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Immunomodulatory effects of novel therapies for stroke

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Title:
Immunomodulatory effects of novel therapies for stroke
Physical Description:
xi, 164 leaves : ill. (some col.) ; 28 cm.
Language:
English
Creator:
Hall, Aaron A
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Subjects

Subjects / Keywords:
Stroke -- therapy   ( mesh )
Stroke -- immunology   ( mesh )
Infarction, Middle Cerebral Artery -- therapy   ( mesh )
Infarction, Middle Cerebral Artery -- immunology   ( mesh )
Nerve Degeneration -- prevention & control   ( mesh )
Cord Blood Stem Cell Transplantation   ( mesh )
Receptors, sigma   ( mesh )
Organ Culture Techniques   ( mesh )
Inflammation -- immunology   ( mesh )
Spleen -- immunology   ( mesh )
Spleen -- blood supply   ( mesh )
Splenectomy   ( mesh )
Immune System   ( mesh )
Cytokines   ( mesh )
Microglia   ( mesh )
Time Factors   ( mesh )
Sigma receptors
Ischemia
Spleen
Inflammation
Microglia
Dissertations, Academic -- Molecular Pharmacology and Physiology -- Doctoral -- USF   ( lcsh )
Genre:
bibliography   ( marcgt )
non-fiction   ( marcgt )

Notes

Summary:
ABSTRACT: Each year, approximately 795,000 people suffer a new or recurrent stroke. About 610,000 of these are first attacks, and 185,000 are recurrent attacks (Carandang et al. 2006). Currently the only FDA approved treatment for ischemic stroke is recombinant tissue plasminogen activator (Alteplase) (Marler and Goldstein 2003). Unfortunately its use is restricted to a short, 4.5 hour, time window. Two promising therapies in the treatment of stroke at delayed timepoints are human umbilical cord blood cells (HUCBC) and the sigma receptor agonist DTG The first series of experiments were conducted to characterize the effects of sigma receptors on various aspects of microglial activation. Sigma receptor activation suppresses the ability of microglia to rearrange their actin cytoskeleton, migrate, and release cytokines. Stimulation of sigma receptors suppressed both transient and sustained intracellular calcium elevations associated with microglial activation.Further experiments showed that sigma receptors suppress microglial activation by interfering with increases in intracellular calcium. An ex vivo organotypic slice culture (OTC) model to was utilized to characterize the efficacy of sigma receptor activation and HUCBC therapy in mitigating neurodegeneration in ischemic brain tissue in the absence of the peripheral immune system. HUCBC but not DTG treatment reduced the number of degenerating neurons and the production of microglia derived nitric oxide in slice cultures subjected to oxygen glucose deprivation (OGD) back to levels seen in the normoxia controls. The final experiments were performed to characterize the effects of the peripheral immune system on the brain over time and identify changes mediated by HUCBC and DTG. Labeled splenocytes were found in spleen, blood, and thymus, but not in the brain in appreciable numbers at any timepoint. IL10 and IFNγ; levels were found to significantly increase by 96hours post MCAO.This increase in IL10 and IFNγ expression was blocked HUCBC or DTG. The experiments described here have shed light on the molecular mechanisms of stroke injury and the relative targets that DTG and HUCBC therapies exploit. These data suggest that the neuroprotection achieved by DTG or HUCBC is mediated by the ability of these treatments to modulate the peripheral immune systems response to injury.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2009.
Bibliography:
Includes bibliographical references.
Additional Physical Form:
Also available online.
Statement of Responsibility:
by Aaron A. Hall.
General Note:
Includes vita.

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aleph - 002067983
oclc - 601567524
usfldc doi - E14-SFE0003109
usfldc handle - e14.3109
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ABSTRACT: Each year, approximately 795,000 people suffer a new or recurrent stroke. About 610,000 of these are first attacks, and 185,000 are recurrent attacks (Carandang et al. 2006). Currently the only FDA approved treatment for ischemic stroke is recombinant tissue plasminogen activator (Alteplase) (Marler and Goldstein 2003). Unfortunately its use is restricted to a short, 4.5 hour, time window. Two promising therapies in the treatment of stroke at delayed timepoints are human umbilical cord blood cells (HUCBC) and the sigma receptor agonist DTG The first series of experiments were conducted to characterize the effects of sigma receptors on various aspects of microglial activation. Sigma receptor activation suppresses the ability of microglia to rearrange their actin cytoskeleton, migrate, and release cytokines. Stimulation of sigma receptors suppressed both transient and sustained intracellular calcium elevations associated with microglial activation.Further experiments showed that sigma receptors suppress microglial activation by interfering with increases in intracellular calcium. An ex vivo organotypic slice culture (OTC) model to was utilized to characterize the efficacy of sigma receptor activation and HUCBC therapy in mitigating neurodegeneration in ischemic brain tissue in the absence of the peripheral immune system. HUCBC but not DTG treatment reduced the number of degenerating neurons and the production of microglia derived nitric oxide in slice cultures subjected to oxygen glucose deprivation (OGD) back to levels seen in the normoxia controls. The final experiments were performed to characterize the effects of the peripheral immune system on the brain over time and identify changes mediated by HUCBC and DTG. Labeled splenocytes were found in spleen, blood, and thymus, but not in the brain in appreciable numbers at any timepoint. IL10 and IFN; levels were found to significantly increase by 96hours post MCAO.This increase in IL10 and IFN expression was blocked HUCBC or DTG. The experiments described here have shed light on the molecular mechanisms of stroke injury and the relative targets that DTG and HUCBC therapies exploit. These data suggest that the neuroprotection achieved by DTG or HUCBC is mediated by the ability of these treatments to modulate the peripheral immune systems response to injury.
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Cord Blood Stem Cell Transplantation.
Receptors, sigma.
Organ Culture Techniques.
Inflammation
immunology.
Spleen
immunology.
Spleen
blood supply.
Splenectomy.
Immune System.
Cytokines.
Microglia.
Time Factors.
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Ischemia
Spleen
Inflammation
Microglia
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Immunomodulatory Effects of Novel Therapies for Stroke by Aaron A. Hall A dissertation in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Molecular P harmacology and Physiology School of Biomedical Sciences College of Medicine University of South Florida Major Professor: Keith R. Pennypacker, Ph.D. Alison Willing, Ph.D. Jay Dean, Ph.D. Lynn Wecker, Ph.D. Javier Cuevas, Ph.D. Date of Approval: April 16, 2009 Keywords: sigma receptors, ischemia spleen, inflammation, microglia Copyright 2009, Aaron A. Hall

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Dedication This dissertation is dedicated to my loving wife Irena and my family, whose love and support have enabled me to complete these studies.

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Acknowledgments I would like to acknowledge all t hose who have contributed to the completion of this work. Part icularly I would like to t hank my mentor Dr. Keith R. Pennypacker, whose encouragement and gui dance have been essential in my maturation and preparation for entrance into research as a career. I would also like to thank Dr. Alison. Willing, whose patience and vast understanding of stroke has been essential for the success of my project. Although this is an individual work, I would like to recognize all of my colleagues who have contributed not only to the intellectual pursuit of the questions raised in this work, but also to the experimentation that was required to colle ct these data, includ ing: Dr. Craig T. Ajmo, for providing assistance with the MC AO surgery technique that led to the completion of aims 3; Dr. Chris Leonardo, for his intellectual contributions and assistance in tissue culture techniques used for aim 2; and Lisa A. Collier, whose intellectual and technical contributions were essential to all parts of these studies. Finally, I would like to extend a special t hank you to all of my family and friends for providing encouragement and support.

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i Table of Contents List of Figures vii Abstract x Chapter 1: Backgroun d and Significance 1 Clinical Impact of Stroke 1 Animal Models of Stroke 3 Failed Therapies for Stroke 6 The Local Response to Ischemia 8 Endogenous Microglial Activation During the Early Inflammatory Response 9 Activation of the Neuro-Imm une and Hypothalamic Pituitary Adrenal Axes 11 The Role of the Peripheral Lymphoid Organs in Stroke 13 The Peripheral Leukocyte Response to Ischemia 14 Current Therapies for Stroke 19 Therapeutic Targets for Stroke at Delayed Timepoints 20 Sigma Receptor Agonists as a Therapy for Stroke 22 Determining the Impact of DT G on the Post Ischemic Inflammatory Response 25 References 27

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ii Chapter 2: Sigma Receptors S uppress Multiple Aspects of Microglial Activation 47 Abstract 47 Introduction 48 Materials and Methods 50 Primary Cultures of Microglia 50 Membrane Ruffling and Quantification 51 Migration Assay 52 ELISA Assay 53 Griess Reaction 54 Calcium Imaging 54 DAF Imaging 55 Data Analysis 55 Reagents 56 Results 56 Sigma Receptor Activation Suppresses Changes in Microglia Morphology in Response to Chemoattractant Stimulation 56 Sigma Receptor Activation Significantly Decreases Membrane Ruffling Induced by ATP 59 The Microglial Migratory Res ponse to Chemoattractant Application is Suppressed by Sigma Receptor Activation 61

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iii The Microglial Inflammatory Response is Suppressed by Sigma Receptor Activation 63 Transient Intracellular Calciu m Signaling is Suppressed by Sigma Receptor Activation 65 Basal Intracellular Calcium Increases are Suppressed by Sigma Receptor Activation 67 Increases in Intracellular Calcium are Sufficient to Induce Membrane Ruffling in Microglia 69 Microglial Migration is a Calc ium Dependent Process Which is not Restored by Ionomycin Treatment 71 Microglial Migration is Independent of Calcium Release from Internal Stores but Requires Calcium Influx 71 Calcium Influx Restores Nit ric Oxide Production Following Sigma Receptor Activa tion in LPS Stimulated Microglia Cells 74 Discussion 77 References 83 Chapter 3: Delayed Treatments for Stroke Differentially affect Neuronal Death in Organotypic Slice Cultures Subjected to Oxygen Glucose Deprivation 89 Abstract 89 Introduction 90 Materials and Methods 94

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iv Animal Care 94 Organotypic Slice Culture 94 Oxygen Glucose Deprivation 95 Fluoro-Jade Staining 95 Nitric Oxide Imaging 96 Immunohistochemistry 96 Results 97 Oxygen Glucose Deprivation Significantly Increases Neurodegeneration and Nitric Oxide Production in OTC 97 HUCBC but not DTG Significantly Reduces Fluoro-Jade Staining in OTC Following OGD 99 DTG Treatment is Ineffectiv e in Reducing Fluoro-Jade Staining at Multiple Concentrations 101 HUCBC Treatment Reduces DAF-FM Staining in OTC Following OGD 102 HUCBC Reduces the Number of Nitric Ox ide Producing Microglia in Ischemic OTC 105 Discussion 107 References 112 Chapter 4: The Spleen Contributes to Stro ke Induced Neurodegeneration in an Indirect Manner 118 Abstract 118 Introduction 119

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v Materials and Methods 121 Animal Care 121 Splenic Injection of CFSE 122 Laser Doppler Blood Flow Measurement 122 Permanent Middle Cerebral Artery Occlusion 123 Brain Extraction and Sectioning 123 Fluoro-Jade Staining 123 Infarct Volume Quantification 124 Immunohistochemistry 124 ELISA assay 125 Results 125 Infarct Volumes Peak by 24 Hours in Rats Subjected to MCAO 125 Post-stroke Shrinkage of t he Spleen is a Transient Phenomenon 126 CFSE Labeled Splenocytes are Present in Various Tissues Following MCAO 128 IL10 Immunostaining is Increas ed in the Brain and Spleen Following MCAO 130 DTG or HUCBC Treatment Reduces IL10 and IFN Following MCAO 132 Discussion 134 References 137

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vi Chapter 5: Conclusions 140 Implications and Future Directions 156 References 159 About the Author End Page

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vii List of Figures Chapter 2 Figure 1. Sigma receptor activa tion suppresses changes in microglia morphology in response to chemoattractant stimulation. 58 Figure 2. Sigma receptor activa tion significantly decreases membrane ruffling in microglia stimulated with ATP. 60 Figure 3. The micr oglial migratory response to chemoattractant application is suppressed by si gma receptor activation. 62 Figure 4. The microglial inflammato ry response is suppressed by sigma receptor activation. 64 Figure 5. Transient intracellula r calcium signaling evoked by ATP was suppressed by sigma receptor activation. 66 Figure 6. Basal intracellular calc ium increases are suppressed by sigma receptor activation. 68 Figure 7. Increases in intrac ellular calcium induce membrane ruffling in microglia. 70 Figure 8. Microglial migration is independent of calcium release from internal stores but requires La3+-sensitive calcium influx. 73 Figure 9. Calcium influx restor es nitric oxide production following sigma receptor activation in LPS stimulated microglia. 76

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viii Chapter 3 Figure 1. Oxygen glucose depriv ation significantly increases Neurodegeneration and nitric ox ide production in OTC. 98 Figure 2. HUCBC but not DTG significantly reduces Fluoro-Jade staining in OTC following OGD. 100 Figure 3. DTG treatment is ineffe ctive in reducing Fluoro-Jade staining at multiple concentrations. 102 Figure 4. HUCBC treatment re duces DAF-FM staining in OTC following OGD. 104 Figure 5. HUCBC reduc es the number of nitric oxide producing microglia in ischemic OTC. 106 Chapter 4 Figure 1. Infarct vo lumes peak by 24 hours in rats subjected to MCAO. 126 Figure 2. The spleen shri nks transiently following MCAO. 127 Figure 3. CFSE labeled splenocytes are present in various tissues following MCAO. 129 Figure 4. IL10 and IFN expression increases over time following MCAO. 131 Figure 5. DTG and HUCBC treatment reduces IL10 and IFN following MCAO. 133

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ix Chapter 5 Figure 1. Sigma re ceptor agonists suppress microglial activation by interfering with intracellular calcium signaling. 146 Figure 2. Changes in spleen size and content over time reflect an evolving interaction between t he brain and immune systems following MCAO. 153 Figure 3. Successf ul therapeutic targets change as the body’s response to stroke changes over time. 158

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x Immunomodulatory Effects of Novel Therapies for Stroke Aaron A. Hall Abstract Each year, approximately 795,000 people suffer a new or recurrent stroke. About 610,000 of these are first a ttacks, and 185,000 are recurrent attacks (Carandang et al. 2006). Currently the only FDA approved treatment for ischemic stroke is recombinant tissue plasminogen activator (Alteplase) (Marler and Goldstein 2003). Unfortunately its use is restricted to a sh ort, 4.5 hour, time window. Two promising therapies in the treatment of stroke at delayed timepoints are human umbilical cord blood cells (HUCBC) and the sigma receptor agonist DTG The first series of expe riments were conducted to characterize the effects of sigma receptors on various aspects of microglial activation. Sigma receptor activation suppresses the ability of microglia to rearrange their actin cytoskeleton, migrate, and release cytoki nes. Stimulation of sigm a receptors suppressed both transient and sustained intracellular calc ium elevations asso ciated with microglial activation. Further experim ents showed that sigma rec eptors suppress microglial activation by interfering with increases in intracellular calcium. An ex vivo organotypic slice culture (O TC) model to was utilized to characterize the efficacy of sigma re ceptor activation and HUCBC therapy in

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xi mitigating neurodegeneration in ischemic brain tissue in the absence of the peripheral immune system. HUCBC but not DTG treatment reduced the number of degenerating neurons and the production of microglia der ived nitric oxide in slice cultures subjected to oxygen gluc ose deprivation (OGD) back to levels seen in the normoxia controls. The final experiments were performed to characterize the effects of the peripheral immune system on the brain ov er time and identify changes mediated by HUCBC and DTG. Labeled splenocytes were found in spleen, blood, and thymus, but not in the brai n in appreciable numbers at any timepoint. IL10 and IFN levels were found to significantly increase by 96hours post MCAO. This increase in IL10 and IFN expression was blocked HUCBC or DTG. The experiments described here have shed light on the molecular mechanisms of stroke injury and the re lative targets that DTG and HUCBC therapies exploit. These data suggest that the neuroprotection achieved by DTG or HUCBC is mediated by the ability of these treatment s to modulate the peripheral immune systems response to injury.

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1 Chapter 1 Background and Significance Clinical impact of stroke Each year, approximately 610,000 people suffer an initial stroke, and 185,000 additional people have a recurrent attack. Every 40 seconds someone in the United States has a stroke making th is disease a leading cause of death and disability in this country (Carandang et al. 2006). While the stroke death rates have decreased due to better treatment and hos pital care, the lack of effective treatments to improve func tional recovery has caused a concomitant increase in the number of people disabled by this diseas e. The estimated direct and indirect cost of stroke in the U.S. is nearly $ 68.9 billion per year (Lloyd-Jones et al. 2009), while the burden on families is unquantifiable and severe. Approximately 87% of stroke cases are due to an ischemic (embolic or thrombotic) stroke with the remainder being hemorrhagic in nature (Lloyd-Jones et al. 2009). Ischemic strokes can be fu rther subcategorized into small vessel and large vessel strokes based on the size of the artery occluded. Small vessel strokes, or lacunar strokes, occur when one of the small penetrating arteries which branch from larger vessels bec omes occluded causing a small (<15mm) lesion in the subcortical regions of the brain (Norrving 2008). This type of lesion

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2 accounts for 25% of ischemic strokes and frequently acute stroke symptoms are not evident with the infarct only pres enting as a small hole or “lacunae” upon necropsy. When acute stroke symptoms do present, they normally present with one of five classical “lacunar syndrom es”. These in clude: pure motor stroke/hemiparesis, ataxic hemiparesis dysarthria/clumsy hand, pure sensory stroke, and mixed sensorimotor stroke. These symptoms typically manifest when the lesion damages major subcortical struct ures such as the internal capsule, basis pontis, thalamus, or the pons. As these strokes do not impact the cortex they do not present with pure cortical symptomology such as aphasia, neglect, and visual field defects (Fisher 1982). Clinical prognosis in the near term for patients with lacunar infarcts is favorabl e as the acute (<30day) survival rate is ~97%, and the chronic (1year and beyond) surviv al rate is 87% (Bejot et al. 2008; Sacco et al. 2006). Acute large vessel strokes typically occur when atherosclerotic plaques develop inside the large arteries feeding the brain causing them to become stenotic and easily occluded by blood clots. These blood clots either break off of the plaque (thrombotic) or are generated elsewhere in the body (embolic). As these large vessels supply a greater porti on of the blood supply to the brain the corresponding infarcts are larger and injure both cortical and subcortical structures. This leads to pathologies wh ich can include all lacunar symptoms in addition to cortical symptomology such as aphasia, neglect, and visual field defects. Clinical prognosis in the near term for patients with large vessel infarcts

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3 is poorer than lacunar pathology as the acute (<30day) survival rate is 85%, and the chronic (1year and beyond) survival rate is 65-70% (Carandang et al. 2006). Small vessel and large vessel strokes respond similarly to currently available interventions such as recomb inant tissue plasminogen activator (rTPA) and aspirin suggesting a common pathological response to the injury (Norrving 2008). Functional MRI studies in large vessel strokes have determined that the infarct increases in size by 21% reaching a maximal value by 74 hrs and subsequently decreases in size during da ys 5-7 (Schwamm et al. 1998). Similar effects on infarct size evolution have been observed in lacunar strokes further suggesting that a common response by t he body to these ischemic injuries exists. Furthermore similar kinetics have been observed rats following middle cerebral artery occlusion (MCAO) (Newcomb et al. 2006). The body’s response to ischemic injury is multifaceted and changes over time. Traditional imaging techniques in humans can provide details on the gross changes in infarct size, neur onal excitability, and cerebr al blood flow. Although new ligands for positron emission tomogr aphy (PET) (Baron 2001) and functional magnetic resonance imaging (fMRI) techni ques are beginning to shed light on the human response to ischemia (Baron 2002), animal models have been required to answer important questions about the cellular response to ischemia. Animal models of stroke Rodent models of stroke have been critical to increasing our understanding of the pathol ogy and progression of ischemia induced brain

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4 lesions (Traystman 2003; Willing 2009). Murine and rat models of stroke are commonly used and can be separated into two categories: global ischemia, in which total perfusion to the brain is blocked, and focal ischemia, in which perfusion of only part of the brain is inhibited. Focal ischemia includes transluminal middle cerebral artery occl usion in which an embolus is advanced into the MCA occluding blood flow, and distal middle cerebral artery occlusion in which the MCA is occluded by a clot which is either injected into the middle cerebral artery (Kaneko et al. 1985) or fo rmed by the photolysis of a dye such as Rose Bengal (Markgraf et al. 1993). Gl obal ischemia is a model which more closely mimics the neuronal damage incurred following a heart attack, while focal ischemia more closely resembles an ischemic stroke. The distal middle cerebral artery occl usion technique lends itself to the study of thrombolytic therapy as the ar tery is occluded by a blood clot; however this technique is more traumatic to the animal as a craniotomy must be performed (Kaneko et al. 1985). The tr ansluminal middle cerebral artery occlusion technique is a commonly empl oyed surgical stroke model. Its advantages include the relative ease at which both permanent as well as transient ischemic events can be induced by merely leaving in or withdrawing the suture used to block the MCA, as well as reproducibility of the infarct size. Due to the large size of the infarct produced, th is model lends itself to investigations of neuroprotective treatments. Furthermore, this stroke model closely mimics large vessel strokes which account for the majori ty of strokes in the human condition.

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5 Experimental rodent models of stroke have yielded a trove of information concerning stroke pathophysiology. There has, however, been substantial difficulty translating therapeutic effica cy in rodents into improved clinical outcomes in stroke patients. This is due in part to inherent differences between human patients and rodent model s. Humans are gyrencephalic organisms (have convoluted cortices) while rodents are li ssencephalic (have smooth cortices). Humans also have a far gr eater proportion of white ma tter than rodents (Dewar et al. 1999; Hagberg et al. 2002). MRI imaging studies have demonstrated a correlation between lesions which impact the descending motor tracts, which are composed of white matter, and poorer functi on outcome in patients (Dawes et al. 2008; Pineiro et al. 2000). White matte r dysfunction in ischemic injury is understudied compared to neuron rich grey matter and is a likely source of the disparity seen between infarct volume reductions in rodents and functional improvements in patients. Another confound between animal models and the human condition is in the age and condition of the subject. Stro ke patients are typically elderly and often present with underlying disease stat es such as high blood pressure, diabetes, atherosclerosis, and heart disease. Animal models, on the other hand, typically utilize healthy adolescent male subjects. Advanced age and underlying pathology can reduce the efficacy in humans of treatments which were protective in rat models. Perhaps the most important sour ce of discrepancy between animal and human models of stroke lies not in the model itself but in the experimental design

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6 utilizing the model. Many researchers negl ect to take into consideration the human conditions when designing in vivo experiments. Administration of experimental neuroprotectants at the time of or prior to the onset of ischemia may be useful for understanding disease pathol ogy but are impractical for use in the clinic. Patients typically arrive at t he ER many hours after stroke, and are in a condition not modeled by t he treatment paradigm. Inde ed many of the preclinical studies targeting excitotoxicity such as NMDA antagonism and calcium channel blockade succumbed to this flaw and the treatments were ineffective clinically. Failed therapies for stroke Attempts to develop neuroprotective treatments for stroke have yielded disappointing results thus far. Therapeutic interventio ns can be grouped into two broad categories: therapies targeting neur ons and the early processes implicated in their demise, and therapies which target the delayed glial/immune response to stroke. The vast majority of trials to date have fo cused on reducing the buildup of intracellular calcium, neuronal firing, and free radicals. These therapies were developed in order to combat a phenom enon called excitotoxicity which is thought to damage neurons (Dirnagl et al. 1999). Neuronal damage in this model is caused by the overactivation of NMDA channels on penumbral neurons due to high levels of glutamate and aspartate released from th e ischemic core. This increased NMDA receptor activation causes calcium to enter the cell where it can activate proapoptotic signaling cascades, c ause free radical buildup, and trigger neuronal death (Dirnagl et al. 1999). Multiple steps in this excitotoxic process

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7 have been targeted for intervention su ch as NMDA receptor antagonism (Selfotel) (Davis et al. 2000), AMPA receptor antagonism (ZK200775) (Elting et al. 2002), calcium channel blockade (F lunarizine) (Limburg and Hijdra 1990), GABA activation (Clomethiazole) (Lyden et al. 2001), sodium channel blockade (fosphenytoin), potassium channel opening (BMS-204352), and free radical scavengers (Lubeluzole) (Diener et al. 2000). While these therapies were effective in preclinical animal studies, none showed significant improvement clinically. Some of the reasons include a short therapeutic time windows (<1 hr) for efficacy, and undesirable side effect s such as hallucination and psychosis (NMDA receptor antagonists) (Davis et al. 2000) and severe reductions in blood pressure (calcium channel blockers) (Horn et al. 2001). Trials which target the glial/immune response to stroke have also been unsuccessful. The earliest of these trials involved the use of high dose dexamethasone to suppress the inflammato ry response following stroke (Mulley et al. 1978). Later trials have focused on preventing immune cell infiltration of the infarct by administering antibodies or other recombinant proteins to bind adhesion molecules and prevent immune cell infiltration following stroke (1999; 2001; Lees et al. 2003). All of these trials failed to show therapeutic efficacy in clinical trials and all but UK-279276 (neutrophil inhibito ry factor) significantly increased post stroke mortality as well. The failures presented here suggest that our knowledge of post-ischemic pathology is incomplete. Therefore it is imperative that novel therapeutic strategies be developed, not only to impr ove the clinical setting, but also to

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8 improve the understanding of the underlying mechanisms of this disease. To develop these therapies the underlying pr ocesses which occur following ischemia must be elucidated. The local response to ischemia Rapidly following the obstruction of blood flow to the brain the tissue parenchyma undergoes significant changes. The area of brain tissue that receives blood supply primarily from t he occluded vessel and has a greater than 75% loss in perfusion is called the core of the infarction. Neurons in the core of the infarct are under the greatest metabolic stress and are the least likely to be responsive to neuroprotective treatm ents (Lipton 1999). The area surrounding the core of the infarction which receives partial blood perfusion from collateral blood vessels is known as the penumbra. Cells in the penumbra while under some metabolic demand are further st ressed by stroke associated phenomena such as anoxic depolarization and in flammation which can cause their destruction at delayed timepoints. T he penumbra is the primary target for neuroprotective therapies since it accounts for a significant area of the infarct and does not die as quickly as the core. When the artery supplying blood flow to brain tissue becomes obstructed, loss of ATP necessary for the maintenance of ionic homeostasis, leads to rapid calcium accumulation in ischemic neurons (Lipton 1999). This process leads to increased in neuronal firing causi ng a phenomenon known as anoxic depolarization. Anoxic depolarization in non-ischemic brain does not cause

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9 neuronal loss, and when induced 3 days prior to ischemia can cause neuroprotection via ischemic precondition ing mechanisms (Obr enovitch 2008). In ischemic brain, however, the incr eased neuronal firing exacerbates the metabolic demand and further decreases energy stores in ischemic neurons leading to their compromise (Takano et al 1996). The increases in intracellular calcium levels induce intracellular phospholipases, proteases, and endonucleases lead to the functional compro mise of the neuron. This results in the production of inflammatory lipid perox idation products such as prostanoids which can exacerbate membrane perm eability and lead to cytotoxic edema (Dirnagl et al. 1999). This leakiness of the neuronal plasma membrane also results in the release of ATP into the ex tracellular space. This release of ATP leads to the early activation of the resi dent microglia and astrocytes inducing the acute inflammatory response. Endogenous microglial activation during the early inflammatory response Microglia are the resident macrophages of the brain, which under normal conditions are kept in a quiescent stat e. Resting microglia exhibit a ramified morphology in vivo characterized by fine processes expressing chemosensitive receptors (Streit et al. 2004). In an uni njured brain these cells are involved essential functions such as synaptic pruning, debris removal, defense against infection, and neurotrophic suppo rt (Streit et al. 2005). Studies using GFP-labeled microglia in vivo demonstrated that microglial processes are highly mobile and quickly mo ve towards ATP released by injured

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10 neurons (Davalos et al. 2005; Haynes et al 2006). Microglia can then assist the damaged neuron by sequestering glutam ate (van Landeghem et al. 2001) in addition to releasing neurotrophins such as BDNF (Coull et al. 2005) and TGF (da Cunha et al. 1997; Lehrmann et al. 1998). In ischemia where the number of injured neurons is large the microglia will then withdraw their processes, assuming an amoeboid “activated” phenoty pe (see Garden et al (Garden and Moller 2006) for review), and can secrete cyto toxins such as nitric oxide, reactive oxygen species, and prostaniods (Gibson et al. 2005). These cells can also promote the inflammatory response by secreting pro-inflammatory cytokines such as IL1 and TNF (Allan and Rothwell 2001; Dirnagl et al. 1999). Whether this causes neuronal death directly however is uncertain as ablation of microglia exacerbates ischemic injury (Lalancette-Hebert et al. 2007). This release of inflammatory cy tokines was originally thought to exacerbate ischemia induced injury based on in vitro studies where direct application of these IL1 and TNF (Jara et al. 2007; Thornton et al. 2006) killed cultured neurons, and in vivo studies where neutralizatio n of these cytokines by endogenous receptor ant agonist (Relton et al. 1996) or soluble receptor (IL1 and TNF respectively) reduced infarct volume s. Whether thes e effects are due to direct neuronal toxicity are unclear as IL1 amplifies the initial immune infiltration by stimulating microglia and astrocytes to produce MMP9. In vivo studies using TNF receptor knockout mice have demonstrated that microglial derived TNF is neuroprotective via activation of the of the p55 subunit of the TNF receptor (Lambertsen et al. 2009). Furthermore ablation of microglia

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11 exacerbates ischemic injury (Lalancette -Hebert et al. 2007). In early ischemic injury, the release of IL1 TNF and IL6 by microglia is extremely important in the transition from a local immune response to a global immune/stress response (Zheng and Yenari 2004). Activation of the neuro-immune and hypothalamic pituitary adrenal axes Local inflammation has both beneficial and deleterious effects on ischemic lesions. When the local inflammation activates the peripheral immune system, however, a much larger inflammatory response is elicited causing collateral destruction of the peri-infarct regions (Offner et al. 2006). Therefore, the peripheral inflammatory response to brain injury is tightly regulated. The brain communicates with the immune sy stem largely via direct innervation of lymphoid tissues and humeral control provided by the hypothalamic-pituitary-adrenal axis (HPA axis) (Chrousos 1995). In flammatory cytokines IL1 TNF and IL6 released in response to brain injury stim ulate centers in the locus ceruleus to induce neural sympathetic activation, as well as cause the release of CRH in the hypothalamus (Chrousos 1992). IL1 and TNF stimulate the production of IL6 which in turn inhibits their production. I L6 acts synergistically with glucocorticoids to induce the release of acute phase react ants from the liver including C-reactive protein. C-reactive protein and IL6 are elevated in the serum of patients with ischemic stroke and are correla ted with poorer outcomes. Stimulation of either the locus ceruleus or the HPA axis activates the other based on neural crosslinks between the lateral paraventricular nucleus and the

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12 locus ceruleus (Saper et al. 1976). On ce these systems are activated, the resulting release of epinephrine from the adrenal glands in addition to the direct secretion of norepinephrine into the lym phoid organs by sympathetic nerve fibers results in a “sympathetic storm” (Cham orro et al. 2007). The increase in circulating catecholamines in addit ion to increased plasma cortisol concentrations suppress immune cell function This acute stress response alters the morphology of the two peripheral lymp hoid organs known to be involved in the progression of stroke: the thymus and the spleen (Elenkov et al. 2000). The thymus and the spleen are dens ely innervated by noradrenergic nerve terminals (Anagnostou et al. 2007), and upon induction of CNS ischemia become reduced in size (Off ner et al. 2006; Vendrame et al. 2006). Multiple groups have associated this decrease in thymus and spleen size with profound leucopenia and an increased susceptibility to infection based a lack of IFN producing T-cells (Prass et al. 20 03; Yilmaz et al. 2006). This immunosuppression, while likely helpful at reducing permanent brain injury, can be reversed by administration of beta-adr energic blockers such as propranolol (Meisel et al. 2005; Prass et al. 2006) The Offner group pos tulated that the decrease in spleen and thymus size wa s due to mass apoptotic cell death in these organs based on TUNEL immunoreactivi ty (Offner et al. 2006). Recent work in our lab has demonstrated that t he spleen shrinkage is due to activation of alpha1 adrenergic receptors located on the smooth muscle capsule and this shrinkage can be blocked by prophylactic ad ministration of prazosin, an alpha1 receptor antagonist.

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13 Human blood samples taken during the PANTHERIS trial (Klehmet et al. 2009) and in other clinical stroke setti ngs (Urra et al. 2009), confirmed the increased catecholamine levels in stroke patients in addition to the leucopenia It is still unclear whether increased susceptibilit y to infection in the clinical setting is a direct product of this immunosuppr ession, or a product of stroke induced complications. These complications in clude dysphasia, which is known to increase pneumonia incidence, and the need for a urinary catheter, which is associated with increased ur inary tract infections (UTIs). Pneumonia and UTIs are common stroke associated nosocomial infections and are the leading causes of delayed (>5 day) stroke mort ality (Aslanyan et al. 2004). The role of the peripheral lymphoid organs in stroke The induction of such profound imm unosuppression indicates that the peripheral lymphoid organs can exacerbat e ischemic injury. The spleen in particular plays a critical role in the re sponse to ischemic injury. Cell tracking studies of intravenously (i.v.) injected of human umbilical cord blood cells (HUCBC) into rats receiving MCAO ident ified the spleen and the infarct as the primary targets of this t herapy (Vendrame et al. 2004a). Studies comparing i.v. injection to striatal injection of HUCBC showed that i.v. injection was superior suggesting that the splenic actions of these cells were important for the therapeutic benefit of this treatment (Willing et al. 2003). Splenectomy significantly reduces ischemic injury in multiple organ systems including liver (Okuaki et al. 1996), GI tract (Savas et al. 2003), and kidney (Jiang et al. 2007).

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14 Recent results in our lab show that spl enectomy prior to stroke reduces infarct volume by 80% (Ajmo et al. 2008); similar to levels of protection seen in HUCBC therapies (Vendrame et al. 2004b). This signi ficant protective effect in multiple tissue types indicates that the spleen cont ributes to ischemic injury in a tissue type independent manner, and this response likely is responsible for infarct expansion and degeneration of the penumbra. The peripheral leukocyte response to ischemia Upon induction of ischemia, endothe lial cells in the infarct begin the process of recruiting per ipheral leukocytes into the damaged parenchyma by upregulating ICAM1 and ICAM2 (Lindsberg et al. 1996; Wang et al. 1994). The first leukocyte subtype to infiltra te the brain is neutrophils, a small polymorphonuclear cell wh ich begins to adhere to the endothelium between 1 and 6 hours following ischemia onset and invade the infarct beginning 6 hours after adherence (Kochanek and Hallenbeck 1992). These cells express selectins and integrins which enable them to extr avate between the endothelial cells and begin degrading the basal lamina th rough the secretion of matrix metalloproteinase and other proteases (J ean et al. 1998; Matsuo et al. 1994). These proteases not only further weaken the blood brain barrier but also contribute to the ensuing inflammation by activating chemokines such as MCP-1 and SDF-1a (Overall et al. 2002) along with proinflammatory cytokines such as IL1 (Schonbeck et al. 1998) and TNF (Gearing et al. 1994). These chemoattractants further serve to recr uit peripheral leukocytes such as

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15 macrophages, T and B lymphocytes, and dendritic cells to the infarcted tissue. Once within the parenchyma, neutrophils release reactive oxygen and nitrogen species in addition to various enzymes such as myeloperoxidase and elastase which are normally sequestered in their cytoplasmic granules (Jean et al. 1998). These observations of potential neur odestructive capabilities of neutrophils, however, must be weighed against the results from clinical trials which show no added clinical benefit to blocking neutrophil migration. These studies used several differing molecula r targets to achieve this such as: neutrophil inhibitory factor (a recombinant glycoprotein with selective binding to the CD11b integrin of MAC-1 (CD11b/CD18)), inhibition of leukocyte adhesion by antibodies against ICAM (Enlimomab Acut e Stroke Trial), or CD18 (HU23F2G Anti-Adhesion to Limit Cytotoxic Injury tr ial). The antibody studies resulted in increased morbidity and mortality and both trials using this rationale were discontinued due to safety concerns. Much of the toxicity a ttributed to neutrophils was based on the activity of the enzyme myeloperoxidase which produc es highly reactive oxygen species and is correlated with both increased infa rct volume and poorer outcome after stroke (Kitamura et al. 1997). Myel operoxidase is expressed not only by neutrophils, but by all cells of myeloi d lineage including macrophages (Lagasse and Weissman 1994). In vivo imaging of myeloperoxidase activity showed peak activation 3 days after MCAO, a timepoint which is well a fter the initial influx of neutrophils and correlates with peak macrophage aggregation (Weston et al. 2007). There is evidence in the literature that ma crophages phagocytose

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16 neutrophils in the ischemic infarct, wit h 50% of neutrophils in the infarct being engulfed three days after MCAO (Meszaro s et al. 1999; Weston et al. 2007). This engulfment is one of the primary mechanisms by resolution of neutrophil based inflammation, with macrophages pref erentially clearing aging neutrophils first (Lagasse and Weissman 1994). Macrophage accumulation following ischem ia is highly correlated with the production of the chemokine MCP-1 (Wang et al. 1995). This chemokine is produced and secreted as a protein precur sor by astrocytes and neurons (Che et al. 2001; Gourmala et al. 1997). This chemokine is activated and released by proteases such as plasmin (Sheehan et al. 2007) and is detected beginning at 6 hours following transient ischemia (Yam agami et al. 1999). This timepoint correlates with neutrophil infiltration and is likely related to production of MMPs by these cells. MCP-1 expression incr eases and reaches peak levels between 2 and three days following MCAO which corre lates with the influx of macrophages to the infarct (Wang et al. 1995). MCP-1 also induces the expression of ICAM-1 mRNA in kidney models (Giunti et al. 2006; Viedt et al. 2002) and likely further increases invasion of the brain parenchyma by peripheral immune cells. Ameboid macrophages are the most abundant immune cells in the infarct following MCAO (Stoll et al. 1998). Whether these cells are microglia which have transformed into an amoeboid phenotype or are infiltrating monocytes is impossible to determine immunohistochemica lly or morphologically. Studies in which peripheral macrophages are label ed with iron particles and tracked following photochemically induced MCAO suggest that peripheral macrophage

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17 recruitment is a delayed phenomenon wh ich peaks at 5 days following MCAO. (Kleinschnitz et al. 2003) These finding suggest that the macrophages which are observed at 3 days following MCAO are microglial in origin. Histochemical studies following ischemia, however, do suggest that a population of CD8+ macrophages do infiltrate the CNS withi n 48 hours following ischemia, though their function is not know n (Jander et al. 1998). Regardless of their or igin, amoeboid macrophage levels peak by 3 days following MCAO and remain elevated for we eks following insult. These cells are involved in tissue destruction, turnover of extracellular matr ix, removal of cell debris, and wound healing (Schilling et al. 2005). Depletion of peripheral macrophages using clodronate liposomes has no effect on infarct volume suggesting that these cells do not directly induce ischemic damage (Schroeter et al. 1997). Sheets of “foamy” macrophages have been described in infarcts of stroke patient’s brains at delayed tim epoints further confirming the phagocytic and tissue removal functions of thes e cells (Petroff et al. 1992). Macrophage depletion impairs remyelination after inju ry (Kotter et al. 2001), demonstrating a complex role in infarct development and resolution. While cells of the innate immune system are the most numerous found in the infarct, cells of the adaptive immune syst em play an important role in stroke pathology. T and B lymphocytes infiltrate the infarct along with neutrophils and macrophages and contribute to the infl ammation and tissue destruction that occurs there. Under normal conditi ons the brain is considered an immune privileged organ. When T-cells enter t he parenchyma following ischemia, they

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18 are exposed to new antigens such as myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG) which they have not been exposed to during the self selection process. Experiments in which mice were tolerized to these proteins prior to MCAO showed a signi ficant reduction in infarct volume demonstrating that adaptive immunity plays a significant part in the post stroke inflammatory response (Frenkel et al. 2003; Gee et al. 2008). Severe combined immunodeficient (SCID) mice and recombinase activating gene 1 (Rag-/-) knockout mice, which lack functional T-and Blymphocytes, had significantly smaller infarcts when compared to their wild type litter mates following transient MCAO (Yilmaz et al. 2006). Further studi es in which mice which lacked CD4+ and CD8+ T-lymphocytes were subjected to transient MCAO showed that these T-lymphocyte subtypes are important fo r the induction of tissue damage following ischemia (Yilmaz et al. 2006). B-lymphocytes are recruited to the br ain following MCAO. Their role once there may be a protective one, as mice which lack B-lymphocytes did not have reduced infarct volumes suggesting that these cells may not directly participate in the acute phase destruction of tissue (Yilmaz et al. 2006). Additionally in human patients, lower B-cell counts, but not other leukocyte subpopulations, in the blood correlated with adverse outcomes upon adm ission and at 2 days after stroke implying a protective effect of these ce lls (Urra et al. 2009). As interactions between B-cells and antigen presenting cells elicit an anti-inflammatory humeral response, reduced numbers of B-cells may correlate with increased inflammation and increased brain damage (Harling-Berg et al. 1999). Alternatively increased

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19 brain damage may induce a stronger stre ss response from the HPA axis causing increased cortisol levels increasing 2-adrenergic receptors which can result in increased apoptosis of B-cells in response to catecholamines. Current therapies for stroke While much has been learned about the mechanisms by which stroke damage occurs, the design of new therapeu tics for ischemic injury has been fraught with failure. The only currently avai lable interventions for stroke sufferers are thrombolytics such as rTPA and me chanical clot removal (Marler and Goldstein 2003). These therapies are aimed solely at restoring blood flow to the infarcted tissue, have no direct neur oprotective properties, and must be administered at similar timepoints. In order to provide benefit these therapies must be administered at a very early ti mepoint prior to the induction of the inflammatory response. The numbers of patients, who arrive at the hospital, receive a CT scan to rule out hemorr hagic stroke, and be administered therapy within the 3-6 hour time window are sma ll, representing only 3-5% of stroke sufferers (Marler and Goldstein 2003). Tissue plasminogen activator is a serine protease produced by macrophages and endothelial ce lls, which converts plasminogen to plasmin, a fibrin cleaving enzyme (Teesalu et al. 2002). Plasminogen, a zymogen secreted by the liver, binds fibrin and is incorporat ed into blood clots. Fibrin is a protein found in blood clots which is composed of chains of fibrin monomers linked through the action of coagulat ion factor III. Once plasminogen is converted to

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20 plasmin, it cleaves fibrin, releasin g soluble fragments and exposing lysine residues which the plasmin binds to prior to cleaving again. Of those patients whom are cand idates for rTPA, only 13% had an improved outcome compared to placebo (Al bers et al. 2004). This benefit must also be weighed against the significantly increased risk for bleeding, a process called hemorrhagic transformation (Toni et al. 1996). In this process, rTPA can activate proMMP9 present in neutrophils granules leading to further degradation of the basal lamina and increased risk for bleeding (Cuadrado et al. 2008). Mechanical clot disruption is a nonenzymat ic method of restoring blood flow by physically removing a clot. In this method, a catheter is introduced into the blocked artery and a device (frequently a co il) is inserted into the clot, the catheter is then removed pul ling the clot free, and allowing recanalization (Noser et al. 2005). This technique can be used in conjunction with rTPA and has shown promising results in phase 2 clinical trials (Kim et al. 2006). Phase three clinical trials such as the IMSIII tr ial are underway and should shed light on the therapeutic efficacy of this technique. Therapeutic targets for stroke at delayed timepoints Until recently it was believed that neurons died very rapidly following stroke onset by these excitotoxic mechanisms. This was based on the observation of neurons which had becom e pyknotic and expressed various markers for apoptosis such as Annexin V, cleaved caspase 3, and TUNEL stains. This belief was challenged on observati ons that HUCBC could reverse these

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21 apoptosis markers resulting in a 50-70% reduction in infarct volume when administered up to 48 hours after undergoi ng permanent MCAO (Vendrame et al. 2004a). HUCBC are the mononuclear fraction of cells isolated from umbilical cord blood collected after birth. This cell population includes approximately 1% CD34+ stem cells with the remainder bei ng lymphocytes at varying stages of development. HUCBC administrati on is associated with profound neuroprotective and anti-inflammatory e ffects when administered in animal models of a variety of disease states su ch as ALS (Garbuzova-Davis et al. 2003), Alzheimer’s disease (Nikolic et al. 2008), Parkinson’s disease (Ende and Chen 2002), spinal crush injury (Saporta et al. 2003), and stroke (Newcomb et al. 2006; Vendrame et al. 2004a). While th is therapeutic does have a stem cell component, cell tracking studies demonstrat ed that very few of these cells actually implanted into the damaged tiss ue suggesting that this is not a cell replacement therapy (Vendrame et al. 2004a). Further studies in our lab have ident ified the sigma receptor agonist 1, 3di-o-tolylguanidine (DTG) as a therapy whic h substantially reduces infarct volume when administered 24 hours following MC AO (Ajmo Jr et al. 2006). The identification of a second unrelated t herapeutic which is neuroprotective at delayed timepoints suggests that ther e is an underlying mechanism which can be harnessed to provide neuroprotection at a ti me when a majority of stroke patients can be seen and treated. Furthermore, th is suggests that excitotoxicity and anoxic depolarizatio n are not as damaging as onc e thought. This finding has

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22 important implications with respect to the treatment of hypoxic ischemic injury in other disease paradigms. Other treatments which suppress in farct volumes at delayed timepoints include ethyl pyruvate (Yu et al. 2005) and granulocyte colony stimulating factor (Sehara et al. 2007),. Thes e treatments also have potent anti-inflammatory properties and show functional recovery in addition to reduced infarct volumes. Sigma receptor agonists as a therapy for stroke Sigma receptors are membrane a ssociated proteins found widely distributed in the mammalian brain, peripheral neurons, and visceral organs. These receptors were first identified by Martin et al ., and have since been classified into two types on the basis of pharmacology, sigma-1 and sigma-2 (Quirion et al. 1992). Both subtypes have high to moderate affinity for antipsychotics, such as haloperidol, and guanidines. Sigma-1 receptors have a higher affinity for (+)-b enzomorphans, such as (+)-pentazocine, than sigma-2 receptors, whereas sigma2 receptors have higher a ffinity for ibogaine and its congeners. Thus far, only the sigma-1 re ceptor has been cloned, and this protein is detected on the membranes of the endoplasmic reticulum and influences calcium release from this organelle (H ayashi and Su 2001; Seth et al. 1998). The sigma-1 receptor, with two put ative transmembrane domains, bears no resemblance to any other known ma mmalian protein. While the endogenous ligand for sigma receptors has not been cl early identified, various candidates have been suggested. N, N-dimethyltrypta mine has been recently described as a

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23 nonsteriodal endogenous ligand for the sigm a-1 receptor (Fontanilla et al. 2009), perhaps explaining the significant anti-anx iolytic properties of sigma receptor agonists. Neurosteroids lik e progesterone were also shown to bind sigma receptors with high affinity, leading to the speculation that some of the physiological actions of progesterone are m ediated by sigma receptors (Su et al. 1990). Other neurosteroids, including pregnenolone and dehydroepiandrosterone, have high a ffinity for sigma receptors. Given the neuroprotective properties of neuroster oids and their abundance in the cortex, these sigma ligands would be obvious candidates for t he possible endogenous neuroprotection by sigma rec eptors report ed here. Numerous in vivo studies have demonstrated a protective role for sigma receptors following ischemia. Rats subj ected to transient MCAO had reduced infarct volume when various sigma ligan ds such as dimemorphan (Shen et al. 2008), (+)pentazosin (Takahashi et al. 1997), and 4-phenyl-1-(4-phenylbutyl) piperidine (PPBP) (Takahashi et al. 1996) were administered during reperfusion. Inhibition of various components of the ear ly response to stroke such as NMDA receptor activation, neuronal nitric oxid e synthesis, and inducible nitric oxide synthesis have been postulated to mediate this neuroprotection. Activation of sigma receptors at 24 hours in the pe rmanent MCAO model by DTG is also neuroprotective (Ajmo et al 2006), though at this timepoi nt it is likely that modulation of the immune system plays a larger role in this effect. The neuroprotective properti es of sigma receptors in vitro have been ascribed to their ability to stabilize intracellular calcium levels due to NMDA

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24 receptor activation and block anoxi c depolarization. Subsequent studies demonstrate that sigma receptor activa tion blocks ischemia induced calcium increases by suppressing calcium influx through multiple pathways (Katnik et al. 2006). Recent work demonstrates that sigma receptor activation suppresses acid sensing ion channel function (Herrera et al. 2008). This family of channels induces calcium influx in the presence of high levels of extracellular protons generated by glycolysis and excessive neur otransmission (Xiong et al. 2007). Pharmacological inhibition of this ion c hannel is protective when administered within 5hrs of MCAO (Xiong et al. 2008). It remains to be determined whether the benefit seen in vivo; due to these effects at the leve ls of the neuron or due to the effects of sigma receptor activa tion on immune system function, or both. A role of sigma receptors in the immune system was first proposed by T.P. Su et al. based on their observation that lym phoid tissues express sigma binding sites (Su et al. 1988). Subsequent studi es have shown that sigma receptor activation can regulate the function of various cells of the immune system. Selective sigma-1 receptor activation is associated with reduced leukocyte invasiveness and decreased levels of inflammatory cytokines in vivo (Zhu et al. 2003). Sigma ligands such as (+) pent azocine, haloperidol, and DTG suppress in vitro murine splenocyte natural killer activity and pol yclonal immunoglobulin production following mitogen stimulati on (Carr et al. 1991). Systemic administration of the sigm a ligand, SR 31747A, inhibits the secretion of TNFand IFN evoked by injection of LPS in rats (Bourrie et al. 2002). This sigma ligand has also been shown to inhibi t NO production in LPS-activated

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25 macrophages (Gannon et al. 2001). In t he CNS, sigma-1 receptors are expressed in microglia (G ekker et al. 2006), and our l aboratory has shown that sigma receptor activation is associated with decreased reactive gliosis following stroke injury in rats (Ajmo et al. 2006). The immunosuppressive effects of cocaine have been attributed to sigma receptor activation via heightened expression of TGF(Gekker et al. 2006), an anti-in flammatory cytokine altered following ischemia. Determining the impact of DTG on the post ischemic inflammatory response-the current project While sigma ligands have been studied in the treatment of ischemia, previous projects have focused on reperfusion injury, neuronal calcium dysregulation, and/or anoxic depolarization. While these studies are useful for understanding sigma receptor biology during early ischemia, they have very little relevance to the clinical condition. The major caveat with attempting to intervene in these pathological systems is that they typically occur at timepoints well before patients are seen for treatm ent. A major finding in our lab, centers around the ability of DTG to impart significant neur oprotection when therapy is administered 24 hours following permanent MCAO. T he only other treatments which have efficacy at such delayed timepoints such as minocycline and HUCBC have immunomodulatory properties. Microglia are the endogenous immune cells of the brain and are intrinsically linked to induction, development, and maintenance of the post

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26 ischemic inflammatory response. The first set of experiments described here were undertaken to test the follow ing hypotheses: (1) sigma receptor activation suppresses microgl ial activation in culture, and (2) suppression of intracellular calcium is the pr ocess which sigma receptor activation targets to suppress microglial activation. Dissociated microglial cultures are a good model for elucidating intracellular signaling cascades which occu r during activation. The relevance of this model to ischemic injury however is low. The organotypi c slice culture model has many advantages over dissociated cell culture. Principal among these is that all cell types found in brain par enchyma are present and many of their functional connections are maintained. When these cultures are subjected to ischemic conditions, microglia are activa ted in conditions similar to those found in vivo. Furthermore as sigma receptor activation modulates microglial and peripheral leukocyte activation this model al lows the exploration of the effect of sigma receptor activation without the i nput of the peripheral immune system. The second set of experiments pr esented here explores the following hypotheses: (1) that ischemia r esults in robust neurodegeneration resulting in microglial activation as measured by nitric oxide production, and (2) sigma receptor mediated s uppression of microglial activation confers neuroprotection. In vivo peripheral immune cells infiltrate the infarct prior to the 24 hour timepoint at which DTG is efficacious. Pe ripheral T-lymphocytes in particular are involved in stroke induc ed neurodegeneration, likely th rough interactions with

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27 antigen presenting cells like macrophages/micr oglia residing in the infarct. One source of T-lymphocytes which contribut es to ischemic neurodegeneration is the spleen. Sigma receptor activation c an modulate T-cell ant igen presenting cell interaction by upregulating the production of anti-inflammatory cytokines IL10 and TGF in T-cells and microglia respectively decreasing peripheral inflammation. Therefore, the third set of ex periments presented here will test the hypothesis that: (1) splenic pe ripheral immune cells infiltrate the infarct following MCAO, and (2) DT G treatment reduces the prevelance of these cells in the infarct References 1999. Hu23F2G. 23F2G, LeukArrest. Drugs R D 1(1):25-6. 2001. Use of anti-ICAM-1 therapy in isc hemic stroke: results of the Enlimomab Acute Stroke Trial. Neurology 57(8):1428-34. Ajmo CT, Jr., Vernon DO, Collier L, Ha ll AA, Garbuzova-Davis S, Willing A, Pennypacker KR. 2008. The spleen contributes to stroke-induced neurodegeneration. J Neurosci Res: (in press, Epub ahead of print). Ajmo CT, Jr., Vernon DO, Collier L, Pennypacker KR, Cuevas J. 2006. Sigma receptor activation reduces infarct si ze at 24 hours after permanent middle cerebral artery occlusion in ra ts. Curr Neurovasc Res 3(2):89-98.

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29 Bourrie B, Bribes E, De Nys N, Esclangon M, Garcia L, Galiegu e S, Lair P, Paul R, Thomas C, Vernieres JC and others. 2002. SSR125329A, a high affinity sigma receptor ligand with pot ent anti-inflammatory properties. Eur J Pharmacol 456(1-3):123-131. Carandang R, Seshadri S, Beiser A, Kelly -Hayes M, Kase CS, Kannel WB, Wolf PA. 2006. Trends in incidence, lifetime risk, severity, and 30-day mortality of stroke over the past 50 years. Jama 296(24):2939-46. Carr DJ, De Costa BR, Radesca L, Blal ock JE. 1991. Functional assessment and partial characterization of [3H](+)pentazocine binding site s on cells of the immune system. J Neur oimmunol 35:153-166. Chamorro A, Amaro S, Vargas M, Obac h V, Cervera A, Go mez-Choco M, Torres F, Planas AM. 2007. Catecholamines infection, and death in acute ischemic stroke. J Neurol Sci 252(1):29-35. Che X, Ye W, Panga L, Wu DC, Y ang GY. 2001. Monocyte chemoattractant protein-1 expressed in neurons and astr ocytes during focal ischemia in mice. Brain Res 902(2):171-7. Chrousos GP. 1992. Regulati on and dysregulation of t he hypothalamic-pituitaryadrenal axis. The corticotropin-rel easing hormone perspective. Endocrinol Metab Clin North Am 21(4):833-58. Chrousos GP. 1995. The hypothalami c-pituitary-adrenal axis and immunemediated inflammation. N Engl J Med 332(20):1351-62. Coull JA, Beggs S, Boudreau D, Boivin D, Tsuda M, Inoue K, Gravel C, Salter MW, De Koninck Y. 2005. BDNF from microglia causes the shift in

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41 Schroeter M, Jander S, Huit inga I, Witte OW, Stoll G. 1997. Phagocytic response in photochemically induced infarction of rat cerebral cortex. The role of resident microglia. Stroke 28(2):382-6. Schwamm LH, Koroshetz WJ, Sorensen AG, Wang B, Copen WA, Budzik R, Rordorf G, Buonanno FS, Schaefer PW, Gonzalez RG. 1998. Time course of lesion development in patients wit h acute stroke: serial diffusionand hemodynamic-weighted magnetic resonance imaging. Stroke 29(11):2268-76. Sehara Y, Hayashi T, Deguchi K, Zhang H, Tsuchiya A, Yamashita T, Lukic V, Nagai M, Kamiya T, Abe K. 2007. De creased focal inflammatory response by G-CSF may improve stroke outcom e after transient middle cerebral artery occlusion in rats. J Neurosci Res 85(10):2167-74. Seth P, Fei YJ, Li HW, Huang W, Lei bach FH, Ganapathy V. 1998. Cloning and functional characterization of a si gma receptor from rat brain. J Neurochem 70:922-931. Sheehan JJ, Zhou C, Gravanis I, Rogove AD, Wu YP, Bogenhagen DF, Tsirka SE. 2007. Proteolytic activation of m onocyte chemoattractant protein-1 by plasmin underlies excitotoxic neur odegeneration in mice. J Neurosci 27(7):1738-45. Shen YC, Wang YH, Chou YC, Liou KT, Yen JC, Wang WY, Liao JF. 2008. Dimemorfan protects rats against isc hemic stroke through activation of sigma-1 receptor-mediated mechani sms by decreasing glutamate accumulation. J Neurochem 104(2):558-72.

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42 Stoll G, Jander S, Schroet er M. 1998. Inflammation and glial responses in ischemic brain lesions. Progre ss in Neurobiology 56:149-171. Streit WJ, Conde JR, Fendrick SE, Fl anary BE, Mariani CL. 2005. Role of microglia in the central nervous syst em's immune response. Neurol Res 27(7):685-91. Streit WJ, Mrak RE, Griffin WS. 2004. Microglia and neuroinflammation: a pathological perspective. J Neuroinflammation 1(1):14. Su TP, London E, Jaffe JH. 1988. Steroid binding at sigma receptors suggests a link between endocrine, nervous, and immune systems. Science 240(4937):1635-1638. Su TP, Shukla K, Gund T. 1990. Steroid binding at sigma receptors: CNS and immunological implications. Ci ba Found Symp 153:107-13; discussion 113-6. Takahashi H, Kirsch JR, Hashimoto K, London ED, Koehler RC, Traystman RJ. 1996. PPBP [4-phenyl-1-(4-phenylbutyl) pi peridine] decreases brain injury after transient focal ischemia in rats. Stroke 27(11):2120-3. Takahashi H, Traystman RJ, Hashimoto K, London ED, Kirsch JR. 1997. Postischemic brain injury is affect ed stereospecifically by pentazocine in rats. Anesth Analg 85(2):353-7. Takano K, Latour LL, Formato JE, Car ano RA, Helmer KG, Hasegawa Y, Sotak CH, Fisher M. 1996. The role of sp reading depression in focal ischemia evaluated by diffusion mapping. Ann Neurol 39(3):308-18.

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43 Teesalu T, Kulla A, Asser T, Koskinie mi M, Vaheri A. 2002. Tissue plasminogen activator as a key effector in neurobiology and neuropathology. Biochem Soc Trans 30(2):183-9. Thornton P, Pinteaux E, Gi bson RM, Allan SM, Rothwell NJ. 2006. Interleukin-1induced neurotoxicity is mediated by glia and requires caspase activation and free radical release. J Neurochem 98(1):258-66. Toni D, Fiorelli M, Bastianello S, Sacc hetti ML, Sette G, Ar gentino C, Montinaro E, Bozzao L. 1996. Hemorrhagic tr ansformation of brain infarct: predictability in the first 5 hours from stroke onset and influence on clinical outcome. Neurology 46(2):341-5. Traystman RJ. 2003. Animal mo dels of focal and global cerebral ischemia. Ilar J 44(2):85-95. Urra X, Cervera A, Villamor N, Pl anas AM, Chamorro A. 2009. Harms and benefits of lymphocyte subpopulations in patients with acute stroke. Neuroscience 158(3):1174-83. van Landeghem FK, Stover JF, Bechmann I, Bruck W, Unterber g A, Buhrer C, von Deimling A. 2001. Early expression of glutamate transporter proteins in ramified microglia after controlled cortical impact injury in the rat. Glia 35(3):167-79. Vendrame M, Cassady CJ, Newcomb J, Butler T, Pennypacker KR, Zigova T, Davis Sanberg C, Sanberg PR, AE W. 2004a. Infusion of human umbilical cord blood cells in a rat model of stroke dose-dependently rescues behavioral deficits and reduces infarct volume. Stroke 35:2390-2395.

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44 Vendrame M, Cassady J, Newcomb J, Butler T, Pennypacker KR, Zigova T, Sanberg CD, Sanberg PR, Willing AE. 2004 b. Infusion of human umbilical cord blood cells in a rat model of stroke dose-dependently rescues behavioral deficits and reduces infarc t volume. Stroke 35(10):2390-5. Vendrame M, Gemma C, Pennypacker KR Bickford PC, Davis Sanberg C, Sanberg PR, Willing AE. 2006. Cord blood rescues stroke-induced changes in splenocyte phenotype and f unction. Exp Neurol 199(1):191200. Viedt C, Dechend R, Fei J, Hansch GM, Kreuzer J, Or th SR. 2002. MCP-1 induces inflammatory activation of human tubular epithelial cells: involvement of the transcription fa ctors, nuclear factor-kappaB and activating protein-1. J Am Soc Nephrol 13(6):1534-47. Wang X, Siren AL, Liu Y, Yue TL, Bar one FC, Feuerstein GZ. 1994. Upregulation of intercellular adhesion molecule 1 (ICAM-1) on brain microvascular endothelial cells in rat ischemic co rtex. Brain Res Mol Brain Res 26(12):61-8. Wang X, Yue TL, Barone FC, Feuerstei n GZ. 1995. Monocyte chemoattractant protein-1 messenger RNA expression in rat ischemic cortex. Stroke 26(4):661-5; discussion 665-6. Weston RM, Jones NM, Jarrott B, Ca llaway JK. 2007. Inflammatory cell infiltration after endothelin-1-induced ce rebral ischemia: histochemical and myeloperoxidase correlation with tem poral changes in brain injury. J Cereb Blood Flow Metab 27(1):100-14.

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45 Willing AE. 2009. Experimental models: help or hindrance. Stroke 40(3 Suppl):S152-4. Willing AE, Lixian J, Milliken M, Poulos S, Zigova T, Song S, Hart C, SanchezRamos J, Sanberg PR. 2003. Intravenous versus intrastriatal cord blood administration in a rodent model of stroke. Journal of Neuroscience Research 73(3):296-307. Xiong ZG, Chu XP, Simon RP. 2007. Acid sensing ion channels--novel therapeutic targets for ischemic br ain injury. Front Biosci 12:1376-86. Xiong ZG, Pignataro G, Li M, Chang SY, Simon RP. 2008. Acid-sensing ion channels (ASICs) as pharmacologi cal targets fo r neurodegenerative diseases. Curr Opin Pharmacol 8(1):25-32. Yamagami S, Tamura M, Hayashi M, E ndo N, Tanabe H, Katsuura Y, Komoriya K. 1999. Differential production of MCP-1 and cytokine-induced neutrophil chemoattractant in the ischemic brain a fter transient focal ischemia in rats. J Leukoc Biol 65(6):744-9. Yilmaz G, Arumugam TV, Stokes KY, Gran ger DN. 2006. Role of T lymphocytes and interferon-gamma in ischemic stroke. Circulation 113(17):2105-12. Yu YM, Kim JB, Lee KW, Kim SY, Han PL, Lee JK. 2005. Inhibition of the cerebral ischemic injury by ethyl pyruvate with a wide therapeutic window. Stroke 36(10):2238-43. Zheng Z, Yenari MA. 2004. Post-ischemic inflammation: molecular mechanisms and therapeutic implications. Neurol Res 26(8):884-92.

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46 Zhu LX, Sharma S, Gardner B, Escuadro B, Atianzar K, Tashkin DP, Dubinett SM. 2003. IL-10 mediates sigma 1 receptor-dependent suppression of antitumor immunity. J Immunol 170:3585-3591.

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47 Chapter 2 Sigma Receptors Suppress Multiple Aspects of Microglial Activation Hall Aaron A., Herrera Yel enis, Ajmo Jr. Craig T., Cuevas Javier, Pennypacker Keith R. Abstract During brain injury, microglia become activated and migrate to areas of degenerating neurons. These microglia, releas e pro-inflammatory cytokines and reactive oxygen species causing additional neuronal death. Microglia express high levels of sigma receptors, howeve r, the function of these receptors in microglia and how they may affect the ac tivation of these cells remain poorly understood. Using primary rat microglia l cultures, it wa s found that sigma receptor activation suppresses the ability of microglia to rearrange their actin cytoskeleton, migrate, and release cytok ines in response to the activators adenosine triphosphate (ATP), monocyte c hemoattractant protein 1 (MCP-1), and lipopolysaccharide (LPS). Next, the role of sigma receptors was explored in the regulation of calcium signalling dur ing microglial activation. Calcium fluorometry experiments in vitro show that stimulati on of sigma receptors

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48 suppressed both transient and sustained intracellular calcium elevations associated with the microglial response to these activators. Further experiments showed that sigma receptors suppress mi croglial activation by interfering with increases in intracellular calcium. In addition, sigma receptor activation also prevented membrane ruffling in a calciu m-independent manner, indicating that sigma receptors regulate the function of microglia via multiple mechanisms. Introduction A principal aspect of the central nervous system (CNS) immune response to injury is the recruitment and activa tion of endogenous microglia. Microglia are the resident macrophages of t he brain, which are kept in a quiescent ramified state under normal conditions. These cells respond to substances released by damaged neurons or invading pathogens by migrating to the site of injury, phagocytosing debris, and releasing pro-in flammatory mediators, such as cytokines and reactive oxygen species (Streit et al. 200 4). In several pathological states of the CNS, this res ponse contributes to the destruction of compromised neurons enhancing neurodegeneration (Dheen et al. 2007; Rogove et al. 2002; Streit et al. 2004). Current research suggests that the inflammatory response after brain injury can be inhibit ed by sigma receptor activation (Ajmo et al. 2006). These receptors are neuroprot ective in several animal models of stroke injury (Ajmo et al. 2006; Haruk uni et al. 1998), and represent a putative

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49 target for neuroprotection at delayed time points ( 24 hrs) following stroke onset (Ajmo et al. 2006). Sigma receptors are membrane a ssociated proteins found widely distributed in the mammalian brain, peripheral neurons, and visceral organs. Two subtypes of sigma receptors, sigm a-1 and sigma-2, hav e been identified based on their pharmacological profiles (Quirion et al. 1992). Thus far, only the sigma-1 receptor has been cloned (Seth et al. 1998) and this protein is detected on the membranes of the endoplasmic reticulum and influences calcium release from this organelle (Hayashi and Su 2001). Sigma receptors were identified in the immune system using radioligand binding studies in lymphoid tissues (Su et al. 1990). Selective sigma-1 receptor activation is associated with reduced leukocyte invasiveness and decreased levels of inflammatory cytokines in vivo (Zhu et al. 2003). Sigma ligands such as (+) pentazocine, haloperidol and 1,3-di-o-tolylguanidin e (DTG) were found to suppress murine splenocyte activity and polyclonal immunogl obulin production following mitogen stimulation in vitro (Carr et al. 1991). Systemic administration of the sigma ligand, SR 31747 A, inhibits the secretion of tumor necrosis factor(TNF) and interferon gamma (IFN) evoked by injection of LPS in rats (Bourrie et al. 2002). This sigma ligand has also been shown to inhibit nitric oxide (NO) production in LPS-activated macrophages (Gannon et al. 2001). In the CNS, sigma-1 receptors are expressed in microglia (Gekker et al. 2006), and our laboratory has shown that si gma receptor activation is associated with decreased reactive gliosis following stroke in jury in rats (Ajmo et al. 2006).

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50 This study investigated sigma receptor modulation of microglial activation. Specifically, experiments were done to char acterize the effects of sigma receptor activation on morphological, migratory, and inflammatory responses of microglia and to determine the role of intracellular calcium concentration on the regulation of these processes. It was found that sigma receptor activation suppressed the morphological, migratory, and inflammatory aspects of microglial activation. Further studies revealed that sigma receptor activation regulates these processes by suppressing calcium increases. However, calcium-independent regulation of microglia activation by sigm a receptors was also noted, indicating that sigma receptors regulate these cells by affecting various signaling pathways. Materials and Methods Primary Cultures of Microglia Primary cultures of mi croglia were prepared from postnatal (2 day) Sprague-Dawley rat pups using a protocol modified from Gottschall et al (Gottschall et al. 1995). Briefly, pups were decapitated, cortices dissected out and dissociated in Hank’s balanced salt solution containing 0.25% Trypsin and 2.21 mM ethylenediaminetetraacetic acid (EDTA) (Mediatech, Manassas, VA). Isolated cortical cells were plated into poly-D-lysine (Sigma-Aldrich, St. Louis, MO) treated tissue culture fla sks. The mixed glial cultur es for the preparation of microglia were maintained in high gluc ose Dulbecco’s Modified Eagle Media (DMEM, Invitrogen, Carlsbad, CA) suppl emented with 10% horse serum, 2.5%

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51 fetal bovine serum, and an antibiotic/ant imycotic cocktail containing 100 I.U. penicillin, 100 g/ml streptomycin, and 0.25 g/ml amphotericin B. The mixed glial cultures were in cubated for 8-10 days at 37 o C. Microglia were then dislodged by vigorous shaking, plated, and used for experiments the following day. Membrane Ruffling and Quantification Microglia plated on poly-D-lysine tr eated glass coverslips were serum starved for four hours in DMEM then stimulated for 5 or 10 minutes with ATP (Sigma-Aldrich, St. Louis, MO) or 10 mi n with MCP-1 (Peprotech Inc., Norwood, MA). Compounds to be tested were incubat ed with the microglia in DMEM for 10 minutes prior to chemoattractant expo sure. In one series of experiments Ca2+free DMEM (Invitrogen) was used. Cytoskeletal changes were visualized using phalloidin conjugated to Al exaFluor 488 (Invitrogen). Multiple photomicrographs (n 4) of fields containing 1-8 cells were acquired for controls (DMEM) and each test group. Morphology of the cells was evaluated by an investigator blind to t he nature of the treatment and a score was given to each cell using the following criter ia: “0” was given to cells which did not display any signs of membrane ruffling and had multiple apparent filopodia; “1” was given to cells which retained filopodi a and lamellipodia, but which also had regions on the cell membrane where ruff ling was present; and “2” was given to cells which had withdrawn all their filopodia and di splayed a fully ruffled phenotype. The scores in each group were then summed and divided by the

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52 number of cells assayed in that group to yield that group’s degree of membrane ruffling which was expressed in arbitr ary units (A.U.). Means for each group were compared and significant differenc es were determined via two-way ANOVA with post-hoc Bonferroni tests. Migration Assay Modified Boyden chambers were used as previously described (McCord et al. 2005). Briefly, 5x105 freshly isolated microglia were applied to the top portion of a Boyden chamber assembled with an 8 m pore polycarbonate membrane. Media (DMEM) containing vehicle or chem oattractant (ATP or MCP-1) with or without compounds to be tested was added to the bottom of the chamber, and the cells incubated fo r four hours at 37oC. The membranes were removed and microglia adhering to the top of the me mbrane were scraped off. The membrane was oriented on a slide such that the ce lls that migrated to the bottom of the membrane were facing up. Vectashiel d Hardset mounting media (Vector Labs, Burlingame Ca) containing 4'-6-diamidi no-2-phenylindole (DAPI) was applied to the membrane and a coverslip affixed to the slide. DAPI positive cells were illuminated at 359 nm and visualized at 461 nm using a Zeiss Axioskop 2 outfitted with a 20X objective. A minimu m of 5 random fields of cells were counted and averaged per membrane, and the results of at least three experiments were averaged.

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53 ELISA assay Plates (96 well) were coated with 2 ng/ ml capture antibody (tumor necrosis factor, MAB510 anti-rat antibody; interleuk in-10, MAB519 anti-rat antibody; R&D Systems, Inc., Minneapo lis, MN) overnight at 4oC. The plates were washed with Tris buffered saline (TBST) consisting of: 20 mM Tris pH 7.5, 150 mM NaCl, and 0.05% Tween 20. The plates were then blocked with 1% bovine serum albumin (BSA) in TBST for 1 hour. A standard curve was prepared using recombinant protein standards. The standar ds as well as the unknowns were incubated for 1hr at room temperature and then washed with TBST. Biotinylated detection antibody (tumor necrosis factor, #BAF510 biotinylated anti-rat antibody; interleukin-10, #BAF519 biot inylated anti-rat IL10 antibody; R&D Systems, Inc., Minneapolis, MN) was added to each well at 100 ng/ml and incubated for 2 hrs at room temperature. After incubation, the plates were washed three times with phosphate buffer ed saline (PBS) containing (in mM): 3.2 Na2HPO4, 0.5 KH2PO4, 1.3 KCl, 135 NaCl (pH 7.4). Streptavidin conjugated to horseradish peroxidase (streptavidin-HRP ) was added to each well followed by 20 min incubation at room tem perature. After washing, 100 l of 3,3’,5,5’tetramethyl-benzidine (TMB) (Sigma-Ald rich, St. Louis, MO) was added and incubated for 5 minutes in the dark. Finally, 50 l of 1 M H2SO4 stop solution was added to each well and optical densit y was measured using a microplate reader set at 450 nm.

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54 Griess reaction Supernatants from treated primary rat microglia cult ures were collected, and clarified by centrifugation. Nitric oxide levels in the supernatant were determined using the Griess Reagent Ki t (Invitrogen) according to the manufacturer’s protocol. Calcium Imaging Intracellular calcium ([Ca2+]i) was measured using the ratiometric Ca2+ sensitive dye, fura-2 as previously de scribed (Katnik et al. 2006). Microglia, plated on coverslips, were incubated for 1 hour at 37oC in DMEM (Mediatech, Manassas, VA) containing 5 M fura-2, acetoxymethy lester (fura-2 AM; Invitrogen) and 0.1 % dimethyl sulfoxide (DMSO). The coverslips were washed in physiological saline solution (PSS) consisting of (in mM): 140 NaCl, 5.4 KCl, 1.3 CaCl2, 1.0 MgCl2, 20 glucose, and 25 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) (p H adjusted to 7.4 with NaOH) prior to the experiments being carried out. A DG-4 high speed wavelength switcher (Sutter Instruments Co., Novato, CA) was used to apply excitation light of alternating wavelength (340/380nm). Fluor escent emission was captured using a Sensicam digital CCD camera (Cook e Corporation, Auburn Hills, MI) and recorded with Slidebook 3.0 software (I ntelligent Imaging Innovations, Denver, CO). Changes in [Ca2+]i were calculated using the Slidebook 3 software.

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55 DAF Imaging Nitric oxide concentration was measur ed using the NO sensitive dye, 4amino-5-methylamino-2' ,7'-difluorofluorescein ( DAF-FM). Microglia plated on coverslips were incubated for 1 hour at 37oC in DMEM (Mediatech) containing 5 M DAF-FM (Invitrogen) and 0.1 % DMSO. The coverslips were washed in PSS prior to NO measurement s. DAF-FM loaded cells were illuminated at 488 nm and visualized at 510 nm, using the same instrumentation described for calcium imaging experiments. Backgr ound fluorescence was determined by measuring fluorescent intensities in ar eas between cells and was subtracted from the raw intensity values. Fluorescent int ensity was expressed as arbitrary units. Data Analysis Analysis of measured intracellular [Ca2+] responses was conducted using Clampfit 9 (Molecular Devices, Union City, CA). Statistical analysis was conducted using SigmaPlot 9 and SigmaSta t 3 software (Systat Software, Inc., San Jose, CA). Two-way ANOVA proc edures with appropriate post-hoc tests were used to analyze data from the calc ium imaging and migration experiments, while a three-way ANOVA procedure wit h post-hoc Tukey’s test was used to analyze data from the NO imaging experiments.

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56 Reagents The sigma ligands DTG and metaphit were obtained from Tocris Bioscience (Ellisville, MO). The microg lial activator MCP-1 was purchased from Peprotech Inc., (Norwood, MA), ATP and LPS from Sigma-Aldrich (St. Louis, MO), thapsigargin from Al omone Labs (Jerusalem, Israel), and ionomycin from Calbiochem (San Diego, CA). All ot her reagents were pur chased from SigmaAldrich and were of anal ytical grade or higher. Results Sigma receptor activation suppresses cha nges in microglia morphology in response to chemoattractant stimulation. ATP released from degenerating neurons activate microglia during brain injury (Davalos et al. 2005). ATP binds purinergic receptors on microglia and causes microglia to change shape and migrat e to the site of injury (Honda et al. 2001). To study these phenom ena, microglia were tr eated with ATP and labeled using phalloidin, which binds to the acti n cytoskeleton. Unstimulated microglia exhibited discrete, actin rich filopodia (F igure 1A and 1D). Upon stimulation with ATP (100 M), for either 5 min (Figur e 1B) or 10 min (Figure 1E) microglia retracted their filopodia and aggregated actin along the leading edge of the cell membrane in a manner consistent with memb rane ruffling. Administration of the sigma receptor agonist DTG (100 M) 10 min prior to and during ATP exposure

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57 decreased the aggregation of actin in the leading edge and promoted the retention of filopodia (Figur e 1C and 1F). DTG treatme nt alone resulted in cells which were morphologically indistinguish able from control ( data not shown). Since ATP-induced retraction of filopodia is an extracellular Ca2+-independent process, experiments were also carri ed out in nominal extracellular Ca2+. In the absence of extracellular Ca2+ microglia exhibited normal morphology, and filopodia were readily observed (Figure 1G). Upon application of ATP (5 min), filopodia were retracted and membrane ru ffling occurred (Figure 1H), and this membrane ruffling was still inhibited by the addition of DTG (Figure 1I).

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58 Figure 1. Sigma receptor activation suppresses changes in microglia morphology in response to chemoattractant stimulation. Photomicrographs of primar y rat microglia cultures exposed to vehicle (0.1% DMSO in DMEM) (A, D, G), 100 M ATP for 5 min (B, H) and 10 min (E), or 100 M ATP for 5 min (C, I) and 10 min (F) in the presence of 100 M DTG. Microglia in A-F were treated in normal DMEM, whereas microglia in G-I were treated in Ca2+-free DMEM. All insets represent magnified image (2.7x). Scale bar = 2 m.

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59 Sigma receptor activation significan tly decreases membrane ruffling induced by ATP. The changes in membrane structure were quantified to determine if activation of sigma receptors altered t he morphological response to ATP. ATP treatment significantly increased the degree of membrane ruffling relative to the DMEM control (Figure 2). Furthermore, wh ile in the control group no cells were found to show complete membrane ruffli ng (n = 63), 62% of cells in the ATP treatment group showed this phenotype (n = 53). DTG treatment alone had no effect on membrane morphology, with 11 of the 12 cells examined showing no evidence of membrane ruffling. Ho wever DTG pretreatment reduced the response to ATP, as evident by the significant reduction in the degree of membrane ruffling induced by the purine (Figure 2). The percentage of cells which exhibited membrane ruffling decr eased to 11% when DTG was co-applied (n = 44).

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60 Figure 2. Sigma receptor activation si gnificantly decreases membrane ruffling in microglia stimulated with ATP. Degree of membrane ruffling assessed as described in the methods section. Microglia were preincubated with vehicle, or DTG (100 M DTG) for 5 minutes then stimulated with DMEM or ATP (100 M). Bars represent means SEM. Asterisks indicate significant differenc es between DMEM and ATP within Control (no DTG, p < 0.001) and DTG groups (p < 0.001). Pound symbol indicates significant difference between Control and DTG within the ATP group (p < 0.001). Significance was determined via two-way ANOVA with post-hoc Tukey’s Test.

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61 The microglial migratory response to chemoattractant application is suppressed by sigma receptor activation. A modified Boyden chamber was used to measure the chemotaxis of freshly isolated microglia in response to ATP, with and without DTG present. ATP (100 M) promoted a ten-fold incr ease in the number of microglia that migrated across the membrane relative to control (Figure 3A). This increase in migration was statistically significant (p < 0.001) and similar to that previously reported (Honda et al. 2001). DTG (300 M) alone had no effect on microglial migration (p = 0.47), but co-application of DTG with ATP significantly (p < 0.0001, n = 3) depressed the ATP-evoked migrati on of microglia (Figure 3A) by 72.68 3.55% relative to control (ATP and no DT G). In the presence of DTG, ATP failed to evoke a statistically significant incr ease in migration above that observed when DMEM was applied (p = 0.076). The chemokine MCP-1, which binds t he CCR2 receptor (Ogilvie et al. 2004), was also tested to determine whether sigma receptor mediated inhibition of migration was limited to the effects elic ited by purinergic receptors. Application of MCP-1 (10 nM) caused a five-fold incr ease in the number of microglia that migrated through the membrane compared to control (Figure 3B). This increase in migration was less than that observed wit h ATP, but consist ent with the 3to 4-fold increase in macrophage and micr oglial migration reported for this concentration of MCP-1 (Dzenko et al. 2001; Peterson et al. 1997). Co-treatment with 300M DTG significantly depressed MCP-1-induced microglial migration relative to the control group (p < 0. 001, n=4). Furthermore, there was no

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62 statistical significance between MCP1 and DMEM-induced migration in the presence of DTG (p = 0.56, Figure 3B). Figure 3. The microglial migratory r esponse to chemoattractant application is suppressed by sigma receptor activation. Microglial migration was assayed us ing a Boyden chamber fitted with a polycarbonate membrane containing 8 m pores. Microglia (500,000 cells) were placed in the upper chamber and control media, 100 M ATP (A) or 10 nM MCP1 (B) were added in the absence and presence of 300 M DTG to the bottom chamber. Microglia were allowed to mi grate for 4 hrs at 37 C, then stained with DAPI and counted. Asterisks indicate a si gnificant difference between ATP (A; p < 0.001, n = 6) and MCP-1 (B ; p < 0.01, n = 4) from DMEM within the Control group (no DTG). Pound symbols indicate si gnificant difference between Control and DTG within the ATP (A; p < 0.001) and MCP-1 (B; p < 0. 01) groups, respectively. In both cases signific ance was determined by two-way ANOVA followed by post-hoc Bonferroni tests.

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63 The microglial inflammatory response is suppressed by sigma receptor activation. To ascertain whether sigma receptor s modulated the pro-inflammatory response of microglia, cytokine, and NO production evoked by LPS were examined after pretreatm ent with DTG. LPS activates microglia and causes robust increases in cytokine producti on (Hoffmann et al. 2003). Microglial cultures were preincubated with va rying concentrations of DTG (50-1000 M) for 30 minutes and the cells were then activated with LPS (1 g/ml). TNF and IL10 levels in the supernatant were meas ured using ELISA and NO levels were quantified with the Greiss reaction. DTG suppressed both cytokine and NO production in a concentration dependent m anner (Figure 4). Fits of the data determined IC50 values of 338.9, 109.6, and 166. 0M for DTG inhibition of TNF IL10, and NO release, respectively (n = 4 for all groups tested).

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64 Figure 4. The microglial inflammato ry response is suppressed by sigma receptor activation. Primary microglial cultures were incubated with LPS (1 g/ml) for 24 hrs and in Levels in the supernatant of TNF and IL10 were measured by ELISA, while NO was measured by the Greiss reaction. Data points represent mean SEM for TNF IL10, and NO levels as a function of DTG concentration (n=4 for all groups). Lines indicate best fits to the data using a Langm uir-Hill equation and yielded IC50 values of 338.9, 109.6, and 166.0M with Hill coefficients of 1.90, 1.94, and 1.4 for TNF IL10, and NO release, respectively.

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65 Transient intracellular calcium signali ng is suppressed by sigma receptor activation. Changes in intracellular calcium are a critical component of the signaling cascade triggering the immune response of microglia. Given that sigma receptors regulate intracellular calcium concentrations ([Ca2+]i) in neurons (Katnik et al. 2006), it seemed prudent to examine the ef fects of sigma receptor activation on changes in [Ca2+]i following microglia stimulation. Focal application of 300 M ATP (10 sec) onto microglia produced intr acellular calcium increases of 140.4 16.2 nM in ~90% of cells tested (Figure 5A). Followi ng pretreatment (10 min) with 100 M DTG, the ATP-elicited increases in [Ca2+]i were reduced by 82 16.4% (n=39), which was statistically si gnificant (p < 0.001, Figure 5C). To confirm that DTG was acting via sigma receptors, cells were preincubated with the irreversible sigma receptor antagonist metaphit. Pretreatment with metaphit (50 M, 1 hr, room temperatur e) abolished the inhibitory effects of DTG on ATPinduced [Ca2+]i increases (Figure 5B), such t hat no statistically significant difference (p = 0.52) exist ed between the ATP-induced [Ca2+]i responses observed in the absence (ATP + Met) and presence of DTG (ATP + DTG + Met) (Figure 5C).

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66 Figure 5. Transient intracellular calcium signaling evoked by ATP was suppressed by sigma receptor activation. Representative traces of [Ca2+]i as a function of time recorded during ATP (300 M) stimulation in the absence (C ontrol) and presence of DTG (100 M), without (A) or with (B) Metaphit (50 M, 1 hr, room temperat ure). C, Mean change in peak [Ca2+]i ( SEM) measured in the above treatment groups. Asterisk denotes significant difference (p < 0.05, n = 25), as measured by one-way ANOVA with post-hoc Dunn’s test using ATP as control.

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67 Basal intracellular calcium increases are suppressed by sigma receptor activation. To determine whether DTG could inhi bit the sustained increase in [Ca2+]i associated with the LPS-induced infla mmatory response (Boddeke et al. 1999; Hoffmann et al. 2003), microglia were tr eated with LPS for 24 hours and then the basal intracellular calcium was measur ed. Treatment with LPS evoked a 46.1 11.0% increase in [Ca2+]i relative to control cells and this increase was statistically significant (p < 0.05, n = 31 Fi gure 6). In contrast, in cells pretreated with 300 M DTG, LPS failed to evoke increases in [Ca2+]i (Figure 6). This inhibition of basal [Ca2+]i by DTG was statistically different from LPS treatment alone (p < 0.05, n = 31). These results are consistent with the hypothesis that sigma receptors inhibit the inflammato ry response elicited by LPS via the inhibition of LPS-induced increases in intracellular calcium.

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68 Figure 6. Basal intracellular calc ium increases are suppressed by sigma receptor activation. Microglia, plated on poly-D-lysine covers lips, were stimulated for 24 hours with LPS (1 g/ml) in the absence (Vehicle) and presence of 300 M DTG. Mean basal calcium levels ( SEM) were meas ured using fura-2 calcium imaging. Values represent measurem ents from three pooled ex periments each having a minimum of 30 cells per group. Asterisks denote statistical difference (p < 0.05, n = 31) between Media and L PS treatment groups withi n the Vehicle group, and pound symbols denote significant differenc e between Vehicle and DTG within the LPS treatment group (p < 0.05, n=31). Statistical difference was determined using a two-way ANOVA with po st-hoc Bonferroni test.

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69 Increases in intracellular calcium are sufficient to induce membrane ruffling in microglia. The calcium ionophore ionomycin was us ed to increase intracellular calcium in microglia via a pathway dist inct from the sigma receptor-sensitive influx pathways activated in respons e to ATP, MCP-1 and LPS. Ionomycin treatment alone altered the morphology of microglia by promoting retraction of filopodia and inducing memb rane ruffling when compared to control (Figure 7A and 7B). Unlike the results obtained w hen ATP was used to stimulate actin rearrangement, DTG was unable to inhibi t ionomycin-induced membrane ruffling (Figure 7C). Quantificat ion of the results obtained with ionomycin and DTG treatment is summarized in Figure 7D Ionomycin evoked a statistically significant increase in the degree of me mbrane ruffling both in the absence and presence of DTG preincubation, and no significant interaction was noted between sigma receptor stimulation and t he response to the ionophore. These data show that increasing [Ca2+]i is sufficient to promote such changes in microglial morphology. Moreover, t he inability of DTG to decrease these ionomycin-evoked changes to the membr ane suggest that sigma receptors are acting at the level of [Ca2+]i and not at downstream tar gets of this molecule.

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70 Figure 7. Increases in intracellular cal cium induce membrane ruffling in microglia. Micrographs of phalloidin stained microg lia treated with vehicle (DMSO, A), 1 M ionomycin for 5 minutes (B), and 1 M ionomycin + 100 M DTG. D, Bar graph of degree of membrane ruffling determined for the indicated conditions. Bars represent means SEM. Asterisks indica te a significant difference between DMEM and Ionomycin treatment groups (p < 0.001). Significance was determined via two-way ANOVA with post-hoc Tukey Test. All insets represent magnified image (2.7x). Scale bar = 2 m.

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71 Microglial migration is a calcium depe ndent process which is not restored by ionomycin treatment. We next investigated whether ionomyci n could overcome the blockade of microglial chemotaxis obs erved following sigma rec eptor activation. The membrane ruffling evoked by ionomycin was not associated with an increase in chemotaxis, and the number of migrating cells observed in response to ionomycin treatment was similar to that obs erved with vehicle alone (Figure 8A). Chemotaxis was observed as before in re sponse to ATP, and it was significantly inhibited by co-administrati on of DTG. However, microg lial migration in response to ATP was disrupted by ionomycin treatment alone (Figure 8A). In contrast to the observed effects on the microglial mo rphological respons e, the addition of ionomycin failed to promote microglial chemotaxis in the presence of ATP and DTG, instead significantly further decreasin g migration relative to that observed in the ATP+DTG group. Microglial migration is independent of calcium release from internal stores but requires calcium influx. To determine the source of increased [Ca2+]i necessary for microglial migration, calcium release from intrac ellular stores and influx from the extracellular media were selectively bl ocked prior to ATP-stimulation. The sarcoplasmic-endoplasmic calcium ATPase inhibitor thapsigargin was used to deplete calcium from the ER by blocki ng reuptake. Pretreatment with 10 M thapsigargin alone, failed to suppress the mi gratory response to ATP (Figure 8B).

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72 In thapsigargin treated samples, 100 M ATP stimulated cell migration averaged 747.0 190 cells per field compared to 683 187 cells per field in control samples (p = 0.67, n = 4) The pan-selective calcium channel blocker La3+ was employed to block calcium channel m ediated influx through the plasma membrane. Lanthanum alone did not promot e a significant change in migration when compared to control (p = 0.4897, n = 5). However, co-application of 50 M La3+ with 100 M ATP significantly suppressed microglial migration from 2069 121 cells per field (ATP alone) to 1636 124 cells per field (ATP + La3+) (Figure 8C, p = 0.022, n = 5). Fi gure 8D summarizes these resu lts, showing that under control conditions ATP induces a 320 67 % increase in cell migration which is reduced to 183 32% in the presence of 50 M La3+. Thus, inhibition of Ca2+ influx to the cell, such as that produced by sigma receptor activation, can prevent microglia chemotaxis.

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73 Figure 8. Microglial migration is i ndependent of calcium release from internal stores but requires La3+-sensitive calcium influx. A, Microglial chemotaxis in response to ATP (100 M) in the absence and presence of DTG (300 M) and DTG with ionomycin ( M). Data were analyzed via two-way ANOVA with post-hoc Tukey te st. Asterisk indicates a statistical difference from respective controls (p <0.001 for all), pound symbol indicates a statistical difference from ATP alone (p <0.001 for all), and dagger indicates statistical significance from ATP+DTG (p <0.05 for all). B, Microglia migration induced by 300 M ATP without and with pretreat ment of the cells with 10 M thapsigargin for 30 min (37 C) (Thapsigargin). Asterisk indicates a statistical difference from control (p <0.05). Ba r represent means SEM, n = 3. C,

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74 Microglia migration evoked by 300 M ATP in the absence (Control) and presence of 50 M La3+. Asterisk indicates a statistical difference from Control (p < 0.001), pound symbol indicates a statisti cal difference from ATP alone (p < 0.01). Bar represent means SEM, n = 3. Statistical significance was determined by two-way ANOVA followed by post-hoc Bonferroni multiple comparison tests. D, Relative ATP induced migration in the absence (Control) and presence of 50 M La3+. Asterisk denotes statistical difference (p < 0.05 by student’s T test). Calcium influx restores nitric oxid e production following sigma receptor activation in LPS stimul ated microglia cells. Increases in basal [Ca2+]i are integral to NO pr oduction in mouse microglia (Hoffmann et al. 2003). Experiments were conducted to determine if ionomycin facilitated Ca2+ influx would overcome the i nhibitory effects of DTG on LPSinduced NO production. Primary microg lia were preincubated (30 min) with vehicle (0.1% DMSO in DMEM), DTG (300 M), ionomycin (1 M), or ionomycin + DTG in the presence and absence of L PS (1 g/ml) for 24 hours. Nitric oxide levels were measured using the intrace llular NO indicator DAF-FM (Figure 9). LPS stimulation alone increased NO levels 138.7 10.87% compared to vehicle (p < 0.001, n = 455). Co-appl ication of DTG with LPS resulted in a statistically significant suppression of NO levels (p < 0.001, n = 461), which were 22.2 6.36% higher than that of the DTG control group. Application of LPS in the presence of ionomycin produced an increase in NO similar to that observed

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75 when LPS was administered alone, such that NO levels were 129.9 10.36% greater than that of the ionomycin contro l group (p < 0.001, n = 446). In contrast to the results observed for ATP-induced mi gration, addition of ionomycin to the LPS + DTG group was able to overcome the inhibitory effects of sigma receptor activation. When ionomycin wa s co-applied with LPS and DTG (LPS+DTG+Iono) intracellular NO levels were increased by 86.1 8.43% from DTG+Iono control (p < 0.001, n = 418). T hese values were significantly greater than those observed for the LPS + DTG gro up (p < 0.001, n = 418), and similar to those elicited by the LPS + I ono group (p = 0.562, n = 446).

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76 Figure 9. Calcium influx restores ni tric oxide production following sigma receptor activation in LPS stimulated microglia. Relative levels of NO produced by micr oglia under control condition (DMEM), in the presence of 300M DTG (DTG), and following application of 1 M ionomycin alone (Iono) and with 300M DTG (Iono + DTG) Microglia were also treated for 24 hrs with LPS (1 g/ml) alone (LPS), LPS and 300 M DTG (LPS+DTG), LPS and 1 M ionomycin, and with a combinat ion of the three compounds (LPS+DTG+IONO). Bars represent mean fl uorescent intensities from DAF-FM loaded cells. Asterisk indicates statisti cal difference from DMEM treated cells, pound symbol denotes statistical significanc e from LPS treated cells, and dagger

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77 indicates significance from LPS+DTG treated cells. In all cases p < 0.001, as determined by three-way ANOVA with post-hoc Tukey test (n 400 cells for all samples). Discussion The salient finding of this study is that sigma receptor activation can suppress multiple aspects of microglial activation, including the morphological, migratory and inflammatory responses to several known microglial activators. The results of this study pr ovide insight into the role of sigma receptors in the regulation of immune system function. Sigma receptor activation can inhibit membrane ruffling and migrati on of microglia, both of which are novel findings to the best of our knowledge. As these pr ocesses are critical to the microglial response to injury, these findings reveal s a novel aspect of the modulation of microglial activation by sigma receptors. Resting microglia exhibit a ramified morphology in vivo characterized by fine processes expressing chemosensitiv e receptors (Glenn et al. 1992). Studies using GFP-labeled microglia in vivo demonstrated that these processes are highly motile and quickly move towards ATP released by injured neurons (Davalos et al. 2005). Upon further activation in vivo these processes are withdrawn and the microglia take on an am oeboid phenotype which is associated with an inflammatory state (Kreut zberg 1996; Lyons et al. 2000). In vitro membrane ruffling decribes the cytoskele tal re-arrangements critical for the

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78 movements of microglial filo podia. Our results demonstr ate that sigma receptor activation blocks the formation of me mbrane ruffles in response to ATP application. Metabotropic P2Y12 recept ors, which evoke increases in [Ca2+]i in microglia (Ohsawa et al. 2007), are critic al to the induction of membrane ruffling by ATP exposure (Honda et al. 2001). ATP also elicits a transient increase in intracellular calcium via the activation of ionotropic P2X4 receptors expressed in microglia. Intracellular calcium is an important co-factor in the process of membrane ruffling, since either depression of intracellular Ca2+ with BAPTA or deletion of the Ca2+-binding domain of Iba1, which is involved in ATP-induced membrane ruffling in microglia (Kanazaw a et al. 2002; Ohsawa et al. 2000), inhibits agonist-elicited ac tin rearrangement in these ce lls (Ohsawa et al., 2000). Elevations in intracellular calciu m have also been implicated in macrophagecolony stimulating factor (M-CSF)-evo ked membrane ruffling in microglia (Ohsawa et al., 2000). Our studi es show that increasing [Ca2+]i in microglia with ionomycin treatment is sufficient to trigger membrane ruffling. Similarly, in rat microglia/astrocyte co-cultures, t he calcium ionophore calcimycine/A23187 evoked a loss of ramifications in microglia (Kalla et al. 2003). Sigma receptor activation suppressed both the ATP mediat ed calcium transient and membrane ruffling which suggests that the calcium in crease is linked to membrane ruffling. Sigma receptor activation was unable to inhibit the ionomycin-induced membrane ruffling, indicating that sigma recept ors are acting upstream of the calcium elevation in this pathway.

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79 A second pathway which is [Ca2+]i-independent is able to induce membrane ruffling in microglia. Previ ous studies have shown that purinergic receptor mediated increases in intracellu lar calcium are necessary for migration but not membrane ruffling in these cells (H onda et al. 2001; Ohsawa et al. 2007). Sigma receptor activation depressed membrane ruffling in the absence of extracellular Ca2+ and in conditions under which the Ca2+-independent membrane ruffling induced by ATP is obs erved (Honda et al. 2001). Thus, sigma receptor activation is able to disrupt membrane ruffling both by preventing [Ca2+]i increases which may stimulate memb rane ruffling and by blocking the Ca2+independent pathway. While both membrane ruffling and mi gration in response to ATP are dependent on P2Y12 receptor activation and intracellular calcium signaling, migration must also incorporate chemoattr actant sensing and di rectionality. In our migration studies we saw robust migr ation in response to ATP and MCP-1, both of which induce migration through a pertussis toxin sensitive Gi/o-protein (Dzenko et al. 2001; Honda et al. 2001; So zzani et al. 1994). Migration induced by both chemoattractants was inhibited by sigma receptor activation, suggesting that sigma receptors inhibit migration eit her by direct interaction with the Gi/oprotein or by interfering with downstream calcium signali ng. Interestingly, unlike membrane ruffling, increasing intracellular calcium levels with ionomycin failed to restore microglial migration in the presence of sigma recept ors. In fact, inclusion of ionomycin effectively suppressed ATP in duced migration. This effect may be due to ionomycin-induced decreases in pur inergic receptor si gnaling, which has

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80 been reported (Hoffmann et al. 2003). Alternatively, the homogeneous elevations in [Ca2+]i produced by ionomycin treatment may saturate and disrupt any directional effects produced by the pur inergic signaling. Similarly, the Ca2+ ionophore, A23187, disrupts microglial migration in response to complement 5a (Nolte et al. 1996). In c ontrast, depletion of intracellular calcium stores with thapsigargin had no effect on migration, whereas the calcium channel inhibitor lanthanum partially suppress ed microglial migration in response to ATP. This latter finding is consistent with ionotropic P2X4 receptor signaling being essential to ATP induced microglial activation, which has been reported (Ohsawa et al. 2007). Given that sigma receptor ac tivation decreased the calcium signaling known to be essential for microglial migrat ion, this is the likely mechanism by which sigma receptor activation suppre sses movement of the cells. Another crucial component of microglial function is the induction of the inflammatory response. Activation of si gma receptors in the peripheral immune system potently suppresses the inflammato ry response to a variety of stimuli (Bourrie et al. 2002). It was proposed that these anti-inflammatory effects were mediated by sigma receptor-induced interl eukin-10 (IL10) production (Zhu et al. 2003). However, studies on sigma re ceptor-mediated immunosuppression in RAW 264.7 macrophages suggested that IL10 is not always involved in these responses (Gannon et al. 2001). This latte r observation is consistent with our finding that IL10 production in response to LPS treatment was decreased with similar kinetics to reductions of TNFand NO production. This observation

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81 implies that sigma receptor activation affects signaling upstream of protein synthesis-dependent processes in t he inflammatory response. Sustained increases in basal intracellula r calcium levels are integral to the induction of the inflammatory response (Hoffmann et al. 2003). Sigma receptor activation reduces the capacity of microg lia to generate this sustained increase in calcium in response to LPS. This is associated with a decreased release of cytokines and NO. Reintroduction of calcium with ionomycin overcame the sigma receptor activation induc ed blockade of NO production. Our results showing that activation of sigma receptors suppresses ATPinduced [Ca2+]i in microglia suggest that si gma receptors are inhibiting P2X receptors in these cells, since these channel s account for most of the increase in [Ca2+]i observed in our experiments (data not shown). Previous studies, including works from our laboratory, hav e shown that sigma receptors couple to both voltage-gated and ligand-ga ted ion channels (Aydar et al. 2002; Herrera 2007; Zhang and Cuevas 2002; Zhang and Cuevas 2005). Thus, this observation is consistent with the hypot hesis that sigma receptors couple to a diverse group of ion channels. While the regulation of these plasma membrane ion channels will affect [Ca2+]i, sigma receptors also affect other proteins which influence [Ca2+]i, including the IP3 receptor of the endoplasmic reticulum and the BiP chaperone found in the mitochondri on-associated ER membrane (Hayashi and Su 2001; Hayashi and Su 2007). Howeve r, the fact that sigma receptors affect membrane ruffling in the absence of Ca2+ signaling indicates that the influence of sigma receptors on microglial function is no t limited to the regulation

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82 of Ca2+ signaling alone. Thus, the mec hanisms by which sigma receptors regulate the activity of microglia remain to be fully elucidated. Activated microglia are associ ated with neurodegenerative disease progression (Streit et al. 2004), and the pres ence of activated microglia is linked to increased neuronal damage. In contra st, ablation of mi croglia is also associated with increased damage (Lalancette-Hebert et al. 2007), which shows that microglia play a complex part in t he etiology of neurodegenerative disease. Our findings support and expand upon the theory that sigma receptors have potent immunoregulatory properties (Bourri e et al. 1995; Bourrie et al. 2002). Prior to our work, the relationship bet ween sigma receptors and microglia had only been studied in an in vitro HIV-1 infe ction model (Gekker et al. 2006). Our studies shed light on the mechanism by which sigma receptors modulate the function of microglia in re sponse to brain injury.

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83 References Ajmo CT, Jr., Vernon DO, Collier L, Pennypacker KR, Cuevas J. 2006. Sigma receptor activation reduces infarct si ze at 24 hours after permanent middle cerebral artery occlusion in ra ts. Curr Neurovasc Res 3(2):89-98. Aydar E, Palmer CP, Klyachko VA, Ja ckson MB. 2002. The sigma receptor as a ligand-regulated au xiliary potassium channel subunit. Neuron 34(3):399410. Boddeke EW, Meigel I, Frentzel S, Gourmala NG, Harrison JK, Bu ttini M, Spleiss O, Gebicke-Harter P. 1999. Cultured rat microglia express functional betachemokine receptors. J Neuroimmunol 98(2):176-84. Bourrie B, Bouaboula M, Benoit JM, Derocq JM, Esclangon M, Le Fur G, Casellas P. 1995. Enhancement of endotoxin-induced interleukin-10 production by SR 31747A, a sigma li gand. Eur J Immunol 25(10):2882-7. Bourrie B, Bribes E, De Nys N, Esclangon M, Garcia L, Galiegu e S, Lair P, Paul R, Thomas C, Vernieres JC and others. 2002. SSR125329A, a high affinity sigma receptor ligand with pot ent anti-inflammatory properties. Eur J Pharmacol 456(1-3):123-31. Carr DJ, De Costa BR, Radesca L, Blal ock JE. 1991. Functional assessment and partial characterization of [3H](+)pentazocine binding site s on cells of the immune system. J Neuroi mmunol 35(1-3):153-66.

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84 Davalos D, Grutzendler J, Yang G, Kim JV Zuo Y, Jung S, Littman DR, Dustin ML, Gan WB. 2005. ATP mediates rapid microglial response to local brain injury in vivo. Nat Neurosci 8(6):752-8. Dheen ST, Kaur C, Ling EA. 2007. Microglial activation and its implications in the brain diseases. Curr M ed Chem 14(11):1189-97. Dzenko KA, Andjelkovic AV, Kuziel WA Pachter JS. 2001. The chemokine receptor CCR2 mediates the bindi ng and internalization of monocyte chemoattractant protein-1 along br ain microvessels. J Neurosci 21(23):9214-23. Gannon CJ, Malone DL, Napolitano LM. 2001. R eduction of IL-10 and nitric oxide synthesis by SR31747A (sigma ligand) in RAW murine macrophages. Surg Infect (Larchmt) 2( 4):267-72; discussion 273. Gekker G, Hu S, Sheng WS, Rock RB, Lokensgard JR, Peterson PK. 2006. Cocaine-induced HIV-1 expression in mi croglia involves sigma-1 receptors and transforming growth factor-beta1. Int Immunopharmacol 6(6):1029-33. Glenn JA, Ward SA, Stone CR, Booth PL Thomas WE. 1992. Characterisation of ramified microglial cells: detailed mo rphology, morphological plasticity and proliferative capability. J Anat 180 ( Pt 1):109-18. Gottschall PE, Yu X, Bing B. 1995. Incr eased production of gelatinase B (matrix metalloproteinase-9) and interleukin-6 by activated rat microglia in culture. J Neurosci Res 42(3):335-42. Harukuni I, Bhardwaj A, Traystman RJ, Crain B, London ED, Kirsch JR. 1998. Neuroprotection from focal ischem ia by 4-phenyl-1-(4-phenylbutyl)

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85 piperidine (PPBP) is dependent on treatment durat ion in rats. Anesth Analg 87(6):1299-305. Hayashi T, Su TP. 2001. Regulating ankyrin dynam ics: Roles of sigma-1 receptors. Proc Natl Ac ad Sci U S A 98(2):491-6. Hayashi T, Su TP. 2007. Sigma-1 receptor chaperones at the ER-mitochondrion interface regulate Ca(2+) signaling and cell survival. Cell 131(3):596-610. Herrera Y, Katnik, C., Hall, A.A., Pennypacker, K.R., Cuevas, J. 2007. Modulation of acid-sensing ion channels by sigma receptors. Society for Neuroscience Abstracts: 551.7. Hoffmann A, Kann O, Ohlemeyer C, Hanisch UK, Kettenmann H. 2003. Elevation of basal intracellular calcium as a cent ral element in the activation of brain macrophages (microglia): suppression of receptor-evoked calcium signaling and control of release f unction. J Neurosci 23(11):4410-9. Honda S, Sasaki Y, Ohsawa K, Imai Y, Nakamura Y, Inoue K, Kohsaka S. 2001. Extracellular ATP or ADP induce chemotaxis of cultured microglia through Gi/o-coupled P2Y receptors. J Neurosci 21(6):1975-82. Kalla R, Bohatschek M, Kloss CU, Krol J, Von Maltzan X, Raivich G. 2003. Loss of microglial ramification in microglia -astrocyte cocultures: involvement of adenylate cyclase, calcium, phosphatase, and Gi-protein systems. Glia 41(1):50-63. Kanazawa H, Ohsawa K, Sasaki Y, Kohsaka S, Imai Y. 2002. Macrophage/microglia-specific prot ein Iba1 enhances membrane ruffling

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86 and Rac activation via phospholipase C-gamma -dependent pathway. J Biol Chem 277(22):20026-32. Katnik C, Guerrero WR, Pennypacker KR, Herrera Y, Cuevas J. 2006. Sigma-1 receptor activation prevents intracellu lar calcium dysregulation in cortical neurons during in vitro ischemia. J Pharmacol Exp Ther 319(3):1355-65. Kreutzberg GW. 1996. Microglia: a sensor for pathological events in the CNS. Trends Neurosci 19(8):312-8. Lalancette-Hebert M, Gowing G, Simard A, Weng YC, Kriz J. 2007. Selective ablation of prolifer ating microglial cells exacerbates ischemic injury in the brain. J Neurosci 27(10):2596-605. Lyons SA, Pastor A, Ohlemeyer C, Kann O, Wiegand F, Prass K, Knapp F, Kettenmann H, Dirnagl U. 2000. Disti nct physiologic properties of microglia and blood-borne cells in ra t brain slices after permanent middle cerebral artery occlusion. J Ce reb Blood Flow Metab 20(11):1537-49. McCord AM, Burgess AW, Whaley MJ, Anderson BE. 2005. Interaction of Bartonella henselae with endot helial cells promotes monocyte/macrophage chem oattractant protein 1 gene expression and protein production and tr iggers monocyte migrat ion. Infect Immun 73(9):5735-42. Nolte C, Moller T, Walter T, Kettenm ann H. 1996. Complement 5a controls motility of murine microglial cells in vi tro via activation of an inhibitory Gprotein and the rearrangem ent of the actin cytoskeleton. Neuroscience 73(4):1091-107.

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87 Ogilvie P, Thelen S, Moepps B, Gierschik P, da Silva Campos AC, Baggiolini M, Thelen M. 2004. Unusual chemokine receptor antagonism involving a mitogen-activated protein kinase pathway. J Immunol 172(11):6715-22. Ohsawa K, Imai Y, Kanazawa H, Sasaki Y, Kohsaka S. 2000. Involvement of Iba1 in membrane ruffling and phagocyt osis of macrophages/microglia. J Cell Sci 113 ( Pt 17):3073-84. Ohsawa K, Irino Y, Nakamura Y, Akazawa C, Inoue K, Kohsaka S. 2007. Involvement of P2X4 and P2Y12 re ceptors in ATP-induced microglial chemotaxis. Glia 55(6):604-16. Peterson PK, Hu S, Salak-Johnson J, Mo litor TW, Chao CC. 1997. Differential production of and migratory response to beta chemokines by human microglia and astrocytes. J Infect Dis 175(2):478-81. Quirion R, Bowen WD, Itzhak Y, Junien JL, Musacchio JM, Rothman RB, Su TP, Tam SW, Taylor DP. 1992. A proposal for the classification of sigma binding sites. Trends P harmacol Sci 13(3):85-6. Rogove AD, Lu W, Tsirka SE. 2002. Microg lial activation and recruitment, but not proliferation, suffice to mediat e neurodegeneration. Cell Death Differ 9(8):801-6. Seth P, Fei YJ, Li HW, Huang W, Lei bach FH, Ganapathy V. 1998. Cloning and functional characterization of a si gma receptor from rat brain. J Neurochem 70(3):922-31. Sozzani S, Zhou D, Locati M, Rieppi M, Proost P, Magazin M, Vita N, van Damme J, Mantovani A. 1994. Rec eptors and transduction pathways for

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88 monocyte chemotactic protein-2 and monocyte chemotactic protein-3. Similarities and differences with MCP-1. J Immunol 152(7):3615-22. Streit WJ, Mrak RE, Griffin WS. 2004. Microglia and neuroinflammation: a pathological perspective. J Neuroinflammation 1(1):14. Su TP. 1991. Sigma receptors. Puta tive links between nervous, endocrine and immune systems. Eur J Bi ochem 200(3):633-42. Su TP, Shukla K, Gund T. 1990. Steroid binding at sigma receptors: CNS and immunological implications. Ci ba Found Symp 153:107-13; discussion 113-6. Zhang H, Cuevas J. 2002. Sigma receptors inhibit high-voltage-activated calcium channels in rat sympathetic and para sympathetic neurons. J Neurophysiol 87(6):2867-79. Zhang H, Cuevas J. 2005. sigma Receptor activation blocks potassium channels and depresses neuroexcit ability in rat intracardiac neurons. J Pharmacol Exp Ther 313(3):1387-96. Zhu LX, Sharma S, Gardner B, Escuadro B, Atianzar K, Tashkin DP, Dubinett SM. 2003. IL-10 mediates sigma 1 receptor-dependent suppression of antitumor immunity. J Immunol 170(7):3585-91. Zwain IH, Yen SS. 1999. Dehydroepiandr osterone: biosynthesis and metabolism in the brain. Endocrinology 140(2):880-7.

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89 Chapter 3 Delayed treatments for Stroke Differe ntially affect Neuronal Death in Organotypic Slice Cultures Subjected to Oxygen Glucose Deprivation A.A.Hall, C.C. Leonardo, L. A.Collier, D.D. Rowe, A.E.Willing, and K.R.Pennypacker Abstract Stroke induced brain injury is the third leading cause of death and disability in the United States. A major limitat ion of current stroke therapies is the need to treat candidate patients within three hours of stroke onset. Human umbilical cord blood cell (HUCBC) and the sigma recept or agonist 1,3, di-o-tolylguanidine (DTG) administration both caused signific ant reductions in brain damage in the rat middle cerebral artery occlusion model of stroke when administered at delayed timepoints. In vivo these treatments suppress the infiltration of peripheral lymphocytes into the brain in addition to decreasing neurodegeneration. It appears that neuroprotection afforded by both treatments

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90 is a product of their actions on the peri pheral immune system as well as a direct effect on the ischemic brain tissue. An ex vivo organotypic slice culture (OTC) model to was utilized to characterize the efficacy of these treatments in mitigating neurodegeneration in ischemic brain tissue in the abs ence of the peripheral immune system. Slice cultures subject ed to oxygen glucose deprivation (OGD) had significantly elevated levels of degenerating neurons and mi croglial nitric oxide production when compared to t heir normoxia counterparts. HUCBC but not DTG treatment reduced the num ber of degenerating neurons and the production of microglia derived nitric oxide in slice cultures subjected to OGD back to levels seen in the normoxia c ontrols. These data show that HUCBC treatment can mediate direct neuroprotec tion and suppress innate inflammation in ischemic brain tissue in the abs ence of the peripheral immune system, whereas DTG requires peripheral effects to mediate neuropr otection. These experiments yield insight into the mechanisms by which neuroprotective treatments function at delayed timepoints following stroke. Introduction Currently the only FDA approved treat ment for ischemic stroke is recombinant tissue plasminogen activato r (Alteplase) (Marler and Goldstein 2003). This treatment works by dissolvi ng the blood clot occluding the offending blood vessel. Unfortunately its use is re stricted to a short, 36 hour, time window. Beyond this time, apoptotic and inflamma tory processes greatly diminish the therapeutic benefits of current treatments. Furthermore this treatment increases

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91 the risk for bleeding and only provides a modest (13%) benefit in mortality and morbidity to patients who are candidates fo r this treatment (Marler and Goldstein 2003). Attempts to develop therapies to increase neurosurvival have not been successful clinically thus far. The vast ma jority of trials to date have focused on reducing the buildup of intracellular calc ium, neuronal firing, and free radicals. These therapies were developed in order to combat a phenomenon called excitotoxicity which is thought to damage neurons (Dirnagl et al. 1999). Neuronal damage due to excitotoxici ty is caused by the release of glutamate which causes overactiva tion of ionotropic AMPA and NMDA glutaminergic receptors on ischemic neurons. The increased AMPA and NMDA receptor activation causes sodium and ca lcium to enter the cell where it can depolarize neurons and incr ease excitability and firi ng. Under ischemic conditions this depolarization results in further metabolic demand and can drop ATP levels below those needed to maintain ionic homeostasis. The resulting anoxic depolarization causes a further calcium influx which activates proapoptotic signaling cascades, causing free radica l buildup, and neuronal death. While these therapies have been successful in vitro their efficacy in animal studies was restricted to early timepoints (<3 hrs). Furthermore reducing excitability by blocking NMDA receptors or calcium c hannels was associated with intolerable side effects such as hallucinations and di zziness (NMDA) (Davis et al. 2000) and reductions in blood pressure (calcium channel blockers) (Horn et al. 2001).

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92 Two experimental treatments which have shown a great deal of promise in experimental rat models of focal ischem ia are human umbilical cord blood cells (HUCBC) (Vendrame et al. 2004) and 1,3-di-o-tolylguanidine (DTG) (Ajmo Jr et al. 2006). The main advantage of these treat ments over previous therapies lies in the fact that they can be given at clinically relevant timepoints (24 and 48 hours post stroke for DTG and HUCBC respec tively). In the permanent middle cerebral artery occlusion (MCAO) model of stroke, injection of HUCBC (106) 48 hours (Newcomb et al. 2006) or administra tion of DTG (15mg/kg) 24 hours (Ajmo Jr et al. 2006) after stroke reduces infa rct volume by up to 80% when compared to vehicle treated control. Originally both treatments were thought to decrease infarct volumes by either reducing neuronal ion imbalances (DTG) (Katnik et al. 2006) or replacing neurons (HUCBC). Fu rther studies have shown each of these treatments had profound effe cts on components of t he immune system. HUCBC have been shown to migrate to injured brain and the spleen (Vendrame et al. 2004). Subsequent studi es showed that infiltration of the HUCBC into the brain was not required for neuroprotection (Borlongan et al. 2004). Sigma receptors ar e also found in the spleen at high densities and are expressed on lymphocytes and macr ophages (Su 1991). The spleen is important in ischemic pathol ogy as splenectomy prior to stroke shows significant reductions in neurodegeneration (Ajmo et al 2008). T-lymphocytes in particular are known to be present in the spleen in large numbers and to contribute to stroke induced neurodegeneratio n (Offner et al. 2006; Yilmaz et al. 2006). Peripheral immune cells such as these mi grate to the infarct and interact with

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93 antigen presenting cells there to promote a proinflammatory immune response. One of the principa l immune cell types present in the infarct and known to serve as antigen presenting cells is mi croglia (Morioka et al. 1993). Microglia, the innate immune cells of the brain, are normally kept in a quiescent ramified state (Streit 2002). Upon stimulation by dying neurons, microglia become activated, assume an am oeboid phenotype, mi grate to the site of injury, and release inflammatory mediator s (Gibson et al. 2005). Nitric oxide is a particular noxious mediator generated by inducible nitric oxide synthase which is upregulated in activated microglia whic h can interfere with neuronal respiration and induce apoptosis. HUCBC and DTG have been shown to reduce microglial activation in vivo (Ajmo Jr et al. 2006; Newc omb et al. 2006). DTG also suppresses microglial activation by bl ocking calcium signaling and reduces lipopolysaccharide (LPS) induced nitr ic oxide by this mechanism in vitro (Hall et al. 2008). The purpose of the present study was to determine whether the efficacy of HUCBC and DTG in vivo at delayed timepoints is solely due to the impact on the peripheral immune system or also involves direct effects on the ischemic brain tissue To study the effects of these therapies on ischemic tissue in the absence of the peripheral immune system; organotypic hippocampal slice cultures were subjected to oxygen glucose depriv ation (OGD) in order to induce neurodegeneration. Fluoro-J ade staining was used as a marker for degenerating neurons, while nitric oxide production wa s used to identify microglia which had differentiated into a neur odestructive phenotype.

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94 Materials and Methods Animal Care All animal procedures were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Anim als with a protocol approved by the Institutional Animal Care and Use Committee at the University of South Florida. Timed pregnant Sprague-Dawley dams we re purchased from Harlan Labs (Indianapolis, IN), maintained on a 12 hr lig ht/dark cycle (7 am – 7 pm) and given access to food and water ad libitum Neonatal rats birthed from time-pregnant dams were used for all experiments. Organotypic Slice Culture Organotypic slice cultures (OTC) we re prepared using a method described (Stoppini et al. 1991), with slight adaptations. Postnatal day 8-10 rat pups were sacrificed by decapitation. Brains were removed and intact hippocampi were isolated in ice cold isotonic buffer (136.89 mM NaCl, 5.37 mM KCl, 169 nM Na2HPO4, 22.04 nM KH2PO4, 27.52 nM glucose, 59.01 mM sucrose). 400 m hippocampal slices were prepared usi ng a McIlwain Tissue Chopper (Mickle Laboratory Engineering Co. Ltd. Gomshall, Surrey, England). Only slices that appeared thin and translucent were selected for culture. Slices were then incubated for 90 min at 4 C. Cultures were maintain ed on Millicell CM (Millipore Corp., Billerica, MA) inserts and placed in 6-well plates containing Neurobasal media supplemented with B27 and 5 mM L-glut amine. Slices were cultured for 14 days in room air supplemented with 5%CO2 maintained at 37 C, receiving partial media changes every 3-4 days prior to experimentation.

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95 Oxygen Glucose Deprivation Organotypic slices were subjected to 48 hrs of normoxia or oxygen glucose deprivation (OGD). Slices were assigned to 1 of 2 exposures (normoxia or OGD) and 1 of 2 treatments (media, or HUCBC). Immediately prior to exposure, inserts were transferred into new 6-well plates containing media, or 106 HUCBC (Sigma Aldrich, St. Louis, MO ). Media consisted of Dulbecco’s Modified Eagles Medium (DMEM; Medi atech, Herndon, VA) for normoxia or DMEM without glucose (Invitrogen, Carl sbad, CA) for OGD. HUCBC (AllCells) were thawed and resuspended in DMEM co ntaining DNase (1ug/ml) to prevent aggregation. HUCBC were washed twice and resuspended at a concentration of 108 cells/ml. HUCBC (106) were added to each well receiving treatment. Cultures were maintained in a standar d tissue culture incubator during the normoxia exposure and a hypox ic chamber flushed with 1%O2, 5%CO2, and balanced N2 (CBS Scientific Co. Inc., Del Mar, CA) held at 37 C during the OGD exposure. Fluoro-Jade Staining Organotypic slice cultures were stained with Fluoro-Jade (Histochem, Jefferson, AR), which labels degenerat ing neurons. This method was adapted from that originally dev eloped (Schmued et al. 1997) and has been described by Duckworth et al. (2005). Tissue was we t mounted and dried to glass slides. Slices were rehydrated by then exposur e to absolute ethanol for 3 min followed by 70% ethanol and deionized water for 1 mi n each. Slices were oxidized using a 0.06% KMnO4 solution for 15 min followed by three rinses of double distilled

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96 water for 1 minute each. Slices were st ained in a 0.001% solution of Fluoro-Jade in 0.1% acetic acid for 30 min. Slides were again rinsed, allowed to dry at 45oC for 20 min, cleared with xylene and co ver slipped with DPX mounting medium (Electron Microscopy Sciences, Ft. Washington, PA). Nitric oxide imaging Nitric oxide levels were measured usi ng the NO sensitive dye, 4-Amino-5methylamino-2',7'-difluorofluorescein ( DAF-FM). Organotypic slice cultures on Millicell inserts were incubated for 1 hour at 37oC in DMEM (Mediatech) containing 5 M DAF-FM (Invitrogen) and 0.1 % DM SO. The slices were washed in PSS prior to NO measurements. DA F-FM loaded slices were imaged using a DG-4 high speed wavelength switcher (S utter Instruments Co., Novato, CA) which applied excitation light at 488 nm. Emitted fluorescence light was collected through the microscope objective (Zeiss; 2,5X Plan-Neofluar) and passed through an emission filter (510 nm) onto a Cooke SensiCam CCD camera (Cooke Corporation, Auburn Hills, MI). Images were recorded with Slidebook 3.0 software (Intelligent Imaging Innovations, D enver, CO). Nitric oxide levels were calculated using the Slidebook 3 software. Fluorescent intensity was expressed as arbitrary units. Immunohistochemistry Slice cultures were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) at 4oC overnight. Slice cultures we re removed from the MilliCell inserts; wet mounted, and allowed to dry at 45oC for 1hr. The slides were rinsed with PBS (pH 7.2), and placed in perm eabilization buffer containing 10% goat

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97 serum, 3% 1M lysine, and 0.3% Tr iton X-100 in PBS for 1 hr at room temperature. Next, sections were incubated overnight at 4 C in a PBS solution with 2% goat serum and 0.3% Triton X100 and primary antibody in a humidified chamber, protected from light when necessa ry. Slides were washed with PBS (3 x 5 min) and incubated with a secondary an tibody solution (PBS, 2% goat serum, 0.3% Triton X-100) in a hum idified chamber pr otected from light. The sections incubated with OX-42 mouse anti-rat (1:3 ,000, Serotec) were incubated with Alexa-Fluor 594 goat anti-mouse secondary (1 :300, Molecular Probes) After final washing the slides were cover slipped with Vectashiel d hard set mounting media with DAPI (Vector Laborat ories, Burlingame, CA). Results Oxygen glucose deprivation signifi cantly increases neurodegeneration and nitric oxide production in OTC. In order to characterize the respons e of the OTC to OGD, slices were subjected to varying OGD durations. The timepoints tested were 0, 12, 24, 36, and 48 hours following induction of OGD. OTC were assessed for neurodegeneration and nitric ox ide production by staining with Fluoro-Jade and DAF-FM, respectively. OGD induced si gnificant increases in Fluoro-Jade staining only in the 48 hour timepoint (p <0.05, n=4) as measured by one-way ANOVA with post-hoc Dunnett’s test using 0 hour as a control (Figure 1A). Significant increases in DAF-FM staini ng were seen in the 12, 24, and 48 hour

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98 timepoints (p<0.01, n=6) (Figure 1B). As the 48 hour timepoint had significant increases in Fluoro-Jade and DAF-FM staini ng it was used for the remainder of this study. Figure 1. Oxygen glucose de privation significantly increases neurodegeneration and nitric oxide production in OTC. Organotypic slice cultures were subjected to varying durations of oxygen glucose deprivation. Neurodegeneration was assess ed using Fluoro-Jade staining while nitric oxide production was determined wit h DAF-FM. Graphs represent mean stained area values +\SEM for Fl uoro-Jade (A) and mean stained intensity values +\SEM for DAF-FM staining (B). Asterisk denotes significance (p<0.05) and (p<0.01) for (A) and (B) respective ly as determined using one-way ANOVA with post-hoc Dunnet’s test using 0hr as control. Scale bars represent 500 m

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99 HUCBC but not DTG signifi cantly reduces Fluoro-Jade staining in OTC following OGD. Fluoro-Jade staining was used to ident ify neuronal damage in each of the treatment groups. There was extensive Fl uoro-Jade staining in all regions of the slice cultures in the OGD group (Fi gure 2D) when compared to the normoxia group (Figure 2A). DTG treatment (100 M) prior to OGD i nduction (Figure 2E) failed to reduce Fluoro-Jade staining wh en compared to OGD alone (Figure 2D). Administration of 106 HUCBC prior to induction of OGD reduced Fluoro-Jade staining (Figure 2F). Normoxic samp les treated with HUCBC (Figure 2C) and DTG (Figure 2B) showed similar levels of Fluoro-Jade staining to the normoxic controls (Figure 2A). To determine w hether treatments ca used a significant decrease in neuronal death, levels of Fl uoro-Jade staining were quantified. Mean levels of Fluoro-Jade staining were significantly increased (p=<0.001, n=9; Figure 2G) (p=<0.0009, n=10; Figure 2F ) in the OGD treated sections when compared to normoxic controls. DTG treat ment did not significantly decrease Fluoro-Jade staining when compared to OGD treatment alone (p=0.5119, n=9) (Figure 2G). HUCBC treatm ent in OGD groups decreased Fluoro-Jade staining significantly from the OGD control (p<0 .05, n=6) (Figure 2G). Statistical significance was determined by two-way ANOVA with post-hoc Bonferroni test.

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100 Figure 2. HUCBC but not DTG significantly reduces Fluoro-Jade staining in OTC following OGD. Photomicrographs depicting OTC under normo xic conditions treated with Vehicle (A), 100 M DTG (B), and 106 HUCBC (C) or OGD conditions treated with Vehicle (D), 100 M DTG (E), and 106 HUCBC (F). (G) Mean Fluoro-Jade positive areas in DTG treated OTC showed no significant differences from vehicle controls in either normoxia or OGD conditions. (F ) Mean Fluoro-Jade positive areas in HUCBC treated OTC showed significantly r eductions in staining when compared to vehicle controls in OGD but not normoxic conditions. Asterisks denote significance (p<0.05) as determined by two-way ANOVA with post-hoc Bonferroni’s test. Scale bars represent 500 m

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101 DTG treatment is ineffective in reduc ing Fluoro-Jade staining at multiple concentrations. T here is likely a discrepancy in the concentrations of DTG between the in vitro OTC and the in vivo MCAO models. Therefor e, a concentration response study was conducted to determine a conc entration at which Fluoro-Jade staining would be reduced following OGD (Figure 3). The concentrations of DTG used were 0, 30, 100, and 300 M. This concentration range has been used to reduce neuronal death and microglial activation in vitro None of these concentrations reduced Fluoro-Jade staining following OGD. The 300 M dose significantly increased Fluoro-Jade staining when co mpared to OGD alone (139.22+/-14.34%, p<0.001, n=6). As only HUCBC show ed efficacy in reducing Fluoro-Jade staining, only the effects of this treatment were ex plored with respect to NO production.

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102 Figure 3. DTG treatment is ineffecti ve in reducing Fluoro-Jade staining at multiple concentrations. Relative levels of Fluoro-Jade stained area in slices incubated in 0, 30, 100, and 300 M DTG prior to the induction of normoxi a or OGD. No concentrations tested decreased Fluoro-Jade st aining; however, the 300 M DTG group subjected to OGD had significantly increased Fluoro-Ja de stained area compared to OGD alone. Asterisk denotes significanc e p<0.001 as determined using one-way ANOVA with post-hoc Dunnet’s test using 0 M DTG subjected to OGD as control. HUCBC treatment reduces DAF-FM st aining in OTC following OGD. The intracellular dye DAF-FM was used to determine the extent of NO production in each of the treatment groups. There was robust NO producti on in all regions

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103 of the slice cultures in the OGD group (Figure 4C) when compared to the normoxia group (Figure 4A). Administration of 106 HUCBC prior to induction of OGD reduced NO production (Figure 4D ). Normoxic samples treated with HUCBC (Figure 4B) showed similar levels of NO production staining to the normoxia controls. To determine whether HUCBC treatment caused a significant decrease in NO production similar to t hat seen with the Fluoro-Jade staining, levels of DAF-FM staining were quantified (Figure 4E). Mean levels of DAF-FM staining were increased 172.01+/-75.21% in the OGD treated sections when compared to normoxic controls. This in crease was significant (p<0.05, n=3). HUCBC treatment in OGD groups decr eased DAF-FM staining 43.3+/-15.86%. This decrease was significant from the OGD control (p<0.05, n=3). Significance was determined by two-way ANOVA wit h post-hoc Bonferroni’s test.

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104 Figure 4. HUCBC treatme nt reduces DAF-FM stai ning in OTC following OGD. Photomicrographs depicting OTC under normo xic conditions treated with vehicle (A), and 106 HUCBC (B) or OGD conditions treated with vehicle (C), and 106 HUCBC (D). (E) Mean DAF-FM in tensities in HUCBC treated OTC showed significantly reductions in staining when compared to vehicle co ntrols in OGD but

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105 not normoxic conditions. Asterisk denotes si gnificance (p<0.05) as determined by two-way ANOVA with post-hoc Bonferr oni’s test. Scale bars represent 500 m. HUCBC reduces the number of nitric oxid e producing microglia in ischemic OTC. Microglia and neurons can produce NO in response to ischemia. To determine whether the observed NO production was du e to activated microglia, DAF-FM stained slices were also stained for t he microglial marker CD11b. In the normoxia slices NO production was observed in cells which did not exhibit CD11b immunoreactivity (Figure 5A). In OGD treated slices NO production was much more prominent and overlapped with CD11b staining (Figure 5C). HUCBC treatment in both normoxia (Figure 5B) and OGD treated (Figure 5D) slices led to similar patterns of staining which wa s indistinguishable from the normoxia controls. Pearson’s correlation coefficient values from multiple images were pooled and averaged to determine whet her the signal overlap between the CD11b and DAF-FM stains were signific antly different between treatment groups (Figure 5E). The Pearson’s correlation coe fficient in the slices subjected to OGD was significantly increas ed 140.21+/-46.99% when comp ared to normoxic control (p<0.0012, n=6). HUCBC treat ment of OGD slices si gnificantly reduced the Pearson’s correlation coefficient (p<0.01, n= 7) such that it was not different from normoxia controls (0.3819, n=5). Si gnificance was determined by two-way ANOVA followed by Bonferroni post-hoc test.

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106 Figure 5. HUCBC reduces the number of nitric oxide producing microglia in ischemic OTC. Confocal micrographs depicting OTC doubl e stained with DAF-FM (NO)(green) and CD11b (microglia)(red) under normoxic c onditions treated with vehicle (A), and 106 HUCBC (B) or OGD conditions treated with vehicle (C), and 106 HUCBC (D). (E) Mean Pearson’s correlation c oefficients demonstrated a significant increase in staining overlap between t he DAF-FM in OTC subjected to OGD (p<0.0012). HUCBC treatment significantly reduced this overlap (p<0.01). Asterisk denotes significance (p<0.05) as determined by two-way ANOVA with post-hoc Bonferroni’s test Scale bars represent 50 m

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107 Discussion HUCBC and DTG have shown great promise in reducing ischemic neurodegeneration at delayed timepoints fo llowing MCAO. The mechanisms by which these treatments are neur oprotective are unclear. In vivo labeling studies have shown that both HUCBC and DTG bind to the br ain and spleen (Su et al. 1988; Vendrame et al. 2004). The spl een is a lymphoid organ which contains peripheral lymphocytes which contribute detrimentally to the progression of ischemic injury (Ajmo et al. 2008). There are both humoral and synaptic crosstalk between the brain and the spl een via the HPA axis and the splenic nerve, respectively (Elenkov et al 2000). To determine whether DTG and HUCBC have direct effects on the loca l microglial mediated inflammation and neurosurvival in brain tissue in the absence of peripheral immune cells; an organotypic hippocampal slice culture model of ischemia was utilized. Organotypic hippocampal slice culture is a superior model for the study of ischemic brain injury than dissociated neuronal or microglial cell cultures owing to the fact that functional connections betw een different cell types are maintained (Noraberg et al. 2005). In order to further characterize th is model with respect to neurodegeneration, slice cultures were subj ected to OGD at di fferent timepoints and stained with Fluoro-Jade. In this model system neurodegeneration increased in a time dependent manner following the induction of OGD with significant increases observ ed at the 48 hr timepoint.

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108 The multiple inflammatory stimuli released by ischemic neurons in the slice cultures activate microglia by a mechanism which mimics the in vivo ischemic microenvironment more closely than traditi onal activators such as lipopolysaccharide. Microglial activation is a hallmark of the neural response to injury (Streit et al. 2004). Early stages of microglial activation, such as phagocytosis of cell debris (Streit et al. 2005) and the release of growth factors such as BDNF (Coull et al. 2005), can be neuroprotective. Further activation of microglia can lead to the production of to xic pro-inflammatory cytokines (Dirnagl et al. 1999; Rothwell 1996) and reactive oxygen species including nitric oxide (Gibson et al. 2005). Nitric oxide can exacerbate the effects of ischemia on neurons by competing for oxygen binding on cytochrome oxi dase (Mander et al. 2005). Subsets of microglia, which produce hi gh levels of nitric oxide, are likely neurotoxic and deleterious in the ischemic microenvironment. In this study the HUCBC treatment was neuroprotective but the DTG treatment was not. This is intriguing as sigma receptor agonists reduce ischemia and acidosis induced death in dissociated neuronal cultures by reducing calcium influx into these neurons (H errera et al. 2008; Katnik et al. 2006). Furthermore sigma agonists suppress spreading depres sion and ionic imbalances in acute hippocampal slice preparations (Anders on and Andrew 2002; Anderson et al. 2005). As spreading depression and ca lcium dysregulation are central components of excitotoxic cell death, blo cking these processes with DTG should have resulted in reduced Fluoro-Jade staining in slices subjected to OGD. This

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109 demonstrates that in this model, excito toxic cell death and anoxic depolarization are not the primary mec hanisms by which neurons die in this model. Sigma receptors also have potent antiinflammatory properties which can reduce inflammation induced neuronal death. In the organotypic slice culture microglia are the only immune cell ty pe present. Sigma receptors reduce microglial activation in vitro by suppressing intracellular calcium signaling (Hall et al. 2008). By blocking intracellular calc ium signals, many aspects of microglial activation such as membrane ruffling, migration, and cytokine production are impaired (Hall et al. 2008). The 300 M DTG concentration, which completely suppressed microglial activation (Hall et al 2008), resulted in the highest levels of Fluoro-Jade staining encountered, sugges ting that some aspects of microglial activation are beneficial. This finding is in agreement with other studies which have showed that ablation of microglia is neurodestructive(Lalancette-Hebert et al. 2007). Sigma receptor activation also supp resses T-cell mediated immunity by upregulating production of the anti-inflamma tory cytokine IL10 (Zhu et al. 2003). As microglia can interact with T-cells as antigen presenting cells (Morioka et al. 1991), it is possible that modul ation of this interaction by sigma receptor agonists such as DTG could result in the robust neuroprotection seen in vivo. The results shown here suggest that the neur oprotection mediated by DTG in vivo at delayed timepoints is mediated by effects on syst ems outside of the CNS such as the peripheral immune system.

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110 While HUCBC treatment has been shown to reduce propridium iodide uptake in organotypic slice cultures, no studies have been performed to our knowledge which directly measure neuronal damage or the ensuing inflammatory response (Vendrame et al. 2005). Here we show that HUCBC can reduce both neurodegeneration and microglial mediated inflammation. HUCBC are composed of a mixture of cell types whic h are mainly leukocytes, with a small (<1%) fraction of stem cells which are al so present (Pranke et al. 2001). The mechanisms by which HUCBC directly pr omote protection are unclear, but may include the secretion of neur otrophic growth factors such as glial cell–derived neurotrophic factor (GDNF), epidermal gr owth factor (EGF), brain-derived neurotrophic factor (BDNF), and fibroblast growth factor (FGF ) (Neuhoff et al. 2007). While cell to cell contact was prohibited by the membranes the slices were grown on, secreted fa ctors such as these grow th factors could decrease neurodegeneration. HUCBC significantly decreased the num ber of microglia which expressed NO. This is in agreement wit h the finding that HUCBC therapy in vivo dramatically reduced the number of microglia present in the infarct (Newcomb et al. 2006). Co-culture experiments in vitro demonstrate this effect is mediated by the production of pro-inflammatory cyt okines by CD8+ T-cells, which induce microglial apoptosis following hypoxia (Ji ang L 2006). Similar experiments using a double transgenic PSAPP/ Tg2576 mouse model of Alzheimer’s disease, demonstrated that HUCBC can decrease microglial activation by reducing CD40/CD40L interactions (Nikolic et al. 2008). Microglial CD 40 is an important

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111 activation signal which is reduced by HUCBC tr eatment (Nikolic et al. 2008). As HUCBC only decreased the incidence of NO producing microglia in the slice, decreasing microglial CD40 expression may be a mechanism by which HUCBC can cause this effect. HUCBC and DTG are both potent suppressors of ischemia induced neurodegeneration in vivo at delayed timepoints (Ajmo Jr et al. 2006; Vendrame et al. 2004). Understanding the proce sses which these treatments modulate, should shed light on the mechanisms for halting the pathophysiological effects of ischemic injury. These studies show t hat sigma receptor activation on ischemic brain tissue is not directly protective, sugges ting that the target of this therapy is peripheral to the CNS. A likely therapeut ic target is the lymphocytes which infiltrate the infarct at delayed timepoint s (Gee et al. 2007). This interaction of peripheral immune cells and injured br ain tissue could also mediate the protective effects of HUCBC seen in this study as they are composed primarily of lymphocytes and monocytes (Pranke et al 2001), and that migration into the CNS is not required for this protection (B orlongan et al. 2004). Further research into the effects of thes e treatments on the interaction between the CNS and the peripheral immune system should yield furt her insight into the mechanisms by which these two treatments reduce stroke induce brain injury.

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112 References Ajmo CT, Jr., Vernon DO, Collier L, Ha ll AA, Garbuzova-Davis S, Willing A, Pennypacker KR. 2008. The spleen contributes to stroke-induced neurodegeneration. J Neurosci Res:(i n press, Epub ahead of print). Ajmo Jr C, Vernon D, Col lier L, Pennypacker K, Cuevas J. 2006. Sigma receptor activation reduces infarct size at 24 hours after permanent middle cerebral artery occlusion in rats. Cur Neurovascular Res 3(2):89-98. Anderson TR, Andrew RD. 2002. Spreading depression: imaging and blockade in the rat neocortical brain slic e. J Neurophysiol 88(5):2713-25. Anderson TR, Jarvis CR, Biedermann AJ, Mo lnar C, Andrew RD. 2005. Blocking the anoxic depolarization pr otects without functional compromise following simulated stroke in cortical brai n slices. J Neurophysiol 93(2):963-79. Borlongan CV, Hadman M, Sanberg CD, Sanberg PR. 2004. Central nervous system entry of peripherally injected umbilical cord blood cells is not required for neuroprotection in st roke. Stroke 35(10):2385-9. Coull JA, Beggs S, Boudreau D, Boivin D, Tsuda M, Inoue K, Gravel C, Salter MW, De Koninck Y. 2005. BDNF from microglia causes the shift in neuronal anion gradient underlyi ng neuropathic pain. Nature 438(7070):1017-21. Davis SM, Lees KR, Albers GW, Diener HC, Markabi S, Karlsson G, Norris J. 2000. Selfotel in acute ischemic stro ke : possible neurotoxic effects of an NMDA antagonist. Stroke 31(2):347-54.

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113 Dirnagl U, Iadecola C, Mosko witz M. 1999. Pathobiology of ischemic stroke: an integrated view. TI NS 22(9):391-397. Duckworth EA, Butler TL, De Mesquita D, Collier SN, Collier L, Pennypacker KR. 2005. Temporary focal ischemia in the mouse: technical aspects and patterns of Fluoro-Jade evident neur odegeneration. Brain Res 1042(1):2936. Elenkov IJ, Wilder RL, Chrousos GP, Vizi ES. 2000. The sympathetic nerve--an integrative interface between two s upersystems: the brain and the immune system. Pharmacol Rev 52(4):595-638. Gee JM, Kalil A, Shea C, Becker KJ. 2007. Lymphocytes : potential mediators of postischemic injury and neuroprotec tion. Stroke 38(2 Suppl):783-8. Gibson CL, Coughlan TC, Murphy SP. 2005. G lial nitric oxide and ischemia. Glia 50(4):417-26. Hall AA, Herrera Y, Ajmo CT, Jr., Cuevas J, Pennypacker KR. 2008. Sigma receptors suppress multiple aspect s of microglial activation. Glia. Herrera Y, Katnik C, Rodriguez JD, Ha ll AA, Willing A, Pennypacker KR, Cuevas J. 2008. sigma-1 receptor modulati on of acid-sensing ion channel a (ASIC1a) and ASIC1a-induced Ca2+ infl ux in rat cortical neurons. J Pharmacol Exp Ther 327(2):491-502. Horn J, de Haan RJ, Vermeulen M, Luit en PG, Limburg M. 2001. Nimodipine in animal model experiments of focal cer ebral ischemia: a systematic review. Stroke 32(10):2433-8.

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114 Jiang L SS, Chen N Sanberg CD, Sanberg PR, and. Willing AE. 2006. Human umbilical cord blood cells depress the microglia inflammatory response in vitro Experimental Neurology 198(2):572. Katnik C, Guerrero WR, Pennypacker KR Herrera Y, Cuevas J. 2006. Sigma-1 receptor activation prevents intracellu lar calcium dysregulation in cortical neurons during in vitro ischemia. J Pharmacol Exp Ther 319(3):1355-65. Lalancette-Hebert M, Gowing G, Simard A, Weng YC, Kriz J. 2007. Selective ablation of prolifer ating microglial cells exacerbates ischemic injury in the brain. J Neurosci 27(10):2596-605. Mander P, Borutaite V, Moncada S, Brown GC. 2005. Nitric oxide from inflammatory-activated glia synergi zes with hypoxia to induce neuronal death. J Neurosci Res 79(1-2):208-15. Marler JR, Goldstein LB 2003. Medicine. Stroke--tPA and the clinic. Science 301(5640):1677. Morioka T, Kalehua AN, Streit WJ. 1991. T he microglial reaction in the rat dorsal hippocampus following transient forebr ain ischemia. J Cereb Blood Flow Metab 11(6):966-73. Morioka T, Kalehua AN, Streit WJ. 1993. C haracterization of microglial reaction after middle cerebral artery occlus ion in rat brain. J Comp Neurol 327(1):123-32. Neuhoff S, Moers J, Rieks M, Grunwald T, Jensen A, Dermietzel R, Meier C. 2007. Proliferation, differentiation, and cytokine secretion of human

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115 umbilical cord blood-derived mononuclear cells in vitro. Exp Hematol 35(7):1119-31. Newcomb JD, Ajmo CT, Sanberg CD, Sanb erg PR, Pennypacker KR, Willing AE. 2006. Timing of cord blood treatment after experim ental stroke determine therapeutic efficacy. Cell Transplant 15(3):213-223. Nikolic WV, Hou H, Town T, Zhu Y, Giunta B, Sanberg CD, Zeng J, Luo D, Ehrhart J, Mori T and others. 2008. Peripherally administered human umbilical cord blood cells reduce parenchymal and vascular beta-amyloid deposits in Alzheimer mice. St em Cells Dev 17(3):423-39. Noraberg J, Poulsen FR, Blaabjerg M, Kristensen BW, Bonde C, Montero M, Meyer M, Gramsbergen JB, Zimmer J. 2005. Organotypic hippocampal slice cultures for studies of brain damage, neuroprotection and neurorepair. Curr Drug Targets CNS Neurol Disord 4(4):435-52. Offner H, Subramanian S, Parker SM, Afentoulis ME, Vandenbark AA, Hurn PD. 2006. Experimental stroke induces massive, rapid activation of the peripheral immune system. J Cereb Blood Flow Metab 26(5):654-65. Pranke P, Failace RR, Allebrandt WF, Steibel G, Schmidt F, Nardi NB. 2001. Hematologic and immunophenotypic characterization of human umbilical cord blood. Acta Haematol 105(2):71-6. Rothwell N. 1996. The role of cytokines in neurodegeneration. In: Rothwell N, editor. Cytokines in the nervous system. Austin: R. G. Landes Co. p 145162.

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116 Schmued L, Albertson C, Slikker W. 1997. Fluoro-Jade: a novel fluorochrome for the sensitive and reliable histoche mical localization of neuronal degeneration. Brain Res 751:37-46. Stoppini L, Buchs PA, Muller D. 1991. A si mple method for organotypic cultures of nervous tissue. J Neur osci Methods 37(2):173-82. Streit WJ. 2002. Microglia as neuroprotec tive, immunocompetent cells of the CNS. Glia 40(2):133-9. Streit WJ, Conde JR, Fendrick SE, Fl anary BE, Mariani CL. 2005. Role of microglia in the central nervous syst em's immune response. Neurol Res 27(7):685-91. Streit WJ, Mrak RE, Griffin WS. 2004. Microglia and neuroinflammation: a pathological perspective. J Neuroinflammation 1(1):14. Su TP. 1991. Sigma receptors. Puta tive links between nervous, endocrine and immune systems. Eur J Bi ochem 200(3):633-42. Su TP, London ED, Jaffe JH. 1988. Steroi d binding at sigma receptors suggests a link between endocrine, nervous and immune systems. Science 240(4849):219-21. Vendrame M, Cassady CJ, Newcomb J, Butler T, Pennypacker KR, Zigova T, Davis Sanberg C, Sanberg PR, AE W. 2004. Infusion of human umbilical cord blood cells in a rat model of stroke dose-dependently rescues behavioral deficits and reduces infarct volume. Stroke 35:2390-2395. Vendrame M, Gemma C, De Mesquita D, Collier L, Bickford PC, Sanberg CD, Sanberg PR, Pennypacker KR, Willing AE. 2005. Anti-inflammatory effects

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117 of human cord blood cells in a rat model of stroke. Stem Cells Dev 14:595604. Yilmaz G, Arumugam TV, Stokes KY, Gran ger DN. 2006. Role of T lymphocytes and interferon-gamma in ischemic stroke. Circulation 113(17):2105-12. Zhu LX, Sharma S, Gardner B, Escuadro B, Atianzar K, Tashkin DP, Dubinett SM. 2003. IL-10 mediates sigma 1 receptor-dependent suppression of antitumor immunity. J Immunol 170:3585-3591.

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118 Chapter 4 The Spleen Contributes to Stroke Induced Neurodegeneration in an Indirect Manner A.A.Hall, D.D. Rowe, C. C. Leonardo, L.A.Collier, A.E.Willing, and K.R.Pennypacker Abstract Stroke induced brain injury is t he third leading cause of death and disability in the United States. Recent st udies have shown that the spleen plays a major role in stroke pat hology. Following stroke, the spleen becomes reduced in size and cellular content. Our lab has show n that removal of the spleen prior to MCAO reduces infarct volume by 80%. This study sought to characterize the changes in spleen and brain over ti me following MCAO, and determine where splenocytes migrate to influence neurodegener ation. To accomplish this, rats received intrasplenic injections of the dy e CFSE five days prior to MCAO surgery, to label splenocytes. Rats were sacrif iced at varying timepoints following MCAO and brains and spleens were harvested for subsequent analysis. The spleens of

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119 animals were found to shrink by 24 hours post MCAO. This effect was transient, with spleens regaining their size by 72 hours post MCAO. Numerous labeled splenocytes were found in spleen, blood, and thymus. Very few splenocytes were found in the brain and were rest ricted to blood vessels. To determine whether the spleen influences the post stroke immune response by secreting cytokines, IL10 and IFN levels were studied in the brain and spleen. IL10 expression was found to significantly increase in the spleen and brain by 96 hours post MCAO. This increase in IL10 and IFN expression was blocked after treatments with HUCBC or DTG and likely reflects modulation of the immune response mediated by these treatments. Introduction Local inflammation has both beneficial and deleterious effects on ischemic lesions. When local inflammation ac tivates the peripheral immune system, however, a much larger inflammatory response is elicited causing collateral destruction of the peri-infarct regions (Offner et al. 2006b). The brain communicates with the immune sy stem largely via direct innervation of lymphoid tissues and humoral control provided by the hypothalamic-pituitary-adrenal axis (HPA axis) (Chrousos 1995). The thymus and the spleen become r educed in size (Offner et al. 2006b; Vendrame et al. 2006) upon induction of CNS ischemia. Multiple groups have

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120 associated this decrease in thymus and spleen size with profound leucopenia and an increased susceptibility to in fection based on a lack of IFN producing Tcells (Prass et al. 2003; Yilmaz et al. 2006). This immunosuppression, while likely helpful at reducing permanent br ain injury, can be reversed by administration of beta-adrener gic blockers such as pr opranolol (Meisel et al. 2005; Prass et al. 2006). The Offner group postulated that the decrease in spleen and thymus size was due to mass apoptotic cell death based on TUNEL immunoreactivity (Offner et al. 2006b). Recent work in our lab has demonstrated that the spleen shrinkage is due, at least in part, to activation of alpha1 adrenergic receptors located on the sm ooth muscle capsule by circulating catecholamines, and this shrinkage can be blocked by prophylactic administration of prazosin, an alpha1 re ceptor antagonist (A jmo Jr. CT 2009). The induction of such profound imm unosuppression indicates that the peripheral lymphoid organs can exacerbat e ischemic injury. The spleen in particular plays a critical role in the re sponse to ischemic injury and its treatment at delayed timepoints. Splenectomy two w eeks prior to stroke effectively reduces infarct volume by 80% demonstrating a crit ical role for this organ in stroke pathology. The importance of the spleen in stroke is also demonstrated by the finding that human umbilical cord blood cells (HUCBC) injected into rats receiving MCAO migrated primarily to the spl een and infarct (Vendrame et al. 2004). HUCBC therapy restores both spleen si ze and cellular content when compared to MCAO alone (Vendrame et al 2005; Vendrame et al. 2006).

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121 The spleen can modulate the peripher al immune system by secreting cytokines which can induce a pro-inflamma tory (TH1) or antiinflammatory (TH2) immune response. Two important cyt okines in this process are IFN and IL10. IFN and IL10 induce TH1 and TH2 responses, respectively, and can serve as surrogate markers for the inflamma tory state following ischemia. Experiments performed here characte rized the post stroke immune response with respect to the spleen over ti me. In this study, the shrinkage of the spleen was shown to be a transient phenom enon. Splenocytes were found in the blood and thymus, but only in very low numbers to the brain. Further analysis demonstrated that IL10 and IFN levels are increased following stroke. Treatment with DTG signific antly decreased IL10 and IFN production in the spleen. These experiments provide evidence that while the acute stress response induces the production of I L10 it cannot completely suppress IFN production. Treatment with DTG signific antly reduces this pro-inflammatory cytokine and decreases infarct volume. Materials and Methods Animal Care All animal procedures were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Anim als with a protocol approved by the Institutional Animal Care and Use Committee at the University of South Florida. Timed pregnant Sprague-Dawley dams we re purchased from Harlan Labs (Indianapolis, IN), maintained on a 12 hr lig ht/dark cycle (7 am – 7 pm) and given

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122 access to food and water ad libitum Neonatal rats birthed from time-pregnant dams were used for all experiments. Splenic injection of CFSE CFSE injections were performed five days prior to MCAO by making a 4 cm dorsal midline skin inci sion at the caudal terminus at the level of the 13th rib. Opening the abdominal wall on the anatomical left, 1.5 to 2.5 cm from midline exposed the spleen. With blunt forceps the organ, (with accompanying blood vessels and adipose tissue), was exterior ized through the incision. CFSE (500 l total volume in DMSO) was injected into 5 evenly spaced sites along the spleen. The spleen was then reinserted into the animal and the wound was closed with sutures. Laser Doppler blood flow measurement Laser Doppler Radar (LDR) was used to monitor blood perfusion (Moor Instruments Ltd, Devon, England). A 2 mm diameter hole was drilled into the right parietal bone (1 mm posterior and 4 mm lateral from bregma), and a guide screw was set. The LDR probe (MP10M200 ST; Moor) was inserted into the guide screw, and the tip of the probe was pl aced against the pial surface of the brain. Rats that did not show >55% reduction in perfusion during MCAO were excluded from the study.

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123 Permanent Middle Cerebral Artery Occlusion MCAO surgery was performed using t he intraluminal method originally described (Longa et al. 1989). Briefly, a filament (4cm long, 6lb test monofilament) was advanced into the inter nal carotid artery by way of the external carotid artery unt il it occluded the origin of the middle cerebral artery, and tied off at the inter nal/external carotid junc tion to produce permanent occlusion. The rat was then sutured, given a 1 ml subcutaneous injection of saline, and allowed to wake in a fresh cage. Brain Extraction and Sectioning The animals were euthanatized with 0. 5 ml of pentobarbital at 96 hours, and perfused with saline and 4% paraformal dehyde. The brains were harvested, post fixed in paraformaldehyde, and imme rsed in 20% followed by 30% sucrose in PBS. Brains were frozen and sliced into 30 m sections with a cryostat. Sections were either thaw mounted on glas s slides or placed in Walter’s Antifreeze cryopreservative. Fluoro-Jade Staining Six coronal brain sections at 1 mm in tervals were cut from 1.7 to -3.3 mm from Bregma for infarct quantification. These sections were stained with FluoroJade (Histochem, Jefferson, AR), which labels degenerating neurons. This

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124 method was adapted from that origina lly developed (Schmued et al. 1997) and has been described by (Duckworth et al 2005). Sections were mounted and dried to glass slides. Slides were rehydrated by then ex posure to absolute ethanol for 3 min followed by 70% ethanol and deionized water for 1 min each. Slides were oxidized using a 0.06% KMnO4 solution for 15 min followed by three rinses of double distilled water for 1 mi nute each. Slides were stained in a 0.001% solution of Fluoro-Jade in 0.1% acet ic acid for 30 min. Slides were rinsed, allowed to dry at 45 oC for 20 min, cleared with xylene and cover slipped with DPX mounting medium (Electron Microscopy Sc iences, Ft. Washington, PA). Infarct Volume Quantification Fluoro-Jade stained tissue was digitally photographed with an Olympus IX71 micr oscope controlled by DP manager software (Olympus America Inc, Melville NY) at a magnification of 1.25X. Images were edited with Jasc Paints hop Pro. Area of neurodegeneration was measured using the NIH Image J software. The area of th e contralateral side of the brain tissue was also measured and used to compensate for possible edema in the ipsilateral hemispheres. Infarct volumes were then calculated by the summation of the infarct areas. Immunohistochemistry The slides were rinsed with PBS (pH 7.2), and then placed in permeabilization buffer containing 10% goat serum, 3% 1M lysine, and 0.3%

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125 Triton X-100 in PBS for 1 hr at room temperature. Nex t, sections were incubated overnight at 4 C in a PBS solution with 2% goat serum and 0.3% Triton X-100 and primary antibody in a humidified c hamber, protected from light when necessary. Slides were washed with PBS (3 x 5 min) and incubated with a secondary antibody solution (PBS, 2% goat serum, 0.3% Triton X-100, biotinylated goat anti rat (1/300))for 1 hour followed by avidin / biotin / horseradish peroxidase complex (Vectast ain Elite ABC kit; Vector) for 1 h. Sections were washed 3x in PBS, and metal-enhanced 3, 3’-diaminobenzidine (Pierce, Rockford, IL) wa s used for color development. ELISA assay READY-SET-GO! ELISA kits (eBiosci ence) for IL10 and interferon-gamma were used to determine levels of these cytokines. ELISAs were run according to manufacturer’s instructions. Results Infarct volumes peak by 24 hours in rats subjected to MCAO To characterize the progression of neurodegeneration following MCAO infarct volumes were determined by Fl uoro-Jade staining at various timepoints following MCAO. The timepoints studied we re 3, 24, 48, 51, 72, and 96 hours post-MCAO. No Fluoro-Jade staining was observed in sham operated animals or animals taken at 3 hours post MC AO. Fluoro-Jade staining reached peak levels 24 hours post MCAO and remained elevated at all subsequent timepoints (Figure 1).

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126 Figure 1. Infarct volumes peak by 24 hours in rats subjected to MCAO Mean infarct volumes plotted over time following MCAO. Infa rct volumes were significantly increased from sham operat ed controls at the 24, 51, 72, and 96hour timepoints. The three hour timepoint did not differ signi ficantly from controls. Asterisk denotes significance at p<0.05 as determined by one-way ANOVA with a post hoc Dunnet’s test. Post-stroke shrinkage of the sp leen is a transient phenomenon The spleen has been shown to shrink following MCAO. As the spleen plays a critical role in infarct dev elopment, the size of the spleen was

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127 characterized over time. The timepoint s studied were 3, 24, 48, 51, 72, and 96 hours post-MCAO (Figure 2). The spleen si gnificantly decreased in size at the 24, 48, and 51 hour timepoints compared to sham operated or 3 hr MCAO timepoint. The spleen regained its size by 72 hours post-MCAO such that it was not different from the sham or 3 hr timepoint (p<0.01). Figure 2. The spleen shrinks transiently following MCAO Mean spleen weights plotted over time following MCAO. Spleen weights were significantly decreased by the 24, 48, and 51hour timepoints. Spleen weights were not significantly different from sham operated controls at the 3 72 and 96hour timepoints. Asterisk denotes significance at p<0.01 as determined by one-way ANOVA with a post hoc Dunnet’s te st using the sham operated animals as controls.

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128 CFSE labeled splenocytes are present in various tissues following MCAO To determine if the shrinkage in spleen size was associated with an increase in spleen derived immune cells in the brain in vivo labeling of splenocytes was performed. CFSE was inje cted into the spleens of animals five days prior to MCAO to label splenocytes. Spleen (Figure 3A), blood (Figure 3B), brain (Figure 3C), and thymus (Figure 3D) of animals at different timepoints were assessed for CFSE positive cells. CF SE staining was abundant in all tissues studied except brain, where CFSE stai ned cells were few and restricted to blood vessels in the infarct.

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129 Figure 3. CFSE labeled splenocytes ar e present in various tissues following MCAO Representative sections from spleen (A), blood (B), brain (C), and thymus (D) at 96 hours following MCAO. CFSE staining was abundant in all tissues studied except brain where CFSE st ained cells were few and restricted to blood vessels in the infarct.

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130 IL10 immunostaining is increased in th e brain and spleen following MCAO IL10 production in the infarct was c haracterized over time in MCAO treated rats. IL10 immunostaining was performed on brain sections from 24 hours (Figure 4A), 51 hours (Figure 4B), 72 hours (Figure 4C), and 96 hours (Figure 4D) following MCAO. Mean values plotted over time demonstrate a significant increase in IL10 at the 96 hour timepoint compared to the 24 hour timepoint (Figure 4E). Mean concentrati ons of IL10 in the spleen over time (Figure 4F) demonstrate a significant (p<0.0 ?) increase in IL10 protein by the 96 hour timepoint. Mean concentrations of IFN in the spleen over time (Figure 4G) demonstrate a significant increase (p<0.05) in IFN protein by the 96 hour timepoint.

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131 Figure 4. IL10 and IFN expression increases over time following MCAO Representative sections stained for IL10 fr om the 24 (A), 51 (B ), 72 (C), and 96 (D) hour timepoints following MCAO. Mean IL10 immunostaining in brain plotted over time (E) reveals a significant in crease at the 96 hour timepoint. Mean concentrations of IL10 in the spleen over time (F) demonstrates a significant increase in IL10 protein by the 96 hour timepoint. Mean concentrations of IFN in the spleen over time (G) demonstr ates a significant increase in IFN protein by the 96 hour timepoint. Asterisk denotes signi ficance at p<0.05 as determined by one-way ANOVA with a post hoc dunnet’s test using the 24 hour timepoint as control.

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132 DTG or HUCBC treatment reduces IL10 and IFN following MCAO As IL10 production is known to be induced by DTG and HUCBC therapies, 96hr timepoint brain sections from ani mals treated with vehicle (Figure 5A), HUCBC (Figure 5B), or DTG (Figur e 5C) were immunostained for IL10 production. Quantification of imm unoreactivity demonstrated that IL10 production was significantly reduced (p<0 .05) in animals treated with DTG and HUCBC when compared to vehicle (Figur e 5D). Mean concentrations of IL10 protein as measured by ELISA show that DTG treatment significantly decreased IL10 production in the spleen (p<0.05) following MCAO (Figure 5E) Mean concentrations of IFN protein as measured by ELI SA show that DTG treatment significantly suppressed IFN production in the spleen (p <0.05) following MCAO (Figure 5F).

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133 Figure 5. DTG and HUCBC treatment reduces IL10 and IFN following MCAO Representative sections from animals tr eated with Vehicle (A), HUCBC (B), and DTG (C) and sacrificed at the 96hr ti mepoint were immunostained for IL10 production. Quantification of imm unoreactivity demonstrated that IL10 production was significant ly reduced in the animals treated with DTG and HUCBC when compared to vehi cle (D). Mean concentrati ons of IL10 protein as measured by ELISA shows that DTG tr eatment significantly decreases IL10 production in the spleen following MCAO (E). Mean concentrations of IFN protein as measured by ELISA shows that DTG treatment significantly suppressed IFN production in the spleen follo wing MCAO (F). Asterisk represents significance at p<0.05 as measured by one-way ANOVA with post hoc Bonferroni’s test. Pound symbol in dicated significance at p<0.05 as measured by students t-test.

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134 Discussion The peripheral inflammatory response to br ain injury is tightly regulated. IL1 TNF and IL6 released in response to brai n injury stimulate centers in the locus ceruleus to induce neural sympathet ic activation. Activity of these cytokines also causes the release of CRH in the hypothalamus (Chrousos 1992). Furthermore reciprocal activation can occur based on neural crosslinks between the lateral paraventricular nucleus and the locus ceruleus (Saper et al. 1976). Release of epinephrine from the adrenal glands in response to CRH, in addition to the direct secretion of nor epinephrine into the lymphoid organs by sympathetic nerve fibers suppresses i mmune cell function and promotes the release of TH2 cytokines such as I L10. Circulating catecholamines and glucocorticoids alter the morphology of peripheral lymphoid organs known to be involved in the progression of stroke, such as the spleen (Elenkov et al. 2000). Experiments were performed to characteri ze the infarct and the size of the spleen over time following MCAO. T he infarct progressed rapidly from essentially no infarction at three hours to 60% of the ipsilateral hemisphere being infarcted by 24 hours. This is reflected in the spleen size as well, with spleens not being significantly smalle r than sham controls at 3 hours while decreased in size by 30% by 24 hours. Interestingly, while infarct volume plateaued at levels seen at the 24 hr timepoint, spleen weight s were recovered by 72hrs such that they were not different from controls. This suggests that the catacholaminergic

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135 stress response is a transient phenom enon which is counteracted by some mechanism by 72 hours following MCAO. As the change in spleen size has been associated with alterations in splenic composition (Offner et al. 2006a), the cellular trafficki ng of splenocytes into the brain was studied. Cells withi n the spleen were labeled with the cell tracer CFSE prior to MCAO. While CFSE labeled cells were found in the blood, spleen, and thymus, at no timepoint observed were labeled cells found in appreciable numbers in the brain parenc hyma. The only CFSE labeling observed was restricted to blood vessels and the number of cells seen was less than 10 per brain section. This was surp rising as removal of splenocytes via splenectomy reduces infarct volume by 80% This suggests that the role of the splenocytes in ischemic injury is indirect. One possible mechanism for splenocytes to alter infarct volume wit hout entering the brai n is through antigen presentation. It is known t hat dendritic cells home to t he spleen upon activation by antigen. As ischemic brain injury releases CNS specific antigens such as myelin basic protein (MBP) and mye lin oligodendrocyte glycoprotein (MOG) dendritic cells could present these proteins to T-cells in the spleen and induce a more potent inflammatory response. Th is theory is supported by studies using mice deficient in T-cells which have ma rkedly reduced infarct volumes (Yilmaz et al. 2006), and in studies in which mice pa ssively tolerized with MOG (Frenkel et al. 2003) and MBP (Gee et al. 2008) show reduced infarct volumes If the response of the spleen to stroke is mediated by modulation of the peripheral immune system, one mechanism by which this could be accomplished

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136 is through the secretion of cytokines such as IL10 and IFN These cytokines can shift the immune system into a TH2 or a TH immune response, respectively. Therefore, the final set of studies examined the producti on of cytokines over time following MCAO. IL10 immunostaining was found to be markedly increased in the infarct beginning at 51 hours and increasin g significantly by 96hours. This immunostaining was primarily neuronal in origin suggesting that these cells are responding to inflammation by releasing th is anti-inflammatory cytokine. IL10 can suppress the release of TNF and IL1 (Chrousos 1995), so the observed increases in the levels of this cytok ine in the brain following MCAO may be responsible for the return of the spleen size to sham control levels. IL10 and IFN production both increased significantly in the spleen over time. This suggests that there is an active inflammato ry response which the body is trying to suppress. Interestingly, both HUCBC and DTG tr eatments significantly reduced IL10 and IFN levels in the brain. Both treatm ents are potently anti-inflammatory and have been associated with increa sed IL10 production. This may be explained by the fact that both treatment s significantly decreased infarct volumes. If IL10 production was a response to inflammation mediated by such as IFN then it is logical that treatm ents which reduce this inflammation would reduce IL10 production in both the brain and the spleen. Ischemic brain injury is a dynamic proc ess. In this study, we characterized the body’s response to ischemia over ti me. The observations gathered here lend insight into the interplay between the brai n and spleen in response to injury. As

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137 the peripheral immune system plays an import ant part in the body’s response to ischemia, an understanding of how the spleen fi ts into this role is important to identify therapeutic targets at delayed timepoints following MCAO References Ajmo Jr. CT CL, Leonardo CC, Hall AA, Green SM, Womble TA, Cuevas J, Willing AE and Pennypacker KR. 2009. BLOCKADE OF ADRENORECEPTORS INHIBITS THE SPLENIC RESPONSE TO STROKE. Experimental N eurology (In press). Chrousos GP. 1992. Regulati on and dysregulation of t he hypothalamic-pituitaryadrenal axis. The corticotropin-rel easing hormone perspective. Endocrinol Metab Clin North Am 21(4):833-58. Chrousos GP. 1995. The hypothalami c-pituitary-adrenal axis and immunemediated inflammation. N Engl J Med 332(20):1351-62. Duckworth EA, Butler TL, De Mesquita D, Collier SN, Collier L, Pennypacker KR. 2005. Temporary focal ischemia in the mouse: technical aspects and patterns of Fluoro-Jade evident neur odegeneration. Brain Res 1042(1):2936. Elenkov IJ, Wilder RL, Chrousos GP, Vizi ES. 2000. The sympathetic nerve--an integrative interface between two s upersystems: the brain and the immune system. Pharmacol Rev 52(4):595-638. Frenkel D, Huang Z, Maron R, Koldzic DN, Hancock WW, Moskowitz MA, Weiner HL. 2003. Nasal vaccination with my elin oligodendrocyte glycoprotein

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138 reduces stroke size by inducing IL -10-producing CD4+ T cells. J Immunol 171(12):6549-55. Gee JM, Kalil A, Thullbery M, Becke r KJ. 2008. Induction of immunologic tolerance to myelin basic protei n prevents central nervous system autoimmunity and improves outcome after stroke. Stroke 39(5):1575-82. Meisel C, Schwab JM, Prass K, Meisel A, Dirnagl U. 2005. Central nervous system injury-induced immune deficiency syndrome. Nat Rev Neurosci 6(10):775-86. Offner H, Subramanian S, Parker SM, Afentoulis ME, Vandenbark AA, Hurn PD. 2006a. Experimental stroke induces massive, rapid activation of the peripheral immune system. J Cereb Blood Flow Metab 26(5):654-65. Offner H, Subramanian S, Parker SM Wang C, Afentoulis ME, Lewis A, Vandenbark AA, Hurn PD. 2006b. Splenic atrophy in experimental stroke is accompanied by increased regulatory T cells and circulating macrophages. J Immu nol 176(11):6523-31. Prass K, Braun JS, Dirnagl U, Meisel C, Meisel A. 2006. Stroke propagates bacterial aspiration to pneumonia in a m odel of cerebral ischemia. Stroke 37(10):2607-12. Prass K, Meisel C, Hoflich C, Braun J, Halle E, Wolf T, Ruscher K, Victorov IV, Priller J, Dirnagl U an d others. 2003. Stroke -induced immunodeficiency promotes spontaneous bacterial in fections and is mediated by sympathetic activation reversal by poststroke T helper cell type 1-like immunostimulation. J Exp Med 198(5):725-36.

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139 Saper CB, Loewy AD, Swanson LW, Co wan WM. 1976. Direct hypothalamoautonomic connections. Br ain Res 117(2):305-12. Schmued L, Albertson C, Slikker W. 1997. Fluoro-Jade: a novel fluorochrome for the sensitive and reliable histoc hemical localization of neuronal degeneration. Brain Res 751:37-46. Vendrame M, Cassady CJ, Newcomb J, Butler T, Pennypacker KR, Zigova T, Davis Sanberg C, Sanberg PR, AE W. 2004. Infusion of human umbilical cord blood cells in a rat model of stroke dose-dependently rescues behavioral deficits and reduces infarct volume. Stroke 35:2390-2395. Vendrame M, Gemma C, De Mesquita D, Collier L, Bickford PC, Sanberg CD, Sanberg PR, Pennypacker KR, Willing AE. 2005. Anti-inflammatory effects of human cord blood cells in a rat model of stroke. Stem Cells Dev 14:595604. Vendrame M, Gemma C, Pennypacker KR Bickford PC, Davis Sanberg C, Sanberg PR, Willing AE. 2006. Cord blood rescues stroke-induced changes in splenocyte phenotype and f unction. Exp Neurol 199(1):191200. Yilmaz G, Arumugam TV, Stokes KY, Gran ger DN. 2006. Role of T lymphocytes and interferon-gamma in ischemic stroke. Circulation 113(17):2105-12.

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140 Chapter 5 Conclusions As our population ages and the quality of medical care improves, a greater proportion of stroke victims survive. M any of these surviv ors are functionally disabled, requiring assistance to perform basic tasks necessary for independence. The costs to the survivor both financ ially and emotionally are devastating. As two-thirds of stro ke survivors are over the age of 65, most of these survivors are enrolled in public m edical programs such as Medicare. The costs to these programs, and thus to the public, are enormous. Currently there is only one FDA a pproved treatment for stroke, tissue plasminogen activator (rTPA). This treatme nt yields significant improvements in stroke outcome with a 13% increase in fa vorable outcome functionally in patients eligible for this treatment. Therein lays the major caveat for this treatment. In order to be eligible for rT PA, patients must get to t he hospital, be diagnosed, and have a CT scan to rule out hemorrhagic stroke within 3 hours of symptom onset in order to be a candidate fo r therapy. Due to this very narrow therapeutic window, only 3-5% of stroke sufferers are eligible for this treatment. Thus, there is an immense need for therapies which can treat stroke effectively at delayed (24-48hr), clinically relevant, timepoints. The heady exuberance with wh ich the stroke field embraced excitotoxicity has largely been replaced with a painful h angover of failed clinical trials and millions of dollars wasted. While it ma y be possible to treat stroke by reducing

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141 excitoxicity from a theoretical standpoint, from a clinical standpoint it is obvious that these processes occur too early on for the patient to arrive for treatment in a timely manner. The goal of stroke treat ment now is to fundamentally alter the body’s response to stroke and unlimber t he potent innate repair mechanisms in order to reverse ischemic brain inju ry and produce functional recovery. To accomplish this it is imperative to look beyond the first 6 hour s of ischemia and gain a deeper understanding of the biology of ischemic injury at these delayed timepoints. Essential to this is the ident ification of treatment s which are effective at delayed timepoints (24-48 hours post stroke). With such treatments it is then possible to identify the systems through wh ich they act to winnow out potential targets for stroke therapy. In order to treat stroke at 24 to 48 hours, it is as important to address the immune response to the infarction as it is to treat the infarction it self. This can be achieved in two ways: a cocktail of drugs targeting both inflammatory and neurodegenerative processes, or a single tr eatment which has multiple effects limiting both the immune response and neurodegeneration. As polypharmacy treatment increases the propensity for si de effects via drug-drug interactions; a single therapy is more attr active for the treatment of patients in a frail state induced by stroke. Two such therapies which have show n promise in the MCAO model of stroke are HUCBC and DTG. Both of these therapies reduce infarct volumes by 50% or more and are effective when administered 24hrs post MCAO. The mechanism by which these therapies ar e neuroprotective is unclear. Both

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142 treatments decrease isc hemic neuronal death and suppress inflammation in vitro and decrease peripheral inflammation in vi vo. As there are multiple systems which could mediate the beneficial effects of these therapies, it is important to identify which system is responsible fo r the observed reductions in infarct volumes. The experiments described her e sought to clarify whether these therapies actively altered inflammatory processes in the CNS or the periphery and the relative contributions of both sources of inflammation on ischemic neuronal death. It was clear from the studies perfo rmed in aim 1 that sigma receptor activation reduces microglial activation at multiple levels, including the morphological, migratory and inflammatory responses to several agents that trigger these responses in microglia. The re sults of this study provide insight into the role of sigma receptors in the r egulation of immune system function. As these processes are critical to the micr oglial response to injury, these findings reveal a novel aspect of the modulati on of microglial activation by sigma receptors. Furthermore, these findings direct ly translate into effects this treatment may have on other monocyte derived cells as well. Blood born monocytes which are precursors to microglia and macrophages can react to cellular debris gener ated by the initial ischemic insult via activation of toll-like receptors. Th is can induce the transformation of these monocytes into dendritic cells whic h can then present CNS antigens to lymphocytes in the spleen and acti vate the adaptive immune system.

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143 Invasion of peripheral macrophages is s een in ischemic injury beginning at 24hours and peaking by 3 days. In both ce ll types sigma receptor activation likely reduces chemokine induced migrati on and activation of toll-like receptor signaling in a similar manner to that seen with microglia. Our results demonstrate that sigma receptor activation blocks changes in actin aggregation in response to AT P application. Metabotropic P2Y12 receptors, which evoke increases in [Ca2+]i in microglia (Ohsawa et al. 2007), are critical to the induction of membrane ruffling by ATP exposure (Honda et al. 2001). In our migration studies we saw robust migration in response to ATP (which is mediated by P2Y12 receptor s as well) and MCP-1, both of which induce migration through a pertussis toxin se nsitive Gi/o-protein (Dzenko et al. 2001; Honda et al. 2001; Sozzani et al. 1994). Migration induced by both chemoattractants was inhibi ted by sigma receptor activation, suggesting that sigma receptors inhibit migration either by direct interaction with the Gi/o-protein or by interfering with downstream calcium signaling. Chemokine signaling is a major mechanism by which local and peripheral immune ells are recruited to sites of inju ry. Microglia, neutr ophils, dendritic cells, lymphocytes and monocytes all use chemoki ne gradients to migrate to sites of injury. Inhibiting chemokine signaling do wnstream of the receptor is a powerful mechanism by which sigma receptor activation can reduce immune cell infiltration into the infarct. P2Y12 receptors not only mediate ATP induced migration in microglia, but they are also found on the surface of pl atelets where they promote adhesion and

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144 clot formation. Inhibition of platelet P2Y12 receptors via clopidogrel reduces the formation of blood clots in patients at ri sk for stroke and myocardial infarction (MI). As stroke and MI survivors are at an increased risk for a subsequent stroke, inhibition of P2Y12 receptors on platelets by DTG would be not only protective acutely but also prophylactically. Activation of sigma receptors in the peripheral immune system has been shown to potently suppress the inflammato ry response to a variety of stimuli (Bourrie et al. 2002). It was proposed that these anti-inflammatory effects were mediated by sigma receptor-induced interl eukin-10 (IL10) production (Zhu et al. 2003a). However, studies on sigma rec eptor-mediated immunosuppression in RAW 264.7 macrophages suggested that IL10 is not always involved in these responses (Gannon et al. 2001). This latte r observation is consistent with our finding that IL10 production in response to LPS treatment was decreased with similar kinetics to reductions of TNFand NO production. This observation implies that sigma receptor activation affects signaling upstream of protein synthesis-dependent processes in t he inflammatory response. Sustained increases in basal intracellula r calcium levels are integral to the induction of the inflammatory response (Hoffmann et al. 2003) in microglia after stimulation with LPS. Stimulation of toll-like receptors by LPS mimics the stimulation of this receptor by cell debris which is present in the infarct. Sigma receptor activation reduces the capacity of microglia to generate this sustained increase in calcium in response to LPS. This is associated with a decreased

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145 release of cytokines and NO. Reintr oduction of calcium with ionomycin overcame the sigma receptor activati on induced blockade of NO production. Our results in aim 1 show that activation of sigma receptors suppresses microglial activation primarily by inhibiting calcium signaling. Calcium is a critical signaling molecule in immune cells s uggesting that the immunosuppressive effects of this receptor are not nec essarily restricted to only microglia. Neurosteroids, such as progesterone and estrogen, bind to sigma receptors with high affinity and are associated with decreased immune responsiveness (Su et al. 1990). Our findings raise the intrigui ng possibility that t hese sigma ligands are exerting central effects via the activati on of sigma receptors (Su 1991). Since astrocytes synthesize dehy droepiandrosterone (Zwain and Yen 1999), another endogenous sigma ligand, the presence of sigma receptors on microglia may represent a pathway by which microglial activation may be suppressed locally by support cells. Activated microglia have been asso ciated with neurodegenerative disease progression (Streit et al. 2004), and the presence of a significant number of activated microglia has been linked to in creased neuronal damage. In contrast, ablation of microglia is also asso ciated with increased damage (LalancetteHebert et al. 2007), which shows that mi croglia play a complex part in the etiology of neurodegenerative disease. As microglia have the capacity to either promote neurodegeneration or neurosurvival, t he microglial response to injury is a highly regulated process. Our findings support and expand upon the theory that sigma receptors have potent immunoregulatory properties (Bourrie et al. 1995;

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146 Bourrie et al. 2002). Interestingly sigma re ceptor activation in microglia has also been associated with increased TGF production (Gekker et al. 2006). TGF-B is an anti-inflammatory cytokine that is important in the induction of tolerance to antigens. As circulating monocytes that enc ounter cell debris mature into antigen presenting dendritic cells fo llowing MCAO, increased TGF signaling may impair this process. Furthermore, TGF is known to enhance wound healing and neurosurvival which could further explain the observed reductions in infarct volume following MCAO.

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147 Figure 1. Sigma receptor agonists suppress microglial activation by interfering with intrace llular calcium signaling. Microglial activation is a complex multi step process. Upon detec tion of a damage signal or invading pathogen via chemoreception, a transient ca lcium increase occurs. Following this, microglia rapidly rearrange their actin cytoskeleton and withdraw their processes. They then migrate to the si te of injury. Upon prolonged stimulation the basal levels of intracellular calcium increase in the microglia, which decreases their ability to generate the lar ge calcium transients in response to the chemoattractant serving as an endogenous stop signal. Once at the site of injury, microglia can then release cyt okines and reactive oxygen species depending on the tissue microenvironment. Sigma receptor activation suppresses the transient and sustained ca lcium increases necessary for these processes, blocking micr oglial activation. In aim 2 organotypic slice cultures were used to study the observed effects of sigma receptor activation on microglia were neuroprotective in the absence of the peripheral immune system. Organotypic hippocampal slic e culture is a superior model for the study of ischemic brain in jury than dissociated cell cultures owing to the fact that all br ain cell types are present with functional connections (Noraberg et al. 2005). The multiple inflam matory stimuli released by ischemic neurons in the slice cultures activate mi croglia by a mechanism which mimics the in vivo ischemic microenvironment more cl osely than traditional activators such as lipopolysaccharide. In order to further characterize this model with respect to neurodegeneration, slice cultures were subj ected to OGD at di fferent timepoints

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148 and stained with Fluoro-Jade. In this model system neurodegeneration increased in a time dependant manner following the induction of OGD with significant increases observ ed at the 48hr timepoint. In this study the HUCBC treatment was neuroprotective but the DTG treatment was not. This is intriguing as sigma receptor agonists reduce ischemia and acidosis induced death in dissociated neuronal cultures by reducing calcium influx into these neurons (H errera et al. 2008; Katnik et al. 2006). Furthermore sigma agonists suppress spreading depres sion and ionic imbalances in acute hippocampal slice preparations (Anders on and Andrew 2002; Anderson et al. 2005). As spreading depression and ca lcium dysregulation are central components of excitotoxic cell death, blo cking these processes with DTG should have resulted in reduced Fluoro-Jade staining in slices subjected to OGD. This demonstrates that in this model, bl ocking excitotoxic cell death, anoxic depolarization, and micr oglial activation directly is not sufficient to protect neurons. Intriguingly, similar effects are noted in vivo Administration of dehydroepiandrosterone, a pot ent sigma receptor agoni st, reduced ischemic infarct volume when administered 6-36 hours following stroke. When DHEA was administered prior to or 1 hour after stroke it significantly increased neurodegeneration. Both effect s were found to be due to activation of the sigma1 receptor. Excitotoxicity associ ated phenomena such as NMDA receptor activation are thought to occur within the fi rst hour of stroke onset. Therefore it seems unlikely that the prevention of exci totoxicity or the bl ockade of microglial

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149 activation is the mechanism by which DTG reduces infarct volume following stroke. Sigma receptors also have potent antiinflammatory properties which can reduce inflammation induced neuronal death. In the organotypic slice culture microglia are the only immune cell ty pe present. Sigma receptors reduce microglial activation in vitro by suppressing intracellular calcium signaling (Hall et al. 2008). By blocking intracellular calc ium signals, many aspects of microglial activation such as membrane ruffling, migration, and cytokine production are impaired (Hall et al. 2008). The 300 M DTG concentration, which completely suppressed microglial activation (Hall et al 2008), resulted in the highest levels of Fluoro-Jade staining encountered, sugges ting that some aspects of microglial activation are beneficial. This finding is in agreement with other studies which have shown that ablation of microglia is neurodestructive (Lalancette-Hebert et al. 2007). While HUCBC treatment has been s hown to reduce propidium iodide uptake in organotypic slice cultures, no studies have been performed to our knowledge which directly measure neuronal damage or the ensuing inflammatory response (Vendrame et al. 2005). HUCBC treatment reduced both neurodegeneration and microglial mediated in flammation. HUCBC significantly decreased the number of microglia which ex pressed NO. This is in agreement with the finding that HUCBC therapy in vivo dramatically reduced the number of microglia present in the infarct (Newcomb et al. 2006). Similar experiments using this model demonstrated that the MMP inhibitor AG3440 also reduced

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150 neurodegeneration in this m odel (Leonardo et al. 2009). MMP9 can reduce neuronal viability directly by degrading the extracellular matrix and inducing anoikis. As these treatments were given at the time of OGD it is unclear whether these treatments reduced microglial activati on directly or indirectly by reducing neuronal death. Based on t he data from DTG it seem s likely that activated microglia are not the cause but a bypr oduct of OGD induced neurodegeneration. The studies from aim 2 show that si gma receptor activation on ischemic brain tissue is not directly protective, sugges ting that the target of this therapy is peripheral to the CNS. Sigma receptor activation suppresses T-cell mediated immunity by upregulating production of t he anti-inflammatory cytokine IL10 (Zhu et al. 2003b). As microglia can interact with T-cells as antigen presenting cells (Morioka et al. 1991), it is possible that modulation of this interaction by sigma receptor agonists such as DTG could re sult in the robust neuroprotection seen in vivo. The results shown here suggest that the neuroprotection mediated by DTG in vivo at delayed timepoints is mediated by effects on systems outside of the CNS such as the peripheral immune system A likely therapeutic target is the lymphocytes which infiltrate the infarct at delayed timepoints (Gee et al. 2007). This interaction of peripheral immune cells and injured brain tissue could also mediate the protective effects of HUCB C seen in this study as they are composed primarily of lymphocytes and m onocytes (Pranke et al. 2001), and that migration into the CNS is not required for this protection (Borlongan et al. 2004). In aim 3, the systemic response to stroke was more closely studied. Experiments were performed to characteri ze the infarct and the size of the

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151 spleen over time following MCAO. T he infarct progressed rapidly from essentially no infarction at three hours to 60% of the ipsilateral hemisphere being infarcted by 24 hours. This is reflected in the spleen size as well, with spleens not being significantly smaller at 3 hour s than sham controls while by 24 hours the spleen decreased in size by 30%. Interestingly while infarct volume plateaued at levels seen at the 24hr tim epoint spleen weights were recovered by 72hrs such that they were not different from controls. This suggests that the catacholaminergic stress response is a transient phenomenon which is counteracted by some mechanism by 72 hours following MCAO. As the change in spleen size has been associated with alterations in splenic composition (Offner et al. 2006), t he cellular trafficking of splenocytes into the brain was studied. Cells within the spleen were labeled with the cell tracer CFSE prior to MCAO. At no timepoint observed were labeled cells observed in appreciable numbers in the brain parenchyma. This was surprising as removal of splenocytes via splenectomy reduces infarct volume by 80%. This suggests that the role of the spleno cytes in ischemic injury is indirect. One possible mechanism for splenocytes to alter infarc t volume without entering the brain is through antigen presentation. It is know n that dendritic cells home to the spleen upon activation by antigen. As ischemic brain injury releases CNS specific antigens such as myelin basic prot ein (MBP) and Myelin oligodendrocyte glycoprotein (MOG) dendritic cells could pr esent these proteins to T-cells in the spleen and induce a more potent infla mmatory response. This theory is supported by studies using mice defic ient in T-cells which have markedly

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152 reduced infarct volumes (Yilmaz et al. 2006), and in studies in which mice passively tolerized with MOG (Frenkel et al. 2003) and MBP (Gee et al. 2008) show reduced infarct volumes The final set of studies examined the production of cytokines over time. IL10 immunostaining was found to be markedly increased in the infarct beginning at 51 hours and increasing sign ificantly by 96hours. This immunostaining was primarily neuronal in origin suggesting that these cells are responding to inflammation by releasing this anti-infl ammatory cytokine. IL10 can suppress the release of TNF and IL1 (Chrousos 1995), so the observed increases in the levels of this cytokine in the brain following MCAO maybe responsible for the return of the spleen size to the pre-stroke condition. IL10 and IFN levels in the spleen also increased over time. As IFN is a very potent activator of lymphocytes and macrophages, the fact that its levels increase over time suggests that there is st ill an ongoing inflammatory response which the body is responding to by upregulating IL10.

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153 Figure 2. Changes in spleen size and content over time reflect an evolving interaction between the brain a nd immune systems following MCAO. The spleen, by virtue of the 1-adrenergic receptors on it s smooth muscle capsule serves as a convenient maker of the acute stress response. Increased circulating catecholamines bind to these receptors and cause the spleen to substantially decrease in size between 3 and 24 hours following MCAO. These same catecholamines act on 2-receptors located on circulating monocytes to induce the release of IL10. Levels of splenic IL10 increase between 24 and 48 hours and remain elevated 96 hours post MCA O. IL10 reduces inflammation in systemically which in turn causes the ac ute stress response to resolve. This likely occurs between 48 and 72 hours followi ng MCAO as the spleen regains its previous size during this interval. Removal of the anti-inflammatory

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154 catecholamine signaling induces a rebound in pro-inflammatory IFN production in the spleen. HUCBC and DTG treatments significantly r educed IL10 levels in the brain. Both treatments are potent ly anti-inflammatory and have been associated with increased IL10 production. This disparity may be explained by the fact that both treatments significantly decreased infarct volumes. If IL10 production by neurons was a response to inflammatory damage, then it is logical that treatments which reduce this inflammation would reduce neuronal IL10 production. IL10 and IFN levels in the spleen were also signif icantly inhibited by DTG 96 hours post MCAO. This likely reflects the fact that the majori ty of catecholamine induced IL10 is produced by monocytes (Woiciecho wsky et al. 1998), a process which is inhibited by DTG (Hall et al. 2008). Taken together the data presented here present an interesting view of stroke induced injury and the relative contributions of the various factors involved. Following the initial inducti on of ischemia excitotoxic processes damage neurons. These neurons are protec ted from immediate neuronal death by the concerted actions of microglia an d astrocytes. If re canalization can be achieved at this point (such as with rT PA administration) the total damage can be significantly reduced. This is effect ive out to approximat ely 6 hours following occlusion. After this ti me, inflammatory cytokines induce the acute stress response via HPA axis activation. This response causes the spleen to shrink and reduces TH1 immune responses. TH2 responses such as IL10 production

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155 increase starting at 24 hours in the spleen and brain. IL10 is released rapidly from blood borne monocytes following 2-adrenergic receptor stimulation (Woiciechowsky et al. 1998). This anti-infl ammatory cytokine likely acts on brain centers in the hypothalamus and brain stem to shut down the acute stress response by 72 hours. The decreased catec holamine signaling al lows the spleen to regain its size and removes the tonic immunosuppressive stimulus on circulating lymphocytes. These lym phocytes have likely been migrating to the infarct and interacting with antigen presentin g cells in the spleen and brain. In the absence of the stress response, t hese cells begin producing TH1 type cytokines. In the spleen this is reflected by the rebound in IFN seen between 48 and 96 hours post MCAO. This correlates with increased IL10 expression in the brain but not with a second induction of t he acute stress respons e. This implies that the inflammatory signaling at this time point is not mediated by TNF IL1 and IL6. This second inflammatory re sponse is likely a T-cell mediated event and likely leads to the cell mediated clear ance of compromised neurons from the infarct. DTG and HUCBC likely interfere with the ability of the peripheral immune response to continue following cessation of the acute stress response. HUCBC can not only suppress inflammation but can also interrupt the acute stress response leading to early recovery of sp leen weight. The therapeutic target for DTG is likely T-cells which upon sigma rec eptor activation switches from a TH1 (IFN producing) to a TH2 (IL10 produci ng) phenotype. Sigma receptor activation likely suppresses macro phage and monocyte function similarly to

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156 microglia. T-cells, however, have been imp licated as the cell type which are necessary for ischemic injury (Yilmaz et al. 2006). Implications and Future Directions The immune system is a promising ta rget for the treatment of stroke induced brain injury. The results from this set of studies further clarify the role of the immune system in ischemic brain injury, and the mechanisms by which delayed treatments for stroke function. Timing of immunomodulatory therapies is critical for their success. HUCBC therapy is good example of this, HUCBC must be transplanted during the acute stress re sponse for efficacy (Newcomb et al. 2006). DTG likely has a similar requirement as sigma receptor activation by DHEA is neuroprotective when administ ered at delayed (6-36 hour) but not hyperacute (<1 hour) times following isc hemia (Li et al. 2009). By studying HUCBC and DTG, and their effect s on ischemic injury both in vivo and ex vivo it was possible to determine that microglial activation following ischemic injury is not necessarily deleterious in the absenc e of the peripheral immune response. This perhaps explains why in a splenecto mized animal there is a greatly reduced infarct. Rats lacking a spleen still have activated microglia in their infarcts, however by 96 hours these cells become ra mified and likely do not contribute to neuronal death (Ajmo et al. 2008). Furthermore, as DTG was neuroprotective only in the presence of the peripheral immune system suggests that mo dulation of this system is an ideal target for the treatment of stroke at delayed timepoints. It is likely that while

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157 HUCBC are neuroprotective in the abs ence of the peripheral immune system, their main mechanism for action is also on the peripheral immune system. Three facts support this contention: 1, HUCBC are more effective when administered intravenously than when administered in trastriataly, 2, HUCBC are most efficacious at 48 hrs a timepoint at which the peripheral immune system is actively infiltrating the brain, and 3, HUCBC do not further decrease infarct volume in splenectomized rats suggesting that the direct ef fects of HUCBC on the ischemic brain tissue is minimal. Further studies will be needed to fully understand the role of the peripheral immune system at delayed timepoints followi ng stroke. One such study would be therapeutic splenectomy. If splenectomy surgery 24 hrs post stroke was as efficacious as two weeks prior to stroke it would demonstrate that excitotoxic processes are not as damaging as previ ously thought. As these processes are still actively being targeted for stroke ther apy, the results of th is type of study would help refine future stro ke therapies and improve t he success rate in stroke treatment.

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158 Figure 3. Successful therapeutic target s change as the body’s response to stroke changes over time. The most important concept in stroke treatment is that stroke therapies must be applied at the proper stage in of the disease state. Therapies which are effective at reducing excitotoxicity or preventing infiltration of the parenchyma are likely are only going to be of benefit very early on in the disease progression, prior to the inducti on of the inflammatory response. Likewise administrations of anti-inflammatory treatments are not likely to be of benefit prior to the induction of the acute stress response. This is because the acute stress response is neurologically protective and is induced by proinflammatory cytokines. Finally treatm ent for stroke at extremely delayed timepoints (>72 hrs) is likely to be limit ed to rehabilitation therapies which use various stimuli to promote axonal r egeneration and partial gain of function.

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About the Author Aaron A. Hall received a Bachelor’s Degree in Microbiology and Cell Science from the University of Florida in 2002. He spent two years engaging in fascinating research on the mitochondrial ri bosome in the laboratory of Dr. T.W. O’brien. He then entered the graduate program in M edical Sciences at the University of South Florida under the tutelage of Dr. Keith Pennypacker and earned a M.S. in 2005. He then matriculat ed directly into the Ph.D. program at the University of S outh Florida in 2005. While in the Ph.D. program at the Un iversity of South Florida, Mr Hall was very active in conducting translational res earch in the field of stroke in the laboratory of Dr. Pennypacker. He has presented his work at multiple international conferences and has author ed six publications in peer reviewed scientific journals