133. Rosenberg LJ , Zai LJ , Wrathall JR (2005) Chronic alterations in the cellular composition of spinal cord white matter following contusion injury. Glia 49:107-120. Ryu JK, Franciosi S, Sattayaprasert P, Kim SU, McLarnon JG (2004) Minocycline inhibits neuronal death and glial activation induced by beta-amyloid peptide in rat hippocampus. Glia 48:85-90. Sadowski T, Steinmeyer J (2001) Effects of tetracyclines on the production of matrix metalloproteinases and plasminogen activators as well as of their natural inhibitors, tissue inhibitor of metalloproteinases-1 and plasminogen activator inhibitor-1. Inflamm Res 50:175-182. Saklatvala J (2004) The p38 M A P kinase pathway as a therapeutic target in inflammatory disease. Curr Opin Pharmacol 4:372-377. Sanchez Mejia RO, Ona VO, L i M , Friedlander R M (2001) Minocycline reduces traumatic brain injury-mediated caspase-1 activation, tissue damage, and neurological dysfunction. Neurosurgery 48:1393-1399; discussion 1399-1401. 179 Sanchez-Gomez M V , Mature C (1999) A M P A and kainate receptors each mediate excitotoxicity in oligodendroglial cultures. Neurobiol Dis 6:475-485. Satake K, Matsuyama Y , Kamiya M , Kawakami H, Iwata H , Adachi R, Kiuchi K (2000) Nitric oxide via macrophage iNOS induces apoptosis following traumatic spinal cord injury. Brain Res Mol Brain Res 85:114-122. Saunders RD, Dugan L L , Demediuk P, Means ED, Horrocks L A , Anderson D K (1987) Effects of methylprednisolone and the combination of alpha-tocopherol and selenium on arachidonic acid metabolism and lipid peroxidation in traumatized spinal cord tissue. J Neurochem 49:24-31. Saville LR, Pospisil C H , Mawhinney L A , Bao F, Simedrea FC, Peters A A , O'Connell PJ, Weaver L C , Dekaban G A (2004) A monoclonal antibody to CD1 l d reduces the inflammatory infiltrate into the injured spinal cord: a potential neuroprotective treatment. J Neuroimmunol 156:42-57. Scarabelli T M , Stephanou A, Pasini E, Gitti G, Townsend P, Lawrence K, Chen-Scarabelli C, Saravolatz L, Latchman D, Knight R, Gardin J (2004) Minocycline inhibits caspase activation and reactivation, increases the ratio of XIAP to smac/DIABLO, and reduces the mitochondrial leakage of cytochrome C and smac/DIABLO. J A m Coll Cardiol 43:865-874. Schutyser E, Struyf S, Van Damme J (2003) The CC chemokine CCL20 and its receptor CCR6. Cytokine Growth Factor Rev 14:409-426. 180 Schwab M E , Bartholdi D (1996) Degeneration and regeneration of axons in the lesioned spinal cord. Physiol Rev 76:319-370. Schwartz G, Fehlings M G (2001) Evaluation of the neuroprotective effects of sodium channel blockers after spinal cord injury: improved behavioral and neuroanatomical recovery with riluzole. J Neurosurg 94:245-256. Schwartz M , Lazarov-Spiegler O, Rapalino O, Agranov I, Velan G, Hadani M (1999) Potential repair of rat spinal cord injuries using stimulated homologous macrophages. Neurosurgery 44:1041-1045; discussion 1045-1046. Sekhon L H , Fehlings M G (2001) Epidemiology, demographics, and pathophysiology of acute spinal cord injury. Spine 26:S2-12. Shuman SL, Bresnahan JC, Beattie MS (1997) Apoptosis of microglia and oligodendrocytes after spinal cord contusion in rats. J Neurosci Res 50:798-808. Skaper SD, Leon A (1992) Monosialogangliosides, neuroprotection, and neuronal repair processes. J Neurotrauma 9 Suppl 2:S507-516. Song Y , Wei EQ, Zhang WP, Zhang L, Liu JR, Chen Z (2004) Minocycline protects PC 12 cells from ischemic-like injury and inhibits 5-lipoxygenase activation. Neuroreport 15:2181-2184. Springer JE, Azbill RD, Knapp PE (1999) Activation of the caspase-3 apoptotic cascade in traumatic spinal cord injury. Nat Med 5:943-946. 181 Springer JE, Azbill RD, Nottingham SA, Kennedy SE (2000) Calcineurin-mediated B A D dephosphorylation activates the caspase-3 apoptotic cascade in traumatic spinal cord injury. J Neurosci 20:7246-7251. Springer JE, Azbill RD, Mark RJ, Begley JG, Waeg G, Mattson MP (1997) 4-hydroxynonenal, a lipid peroxidation product, rapidly accumulates following traumatic spinal cord injury and inhibits glutamate uptake. J Neurochem 68:2469-2476. Sribnick EA, Wingrave JM, Matzelle DD, Ray SK, Banik N L (2003) Estrogen as a neuroprotective agent in the treatment of spinal cord injury. Ann N Y Acad Sci 993:125-133; discussion 159-160. Stambe C, Atkins RC, Tesch GH, Kapoun A M , Hi l l PA, Schreiner GF, Nikolic-Paterson DJ (2003) Blockade of p38alpha M A P K ameliorates acute inflammatory renal injury in rat anti-GBM glomerulonephritis. J A m Soc Nephrol 14:338-351. Stirling D, Plunet, W, Khodarahmi, K, Liu, J, Becker, K G, Dyer, J K, McBride, CB, Steeves, JD, Tetzlaff, W. (2003) Minocycline treatment reduces p38 M A P Kinase expression within microglia/macrophages and improves functional outcome after spinal cord injury. In: Program No. 76.9. 2003 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience. Online. Stirling DP, Khodarahmi K, Liu J, McPhail LT, McBride CB, Steeves JD, Ramer MS, Tetzlaff W (2004) Minocycline treatment reduces delayed oligodendrocyte death, attenuates axonal dieback, and improves functional outcome after spinal cord injury. J Neurosci 24:2182-2190. 182 Stirling DP, Plunet W, Khodarahmi K, Liu J, Becker K G , Dyer JK, McBride CB, Steeves JD, Tetzlaff W (2003) Minocycline treatment reduces p38 M A P kinase expression within microglia/macrophages and improves functional outcome after spinal cord injury. SFN:Program No. 76.79. Abstract: online. Stokes BT, Noyes D H , Behrmann D L (1992) A n electromechanical spinal injury technique with dynamic sensitivity. J Neurotrauma 9:187-195. Streit WJ, Semple-Rowland SL, Hurley SD, Miller RC, Popovich PG, Stokes BT (1998) Cytokine mRNA profiles in contused spinal cord and axotomized facial nucleus suggest a beneficial role for inflammation and gliosis. Exp Neurol 152:74-87. Suk K (2004) Minocycline suppresses hypoxic activation of rodent microglia in culture. Neurosci Lett 366:167-171. Suzuki K , Hino M , Hato F, Tatsumi N , Kitagawa S (1999) Cytokine-specific activation of distinct mitogen-activated protein kinase subtype cascades in human neutrophils stimulated by granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-alpha. Blood 93:341-349. Tajouri L, Mellick AS, Ashton KJ , Tannenberg A E , Nagra R M , Tourtellotte WW, Griffiths L R (2003) Quantitative and qualitative changes in gene expression patterns characterize the activity of plaques in multiple sclerosis. Brain Res Mol Brain Res 119:170-183. Takagi T, Takayasu M , Mizuno M , Yoshimoto M , Yoshida J (2003) Caspase activation in neuronal and glial apoptosis following spinal cord injury in mice. Neurol Med Chir (Tokyo) 43:20-29; discussion 29-30. 183 Takami T, Oudega M , Bethea JR, Wood P M , Kleitman N , Bunge M B (2002) Methylprednisolone and interleukin-10 reduce gray matter damage in the contused Fischer rat thoracic spinal cord but do not improve functional outcome. J Neurotrauma 19:653-666. Taoka Y , Okajima K (2000) Role of leukocytes in spinal cord injury in rats. J Neurotrauma 17:219-229. Taoka Y , Okajima K , Murakami K, Johno M , Naruo M (1998) Role of neutrophil elastase in compression-induced spinal cord injury in rats. Brain Res 799:264-269. Tator C H (1995) Update on the pathophysiology and pathology of acute spinal cord injury. Brain Pathol 5:407-413. Tator C H , Fehlings M G (1991) Review of the secondary injury theory of acute spinal cord trauma with emphasis on vascular mechanisms. J Neurosurg 75:15-26. Teng Y D , Wrathall JR (1997) Local blockade of sodium channels by tetrodotoxin ameliorates tissue loss and long-term functional deficits resulting from experimental spinal cord injury. J Neurosci 17:4359-4366. Teng Y D , Choi H , Onario RC, Zhu S, Desilets FC, Lan S, Woodard EJ, Snyder E Y , Eichler M E , Friedlander R M (2004) Minocycline inhibits contusion-triggered mitochondrial cytochrome c release and mitigates functional deficits after spinal cord injury. Proc Natl Acad Sci U S A 101:3071-3076. 184 Thomas A J , Nockels RP, Pan HQ, Shaffrey CI, Chopp M (1999) Progesterone is neuroprotective after acute experimental spinal cord trauma in rats. Spine 24:2134-2138. Thomas M , Le W D (2004) Minocycline: neuroprotective mechanisms in Parkinson's disease. CurrPharmDes 10:679-686. Thomas M , Le WD, Jankovic J (2003) Minocycline and other tetracycline derivatives: a neuroprotective strategy in Parkinson's disease and Huntington's disease. Clin Neuropharmacol 26:18-23. Tikka T, Fiebich B L , Goldsteins G, Keinanen R, Koistinaho J (2001) Minocycline, a tetracycline derivative, is neuroprotective against excitotoxicity by inhibiting activation and proliferation of microglia. J Neurosci 21:2580-2588. Tikka T M , Koistinaho JE (2001) Minocycline provides neuroprotection against N-methyl-D-aspartate neurotoxicity by inhibiting microglia. J Immunol 166:7527-7533. Tilley BC, Alarcon GS, Heyse SP, Trentham DE, Neuner R, Kaplan DA, Clegg DO, Leisen JC, Buckley L , Cooper SM, Duncan H , Pillemer SR, Tuttleman M , Fowler SE (1995) Minocycline in rheumatoid arthritis. A 48-week, double-blind, placebo-controlled trial. MIRA Trial Group. Ann Intern Med 122:81-89. Toffano G, Savoini G, Aldinio C, Valenti G, Dal Toso R, Leon A, Calza L , Zini I, Agnati LF, Fuxe K (1984) Effects of gangliosides on the functional recovery of damaged brain. Adv Exp Med Biol 174:475-488. 185 Tomas-Camardiel M , Rte I, Herrera A J , de Pablos R M , Cano J, Machado A , Venero JL (2004) Minocycline reduces the lipopolysaccharide-induced inflammatory reaction, peroxynitrite-mediated nitration of proteins, disruption of the blood-brain barrier, and damage in the nigral dopaminergic system. Neurobiol Dis 16:190-201. Tsai EC, Tator C H (2005) Neuroprotection and regeneration strategies for spinal cord repair. Curr Pharm Des 11:1211 -1222. Tsuda M , Mizokoshi A , Shigemoto-Mogami Y , Koizumi S, Inoue K (2004) Activation of p38 mitogen-activated protein kinase in spinal hyperactive microglia contributes to pain hypersensitivity following peripheral nerve injury. Glia 45:89-95. Van Den Bosch L, Tilkin P, Lemmens G, Robberecht W (2002) Minocycline delays disease onset and mortality in a transgenic model of A L S . Neuroreport 13:1067-1070. Vartanian T, L i Y , Zhao M , Stefansson K (1995) Interferon-gamma-induced oligodendrocyte cell death: implications for the pathogenesis of multiple sclerosis. Mol Med 1:732-743. Vera Y , Rodriguez S, Castanares M , Lue Y , Atienza V , Wang C, Swerdloff RS, Sinha Hikim AP (2005) Functional role of caspases in heat-induced testicular germ cell apoptosis. Biol Reprod 72:516-522. Viviani B, Bartesaghi S, Corsini E, Galli CL, Marinovich M (2004) Cytokines role in neurodegenerative events. Toxicol Lett 149:85-89. Wada T, Penninger J M (2004) Mitogen-activated protein kinases in apoptosis regulation. Oncogene 23:2838-2849. 186 Wang A L , Y u A C , Lau LT, Lee C, Wu L M , Zhu X , Tso M O (2005) Minocycline inhibits LPS-induced retinal microglia activation. Neurochem Int. Wang C X , Yang T, Shuaib A (2003a) Effects of minocycline alone and in combination with mild hypothermia in embolic stroke. Brain Res 963:327-329. Wang J, Wei Q, Wang C Y , Hi l l WD, Hess DC, Dong Z (2004a) Minocycline up-regulates Bcl-2 and protects against cell death in mitochondria. J Biol Chem 279:19948-19954. Wang X , Zhu S, Drozda M , Zhang W, Stavrovskaya IG, Cattaneo E, Ferrante RJ, Kristal BS, Friedlander R M (2003b) Minocycline inhibits caspase-independent and -dependent mitochondrial cell death pathways in models of Huntington's disease. Proc Natl Acad Sci U S A 100:10483-10487. Wang X , Arcuino G, Takano T, Lin J, Peng W G , Wan P, L i P, X u Q, Liu QS, Goldman SA, Nedergaard M (2004b) P2X7 receptor inhibition improves recovery after spinal cord injury. Nat Med 10:821-827. Warden P, Bamber NI, L i H , Esposito A , Ahmad K A , Hsu C Y , X u X M (2001) Delayed glial cell death following wallerian degeneration in white matter tracts after spinal cord dorsal column cordotomy in adult rats. Exp Neurol 168:213-224. Waxman SG (1992) Demyelination in spinal cord injury and multiple sclerosis: what can we do to enhance functional recovery? J Neurotrauma 9 Suppl LSI05-117. Waxman SG (1993) Aminopyridines and the treatment of spinal cord injury. J Neurotrauma 10:19-24. 187 Wei X , Zhao L, Liu J, Dodel RC, Farlow MR, Du Y (2005) Minocycline prevents gentamicin-induced ototoxicity by inhibiting p38 M A P kinase phosphorylation and caspase 3 activation. Neuroscience 131:513-521. Wells JE, Hurlbert RJ, Fehlings M G , Yong V W (2003a) Neuroprotection by minocycline facilitates significant recovery from spinal cord injury in mice. Brain 126:1628-1637. Wells JE, Rice TK, Nuttall RK, Edwards DR, Zekki H , Rivest S, Yong V W (2003b) A n adverse role for matrix metalloproteinase 12 after spinal cord injury in mice. J Neurosci 23:10107-10115. Werner P, Pitt D, Raine CS (2000) Glutamate excitotoxicity~a mechanism for axonal damage and oligodendrocyte death in Multiple Sclerosis? J Neural Transm Suppl:375-385. Williams AS, Mizuno M , Richards PJ, Holt DS, Morgan BP (2004) Deletion of the gene encoding CD59a in mice increases disease severity in a murine model of rheumatoid arthritis. Arthritis Rheum 50:3035-3044. Winkler T, Sharma HS, Stalberg E, Badgaiyan RD, Gordh T, Westman J (2003) A n L-type calcium channel blocker, nimodipine influences trauma induced spinal cord conduction and axonal injury in the rat. Acta Neurochir Suppl 86:425-432. Wrathall JR, Choiniere D, Teng Y D (1994) Dose-dependent reduction of tissue loss and functional impairment after spinal cord trauma with the AMPA/kainate antagonist N B Q X . J Neurosci 14:6598-6607. 188 Wrathall JR, Teng Y D , Choiniere D (1996) Amelioration of functional deficits from spinal cord trauma with systemically administered N B Q X , an antagonist of non-N-methyl-D-aspartate receptors. Exp Neurol 137:119-126. Wrathall JR, Teng Y D , Choiniere D, Mundt DJ (1992) Evidence that local non-NMDA receptors contribute to functional deficits in contusive spinal cord injury. Brain Res 586:140-143. Wu DC, Jackson-Lewis V , Vila M , Tieu K , Teismann P, Vadseth C, Choi DK, Ischiropoulos H, Przedborski S (2002) Blockade of microglial activation is neuroprotective in the 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine mouse model of Parkinson disease. J Neurosci 22:1763-1771. X u G Y , Hughes M G , Ye Z, Hulsebosch CE, McAdoo DJ (2004a) Concentrations of glutamate released following spinal cord injury kil l oligodendrocytes in the spinal cord. Exp Neurol 187:329-336. X u L, Fagan SC, Waller JL, Edwards D, Borlongan C V , Zheng J, Hi l l WD, Feuerstein G, Hess DC (2004b) Low dose intravenous minocycline is neuroprotective after middle cerebral artery occlusion-reperfusion in rats. B M C Neurol 4:7. Yong C, Arnold P M , Zoubine M N , Citron B A , Watanabe I, Berman NE, Festoff B W (1998) Apoptosis in cellular compartments of rat spinal cord after severe contusion injury. J Neurotrauma 15:459-472. Yong V W (2004) Prospects for neuroprotection in multiple sclerosis. Front Biosci 9:864-872. 189 Yong V W , Wells J, Giuliani F, Casha S, Power C, Metz L M (2004) The promise of minocycline in neurology. Lancet Neurol 3:744-751. Yoshino O, Matsuno H, Nakamura H , Yudoh K, Abe Y , Sawai T, Uzuki M , Yonehara S, Kimura T (2004) The role of Fas-mediated apoptosis after traumatic spinal cord injury. Spine 29:1394-1404. Yoshioka A, Bacskai B, Pleasure D (1996) Pathophysiology of oligodendroglial excitotoxicity. J Neurosci Res 46:427-437. Yoshioka A , Hardy M , Younkin DP, Grinspan JB, Stern JL, Pleasure D (1995) Apha -amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors mediate excitotoxicity in the oligodendroglial lineage. J Neurochem 64:2442-2448. Young W (2002) Spinal cord contusion models. Prog Brain Res 137:231-255. Young W, Flamm ES (1982) Effect of high-dose corticosteroid therapy on blood flow, evoked potentials, and extracellular calcium in experimental spinal injury. J Neurosurg 57:667-673. Young W, DeCrescito V , Flamm ES, Blight AR, Gruner JA (1988) Pharmacological therapy of acute spinal cord injury: studies of high dose methylprednisolone and naloxone. Clin Neurosurg 34:675-697. Yrjanheikki J, Keinanen R, Pellikka M , Hokfelt T, Koistinaho J (1998) Tetracyclines inhibit microglial activation and are neuroprotective in global brain ischemia. Proc Natl Acad Sci U S A 95:15769-15774. 190 Yrjanheikki J, Tikka T, Keinanen R, Goldsteins G, Chan PH, Koistinaho J (1999) A tetracycline derivative, minocycline, reduces inflammation and protects against focal cerebral ischemia with a wide therapeutic window. Proc Natl Acad Sci U S A 96:13496-13500. Y u Y , Matsuyama Y , Nakashima S, Yanase M , Kiuchi K , Ishiguro N (2004) Effects of MPSS and a potent iNOS inhibitor on traumatic spinal cord injury. Neuroreport 15:2103-2107. Yune TY, Kim SJ, Lee S M , Lee Y K , Oh Y J , Kim Y C , Markelonis GJ, Oh TH (2004) Systemic administration of 17beta-estradiol reduces apoptotic cell death and improves functional recovery following traumatic spinal cord injury in rats. J Neurotrauma 21:293-306. Zemke D, Majid A (2004) The potential of minocycline for neuroprotection in human neurologic disease. Clin Neuropharmacol 27:293-298. Zhang SC, Goetz BD, Duncan ID (2003a) Suppression of activated microglia promotes survival and function of transplanted oligodendroglial progenitors. Glia 41:191-198. Zhang W, Narayanan M , Friedlander R M (2003b) Additive neuroprotective effects of minocycline with creatine in a mouse model of A L S . Ann Neurol 53:267-270. Zhang X Y , Zhou CS, Jin A M , Tian J, Zhang H, Yao WT, Zheng G (2003c) Effect of aminoguanidine on the recovery of rat hindlimb motor function after spinal cord injury. Di Y i Jun Y i Da Xue Xue Bao 23:687-689. Zhu S, Stavrovskaya IG, Drozda M , Kim B Y , Ona V , L i M , Sarang S, Liu AS , Hartley D M , Wu du C, Gullans S, Ferrante RJ, Przedborski S, Kristal BS, Friedlander R M (2002) 191 Minocycline inhibits cytochrome c release and delays progression of amyotrophic lateral sclerosis in mice. Nature 417:74-78. Zink M C , Uhrlaub J, DeWitt J, Voelker T, Bullock B, Mankowski J, Tarwater P, Clements J, Barber S (2005) Neuroprotective and anti-human immunodeficiency virus activity of minocycline. Jama 293:2003-2011. Zu Y L , Qi J, Gilchrist A , Fernandez GA, Vazquez-Abad D, Kreutzer DL, Huang C K , Sha'afi RI (1998) p38 mitogen-activated protein kinase activation is required for human neutrophil function triggered by TNF-alpha or F M L P stimulation. J Immunol 160:1982-1989. 192 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0092356/manifest

Comment

Related Items