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Role of Microglia during Motoneuron Regeneration and Degeneration: Relevance for Pathogenesis and Treatment of Amyotroph...

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ROLE OF MICROGLIA DURING MOTONEURON REGENERATION AND DEGENERATION: RELEVANCE FOR PATHOGENESIS AND TREATMENT OF AMYOTROPHIC LATERAL SCLEROSIS By SARAH EMILY FENDRICK A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by Sarah Emily Fendrick

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This dissertation is dedicated to my family and friends for their support and encouragement throughout graduate school.

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ACKNOWLEDGMENTS I would like to thank my mentor, Dr. Wolf gang Streit, for his guidance, expertise and willingness to teach me. I also tha nk my committee members Dr. John Petitto, Dr. William Millard and Dr. Paul Reier for their time and support needed to successfully complete my dissertation. A special thank you is extended to all the Streit lab members. In particular, I would like to thank Kelly Miller, for her early mo rning help in the perfusion room and her endless support, Chris Mariani for his constant advice on technique and the competition that motivated me to graduate, and Kryslaine Lopes for being a supportive lab member and friend. I thank BJ Streetman and John Neely in the neuroscience office both of whom made the administrative aspect of graduate school a simple one. Finally, and most of all I would like to thank my friends and family for being supportive and encouraging through out my time in graduate school. I thank my parents for providing me with the love and support that I have needed to succeed both in and out of school. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................iv LIST OF TABLES ............................................................................................................vii LIST OF FIGURES .........................................................................................................viii ABSTRACT .........................................................................................................................x CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW....................................................1 Microglia: An Overview ...............................................................................................1 Introduction ...........................................................................................................1 Microglia Are Neurosupportive ............................................................................2 Facial Nerve Axotomy ..................................................................................................3 Amyotrophic Lateral Sclerosis .....................................................................................4 Microglia in ALS ..........................................................................................................7 Neuroinflammation .......................................................................................................8 Microglia Dysfunction ................................................................................................10 Minocycline ................................................................................................................12 2 MINOCYCLINE DOES NOT INHIBIT MICROGLIA PROLIFERATION OR NEURONAL REGENERATION IN THE FACIAL NUCLEUS FOLLOWING A FACIAL NERVE CRUSH.........................................................................................14 Introduction .................................................................................................................14 Materials and Methods ...............................................................................................15 Animals and Diet .................................................................................................15 Facial Nerve Axotomy ........................................................................................16 3H-Thymidine Injections and Radioactive Perfusions ........................................16 Tissue Processing for Audoradiography .............................................................17 Audoradiography .................................................................................................18 Quantitative Analysis for 3H-thymidine Labeled Microglia ...............................18 Fluorogold Labeling ............................................................................................19 Perfusion and Tissue Proce ssing for Fluorogold Labeling ..................................19 Quantitative Analysis of Fluorogold Labeling ....................................................19 Results .........................................................................................................................19 v

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Discussion ...................................................................................................................22 3 DETERMINATION OF MINOCYCLINE CONCENTRATION IN THE BRAIN AFTER DRUG ADMINISTRATION IN DIET........................................................25 Introduction .................................................................................................................25 Materials and Methods ...............................................................................................26 Reagents ..............................................................................................................26 Animals ................................................................................................................27 Extraction ............................................................................................................27 Equipment ............................................................................................................27 Results .........................................................................................................................28 Discussion ...................................................................................................................29 4 TIMELINE OF MICROGLIA PR OGRAMMED CELL DEATH IN THE FACIAL NUCLEUS FO LLOWING INJURY..........................................................31 Introduction .................................................................................................................31 Materials and Methods ...............................................................................................32 Animals and Tissue Processing ...........................................................................32 TUNEL and DAPI Staining .................................................................................33 Quantitative Analysis ..........................................................................................33 Results .........................................................................................................................33 Discussion ...................................................................................................................36 5 MICROGLIA UNDERGO MORPHOLOGICAL AND FUNCTIONAL ABNORMALITIES IN THE SUPE ROXIDE DISMUTASE 1 RAT........................38 Introduction .................................................................................................................38 Materials and Methods ...............................................................................................39 Animals and Surgery ...........................................................................................39 OX-42 and OX-6 Immunohistochemistry ...........................................................40 Quantification of Immunohistochemistry Labeling in the Ventral Spinal Cord .41 TUNEL labeling and cell identification in the spinal cord ..................................42 Lectin histochemistry ..........................................................................................42 TUNEL and Lectin Double Labeling ..................................................................43 Brown and Brenn Gram Stain .............................................................................43 Results .........................................................................................................................44 Discussion ...................................................................................................................56 6 CONCLUSION...........................................................................................................64 LIST OF REFERENCES ...................................................................................................70 BIOGRAPHICAL SKETCH .............................................................................................85 vi

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LIST OF TABLES Table page 2-1. Average intake of minocycline during experiments. .................................................16 5-1. Age and corresponding disease stage for animals used in experiments. ...................40 vii

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LIST OF FIGURES Figure page 1-1. Facial nerve diagram ...................................................................................................4 1-2. Proposed mechanisms of ALS .....................................................................................6 2-1. Quantitative analysis of microglial prolif eration reveals no difference in the facial nucleus in minocycline treat ed versus control animals. ...........................................20 2-2. Photomicrographs of tritiated thymid ine labeled cells in the lesioned facial nucleus.. ....................................................................................................................21 2-3. Motor neuron regeneration in the faci al nucleus following injury is unaffected following minocycline treatment .............................................................................21 2-4. Fluorogold labled neurons within the lateral and ventral intermediate sections of the injured facial nucleus.. ........................................................................................22 3-1. Chemical structures of in ternal standard and minocycline. .......................................26 3.2. Minocycline concentrations f ound through HPLC/MS/MS analysis. .......................28 4-1. A time line of TUNEL positive microglia in the facial nucleus following injury. ....34 4-2. Non-classical TUNEL positive cells we re found throughout the neuropil of the injured facial nucleus 14 days post-injury. ...............................................................35 4-3. Flourogold labeled neurons at various survival times following a crush injury in the rat facial nucleus. ................................................................................................36 5-1. Photomicrographs of micr oglia labeling in the cortex of SOD1 rats revealing no abnormal microglia mophology or activation ..........................................................44 5-2. Photomicrographs representing aberrant microglial activation at the level of the red nucleus in early onset SOD1 animals. ................................................................47 5-3. Abnormal morphological changes seen at the level of the facial nucleus in SOD1 rats.. ..........................................................................................................................48 5-4. Pathological changes occurring in early SOD1 symptomatic animals. .....................49 viii

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5-5. Percentage of area covered by OX-6 i mmunoreactive cells in the ventral horn of lumbar spinal cord of SOD1 transgenic rats and age-ma tched control rats from 74 days to156 days. ..................................................................................................49 5-6. Photomicrographs demonstrating change in OX-6 expression with age ...................50 5-7. Photomicrographs demonstrating change in OX-42 expression with age .................51 5-8. Percentage of area covered by OX-42 i mmunoreactive cells in the ventral horn of the lumbar spinal cord of SOD1 transgenic rats and age-matched control rats from 74 days to156 days. .........................................................................................52 5-9. Microglial response a nd changes in SOD1 animals in the ventral spinal cord .........53 5-10. TUNEL positive cells in the ventral lumbar spinal cord in ALS animals. ..............54 5-11. Number of TUNEL positive cells in facial nucleus 14 days post axotomy in non-symptomatic transgenic animals was signifcantly less when compared to and age-matched control animals. ............................................................................55 5-12. Apoptotic microglial cells in the facial nucleus following injury. ..........................58 ix

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Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ROLE OF MICROGLIA DURING MOTONEURON REGENERATION AND DEGENERATION: RELEVANCE FOR PATHOGENESIS AND TREATMENT OF AMYOTROPHIC LATERAL SCLEROSIS By Sarah Emily Fendrick August 2006 Chair: Wolfgang J. Streit Major Department: Neuroscience Recently, microglial activation has been id entified as a contributing factor in a number of neurodegenerative diseases and b een targeted for therapeutic treatment. However, this view fails to consider the neuroprotective role of microglia observed in injury models where microglial activation a ccompanies neuronal regeneration. The main goal of this study was to investig ate the role of microglia activ ation in an injury model as well as the transgenic SOD1 rat model and characterize the accompanying neuronal responses. In order to determine if microglia l activation in the facial nerve paradigm is beneficial, minocycline, a drug shown to i nhibit microglial activation, was utilized allowing neuronal regeneration to be assessed in the absence of mi croglial activation. Microglial activation was assessed in the SOD1 transgenic rat mode l to characterize the role of microglial activation in a neurodegenerative disease. In this study we investig ated the effect of minocycline specifically on microglial mitotic activity and neuronal regeneration within the facial nucleus following a nerve x

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crush injury. No significant differenc e was found between minocycline treated and control rats when comparing the 3 H-thymidine labeled microglial cells or fluorogold labeled neurons at all post-inj ury time points investigated. To assess microglial activation in the ALS rat model, microglial morphology and activation were assessed in various brain re gions at three stages of the disease, asymptomatic, onset of symptoms and end stag e. Microglia were found to have a normal resting morphology in the motor cortex at all time points assessed. In the ventral spinal cord and brainstem there were signs of intense microglial acti vation. In addition, microglia fusions and multinucleated giant cells were seen dispersed throughout the brainstem and ventral horn of the lumbar spinal cord. To further assess microglial response and function in the transgenic mode l a facial nerve axotomy was performed and apoptotic microglial were quantified. Transgenic animals were found to have significantly reduced numbers of apoptotic mi croglial when compared to the age-matched controls 14 days post axotomy. The findings in the current study suggest that microglia may undergo both functional and morphological changes as a result of mutant SOD1 contributing to the disease. xi

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CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW Microglia: An Overview Introduction Microglia, the resident central nervous system (CNS) macrophage, represent about 10% of the adult brain cell population (Lawson et al., 1990). Historically, microglia research focused on the ontogeny of microglia with two conflic ting hypotheses. One hypothesis states microglia precursors are cell s of neuroectodermal origin (Kitamura et al., 1984; Fedoroff and Hao, 1991; Hao et al ., 1991; Fedoroff et al., 1997); the other proposes they proceed from mesodermal cells and originate outside of the developing CNS (Perry and Gordon, 1991; Ling and W ong, 1993; Cuadros and Navascues, 1998). The latter view is currently the most accep ted by those in the field who believe that microglia derive either from monocytes th at leave the blood stream and populate the brain parenchyma or from primitive hemopoie tic cells that differentiate as microglial cells within the CNS. The presence of prim itive microglia in the developing CNS can be detected prior to the appearance of monocyt es causing speculation on the theory that microglia precursors are monocytes (Hurley and Streit, 1996; Alliot et al., 1999). An alternative precursor for microglia is prim itive hemopoietic cells, also called fetal macrophages (Streit, 2001). Fe tal macrophages form in the blood islands of the yolk sac, enter the CNS via the meninges by transversing the pial surface (Navascues et al., 2000) and expand until assuming the fully differen tiated, ramified adult microglial cells. 1

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2 In the healthy adult CNS, microglia c onstitute a stable cell population, which maintains itself by proliferation of resident microglia or recruitment of bone-derived cells (Barron, 1995; Kreutzberg, 1996; Simard and Rivest, 2004; Bechmann et al., 2005). Resident microglia in the healt hy brain are termed r esting however this is far from their actual dynamic and motile func tions performed. Resident microglia undergo constant structural changes allowing microglia to effectively survey and maintain the CNS environment by sampling the environment with highly motile protrusi ons (Nimmerjahn et al., 2005). Upon injury, microglia under go morphological and phenotypic changes specifically cells undergo hypert rophy, proliferate and up regul ate surface antigens and various cytokines to tr ansform into an activated state (G raeber et al., 1988a; Streit et al., 1989b, 1989a). Microglia Are Neurosupportive Many studies have demonstrated the ability of microglia to produce growth factors (transforming growth factor (TGF-), IL-, and nerve growth f actor) (Giulian et al., 1986; Kreutzberg, 1996; Nakajima et al., 2001) that aid in neurona l survival during development and following injury. Further evidence for a neurosupportive role of microglia is seen in vitro studies in which cultured ne ocortical and mesencephalic neurons show enhanced survival and ne urite outgrowth following treatment with conditioned microglial medium (Nagata et al., 1993). In vivo microglial activation in the facial nerve paradigm accompanies neuron al regeneration following injury whereas a central axotomy, such as a transaction of the r ubrospinal tract in the cervical spinal cord, does not result in regeneration and elicits onl y a minimal microglial response (Barron et al., 1990; Tseng et al., 1996; Streit et al., 2000). These in vivo observations clearly support that microglial activation is required to facilitate regeneration. More direct

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3 evidence is seen in transplantation studies that show cultured microglial cells engrafted into the injured spinal cord promote neurit e outgrowth (Rabchevsky and Streit, 1997). Another function of microglia is to ma intain the environment in the CNS by removing cellular debris and dysfunctional cells In the presence of degenerating neurons microglia transform into phagocytic cells th at remove damaged cells eliminating the potential for toxic products to be rele ased into the CNS environment. Facial Nerve Axotomy The facial nucleus is the largest brai nstem motor nucleus in the rat with approximately 3000-5000 motoneurons innervat ing muscles controlling facial movement, including whisker movement. The motoneuron s are organized into several muscle and region specific nuclei located in the ipsilateral brainstem. Axotomy of the facial nerv e is a well-established paradigm for the study of microglial activation in response to injury. Peripheral injury of cranial nerve VII generates a response in the CNS while maintain ing the integrity of the blood brain barrier restricting leukocyte inf iltration. In addition, this model has proven useful in microglia research because the injury is reproducible a nd can be done in the absence of direct brain manipulation. The microglial response is well -documented following facial nerve injury. Early activation is seen within 24 hours of injury and is characterized by an increase in molecules with an immune function as we ll as up regulation of OX-42 immunoreactivity (Graeber et al., 1988a; Kreutzbe rg et al., 1989). The next stage of microg lial activation occurs 2-4 days post injury when microglia proliferate and begin to home and adhere to the axotomized motoneurons allowing microglia l processes to strip away afferent axon terminals (Blinzinger and Kreutzberg, 1968) Following nerve reinnervation which occurs 2 weeks post injury in the rat, microglia decline in number and return to a resting

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4 state. It has been proposed that microg lia undergo cell death fo llowing recovery of axotomized neurons, possibly through apoptos is (Gehrmann and Banati, 1995; Jones et al., 1997). Figure 1-1. Lateral view of the facial nerv e (7N) with its upper (UP) and lower (IP) peripheral branches that innervate the vi brissae follicular muscles. Arrowhead identifies the location of crus h site. (Kamijo et al., 2003) Amyotrophic Lateral Sclerosis Amyotrophic Lateral Sclerosis (ALS) is one of the most common adult-onset neurodegenerative disease affecting ~5 pe r 100,000 individuals. First described by Charcot in 1869, ALS is characterized by th e selective loss of upper and lower motor neurons invariably progressing to paralysis and death over a 1-5 y ear time course. Its etiology is still poorly understood; however a major breakthrough in the field occurred with the discovery that mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene affect approximately 20% of patients with familial ALS (Rosen, 1993; Siddique and Deng, 1996). This discovery allowed for generation of transgenic an imal models which

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5 closely resemble the motor weakness and de generation seen in human disease (Gurney, 1994; Wong and Borchelt, 1995; Bruijn et al., 1997; Nagai et al., 2001; Howland et al., 2002). Prior to animal models, it was proposed the mutations in SOD1 reduced enzyme activity causing decreased free radical scaven ging activity and an increase in oxidative stress. Contradicting evidence emerged fr om transgenic SOD1 mice that developed progressive motor neuron disease despite possessing two normal mouse SOD1 alleles (Bruijn et al., 1998; Jaarsma et al., 2001). In addition, SOD1 knockout mice live until adulthood and do not develop motor neuron disease (Reaume et al., 1996) indicating that the mutation in SOD1 causes a toxic gain of function rather then a loss of dismutase activity. The mutant SOD1 may catalyze abe rrant biochemical reactions which result in production of damaging reactive oxygen species (ROS) such as the superoxide anion, the hydroxyl radical, hydrogen peroxide and pero xynitrite (Cluskey and Ramsden, 2001). Misfolding of the mutant protei n causes copper at the active s ite to be less tightly bound increasing the release of copper. The uno ccupied active site is more accessible to abnormal substrates such as peroxynitrite whic h leads to nitration of tyrosine residues (Beckman et al., 1993) and hydrogen peroxide wh ich generates hydroxyl radicals that can damage cellular targets (Wiedau-Pazos et al., 1996). In addition, to aberrant chemical reactions occurring at the active site, the SOD1 toxic gain of function may be due to its participation in formation of protein aggregat es. Protein aggregates or inclusion bodies intensely immunoreactive for SOD1 are found in motor neurons of the mouse model of ALS (Bruijn et al., 1997). Aggregates may be toxic due to additional proteins associating with them thereby depleting pr otein functions that may be essential for neuronal survival. Another hypothesis is that by repetitively misfol ding, mutant proteins

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6 are reducing availability of chaperones for proteins required for normal cell function. A final hypothesis relating to aggreg ates and their role in ALS is that SOD1 mutants reduce proteasome activity needed for normal protei n turnover. SOD1 aggregates are very stable and even with treatment using strong de tergents and reducing ag ents the aggregates are not easily dissociated. Formation of SO D1 aggregates disrupts normal balance of protein synthesis and degradation interrupting normal degradation of misfolded proteins critical to cell survival. While familial ALS has been attributed to mutations located within the SOD1 gene, mechanisms underlying onset and progression of sporadic ALS, which accounts for the largest percentage of cases, are still largely unknown. It has been hypothesized that ALS is due to excitotoxcity, protein aggregation, mitochondrial dysfunction, and recently it has been proposed microglial activation is a co ntributing factor in th e disease process. Figure 1-2. Proposed mechanisms of ALS. (Cleveland, 1999)

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7 Microglia in ALS Glial cell involvement in ALS pathology is unknown; however recent studies have presented strong evidence for non-neuronal ce ll involvement in ALS. A series of experiments revealed that SOD1 mutations ar e not directly toxic to motor neurons, but rather exert their neurotoxic effects in a non-cell autonomous fashion. Initial experiments showed expression of mutant SOD1 in neur ons (Pramatarova et al., 2001; Lino et al., 2002) or astrocytes (Gong et al., 2000) alone failed to indu ce motor neuron degeneration. Clements et al., further implicated glial cells in ALS pathology by showing neuronal degeneration is dependent not on the type of cell carrying the mutant SOD1 gene, but rather the number of cells. This conclusi on was reached from an experiment in which mutant SOD1 expression in individual ne urons surrounded by wild type glial cells allowed neuronal populations to remain healt hy whereas when the reverse occurred with wild type neurons surrounded by mutant SOD1 glial cells the result was neuronal degeneration (Clement et al ., 2003). The role of glial cells in ALS is clearly demonstrated in experiments manipulating e xpression of mutant SOD1 in various cell types however further experime ntation has specifically shown a role for microglia in ALS pathology. Cell-specific knock down of mutant SOD1 in microglia and macrophages in transgenic mice cause increased life span (C leveland, 2004). Further evidence is seen in a study conducted by Weydt where microglia isolated from transgenic mice showed significantly higher levels of tumo r necrosis factor alpha (TNF) when compared to agematched controls (Weydt et al., 2004). Cellular evidence of microglia involvement can be seen in histological studies of both human and animal tissue where microglial activation and proliferat ion is seen in regions of motor neuron loss such as the spinal cord, brainstem and primary motor cortex (Kawamata et al., 1992; Hall et al., 1998;

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8 Almer et al., 1999; Alexianu et al., 2001; Henkel et al., 2004). Microglial activation temporally corresponds to onset of motor weakness and neuronal loss. Accordingly, increased microglia production of TNF, IL-1, iNOS, and COX-2 all pro-inflammatory mediators are present in ALS patients suggest ing a likely role of neuroinflammation in ALS etiology (Poloni et al., 2000; Elliott, 2001; Nguyen et al., 2001; Olsen et al., 2001; Hensley et al., 2002; Yoshihara et al., 2002). In addition to the products listed above, there are a number of microglial activating f actors found to be elevated in ALS patients providing a plausible source for persistent microglial activ ation. It is unknown if microglial activation is triggered as a res ponse to neuronal death or some other manner however it appears that once act ivated they are able to self-activate furthering an inflammatory environment. It should be noted that unlike other neurodegenerative diseases, influx of peripheral immune cells is a rare occurrence only seen at the end stage of ALS (Kawamata et al., 1992; Bruijn et al., 2004). Therefore, in ALS inflammatory reactions are generated and su stained only by CNS cells most likely microglia since they are the major immunocompetent cells of the CNS. Evidence to date strongly supports active participation of micr oglial cells in ALS pathogene sis however, it still remains unknown in what manner microglia exert thei r detrimental effects and if microglial activation is a secondary effect of the disease process or an initial contributing factor. Neuroinflammation Classically, inflammation is a complex re sponse which aims to repair tissue damage and is accompanied by the cardinal points described by Celsus: pain, tumor, rubor and heat. Under normal conditions in flammation is a tightly controlled process maintained until the initial stimulus is repaired or eliminated however the inability of the immune system to clear the foreign target or repair the existing stimuli results in chronic

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9 stimulation of immune cells thereby resu lting in damage to tissue (Nathan, 2002). Chronic inflammation in the CNS, often referred to as neuroinf lammation, is defined by the presence of activated micr oglia, reactive astrocytes and inflammatory mediators with activated microglia being the central component. Recently, neuroinflammation has been implicated in a number of neurodegenerative diseases such as Parkinsons, Alzheimers and ALS. Microglial activation and its role in neurodegenerative diseases is a highly debated topic in that it still remains unclear whether microglial activation is beneficial or detrimental. In the non-diseased brain micr oglia are found in a resting ramified state where upon acute injury microglia prolifer ate and undergo morphological changes to attain a state of activation to aid in repa ir of damaged tissue. Following recovery microglia return to a resting state however in neurodegenerative di seases microglia are thought to maintain a persistent state of activation. It is proposed that neuroinflammation may drive a self-propagating toxic cycle of mi croglia in which several factors of disease such as protein aggregates, injured ne urons, and AB plaques activate microglia exacerbating neuronal death through production of pro-inflammatory products which in turn further increases levels of microg lial activation. Evidence supporting proinflammatory properties of mi croglia is extensive in neurodegenerative diseases however whether the microglial reaction is a secondary response to neuronal death or a causative factor still remains unresolved. Nonetheless, therapeutic treatments targeting inflammation in a number of ne urodegenerative diseases ar e currently un derway with variable results. COX-2 inhibi tor, rofecoxib, has been used for treatment of Alzheimers and Parkinsons however clinical trials failed to demonstrate a benefici al effect (Aisen et al., 2003; Przybylkowski et al., 2004; Reines et al., 2004). These fi ndings suggest that

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10 inflammation may be a secondary cause of ne urodegenerative diseases due to the failure of treatment to halt disease progression. Microglia Dysfunction An alternative to neuroinflammation is that microglial cells undergo functional changes in the diseased brain where they acqui re toxic functions or become incapable of performing normal functions in the CNS. Th e inability of microglia to perform their normal function may have detrimental conseq uences for neurons thereby propagating neuronal degeneration. Microg lia dysfunction has been observed in HIV patients, the normal aging brain and neurodegenerative diseases. HIV-1 associated dementia (HAD) is a syndrome of motor and cognitive dysfunction in 10% of patients infected with HIV-1 with acquired immune deficiency syndrome (McArthur et al., 1993; Sacktor et al., 2001). Neurons are not productively infected with the virus sugges ting infected cells in the CN S, in particular microglia, release signals that l ead to secondary neuronal injury. Microglia can be activated by HIV infection itself, by interaction with viral protei ns or by immune stimulation in response to factors released from other infected cells (Lipton and Gendelman, 1995). In response to activation, microglia have increased production and release of neurotoxic immunomodulatory factors such as pro-in flammatory cytokines, free radicals and neurotoxic amines (Genis et al., 1992; Achim et al., 1993; Bukrinsky et al., 1995; Giulian et al., 1996; Zhao et al., 2001). Further evidence of abnormal mi croglia is the presence of multinucleated giant cells which are found in close proximity to apoptotic neurons. Phenotypic changes have been shown in micr oglia with aging such as up regulated expression of MHC II (Perry et al., 1993; O gura et al., 1994; DiPatr e and Gelman, 1997; Streit and Sparks, 1997; Morgan et al ., 1999), greater exhibition of phagocytic

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11 morphology and IL-1 immunoreactivity (Sheng et al., 1998). In addition, several studies have reported significant changes in microglia morphology, including cytosolic inclusions (Peinado et al., 1998), higher in cidence of clumping in and around white matter (Perry et al., 1993) and structural changes. Morphological abnormalities in microglia have been identif ied in the Huntingtons mous e model (Ma et al., 2003) and human Alzheimers tissue. Structural cha nges included bulbous swellings, long stringy processes, cytoplasmic fragmentation and deramified processes. Corresponding to phenotypic changes microg lia appear to undergo functional changes due to aging. In cultu re studies and in the facial nerve paradigm aged microglia display an increased proliferative response (Rozovsky et al., 1998; Conde and Streit, 2005). In vitro microglial proliferation progressive ly increases with donor age up to 400% greater at 24 months vs. 3 months (R ozovsky et al., 1998). Following a facial nerve axotomy microglial proliferation in ag ed rats is significantly higher 4 days following axotomy (Conde and Streit, 2005). The enhanced proliferation of aged microglia may be due to a loss of response to regulatory mechanisms which is supported by culture experiments where aged microglia did not respond to TGFin contrast to young microglia that showed an inhibition of proliferation following TGFtreatment (Rozovsky et al., 1998). This lo ss of sensitivity to TGFwith increased age was also demonstrated in the regulation of prolactin in rat anterior pitu itary cells (Tan et al., 1997). The desensitization to anti-proliferative proper ties of TGF -1 provides a plausible cause for the increased proliferative response of aged microglia. Further impairment of TGF1 regulatory mechanisms is seen in cu ltured microglia. Lipopolysaccharide (LPS) treatment induces NO production in all donor age cultures ho wever in young donor

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12 cultures NO production is inhibited following TGF-1 but TGF-1 has no effect on NO production in aged donor cultures (Rozovsky et al., 1998). Aging-related changes in microglia appear to affect the regulatory m echanism of microglial activation causing the homeostasis of the local CNS environment to be disrupted. Minocycline Minocycline is a second generation tetracyc line with antibiotic activity against a broad-spectrum of bacterial types incl uding both Gram-positive and Gram-negative bacteria. Completely separate and distinct from its antimicrobial activity, minocycline exhibits anti-inflammatory effects that are proven to be neuroprotective in a number of neurodegenerative diseases and brain ischemia (Yrjanheikki et al., 1998; Yrjanheikki et al., 1999; Du et al., 2001; Kriz et al., 2002; Wu et al., 2002). Although other tetracyclines can diffuse across the blood-brai n barrier into the CNS in small amounts, the lipophilicity of minocycline allows it to a ttain significantly higher levels in the CNS furthering its therapeutic potenti al in neurodegenerative dis eases (Barza et al., 1975). One proposed mechanism of minocycline is a direct neuroprotective action in which caspases are inhibited by preventing release of mitochondrial cytochrome c (Zhu et al., 2002; Teng et al., 2004) Another proposed action of the drug is deactivation of microglial cells indirectly accounting for the observed neuroprotecti on (Yrjanheikki et al., 1999; Du et al., 2001; He et al., 2001; Kriz et al., 2002; Wu et al., 2002). Microglia deactivation occurs through inhibition of p38 MAPK which is thought to mediate the inflammatory process within microglia by i nducing transcription f actors that positively regulate inflammatory genes (Tikka et al., 2001; Koistinaho and Koistinaho, 2002). Minocycline treatment administered to S OD1 mice delays onset of motor neuron degeneration and increases longevity of SOD1 mice lifespan (Kriz et al., 2002; Van Den

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13 Bosch et al., 2002; Zhu et al., 2002). Currentl y, clinical trials are underway to determine the benefits of minocycline treatment in human ALS patients. Minocycline appeared to hold great therap eutic treatment for neurodegenerative disease however recent findings showed a deleterious effect in Parkinsons (PD), Huntingtons (HD) and hypoxic-ischemia (HI) animal models (Smith et al., 2003; Yang et al., 2003; Tsuji et al., 2004). In a PD model minocycline increased 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) toxicity and s howed no effect in the transgenic mouse model of HD (Smith et al., 2003; Yang et al., 2003). It was also reported that in two chronic models: the MPTP-intoxicated nonhuman primate model of PD and the 3nitropropionic acid (3-NP) intoxicated model of HD minocyc line treatment resulted in earlier clinical motor symptoms during toxic treatment, decreased motor performance and greater neuronal loss when compared to controls (Diguet et al., 2003; Di guet et al., 2004). In addition, minocycline is proposed to exacerbate hypoxic-ischemia br ain injury in the immature mouse cortex, thalam us and striatum while neuropr otective in the immature rat brain (Arvin et al., 2002; Tsuji et al., 2004). In models of HI, deleterious effects of minocycline may be due to the reduction in compensatory angiogenesis after HI by inhibiting endotheli al proliferation.

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CHAPTER 2 MINOCYCLINE DOES NOT INHIBIT MICROGLIA PROLIFERATION OR NEURONAL REGENERATION IN THE FACIAL NUCLEUS FOLLOWING A FACIAL NERVE CRUSH Introduction Minocycline is a second-generation tetr acycline reported to have an antiinflammatory activity independe nt of its antimicrobial f unction (Amin et al., 1996). Recently, minocycline has been shown to i nhibit microglial activation and promote neuronal survival in animal models of neur odegenerative disease and stroke (Yrjanheikki et al., 1999; Du et al., 2001; He et al., 2001; Kriz et al., 2002 ; Wu et al., 2002). It has been hypothesized that in neurodegenerati ve disease microglia undergo detrimental activation characterized by in creased production and rel ease of neurotoxins that contribute to neuronal cell deat h. Accordingly, minocycline has been proclaimed as a potential treatment for neurodegenerative dise ases such as amyotrophic lateral sclerosis (ALS) and Parkinsons disease which are thought to have a neuroinflammatory component in their pathogenesis (Du et al ., 2001; Kriz et al., 2 002; Wu et al., 2002). On the other hand, experimental studies afte r acute brain injury show that microglial activation is a consequence of ne uronal injury rather than the cause of it. In particular, experimental paradigms i nvolving neuron regeneration su ch as motoneuron axotomy, show that microglial activation precedes successful regenerati on of severed axons suggesting that activated microglia ar e neuroprotective and support motoneuron regeneration (Streit, 1993, 2002, 2005). Fo llowing axotomy, greater numbers of microglia are generated through local prolifer ation (Graeber et al ., 1988b) and these cells 14

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15 encircle the injured neurons in a manne r that suggests neuroprotection through displacement of afferent synapses and cl ose glial-neuronal apposition which may allow for targeted delivery of microglia-derived growth factors, such as TGF-beta (Mallat et al., 1989; Martinou et al., 1990; Araujo and Cotm an, 1992; Elkabes et al., 1996; Lehrmann et al., 1998). Furthermore, there is little if any neuronal cell death with in the facial nucleus of the rat following axotomy (Johnson and D uberley, 1998) suggesting that microglia are aiding with recovery of damaged neurons ra ther than harming them (Lieberman, 1971; Streit and Kreutzberg, 1988; Stre it, 1993; Kuzis et al., 1999). To directly test the hypothesis that mi nocycline inhibits microglia activation in vivo we have quantified microglial proliferati on in the axotomized facial nucleus. To determine if there are functional consequen ces for neurons of this potential microglial inhibition, we have also quantified and comp ared numbers of regenerating motoneurons between minocycline treated and non-treated animals. Materials and Methods Animals and Diet Animal use protocols were approved by the University of Florida Institutional Use and Care of Animals Committee (IUCAC). Young adult male Sprague Dawley rats were divided into two groups, one r eceiving a standard rodent di et while the second group was fed a diet enriched with minocycline (1gr am/kilogram) obtained from Harlan Tekland (Madison, WI). The diets were implemented one week prior to surgery and continued throughout the remainder of the experiment. Levels of food intake were recorded for each cage of animals. Both treatment gr oups underwent a unilateral facial nerve axotomy.

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16 Table 2-1. Average intake of minocycline during experiments. Survival Time Point for Proliferation Studies Number of Days on Diet Average Drug Intake/Animal (grams) 2 days post-axotomy 9 0.19286 3 days post-axotomy 10 0.24166 4 days post-axotomy 11 0.27190 Survival Time point for Flurogold Studies 7 days post-axotomy 14 0.30158 14 days post-axotomy 21 0.50119 21 days post-axotomy 28 0.63218 Facial Nerve Axotomy Animals were anesthetized with isoflurane using a precision vapor machine with gas scavenging system attached. Animals were placed in an inducing box where isoflurane was administered until the pe dal and palpebral reflexes we re absent. Animals were transferred to a nose cone where isoflurane was administered to the animal for the remainder of surgery. Upon full sedation, a sm ade directly behind the right ear. Using a pair of angled scissors, th e superficial levels of the muscle tissue were cut until the facial nerve was exposed. Both branches of the facial nerve were separated from the surrounding tissue and crushed with of hemostats for 10 sec. The incision was closed with a surgical staple and animal s were removed from isoflurane and closely monitored until fully recovered. The absencker movement was assessed to confirm that both nerve branches were compcrushed. 3H-Thymidine Injections and Radioactive Perfusions Microglial proliferation was assessed at 2, 3 and 4 days after facial nerve crush since it is known that the burst of mitotic ac tivity occurs during this time period. Each time point included ten animals, five in eachgroup. Animals were weighed and given an intraperitoneal (i.p) injection of 3 Ci per gram body weight of [methyl-3H] all incision was m a pair s e of whi letely treatment

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17 thymidine (Amersham Pharmacia Biotech) tw prior to perfusion. Animals were caged in radioactive labeled cages until perfu Two hours following the 3H-Thymidin each animal was given a lethal ose of sodium pentobarbital (150 mg/kg; i.p.). In the absence of pedal and palpebral scardially perf used with phosphate buffered saline (PBS) fol s dry sity of ain sections were placed in cassette holders. Cassette holde rs o hours sion. e injection d reflexes animals were tran lowed by 4% paraformaldehyde (in PBS). Following perfusion brains were dissected out and placed in 4% paraformaldehyde overnight at room temperature. Each animal wa assigned a random number to maintain objectivit y in quantitative analys is. All liquid and animal waste were labeled as radioactive and disposed according to the Univer Florida waste management protocol. Tissue Processing for Audoradiography Following overnight fixation, a coronal se ction of the brainstem containing the facial nucleus was dissected out and rinsed in PBS. E ach section was processed for paraffin embedding by slowly dehydrating th rough an ascending series of alcohol beginning with 70% ethanol for 2h, 45 min for 70%, 90%, 95%, 100% ethanol then 100% ethanol overnight. Following overnight in cubation, br two changes of xylenes for 2h and transferred to paraffin rs were immersed in 2 changes of Surgipath Formula R paraffin (Surgipath, Richmond, IL) at 60C for 2h each. Lastly, th e tissue was removed from cassette holde and embedded in Surgipath Tissue Embedding Medium paraffin (Surgipath, Richmond IL) and allowed to cool. The facial nucleus was serially cut (7m sections) from rostral to caudal on a microtome and collect ed on Superfrost Plus slides.

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18 Audo he c ounterstained with 0.5% cresyl violet, ount mounting mediu e facial 6 lined pared treated and non-treated animal s at each time point with a t test using Graph radiography Immediately before beginning the developi ng process, slides were deparaffinized and hydrated through a series of descending al cohols then rinsed in PBS for 5min. T sections were dipped in a 50% solution of NTB-2 emulsion (Eastman Kodak) and allowed to air dry in a darkroom with a sa felight with Kodak #2 filter. Slides were exposed in lightprotected slide boxes with desiccant at 4C for 5 weeks and developed with 50% Dektol developer for 2.5min (Eastman Kodak), rinsed in ddH 2 O for 10dips, fixed in Kodak fixer for 5min, washed, dehydrated through alcohols and xylenes a nd coverslipped using Perm m (Fisher Scientific). Quantitative Analysis for 3 H-thymidine Labeled Microglia For quantitative analysis of proliferating microglia ever y fourth section of th nucleus was counted for 3 H-thymidine labeled microglial cells. The sections were viewed using a Zeiss Axiophot microscope with a Sony DXC970 camera attached. In the selected sections the facial nucleus was outlined and the area measured using MCID software (Imaging Research, St. Catherines, Ontario). The labeled cells in the out area were manually counted under 40X magni fication. Labeled cells were pooled together for each animal and divided by th e pooled area measured for each animal to determine a population density of proliferating microglia in the facial nucleus. Results are represented as mean values SEM. Th e density of dividing microglia was com among minocycline Pad Prism software (GraphPad Software San Diego, CA). A significance level of p<0.05 was used.

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19 Fluorogold Labeling Regeneration of motor neurons following minocycline treatment was investigated at 7, 14, and 21 days after facial nerve crush. Each time point included ten animals, fiv in each treatment group. Three days before perfusion, animals were given two 1 injections of 4% fluorogold (in saline): one into the whisker pad and one directly underneath the eye. e 0 L ng lowed and rogold labeled cells were compared among minocycline treated and each time point with a t test. A significance level of p<0.05 was used. 3, and 4 days post facial nerve axotomy. Our objective was to determine in vivo if Perfusion and Tissue Processing for Fluorogold Labeling The transcardial perfusion was performed as detailed in the previous radioactive perfusion section, however in the absence of radioactive precauti ons. Following the perfusion brains were dissected out and placed in 4% paraformaldehyde until sectioning. The brainstem containing the facial nucleus was dissected out and mounted onto a cutti block. The facial nucleus was sectioned caudal to rostral on a vibratome in 50 m sections and collected on SuperFrost slides. Following collection the slides were al to air dry for one hour, dehydrated through a series of ascending alc ohols and xylenes coverslipped with Permount mounti ng medium (Fischer Scientific). Quantitative Analysis of Fluorogold Labeling For quantitative analysis of regenerating neurons the en tire facial nucleus was counted for fluorogold labeled cells. Results are represented as mean values SEM. The number of fluo non-treated animals at Results Microglia proliferation is not inhibited in minocycline treated animals at 2,

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20 minocycline could attenuate microglial activatio n, and to this end we decided to m cell proliferation, whi easure ch is a reliable, quan tifiable parameter of microglial activation. cells at all three time points exam ring Consistent with pr ior reports, we found 3 H-thymidine-labeled ined. However no statistically signi ficant difference between the number of 3 Hthymidine cells was found in the injured faci al nucleus at any time point when compa the control rats and the rats fed a diet enri ched with minocycline in the injured facial nucleus at all three time poi nts. No cells labeled with 3 H-thymidine were observed on the uninjured side of the facial nucle us within either treatment group. Figure 2-1. Microglial prolifer ation in the facial nucleus. There is no statistically is 1.12 significant difference in the cell proliferation between the control animals and the animals fed a diet enriched with minocycline at any post-axotomy time point. (Difference at day two is 1.1 4 1.171 (p = 0.3587), at day three 0.5775 (p = 0.1207), and difference at day four is 0.29 0.1922 (p = 0.2364)).

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21 the Figure 2-2. Photomicrographs of 3H-thymidine labeled cells in the injured facial nucleus. A) Control rat 3 days post-injury. B) Minocycline treated rat 3 days postinjury. Note that 3H-thymidine labeled microglia are in close proximity to regenerating neurons Magnification 250x. Figure 2-3. Motor neuron regeneration in the facial nucleus. There is no statistically significant difference in the number of fluorogold labeled neurons between the control animals and the animals fed a diet enriched with minocycline at 14 and 21 days after nerve crush. Differe nces between treatment groups at day 14 is 6 8.051 (p = 0.4774) and at day 21 is 10.8 32.12 (p = 0.7454) Neuronal regeneration is not inhi bited by minocycline treatment. Since any attenuation of microglial act ivation by minocycline could have an effect on neuronal

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22 regenerative ability after nerve crush we performed fluorogold injections 7, 14 and 21 ing a unilateral facial nerve crus h to determine the number of neurons that days follow underwent successful reinnervation. No fl uorogold labeled neurons were found on day 7 after nerve crush; they first became appare nt at 14 days and became more numerous by day 21. The results show that successful re innervation of facial muscles takes place between 2 and 3 weeks (Soreide, 1981; Fawce tt and Keynes, 1990). The counts of regenerating neurons throughout the facial nucleus show that minocycline did not influence regeneration of neurons between the control and minocyc line treated groups. ions s Figure 2-4. Fluorogold labl ed neurons within the lateral an d ventral intermediate sect of the injured facial nucleus. A) Contro l rat 21 days post-facial nerve injury. B) Minocycline treated rat 21 days post-facial nerve injury. Bar = 100 m. Discussion In this study we report that microglial proliferation in vivo is not inhibited by minocycline within the facial nucleus followi ng a nerve crush. These results differ from those reported in previous studies, which found that minocycline significantly inhibit

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23 microglial proliferation in vitro (Tikka et al., 2001; Tikka and Koistinaho, 2001). T reconcile this apparent contra diction in experimental finding s, one needs to consider the o ct that there are fundamental differences between microglia in vitro and in vivo (Streit, al., 2001; Kriz et al., 2002; Tomas-Camardiel et al., 2004). In these studies a decrease in expression of OX-6, OX-42 or Mac-2 w lowing minocycline treatment which, in tur e is fa 2005). Specifically, with regard to cell division, it is impor tant to note that microglial cells in culture undergo mitosis constitutively and spontaneously, because they exist in a permanent state of activation in vitro This differs from resting microglia in the CNS, which only become activated and proliferate in the presence of a stimulus, such as acute CNS injury. This mismatch of activation stat es between cultured microglia and microglia in the brain likely accounts for the differe nces in proliferation dynamics observed after minocycline treatment in vitro and in vivo In addition, previous studies conducted in the SOD1 and Parkinsons mice (LPS and 6-hydrox ydopamine induced) classified activated microglia by the presence of cell surface markers such as OX-6, OX-42 and Mac-2 (He et as f ound fol n, led to the conclusion that by decr easing microglial activation life span in the SOD1 mouse is increa sed and dopaminergic cells become partially protected in the Parkinsons mouse. However, the exclusive use of cell surface markers is not a reliabl method for quantification of ac tivated microglia. OX-6 labeli ng of MHC II expression not always indicative of microglial activation considering that not all activated microglia are MHC II positive and MHC II expression can be found on resting microglial cells in the normal rodent brain, often in the white matte r (Streit et al., 1989a). The decrease in expression of OX-42 and Mac-2 following minoc ycline treatment could be a result of a direct neuroprotective effect of the drug which may allow greater neuronal survival

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24 thereby decreasing the intens ity of the reactive microglio sis. It has been shown in vitro and in vivo that minocycline is able to inhibit cyctochrome c release as well as activation of downstream caspase dependent and independ ent cell death pathways (Zhu et al., 2002 Wang et al., 2003). Presently, c linical trials are underway to test th e effectiveness of minocycline vs. placebo in the treatment of ALS patients. These trials resulted after minocycline was found to increase life span in animal models representing familial ALS. It is hypothesized that mi nocycline increased life span in SOD1 mutant mice by inhibiting microglial ac tivation through the p38 MAPK pathway. However, as we show in this study minocycline fails to inhibit microglial proliferat ion, a key charac teristic o microglial activation. Perhaps, minocycline is effective in the SOD1 mouse by bein directly neuroprotective. In the facial nucleus following injury the number of regenerating neurons was unchanged between the minocycline treated animals and the control group as was the number of proliferating microglial. This finding offers further support for the idea that microglial activati on after CNS injury is a neuroprotective mechanism. ; f g

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CHAPTER 3 DETERMINATION OF MINOCYCLINE CONCENTRATION IN THE BRAIN AFTER DRUG ADMINISTRATION IN DIET Introduction The semisynthetic second-generation minoc ycline has neuroprote ctive effects in models of cerebral ischemia (Yrjanheikki et al., 1998; Yrjanheikki et al., 1999; Tikka et al., 2001). Minocycline has also been show n to prevent nigrostriatal dopaminergic neurodengene ration in mouse models of Parkin sons disease (Du et al., 2001; Wu et al., 2002), delay disease progression in a transgen ic model of Huntingt on disease (Chen et al., 2000) and increase life span in a mouse model of ALS (Kriz et al., 2002; Van Den Bosch et al., 2002; Zhu et al., 2002). While all these studies s uggest that minocycline might offer a useful pharmological a pproach for treatment of numerous neurodegenerative disease it is still unknown on the concentrations reached in the brain and how they relate to blood concentrations in animals. Analyses based on simple protein precipitation (Birminham et al., 1995), liquid-liquid (Mascher, 1998; Araujo et al., 2001) or solid-phase extraction procedures (Wrightson et al., 1998; Orti et al., 2000) followed by high-performance liquid chromatography (HPLC) with ultraviolet detection have been reported for the determination of minocycline in biological samples. The current study will inve stigate mincoycline concen trations in the brain following drug treatment administered through diet. The absorption of minocycline is believed to be affected by simultaneous ad ministration with food (Leyden, 1985; Meyer, 1996) therefore, to confirm that the lack of inhibition of microglia proliferation in chapter 25

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26 2 was not a result of insufficient levels of minocycline in the brain, HPLC MS/MS will be utilized to measure minocycline concen trations in the brain following drug intake through diet. Reagents Materials and Methods Minocycline hydrochloride and tetracycline hydrochloride were obtained from Sigma-Aldrich. Stock solutions were prepared by dissolving tetracyclines in methanol at a concentration of 1 mg/mL. Working sta ndard solutions were prepared from stock solutions by dilution with methanol. To prep are standards for the concentration curve, three concentrations of the analyte were prep ared in methanol; 0.05 g/mL, 0.5 g/mL and 2.5 g/mL. Figure 3-1. Chemical structures of internal standard and minocycline.

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27 Animals Two male Sprague Dawley rats weighing 200-250 g were used, one was fed a d enriched with m iet inocycline (1 gram/kilogram) for one week prior to analysis while the other rat was fed a standard rode ed by decapitation under deep brains were rapidly removed a nd blotted to remove excess surface blood and stored dy. rom o 4 were added, vortex mixed and centrifuged at 2000rpm at 4C for 30min. The precipitate was re of 0.01M phosphate buffer, pH 7.4 and centrifuged. Following centrifugation, th bined. To clean up supernatants 1m artri dges (Waters) were used. Prior to applying the hom le cartridges were preconditioned with 1 mL of methanol f ples were loaded into columns, the column thanol solution, and dried for 10 min through suction. The sam thanol. A blank sample was processed in tandem with brain sam ncern of contamination during extraction. Equipment tography was an Agilent p (Palo Alto, CA) equi pped with an Agilent 1100 UV/V detector nt diet. Animals were kill anesthesia, at -20C until analysis. Water and f ood were freely availabl e throughout the stu Extraction A 200 mg section of the brainstem containi ng the facial nucleus was removed f the whole brain and homogenized in 1 mL of 0.01M phosphate buffer pH 7.4, on ice. T 1 mL of the resulting homogenate 25 L of the internal standard tetracycline (10 g/mL) and 20 L of H 3 PO -dissolved in 1 mL e supernatants were com l/30mg Oasis HLB extraction c ogenized ti ssue samp ollowed by 1 mL of di stilled water. Sam s were rinsed with 1 mL of 5% me ples were eluted with 1 mL of me ple s to eliminate co The apparatus used for High Performan ce Liquid Chroma 1100 series binary pum

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28 set at 353 nm. Separation was carried out on a Pehnomenex Synergi 4u Hydro-RP 80A (Torr ace, CA) (2 x 150 mm, 4 um) plus C18 guard column (2 mm x 4 mm). Mass spectrometric detection was performed using a TehermoFinnigan LCQ (San Jose CA) with electrospray ionization (ESI). the minocycline treated animal there wa s a peak area showing a presence of there was no peak area corresponding to minocycline. Results Minocycline was found at a significant concentration in the treated animal. In order to confirm that minocycline had cro ssed the blood brain barrier, HPLC MS/MS performed on brain Figure 3.2. Minocycline concentrations found through HPLC/MS/MS analysis. A) In minocycline at a concentration of 0.4968 g/mL. B) In the control sample was stem tissue from a c ontrol animal and from an animal fed This showed a peak area for the minocycline compound in the treated anima extraction procedure, the concentration of minocycline was determined and was found at minocycline. l but not in the control animal. The extract of the minocycline-treated animal was analyzed twice. From the concentrati on in the methanol extract and knowing the

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29 a concentration of 0.4968 g/mL in the treated animal. Using the area of the standard tetracycline a calibratio n curve was created. A linear re gression yielded the equation of ent of this equation yielded the equation to determ 120m odel the 2.484 g/g found in the rat brains assessed in this study. Accordingly, it appears the lack of inh result of in f effect of minocycline on microglial activation. ine the in the the line: y = 9.338x 0.0314 where y = the area ratio Minocycline/Tetracycline and x is the [Minocycline] in g/mL. Rearrangem ine the [M] in the unknown: [M] = {(M/T) + 0.0314} /9.3338. Discussion The current study demonstrates that significant levels of minocycline were detected in the brain following drug administration in rat rodent diet. A study conducted by Du and colleagues found that minocycline orally administered at a concentration of g/kg in the 1-methyl-4-phenyl-1,2,3,6-te traydropyridine (MPTP) Parkinsons m provided neuroprotection to midbrain dopamine neurons from the toxic effects of MPTP. Minocycline levels were assessed using liquid chromatography and mass spectral detection and found mino cycline concentration in the midbrain at 0.32 g/g compared to ibition of microglia proliferation in the study described in chapter 2 is not a significant levels in the brain but rather an actual lack o A number of studies using intraperitoneal (i.p.) injection for drug delivery have claimed minocycline as a neuroprotective agent in neurodegenerative diseases (Van Den Bosch et al., 2002; Zhu et al., 2002; Wang et al., 2003). It is diffi cult to determ levels of minocycline in the br ain using this route of admini stration due to the lack of studies investigating minocyc line concentrations in brai n tissue following an i.p. injection. The majority of HPLC studies concentrate on the levels of minocycline plasma rather than actual brain tissue.

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30 It can be concluded, that oral administra tion of minocycline is an effective metho for drug delivery and that failure of minocyc line to inhibit microglial activation in the facial nucleus is not a result of low levels of minocycline in the brain. d

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CHAPTER 4 TIMELINE OF MICROGLIA PROGRA MMED CELL DEATH IN THE FACIAL NUCLEUS FOLLOWING INJURY Introduction ctivation and proliferation of microglia is one of the earliest and most common glial reactions in the injured brain. Upon inju ry in the CNS, specifi cally in the facial nucleus, microglial cells dramatically in crease in cell number (Cammermeyer, 1965; Graeber et al., 1988b; Raivich et al., 1994) and are recruited to perineuronal sites where it is suggested they displace afferent synaptic terminals (Blinzinger and Kreutzberg, 1968). Following the initial microglial response and regeneration of motoneurons, activated microglia migrate into the nearby parenchym a (Angelov et al., 1995) and decline in number often reaching baseline levels several weeks following injury (Streit et al., 1988; Raivich et al., 1993). Initially, the mechan ism used to maintain homeostasis of microglial cell numbers was attributed to migration of activated microglia to blood vessels where microglia exit through the wa lls (Del Rio-Hortega, 1932; Cammermeyer, 1965) however, recent studies suggest that microg lia regulation is controlled by a form of programmed cell death (Gehrmann and Ba nati, 1995; Jones et al., 1997). Programmed cell death is an important m echanism used to control cell population during development, growth and in regulation of the immune response (Allen et al., 1993; Bortner et al., 1995; Majno and Joris, 1995). Apoptosis is the most common type of programmed cell death inve stigated in the literature and is characterized by chromatin condensation, DNA fragmentation, membrane bl ebbing and caspase induced (Gavrieli et A 31

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32 al., 1992; Bohm and Schild, 2003; Jaattela and Tschopp, 2003). Apoptotic cells are quickly phagocyctosed without induction of an inflammatory response allowing homeostatic regulation of the CNS. More recently, a number of reports have described a cas proteases and changes in mor phology not consistent with clas sical apoptosis (Jaattela and Tscho g be als per survival time were euthanized at 10, 14, 17 pase-independent form of programmed cell death that display activation of other pp, 2003; Nagy and Mooney, 2003; Lockshin and Zakeri, 2004). The main objective of the study in the current chapte r is to compare the postmitotic turnover of microglia at different post-injury time points using the ApopTag assay, a kit variation of the terminal deoxynucleotidyl tran sferase-mediated deoxyuridine triphosphate nick end labeling method. The ApopTag kit labels DNA fragmentation, a key component of apopotsis, by detecting DNA strand breaks by enzymatically labelin the free 3-OH termini with modified nucleotides. The findings from this study will used for further experimentation in ALS anim al models to determine key differences in post-mitotic microglia turnover in di seased and non-diseased animals. Materials and Methods Animals and Tissue Processing Male Sprague Dawley rats weighing 200-250 grams were used. Animals underwent a unilateral facial nerve crush as described in chapter 2. Four anim and 21 days post-injury by a transcardial perfusion as described in chapter 2 without radioactive precautions. Briefly, animals were given an overdose of sodium pentobarb ital and transcardially perfused with 0.1 M PBS pH 7.4 followed by 4% paraformaldehyde. Immediately following perfusion, brains were quickly removed and stor ed in 4% paraformaldehyde fo r 2h and then transferred to

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33 PBS. Brainstem containing the facial nucle us was paraffin processed as described in chapter 2 and 7 m sections were cut on a microtome. TUNEL and DAPI Staining To assess apoptotic cells, TUNEL labeling was performed on the processed tis Sections were deparaffinized through xyl enes (2 changes for 15 min each) and descending alcohols for 2 min each (100%, 100%, 95%, 90%, 70%, 70% sue. ) and rinsed in PBS f d Eight sections containing d for TUNEL quantification. t RT digital camera (Diagnostic Instruments, Sterling Heigh lls ed or 5 min. The ApopTag Red In Situ detection kit (Ser ologicals Corporation, Norcross, GA) was used as described in the manufacturers protocol however the pretreatment step was omitted. Negative controls omitted the terminal deoxynucleotidyl transferase (TdT) enzyme. Following TUNEL labeling, slides were coun terstained with DAPI. Slides were incubated with DAPI at a concentration of 1: 333 for 5 min in a light protected box, rinse in PBS and coverslipped. Quantitative Analysis th e facial nucleus were use Sections were imaged using a Spo ts, MI) attached to a Zeiss Axios kop microscope. All TUNEL positive ce located in the facial nucleus were counted a nd pooled together per time point and divid by the total number of sections counted to gi ve an average. Results are represented as mean values SEM. Results Differences in density of TUNEL positive cells in the injured facial nucleus were seen at different post-axotomy time points. TUNEL positive cells were found at

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34 all time-points investigated in the injured facial nucleus. There were differences seen the number of TUNEL labeled cells at 10, 14, 17 and 21 in days (22 1.194, 51 3.061, th the greatest de nsity of TUNEL positive cells present at 14 da 10 0.064, and 13 0.832) wi ys post-axotomy (Fig. 4-1). No TUNEL labeled cells were found in the contralateral, unoperated facial nucleus (Fig. 4-2d). 0 10 30 60Days Post AxotomyNur of Apopt Bo 194, TUNEL labeled microglia display abnormal cytoplasmic staining at all time points post-axotomy. The majority of TUNEL positive microglia showed staining diffusely dispersed throughout the cytoplasm however, lacked intens e nuclear staining commonly found with classic apoptosis. Th microglial cells stained with microglia ce ll surface markers (Fig. 4-2c). A small 40 50oticdies 20mbe 10 14 17 21Figure 4-1. A time line of TUNEL positive microglia in the facial nucleus following injury. Results are represented as mean values SEM. (Day 10, 22 1. Day 14, 51 3.061, Day 17, 10 0.064, and Day 21,13 0.832) e cytoplasmic staining revealed ramified processes and a perineuronal location of the TUNEL positive cells similar to that seen of

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35 population of TUNEL cells displayed both cytoplasmic and nuclear staining revealing an overlap between TUNEL and DAPI staining (Fi g. 4-2a,b). Cytoplas mic staining did not the TUNEL labeled cells, previous appear to be contained in lytic vesicles characteristic of phagocytosed debris making it unlikely to be degraded DNA from adjacent dying cells. While no co-labeling was perf ormed to identify studies have identified microglia as the only cell to die during the regenerative process in the facial nucleus (Gehrmann and Banati, 1995). In addition, the morphology of the TUNEL cells is identical to microglia morphology. the injured facial nucleus 14 days po st-injury. A) TUNEL labeling in the cells. B) Section of double labeled TUN EL/DAPI cells. The arrows identify Figure 4-2. Non-classical TUNEL positive cells were found throughout the neuropil of facial nucleus displaying cytoplasmic staining representative of microglial nuclear and cytoplasmic staining of micr oglia. C) Facial nucleus 14 days postaxotomy with arrows identifying the pe rineuronal position of microglia. D) Contralateral, unoperated f acial nucleus. Bar = 80 m

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36 Discussion Axotomy of the rat f acial nucleus leads to mitotic division of microglial cells leading to an increase in cell number (G raeber et al., 1988b; Gehrmann and Banati, 1995). Upon recovery of motoneurons, micr oglial are tho ught to undergo programmed cell d ccessful reinnerv ation of the facial m eath to return to normal homeostatic levels (Geh rmann and Banati, 1995; Jones et al., 1997). The current study inve stigated the time course of microglial turnover in the facial nucleus following a crush injury. The greatest density of TUNEL positive microglia was seen at 14 days post axotomy wh ere su muscles has occurred (Kamijo et al., 2003). The correlation between regeneration and icroglia turnover further supports the esse ntial role of progr ammed cell death in regulating the immune response in the CNS following an injury in order to maintain the homeostatic CNS environment (Fig 4-3). Flourogold labeled neurons at different surviv al times following a crush injury in the rat facial nucleus. Th e regenerative patterns of neurons shows a positive correlation with microglia turnover (Kamijo et al., 2003). Figure 4-3

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37 TUNEL labeling in the facial nuc trated diffuse cytoplasmic and nucle l 97). nver, this is an unlikely explanation for the al structures. Furtherm inating the y croglial phagocytosis but 1997). leus demons ar staining of microglial cells, similar to previous studies that found non-typica cytoplasmic staining of microglial cells followin g facial nerve injury (Jones et al., 19 To dismiss handling artifact, the study conducted by Jones and collages examined different fixations, pretreatment and stai ning methods where it was found cytoplasmic staining was present under all experimental conditions. The authors concluded that the non-typical TUNEL staining suggested micr oglial turnover oc curred through a no classical form of programmed cell death. As the only cell in the CNS capable of transforming into phagocytic cells, microglia can engulf extracellular and neuronal debris which may contain degraded DNA. In vivo it is known that microglia are recrui ted to neuronal debris and responsible for their removal (Moller et al., 1996) howe diffuse cytoplasmic staining seen since there was no evidence of phagosom ore, facial nerve injury in the rat does not induce neuronal death elim phagocytic properties of microglia (Moran a nd Graeber, 2004). However, ricin/axotom model leads to extensive neuronal degenera tion followed by mi does not show a significant increase in the number of TUNEL labeled cells (Jones et al.,

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CHAPTER 5 MICROGLIA UNDERGO MORPHOLOGICAL AND FUNCTIONAL ABNORMALITIES IN THE SUPEROXIDE DISMUTASE 1 RAT Introduction Amyotrophic lateral sclerosis (ALS) is an adult onset neurodegenerative disease characterized by selective loss of upper and lower motor neur ons. Loss of motor neu results in muscle paralysis and ultimately d rons eat h due to respiratory failure. 5-10% of ALS cases 6; Deng % ion of mutant SOD1 limited to motor neurons is insufficient to cause motor neuron degeneration supporting g lial cell involvement in ALS (Pramatarova et al., 2001; Lino et al., 2002; Clement et al., 2003). In vitro studies found differences in TNFlevels secreted following stimulation with LPS when comparing microglia isolated from transgenic and wild type mice at day 60 (Weydt et al ., 2004). Transgenic microglia produced higher levels of TNF. In addition, increased leve ls of microgl ial activation are familial, inherited in an autosomal dominant pattern (Mulder et al., 198 et al., 1993; Rosen et al., 1993; Siddique and Deng, 1996) Of familial ALS cases, 20 have been linked to mutations located in th e Cu/Zn superoxide dismutase 1 (SOD1) gene (Rosen et al., 1993; Siddique and Deng, 1996). The identification of SOD1 gene mutations has provided insight into understa nding the molecular pathology of ALS. Specifically, transgenic rodent models expr essing SOD1 mutant G93A have provided a model that closely resembles the human form of the disease, however to date there is no single mechanism that can be identified in th e etiology of ALS. Recent literature has focused on non-neuronal cells in the propagati on of ALS. Several studies have shown express 38

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39 are readily discernible in affected areas in both human and animal models of the disease (Kawamata et al., 1992; Hall et al., 1998; Alexianu et al., 2001). Thus, from current evidence it may be proposed that mutant alities in microglial cells in A o functional changes that result in incre totoxicity further propagating diseas d at three stages of the disease: asymptomatic where animals had no visual muscle SOD1 may cause abnorm LS that alter normal cell func tion. The abnormal microglia could underg ased levels of cy e. Alternatively, microglial may become senescent or dysf unctional due to the SOD1 mutation thereby reducing the number of functional microglia able to provide trophic support to motor neurons furthering neuronal cell death. The aim of the present study is to character ize microglia in the mutant G93A SOD1 transgenic rat, specifically the morphological changes that occur throughout the brain an spinal cord at specific stages of the dis ease. In addition, this study will investigate microglia turnover in the spinal cord and follo wing a facial nerve in jury to determine if mutant SOD1 expression in microglia cause s the cells to be more susceptible to apoptosis causing fewer microglial to be r eadily available in maintaining a healthy environment for neurons. Materials and Methods Animals and Surgery Animal use protocols were approved by the University of Florida Institutional Use and Care of Animals Committee (IUCAC). All transgenic animals used in this study were male Sprague Dawley NTac:SD-TgN(SOD1G93A)L26H rats obtained from Taconic Farms. Animals were monitored daily to assess muscle weakness and to record disease progression. To examine microglial morphology OX-42, OX-6 and TUNEL staining were used

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40 weakness, onset of symptoms where animals first showed evidence of weakness in the hind limb and end stage where animals were no l onger able to right themselves after 3 Animals were sacrificed by tran scardial perfusion as detailed in Chapter 2 at the specified stage of the disease. Immediately followi ng perfusion brains and spinal cords were removed and fixed in 4% paraformal dhyde for 2 h. For OX-42, OX-6 and TUNEL labeling the tissue was placed in 30% sucrose until the tissue was saturated. All time points included 4 transgenic animals and 4 age-matched wild type controls. To assess microglial turnover in the f acial nucleus, 6 non-s 0 s. ymptomatic SOD1 transg y a unted on SuperFrost Plus s lides. A number of slides from these animals were labeled with ectin histochemistry section h in th e materials and methods section. Table Age of Age of Onset Age of OX-42/OX-6 74-84 days 113-117 days 135-140 days -114 N/A N/A enic and 6 age-matched control anim als underwent a facial nerve axotomy where under isoflurane anesthesia, th e right facial nucleus was e xposed at the exit from the stylomastoid foramen and crushed with a he mostat for 10 s. At 14 days post-axotom animals were sacrificed by tran scardial perfusion as describe d in Chapter 2. The brains were immediately removed and placed in 4% fixative overnight. Tissue sections containing the facial nucleus were paraffin embedded as detailed in Chapter 2, cut on microtome at 7 m, and mo lectin as de scribed in the l found in a later paragrap 5-1. Age and correspondi ng disease stage for animals used in experiments. Asymptomatic Animals Animals Endstage Animals Labeling TUNEL/Lectin Labeling 107

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41 OX-42 and OX-6 Immunohistochemistry Lumbar spinal cord, cortical, and brains tem sections were serially cut at 20 m on a cryostat, mounted on Superfrost Plus slides and air dried for one hour. Sections were pretreated in 0.5% PBS-Triton for 15 min, bl ocked in 10% normal goat serum for 30 m and incubated overnight at room temperature in the primary antibody diluted in buff The primary antibodies included MRC OX-42 (Serotec, Cambridge, UK) and MRC OX (Serotec, Cambridge, UK) at 1:500. The slid es were rinsed in PBS and incu in er. -6 bated in secon in ted e r as a percentage of total t was performed to determine statistical signifi cance between transgenic SOD 1 and control animals at each time point. A one-way ANOV A was performed to compare differences among the transgenic animals followed by a Tukey multiple comparison test. A significance level of p<0.05 was used. dary antibody (1:500) for 1h. Following in cubation, slides were rinsed for 9 m and Horseradish Peroxidase Avidin D was a pplied (1:500) (Vector, Burlingame,CA) and incubated for 30 min. Slides were washed and immunoreactivity was visualized with 3,3-diaminobenzidine (DAB)-H 2 O 2 substrate. After a brief ri nse, slides were dehydra in increasing concentrations of ethanols, cleared in xylene, and coverslipped using Permount mounting medium (Fisher Scientific). Quantification of Immunohistochemistry Labeling in the Ventral Spinal Cord OX-42 and OX-6 expression in the ventral sp inal cord was quantified using Imag Pro Plus software. The area occupied by labe led cells was highlighted and measured fo each section of spinal cord (6 sections per animal) then expressed area of ventral spinal cord. Using GraphPad Prism software (San Di ego, CA) a t-tes

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42 TUNEL labeling and cell identification in the spinal cord Lumbar spinal cord sections were seria ounted on Superfrost Plus slides and air dr ied for one hour. The ApopTag Red In Situ Apoptosis Detection Kit (Serologicals Corporation, Norc ross, GA) was used as described in the manufacturers protocol omitting the pretreat ment step. To identify the cell type of TUNEL positive cells RIP1 (oligodendrocytes ), GFAP (astrocytes), OX-42 and OX-6 (microglia) were used. Following the TUNEL procedure, sections were pretreated in 0.5% PBS-Triton for 15 min, blocked in 10% normal goat serum for 30 min and incubated overnight at room temperature in the primary antibody diluted 1:500 in buffer. The slides were rinsed in PBS and inc ubated in secondary an tibody (1:500) for 1h. Following incubation, slides were rinsed for 9 min and FITC-Avidin D was applied (1:500) (Vector, Burlingame,CA) and incubated for 30 min. S lides were rinsed briefly and coverslipped. Lectin histochemistry Prior to lectin staining, sections were deparaffinized through xylenes, graded alcohols and rinsed in PBS. Next, the slid es were trypsin treated (0.1% trypsin, 0.1% CaCl2) for 12 min at 37C. Following a 10 min wa sh the slides were incubated overnight at 4C in lectin GSA I-HRP (Sigma Chem ical Co.) diluted 1:10 in PBS containing cations (0.1 mm of CaCl2, MgCl2 and MnCl2) and 0.1% Triton-X100. After overnight incubation slides were briefl y rinsed in PBS and visualized with 3,3-diabimobenzidine (DAB)H O substrate. Sections were counters tained with cresyl violet, dehydrated through ascending alcohols, cleared in xylen es and coverslipped with Permount. lly cut at 20 m on a cryostat, m 2 2

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43 TUNEL and Lectin Double Labeling Microglial cell death was visualiz ed in th e facial nucleus us ing TUNEL on tissue tions were deparaffinized, washe d re paired enic etailed for the TUNEL la beling in the facial nucleus. Slides were ted to distilled water. Next 1 mL of crysta l violet solution and 5 drops or n ely sections from animals 14 days post-facial nerve axotomy. Sec d in PBS, trypsin treated (0.1% trypsin, 0.1% CaCl 2 ) for 12 min at 37C and rinse two times in PBS for 5 min each. The ApopTag Red In Situ Apoptosis Detection Kit (Serologicals Corporation, Norcross, GA) was then used as described in the manufacturers protocol omitting the pretreat ment step. Following TUNEL, sections were stained using GSA I-FITC (Sigma, St.Louis, MO) according to protocol described in the previous lectin section. Six sections containing the facial nucleus per animal we manually counted for TUNEL labeled cells and statistically evaluate d using an un t test. Brown and Brenn Gram Stain A gram stain was performed on tissue containi ng the facial nucleus from transg SOD1 animals 14 days post-axotomy. The tissue was handled and processed following the same protocol d deparaffinized and hydra of 5% sodium bicarbonate solution were added to sections a nd allowed to sit f one minute then rinsed in tap water. The slid es were decolorized with acetone, rinsed i water, flooded with basic fuchsin working so lution for 1 min, rinsed again and placed in water. Each slide was indi vidually dipped in acetone to start reaction and immediat differentiated with picric acid-acetone solu tion until turning a yellowish pink. Finally slides were quickly rinsed in acetone, th en in acetone-xylene solution, cleared in 2 changes of xylene and mounted with resinous medium.

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44 Results e on occurred glia in the cortex of ALS rats at all stages of the disease (Fig. 5-1b). No change in microglia morphology or activation was seen in the cortex of SOD1 animals. To investigate changes in microglia morphology in the transgenic SOD1 rat, a number of microglia markers were used for staining at various time points throughout disease progression. Numerous CNS regions were investigated to determin if microglia change was limited to areas wh ere extensive neuronal degenerati or rather was a widespread effect. The cortex revealed OX-42 positive micr oglia with a normal resting morphology distributed evenly throughout the cortex (Fig. 5-1a). OX-6 staining labeled a small number of microglial cells in the gray ma tter indicating an absence of MHC II positive micro 42 labeled cells in animals with early ons et of symptoms displaying a ramified stain worked in the cortex. Bar = 40 m s (Fig. 5-2a,d) and persisted until Figure 5-1. Photomicrographs of microglia labeling in the cortex of SOD1 rats. A) OX morphology representative of a resting stat e. B) Cortex of end stage animal labeled with OX-6 showing that there is a lack of MHCII expression in SOD1 animals. The arrow identifies a labeled microglia confirming that the OX-6 Abnormal microglial fusion and activatio n was present at the level of the red nucleus. The brainstem portrayed drastically di fferent characteristics of microglia throughout disease progression. Morphological changes of microglial cells were seen prior to onset of symptoms at the level of the red nucleu

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45 end stage. OX-42 staining revealed inten glial activation within the red nucleus at all e level of the red nucleus were seen with the presence of mi s er disease nucleus. W the red nuc howe ions se micro 3 stages of the disease. A disti nguishable border was present between the red nucleus and the surrounding tissue clearly demonstrating that intense microglial activation was contained to the red nucleus (Fig. 5-2e). It ap pears that activation occurred prior to neuronal degeneration si nce neuronal populations of the red nucleus were similar between control and transgenic animals in asymptomatic animals. Further evidence of microglial changes at th croglial fusions (Fi g. 5-2f). The majority of fusions were located within the nucleu and contained a large number of microglial cells clumped together. Upon close observation, it is evident that the fusions were microglia because the cells were OX-42 positive and displayed a morphology representati ve of microglia. With furth progression the fusions were dist ributed throughout the entire level of the red nucleus. In the same sections containing the re d nucleus, the oculomotor nucleus and substania nigra were investigated and found to have no microglial fusions and a population of evenly dispersed ramified microglial cells which remained throughout disease progression (Fig. 5-2b,c). SOD1 animals present abnormal microglia fusions at the level of the facial hen looking at the level of the facial nucleus similar changes were seen as in leus. Asymptomatic animals showed a small number of fused microglia ver no other abnormalities or activation were seen. With the appearance of muscle weakness through the end stage of the disease, increasing nu mbers of microglial fus were present with the majority of fusions distributed outside the facial nucleus. The fusions varied in appearance with some displaying a long string of microglial cells

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46 whereas others were rounded fusions of micr oglial cells representing multinucleated gian cells (Fig. 5-3a,b,c). The giant cells had micr oglial nuclei orientated in a circle and w intensely stained with both OX-42 and lectin (Fig. 5-3d). Unlike the red nucleus, facial nucleus did not have increased le vels of microglial activation throughout the disease process. The microglial population in the nucleus was found to have a ramified morphology indicative of a resting state. An unexpected finding was seen in one animal where a nidus of bacillus bacteria was detect ed within the tissue section (Fig. 5-4d). However, when trying to confirm the presence bacteria using a gram stain there was no evidence of bacteria. In addition to microglial abnormalities ther e was evidence of pathological change of neurons. It appears that neuronal bodies are undergoing degenerative changes as seen by the separation of dendrites from their neur onal bodies (Fig. 5-4a,b) The majority of t ere the s fragm the ord s. Microglial response to disease progression in the ventra ion ented dendrites were loca ted in vacuoles that become increasingly apparent in diseased brainstem (Fig. 5-4c). Microglial activation and abnormalities w ere evident in the ventral spinal c prior to onset of muscle weaknes l spinal cord was assessed usi ng OX-42 and OX-6 marker s. OX-6 labeling revealed an increase in density of MHC II positive labeling with progression of the disease and was significantly higher in tr ansgenic animals when compared to agematched controls after the occu rrence of symptoms (Figs.55, 5-6). OX-42 density in the ventral spinal cord initially increased with the occurrence of symptoms however decreased in number at the end stage of the disease. Elevated leve ls of OX-42 express

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47 were found in the transgenic SOD1 animals at all time points when compared to agematched controls (Figs.5-7, 5-8). Figure 5-2. Photomicrographs representing aberrant microglial activation at the level of with OX-42. A) Midbrain section displaying intense microglial activation located microglial activation. C) Subtania nigr a displaying microglia with a ramified se in the red nucleus. (A-D) Bar = 160 m E) The border separating the microglial Presence of fused microglia located in the red nucleus intensely stained with OX-42. Bar = 40 m the red nucleus in early onset SOD1 anim als. All sections were stained in the red nucleus. B) Oculomotor nucleus showing a lack of aberrant resting morphology. D) Higher magnification of the microglial respon activation in the red nucleus from the surr ounding tissue. Bar = 80 m F)

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48 Figure 5-3. Abnormal morphological changes seen at the level of the facial nucleus in SOD1 rats. A) Microglial fusion di splaying a long rod-li ke structure in asymptomatic animals (day 74) labeled with OX-42. Note the individual microglial cells and the ability to observe the processes of microglia. B) Microglial fusion displayi ng a rounded structure in asymptomatic animals labeled with OX-42. C) Rod-like microglia l fusions (lectin and creysl violet labeled) in day 107 animals displaying hind limb weakness. (A-C) Magnification 250x. D) Multinucleated gian t cell of the Langhans type (lectin labeled) in animals with hind limb weakness. Magnification 630x. Microglial fusions as well as multinucleated giant cells were present in the gray matter of the spinal cord with the majority distributed in the ventral horn (Fig.5-9b). Th fusions were identical to those seen in the brainstem and were in addition to phagocytic clusters that were present as a direct result of neuronal degeneration (F ig. 5-9a). The two e neurons wh was locate normal. In ramified m integrity was impaired (Fig. 5-9c). OX-6 la beled microglial were often associated with were distinguishable because the clusters were limited to areas surrounding ventral horn ereas the fusions were not always in the vicinity of neur ons. Only one fusion d in the white matter, with the ma jority of the microg lial population appearing the end stage animals, OX-42 st aining revealed swollen, fragmented, nonicroglial cells dispersed throughout the ventral horn suggesting that cell

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49 multinucleated giant cells at the end stag e, however also revealed a population of fragme nted swollen micr oglial cells (Fig. 5-9d). Pathological changes occurring in early SOD1 symptomatic animals. A) Neuronal fragmentation due to vacuoliz ation. B) Dendrite s separated from neuronal bodies located in vacuoles. C) Vacuole c ontaining a lectin labeled cell body. D) Evidence of bacillus bact eria suggesting an impaired immune response. Bar = 16 m Figure 5-4. 0 0.005 0.01 0.015 0.03Stages of DiseasePercntage of Lbelerea 0.02 0.025e ad A SOD1 Transgenic Animals Age-matched Control Animals** # Figure 5-5. Percentage of area covered by OX-6 immunoreactive cells in the ventral h from 74 days to156 days. Columns represent mean S.E.M of 4 animal control rats and #P<.05 with respect to presymptomatic transgenic rats. **orn of lumbar spinal cord of SOD1 transgenic rats and age-matched control rats s for each time point. **P<.001 with resp ect to the corresponding age-matched

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50 OX-6 labeled microglia in the ventra l spinal cord of SOD1 animals Figure 5-6. Photomicrographs de monstrating change in OX-6 e xpression with age. A-C) throughout the disease progression. OX-6 density increased throughout the disease progression. Magni fication 60x. D-F) OX-6 la beled microglia in the ventral spinal cord in age-matched control animals. Magnification 125x.

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51 Figure 5-7. Photomicrographs demonstrating change in OX42 expression with age. AC) OX-42 labeled microglia in the vent ral spinal cord of SOD1 animals s. D-F) throughout the disease progres sion. An initial increas e of OX-42 density is seen in with the onset of symptoms that declines in end stage animal OX-42 labeled microglia in the ventral spinal cord in age-matched control animals. Bar = 160 m

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52 0 0.05 0.1 0.15 0.2 0.25 0.3Stages of DiseasePercentage of Labeled Area SOD1 Transgenic Animals Age-Matched Control Animals* ** ** # #Figure 5-8. Percentage of area covered by OX-42 immunoreactive cells in the ventral horn of the lumbar spinal cord of SOD1 transgenic rats and age-matched control rats from 74 days to156 days. Columns represent mean S.E.M of 4 animals for each time point. *P<0.05 and **P<0.001 with respect to corresponding age-matched control rats and #P<0.05 with respect to transgenic onset of symptom rats. TUNEL positive cells were dispersed th roughout the lumbar ventral spinal cord in ALS animals. TUNEL labeling was performed on spinal cord sections to determine if microglial cells were undergoi ng apoptosis due to ce llular senescence. TUNEL positive cells were found at all stages of the disease with the majority located in the ventral area of the spinal cord displaying classic nucl ear staining. A few isolated TUNEL positive cells in asymptomatic and ear ly symptomatic animals portrayed nuclear and diffuse ied as microgl similar occ uclear st ained TUNEL positive cells suggesting that microglial turnover in the spinal cord regulat ed by apoptosis is unchanged in transgenic cytoplasmic staining similar to the TUNEL positive cells that were identif ial cells in the faci al nucleus (Fig. 5-10a). Cont rol spinal cord sections had urrence of cytoplasmic/n

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53 animals (Fig. 5-10c). End stag e animals displayed nuclear staining orientated in a string of labeled cells not represen tative of microglial cells (Fig 5-10b). Co-labeling with markers for each CNS cell type revealed no overlap between cell-sp ecific markers and TUNEL labeled cells not allowing conclusi ve identification of apoptotic cells. Figure 5-9. Microglial response and changes in SOD1 animals in the ventral spinal cord. d in the ventral horn of asymptomatic an imals (OX-42 labeled). B) Rod-like ons are similar to those seen in the brainstem in SOD1 animals. C) OX-42 labeled representative of degenerative changes. D) OX-6 labeling of giant cells in end expression. Magnification A,B,D 125x and C 630x. TUNEL positive cells were found at significantly lower numbers in the SOD1 facial nucleus 14 days post-axotomy. TUNEL-positive cells in the SOD1 transgenic and wild type axotomized facial nucleus l acked the classic nuclear staining instead having cytoplasmic staining similar to previous studies of apoptotic microglia (Gehrmann and Banati, 1995; Jones et al., 1997). In a ddition, double-labeling re vealed an overlap A) Microglial activation seen in close proximity to motor neurons locate microglial fusions stained with OX-42 in asymptomatic animals. The fusi microglia in end stage animals portr aying an abnormal swollen morphology stage animals suggesting a relations hip between giant cells and MHCII

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54 between lectin and TUNEL staining furthe r identifying TUNEL-labeled cells as microglia (Fig. 5-12). TUNEL/lectin positive cells distribution was limited to the fa nucleus. cial Figure 5-10. TUNEL positive cells in the ventra l lumbar spinal cord in ALS animals. A) 74). B) End stage TUNEL labeling with exclus ive nuclear staining orientated in a and nuclear staining similar to TUNEL positive cells seen in asymptomatic Diffuse cytoplasmic and nuclear staining in asymptomatic animals (Day string of positive cells. C) Control TUNEL labeling revealing cytoplasmic and early symptomatic animals. Bar = 80 m.

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55 The number of TUNEL labeled cells in the injured facial nucleus was significantly lower (p<0.001) in the transgenic animals when compared to age matched controls ( (Fig. 5-11) SOD1 transgenic animals 0.5278 0.1847, control animals 5.357 0.5058)). No TUNEL-positiv ucleus in both anim r numbers of nuclei in the transgenic and control anim response 14-days post axotomy appeared to be sim als both displaying perineruonal mi density of lectin labeling within the nucleus. e cells were found in the unoperated facial n al groups. Qualitative analysis of a niss il stain revealed simila als. In addition, the microglial ilar between c ontrol and transgenic anim croglia and quali tatively revealing a similar 0 1 2 3 4 5 6 7Number of Tunel Postive Cells/Nucleus SOD1 Transgenic Animals Age-matched Control Animals Days Post Axotomy 14 Days **er of TUNEL positive cells in facial nucleus 14 days p Figure 5-11. Numb ost axotomy in non-symptomatic transgenic and agematched control animals. Columns represent mean S.E.M of 6 animals for each time point (**P<0.001).

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56 Discussion The current study demonstrated for the fi rst time a presence of microglial fusions and Langhans giant cells in the ALS rat mode l. Additional pathological changes were observed at the levels of the red and facial nucleus in the brainstem where aberrant microglial activation and neuronal fragment ation was seen. Evidence of abnormal microglial function was found in the T UNEL studies where SOD1 animals had significantly reduced levels of TUNEL positive cells in the facial nucleus compared to age matched controls 14 days post-axotomy. The current findings demonstrate that microglial cells unde rgo morphological a nd functional changes during the disease lymphocyte recruitment and release of cytokine s some of which cause further recruitment of macroph (Lee et al., 1993). In vitro studies have found that in terleukin-3 and interferoninduced Langhans multinucleated giant cell formation in the presence of granulocyte macrophagecolony stimulating factor (G M-CSF) (McNally and Anders on, 1995). The characteristics progression in ALS animals. The presence of multinucelated giant cel ls was an unexpected finding since these cells are often only associated with viral and bacterial infections, wh ich to date have not been reported in ALS animal models. The current study demonstrated a large number of multinucleated giant cells of the Langhans t ype throughout the CNS of the rat transgenic model. Langhans giant cells are characterized by nuclei or ientated around the periphery of the cell and most often associated with granulomat ous reactions, specifically Mycobacterium tuberculosis The presence of indigestible particles of an organism causes macrophages to aggregate at the site and engulf the foreign particle. During the ensuing days, a cell-mediated immunity to the bacterium develops leading to T ages and cell fusion through inducti on of cell surface a dhesion molecules

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57 of Langhans giant cell formation sugge ce of bacteria in the ALS rat model which of lls t changes (Nottet et al., 1997; Zheng and Gendelman, 1997; Kaul roglial id sts the presen was supported by the observed nidus of b acillus bacteria in a tissue section of a transgenic animal. The fact that we detect ed bacilli and giant cells in animals that develop neurodegenerative disease leads us to conclude that a common link for the development of brain infection and neurode generation may be found in dysfunction microglia that results in im paired immunological defense mechanisms and diminished neuroprotection. Further evidence of th is novel theory is found in human immunodeficiency virus encephalitis (HIVE) cas es where viral infected microglial ce portray altered cytokine production and formation of mulit nucleated giant cells tha accompany neurodengerative et al., 2001). In ALS, microglial dysfunction may be a direct result of the SOD1 mutation whereas in HIV encephalopathy microgl ial dysfunction may be a result of viral infection both rendering detrimental consequences for the neuronal cell population. An additional link between ALS and HIV is seen in a number of HIV patients that were clinically diagnosed with ALS suggesting a related mechanism of disease propagation (Verma et al., 1990; Huang et al., 1993; Casado et al., 1997; Ga lassi et al., 1998; MacGowan et al., 2001; Moulignier et al., 2001; Zoccolella et al., 2002). In addition to the presence of giant cells, intense microglial activation was seen throughout the brainstem and in the ventral horn of the lumbar spinal cord. Mic activation was seen in the sp inal cord corresponding to neuronal degeneration, however in the brainstem the activation is not a direct result of neuronal degeneration but may be due to elevated levels of microglial-activati ng factors identified in cerebral spinal flu and serum of ALS patients.

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58 Figure 5-12. Apoptotic microglial cells in the facial nucleus following injury. A) Lectin confirming TUNEL labeled cells are microglial cells. Bar = 40 m. labeled perineuronal cell. B) TUNEL labeled cell. C) Merged images

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59 Macrophage colony stimulating factor (M-C SF), monocyte chemoattr act protein 1 (MCPor necrosis factor alpha (TNF), and transforming growth factor beta 1 (TGF-) have all been found at elevated levels in AL S when compared to controls (Poloni et al., 2000; Elliott, 2001; Hensley et al., 2002; Ilzecka et al., 2002; Yoshihara et al., 2002; Hensley et al., 2003; Wilms et al., 2003; Henkel et al., 2004). The mechanism in which croglia activating factors are produced is unclear, but may be re leased by glial cells selves. The glial cells may be aff ected by the mutant SOD1 thereby becoming neurotoxic and increasing production of pro-inflammatory molecules. In addition, to ating microglia the elevated levels of inflammatory molecules may further affect the function of microglia propagating the diseas e. Chronic expression of MCP-1 in the central nervous system causes impairment of microglia func tion in mice, specifically the ability of microglia to respond to envir onmental stimuli (Huang et al., 2005). It is unclear the cause for isolated mi croglia activation in the red nucleus, but absent in other motor nuclei such as the oc ulomotor and facial nucleus. The aberrant croglia activation may be dependent on the pr ojection of cells in the specific nuclei. Cells in the red nucleus are th e only cells that directly proj ect to spinal cord levels whereas those in the oculomotor, facial and s ubstania nigra have no di rect projections to al cord. Initial observations in the spinal cord confirmed prior re ports of prominent 1), tum mi them activ mi the spin microglial activation in areas of motor ne uron degeneration. Micr oglial activation was assessed in n between OX-42 and OX-6 was observed. OX6 expression transiently increased throughout the disease whereas OX-42 levels we re increased in asymptomatic animals, the ventral spinal cord usi ng OX-42 and OX-6. A vari ation in expressio

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60 continued to increase with th e onset of symptoms followed by a decline in density in end stage animals. OX-6 is often used as a ma rker of microglial activation which suggests that microglial activation occurs in response to neuronal degene ration in the spinal cord. However, MHC II is not always indicative of activation since non-activated ramified microglia present in young and non-diseased human subjects are posit ively stained with MHC II (Streit and Sparks, 1997; Streit et al ., 2004). Alternatively, MHC II may be a marker of microglia maturation and an early stage of cellular se nescence which is supported by the increasing number of MHC II positive microglial cells from infancy old age (Streit and Sparks, 1997). Therefore, the progressive increa se in OX-6 in SOD1 animals may be a result of the SOD1 mutation negatively effecting microglial cells causing increased cellular dysf unction and senescence. The conflicting decline of OX-42 density in end stage animals ma y be attributed to differen tial regulation of cell s to urface marke end ented, ever, the lack of increased levels of apoptot ic microglia observed in the transg rough e a rs dependent on the life stage of microglia. OX-6 labeli ng remains elevated in stage animals because the remaining micr oglial cells are senescent whereas OX-42 density declines as a result of microglial de generation causing a decrease in the overall microglia population. Evidence of abnormal microglia structur e as seen with fragm swollen OX-42 positive cells in end stag e animals is suggestive of microglial degeneration how enic spinal cord suggest s that if microglia l are under going cell death it is th an alternative pathway. To investigate microglial function in the SOD1 transgenic animal, this study utilized the facial nerve injury model. Peripheral nerve lesions like the facial nerv axotomy maintain the integrity of the blood brain barrier thus allowi ng resident microgli

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61 to be studied in the absence of infiltrating blood-derived cells. In non-diseased animals microglia proliferate in response to facial nerve injury (Kreutzberg, 1968; Graeber et al. 1988; Gehrmann and Banati, 1995) reaching a peak at 3 days and gradually decrease in number (Streit et al., 1988; Raivich et al., 1993). The decline in microglia has been attributed to non-classical pr ogrammed cell death, a possible mechanism used to regulate post-mitotic microglia populations through elimination of ac tivated microglia (Gehrma and Banati, 1995; Jones et al., 1997). The lack of TUNEL-positive microglia in transgenic animals may be a mark of abnorma lities in microglial regulatory mechanisms. If microglia fail to undergo pr ogrammed cell death the levels of microglia will remain elevated and may maintain an activated stat e causing a cytotoxic environment. Evid for dysfunctional microglial func tion in ALS has been seen in the study by Weydt et al. where microglia isolated from the transgen ic SOD1 mouse produced higher levels of TNF. Another explanation for the lack of TUNEL positive cells in the transgenic animals is that post-mitotic tu rnover of microglia is controlle d by an alternative type programmed cell death. Neuronal abnormalities must also be addre ssed as a factor in the reduction of microglia turnover in transgenic animals. The motoneuron population in the fa nucleus may be affected by the disease process resulting in reduced numbers of motoneurons thereby upon injury a diminished neuronal response is present to induce microglia activation requiring less cell turnov nn ence of cial er in order to maintain a resting CNS enviro facial nment. However, qualitative analysis of nissl staining of mot oneurons in the nucleus revealed similar numbers between tr ansgenic and control animals, which is further supported by magnetic imaging and histochemical studies that establish

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62 motoneuron populations in the f acial nucleus remain unaffected in mice prior to onset of symptoms (Nimchinsky et al., 2000; Haenggeli and Kato, 2002; Angenstein et al., 2004). Although, neuronal numbers remain unchanged, previous findings have shown an impaired neuronal response to peripheral inju ry in the transgenic SOD1 mouse (Mariotti et al., 2002). Following a facial nerve a xotomy, levels of NOS immunoreactivity was significantly reduced in the axotomized facial nucleus of transgenic animals. The precise function of NOS induction in the facial nucleus is debated however in cranial motoneurons it has been identified as an indi cator of the cell body res ponse to both lethal (Wu, 2000) and non-lethal (Mario tti et al., 2001) peripheral in juries. The lack of NOS induction in the transgenic animals may im pair microglia-neuronal signaling thereby reducing the microglia response following in jury diminishing microglial turnover. Contradicting findings on levels of TUNEL labeled cells were seen in the spinal cord and facial nucleus. The facial nucleu s showed decreased levels of TUNEL labe microglia whereas the spinal cord had similar levels of TUNEL labeled microglia as the controls. The discrepanc led y may be due to regi onal differences as well as activation levels of the aired ong microglial population. The spinal co rd presents intense neuronal degeneration accompanied by an intense microglial respon se whereas the facial nucleus had no evidence of neuronal death. The current findings suggest an aberra nt microglial response in the SOD1 transgenic animal which may be a direct resu lt of the SOD1 mutant resulting in imp function of microglial cells. Previous studi es have found that exclusive expression of SOD1 mutant in neurons (Pramatarova et al ., 2001; Lino et al., 2002) or astrocytes (G

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63 et al., 2000) is insufficient to cause neurona l degeneration indicati ng that the mutation must be affecting both the ne uronal and glial population.

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CHAPTER 6 CONCLUSION spite of the larg e body of evidence indicating th at microglial activation might influence the pathogenesis of degenerative diseases, there is considerable debate garding whether microglial activation is beneficial or harmful. To address this ndamental question we decided to examin e the effect of mino cycline on microglial activation and neuronal regene ration. Initially, it was thought that minocycline would inhibit microglial activation, as seen in a number of previ ous studies, allowing neuronal regeneration to be investigated in the abse nce of a robust microglial response providing evidence for or against a pro-regenerative role of microglial activation. However, there was no difference seen in proliferating microgl ia in the facial nucleus 2, 3, and 4 days post-axotomy between minocycline treated and control animals. The unexpected effect of minocycline did not allow th e role of microglial activati on in neuronal regeneration to be addressed. Nonetheless, th e results provided important insight to the mechanism in which minocycline provides neur oprotection in a number of neurodegenerative and injury models. From this study it appears that minocycline does not provide neuroprotection as a result of microglial deactivation. Further experiments in a lethal motor neuron injury model would address if minocycline functions by providing direct neuroprotection to injured neurons. The conflicting findings of minocycline on microglial activation may be attributed to the model used as well as the marker used to assess microglial activation. The facial nerve model may elicit a differe nt microglial respons e than that in a neurodegenerative model since motor neurons in the facial regenera te and fully recover In re fu 64

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65 whereas the neurons in a neurodegenerative model degenerate providing a chronic source for microglial activation. Future studies need to address m minocycline treatment to assess if m n is truly i nhibited, specifically, pro-in native and e imal i croglia cytokine production following icroglial activatio flammatory molecules that have been suggested to be detrimental in the CNS. In addition, a different route of drug administ ration should be tested to determine if minocycline protection is dependent on the rout e of drug administrati on. An alter to oral drug administration would be an i .p. injection of a corr esponding dose of drug then assessment of microglial proliferation in the facial nuc leus following axotomy. An i.p. injection would determine if the facial nerve paradigm portrays a different response than that seen in neurodegenerative models following an i.p. injection of minocycline. To confirm that the lack of effect of minocycline on microglial activation was not a result of insufficient drug levels in the brain, we performed HPLC/MS/MS to determin levels of minocycline in the brain following drug administration through oral diet. HPLC/MS/MS revealed minocyc line levels similar to t hose found in studies where microglial activation was significantly inhi bited following minocycline treatment. The second aim of the current study investigated changes in microglia morphology and activation in the rat transg enic model of ALS to assess the role of microglia in disease progression. Morphological change s of microglia were observed in the brainstem and spinal cord of ALS transgenic animals at all stages of the disease. Included in the morphological changes was th e formation of giant cells and microglial clusters. The giant cells por trayed the classic characteris tics of Langhans giant cells suggesting a bacterial infection accompanyi ng neurodegeneration in the ALS an

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66 model. This finding was further supported by th e presence of bacilli bacteria seen in a symptomatic animal. How ever, a gram stain failed to identify bacteria in brain tissues from e to the a es in the spinal in in the nuous s. ALS animals. The failure of the gram stain to identify bacteria may be du short time frame in which the bacteria are pres ent prior to their clearance or the bacteri present in the brain are acid-f ast thereby causing them to be gram resistant. To further assess the presence of bacteria l infections in ALS transgenic animals, future studi should directly culture brains to determin e the presence of bacteria as well as performance of a broader range of bacterial stains to include all types of bacteria. Further aberrant microglial activation was present in the red nucleus where intense activation was seen at all stages of the disease. However, in the same animals the oculomotor nucleus remained unaffected. The discrepancy between microglial activation two motor nuclei may be a direct result of the nucleuses cell projections. Cells in the red nucleus directly proj ect into cervical, thoracic and lumbar levels whereas the oculomotor has no direct pathways to the spinal cord. Degenerative changes in the cord affecting the rubrospi nal tract may cause neuronal changes in the red nucleus eliciting a microg lial response. Levels of microglial activation in the vent ral spinal cord were investigated using OX-42 and OX-6 to determine a time line of micr oglial activation in the spinal cord response to disease progression. OX-6 expressi on was found at increased levels transgenic animal when compared to the ag e-matched controls and showed a conti increase in density that corresponded with disease progression. OX-42 density initially increased with the onset of hind limb weakness however declined in end stage animal The differences in OX-42 and OX-6 expressi on may be due to the heterogeneity of

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67 microglia cells found in the CNS and the di fferential regulation of cell surface markers during microglia development. OX-6 positive microglial cells may be in early stages of cellular senescence as a direct result of the SOD1 mutation thereby increasing in numbe during the disease process. OX42 labels the majority of microglial cells representing the overall response of mi croglial cells which declines through microglia degeneration in en stage animals. Therefore, MHC II positive cells are indicative of late stages of micro life cycle thereby remaining elevated in end stage animals whereas the majorit cells have undergone structural changes indicating degenerative ch anges as a result r d glial y of OX-42 of cellul ers ptotic cells with of a wider range of microglial markers. Rathe d rther ar senescence therefore, no longer staining with MHC II. To determine if microglial cells in the ALS model were more susceptible to apoptosis due to cellular se nescence TUNEL labeling was ut ilized to assess apoptotic microglia. In the spinal cord TUNEL labeled cells had a morphology that was representative of microglia how ever failed to co-label with any of the microglial mak used. The failure to co-label may be due to differential expression of cell surface markers on cells undergoing programmed cell death. Future experiments need to further investigate the identity of the apo r than using a surface antigen, a nuclear stain allowing identification of fragmente cells should be tested. In the facial nucleus, TUNEL labeled microglial were significantly fewer in number in the transgenic animals 14 days pos t-axotomy. The findings seem to contradict the hypothesis of cellular senes cence and favor dysfunction of transgenic microglia. In chapter 5, a number of theories were postulated to explain th e findings of fewer apoptotic microglia in the transgenic animals however until the initial microglia l response is fu

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68 investigated little speculation can be made on the cause behind the lack of microglial turnover. In order to investig ate the initial microglial respon se, the microglial response a 4 days post-axotomy in the facial nucleus should be compared between asymptomatic ALS animals and age-matched controls. This experiment will determine if the lack of turnover is due to a diminished microglial response or is in fact dysregulation of deactivating mechanisms in microglia. A follo w-up to the above experiment would be to measure microglia-neuronal sign als to determine if the lack of response is due to neuronal dysfunctions or microglia dysfuncti ons. The cytokine interleukin-6 (IL-6) has been suggested to be a potential signaling molecule between neu t rons and microglia and fracta ons al 98). Both ve stress has been shown to be a key contributing factor in famil a lkine is a chemokine that is found to be constitutively expressed on CNS neur while the corresponding receptor CX3CR1 is found on microglial cells another potenti signal between microglia and neurons (Harrison et al., 1998; Nishiyori et al., 19 of the potential microglia-neuron signals would be key candidates to investigate microglia-neuron signaling in the facial nucleus. In conclusion, the microglial cells in the ALS rat appear to be abnormal and undergoing functional changes due to the SOD1 mutation. It is still unclear in what manner the mutation renders the microglia dys functional, but may be attributed to oxidative stress. Oxidati ial ALS where the mutation catalyzes abe rrant chemical reactions that initiate cascade of oxidatively damaged products. Experiments addressing oxidative damage directly effecting the microglial cell population would provide gr eat insight to the functional and morphological chan ges of microglia in the ALS rat. Furthermore, the dysfunction of microglia appears to compromise the integrity of the immune system in

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69 the CNS allowing for a bacterial infec tion to occur and may contribute to neurodegeneration due to diminished neuroprotective properties.

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84 Yrjanheikki J, Tikka T, Keinanen R, Gold steins G, Chan PH, Koistinaho J (1999) A tetracycline derivative, minocycline, redu ces inflammation and protects agai focal cerebral ischemia with a wide th erapeutic window. Proc Natl Acad Sci U S A 96:13496-13500. nst e itis. J Neuroimmunol 115:182-191. Zheng lic clear phagocytes. Curr Opin Neurol 10:319-325. Zhu S, r RM tochrome c release a nd delays progression of amyotrophic lateral sclerosi s in mice. Nature 417:74-78. Zoccol Bruno F, Ferranni ni E, Iliceto G, Serlenga L, Lamberti P (2002) A case of concomitant amyotrophic lateral Zhao ML, Kim MO, Morgello S, Lee SC (2001) Expression of inducible nitric oxid synthase, interleukin-1 and caspase-1 in HIV-1 encephal J, Gendelman HE (1997) The HIV-1 a ssociated dementia complex: a metabo encephalopathy fueled by viral replicati on in mononu Stavrovskaya IG, Drozda M, Kim BY Ona V, Li M, Sarang S, Liu AS, Hartley DM, Wu du C, Gullans S, Ferrante RJ, Pr zedborski S, Kristal BS, Friedlande (2002) Minocycline inhibits cy ella S, Carbonara S, Minerva D, Pala gano G sclerosis and HIV infection. Eur J Neurol 9:180-182.

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BIOGRAPHICAL SKETCH ily Fendrick w Sarah Em as born in Mansfield, Ohio and remained in Ohio until her sophom she graduated from high school in 1998. Fo r her undergraduate education she attended the Uni Howev sion to follow a path ntil she fina genetics. While in college, her first experience in the field of research was through employment in a stem cell lab. Her time spent in the stem cell lab was short due to her dislike of working in close proximity to m onkeys. She then obtained employment in a functional genomics lab where she spent the two years working on a project that did functional genomics within the E. coli genome. The summer of her junior year Sarah was selected for an internship funded th rough the National Science Foundation at the Whitney Lab in St. Augustine, Florida. Du ring her internship, Sarahs fondness for the warm weather and sunny days persuaded her to continue her edu cation in Florida. In 2002, she began her graduate educati on at the University of Florida in the Interdisiplinary Program in Biomeidcal Rese rach. Entering into graduate school, her interest was in neuroimmunlogy which caused her to immediatel y pursue Jake Streit to be her mentor. Upon active persuasion, Jake accep ted Sarah into his lab where her research focused on the role of microg lial activation in ALS. Afte r completing her Ph.D. Sarah ore year in high school. She then relo cated to Minnesota with her family where versity of Wisconsin-Madison where she wanted to pursue a career in journalism. er, when attending orientation she ma de a last minute deci that would allow her to attend medical school. Her mind changed a number of times u lly decided that she would like to pursue her degree in 85

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86 would like to pursue a career in patent law re lated to biomedical research and plans to begin law school in the Fall of 2006 at The Ohio State University.


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ROLE OF MICROGLIA DURING MOTONEURON REGENERATION AND
DEGENERATION: RELEVANCE FOR PATHOGENESIS AND TREATMENT OF
AMYOTROPHIC LATERAL SCLEROSIS















By

SARAH EMILY FENDRICK


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2006

































Copyright 2006

by

Sarah Emily Fendrick

































This dissertation is dedicated to my family and friends for their support and
encouragement throughout graduate school.















ACKNOWLEDGMENTS

I would like to thank my mentor, Dr. Wolfgang Streit, for his guidance, expertise

and willingness to teach me. I also thank my committee members Dr. John Petitto, Dr.

William Millard and Dr. Paul Reier for their time and support needed to successfully

complete my dissertation.

A special thank you is extended to all the Streit lab members. In particular, I would

like to thank Kelly Miller, for her early morning help in the perfusion room and her

endless support, Chris Mariani for his constant advice on technique and the competition

that motivated me to graduate, and Kryslaine Lopes for being a supportive lab member

and friend.

I thank BJ Streetman and John Neely in the neuroscience office both of whom

made the administrative aspect of graduate school a simple one.

Finally, and most of all I would like to thank my friends and family for being

supportive and encouraging throughout my time in graduate school. I thank my parents

for providing me with the love and support that I have needed to succeed both in and out

of school.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TABLES ......................... .......... ... ...... .... ...... .. vii

LIST OF FIGURES ................................................. ... ........... viii

A B STR A C T ................................................. ..................................... .. x

CHAPTER

1 INTRODUCTION AND LITERATURE REVIEW ....................................................1

M icroglia: A n O v erview .................................................................... .................. .
Introduction ................. ...................... ...............1
M icroglia A re N eurosupportive ........................................ ........................ 2
Facial N erve Axotom y ............................................. .. ...... ................ .3
A m yotrophic Lateral Sclerosis .............................................................................. 4
M icroglia in A L S ............................................................... .. ... ......... 7
Neuroinflammation ............ ........... .................. 8
M icroglia D dysfunction .................. ................. .................... ................. 10
M inocycline ......... ....................................... .. ....... .. ... 12

2 MINOCYCLINE DOES NOT INHIBIT MICROGLIA PROLIFERATION OR
NEURONAL REGENERATION IN THE FACIAL NUCLEUS FOLLOWING A
FA C IA L N E R V E C R U SH .............................................................. .....................14

In tro d u ctio n ...................................... ................................................ 14
M materials and M methods ....................................................................... .................. 15
A nim als and D iet............. .... .................................................... ......... .. 15
Facial N erve A xotom y .................................................................. ............... 16
3H-Thymidine Injections and Radioactive Perfusions ........................................16
Tissue Processing for Audoradiography .................................. ............... 17
A udoradiography ................1.. .... ........ .......... .... .................... .............. 18
Quantitative Analysis for 3H-thymidine Labeled Microglia.............................18
Fluorogold Labeling ............... .... .... ............................. ...... ... ...... 19
Perfusion and Tissue Processing for Fluorogold Labeling...............................19
Quantitative Analysis of Fluorogold Labeling..................................................19
R e su lts ...................................... .................................................... 1 9









D iscu ssio n ...................................................... ................ 2 2

3 DETERMINATION OF MINOCYCLINE CONCENTRATION IN THE BRAIN
AFTER DRUG ADMINISTRATION IN DIET ................................................25

Intro du action ...................................... ................................................ 2 5
M materials and M methods ....................................................................... ..................26
R agents ........................................... ........................... 26
A n im als................................. ..................................................... ............... 2 7
E x tra ctio n ................................................................2 7
E quipm ent .............................................. 27
R e su lts ...........................................................................................2 8
D isc u ssio n ............................................................................................................. 2 9

4 TIMELINE OF MICROGLIA PROGRAMMED CELL DEATH IN THE
FACIAL NUCLEUS FOLLOWING INJURY ................. ................. ..........31

In tro d u ctio n .......................................................................................3 1
M materials an d M eth od s ......................................................................................... 32
Animals and Tissue Processing ..................................... ............................ 32
TUNEL and DAPI Staining ............................................. 33
Q uantitative A analysis .............................................................. 33
R results ........................................................................................... .................... 33
D isc u ssio n ............................................................................................................. 3 6

5 MICROGLIA UNDERGO MORPHOLOGICAL AND FUNCTIONAL
ABNORMALITIES IN THE SUPEROXIDE DISMUTASE 1 RAT ........................38

In tro d u ctio n .......................................................................................3 8
M materials an d M eth od s ......................................................................................... 39
A n im als an d Su rg ery ..................................................................................... 3 9
OX-42 and OX-6 Immunohistochemistry ................. ...............................40
Quantification of Immunohistochemistry Labeling in the Ventral Spinal Cord .41
TUNEL labeling and cell identification in the spinal cord...............................42
Lectin histochem istry .............................................................. 42
TUNEL and Lectin Double Labeling ........ ................. .... ......... 43
B row n and B renn G ram Stain ....................................................... 43
Results ................... .......................................44
D iscu ssio n ............................................................................................. 5 6

6 C O N C L U SIO N ...............................................................................................64

L IST O F R EFER EN C E S ......... ..... .... ........................................ ..........................70

B IO G R A PH ICA L SK ETCH .......................................................................... ..85


















LIST OF TABLES

Table page

2-1. Average intake of minocycline during experiments.............................................. 16

5-1. Age and corresponding disease stage for animals used in experiments ..................40
















LIST OF FIGURES


Figure page

1-1. Facial nerve diagram ...................... .................. ......................... .... .4

1-2. Proposed m mechanism s of AL S.......................................................... ............... 6

2-1. Quantitative analysis of microglial proliferation reveals no difference in the facial
nucleus in minocycline treated versus control animals..................... ..............20

2-2. Photomicrographs of tritiated thymidine labeled cells in the lesioned facial
n u c leu s ................................. ........................................................... ............... 2 1

2-3. Motor neuron regeneration in the facial nucleus following injury is unaffected
follow ing m inocycline treatm ent ........................................ ........................ 21

2-4. Fluorogold labled neurons within the lateral and ventral intermediate sections of
th e inju red facial nu cleu s............................................................... .....................22

3-1. Chemical structures of internal standard and minocycline .............. ...............26

3.2. Minocycline concentrations found through HPLC/MS/MS analysis. ......................28

4-1. A time line of TUNEL positive microglia in the facial nucleus following injury.....34

4-2. Non-classical TUNEL positive cells were found throughout the neuropil of the
injured facial nucleus 14 days post-injury....................... ..................... 35

4-3. Flourogold labeled neurons at various survival times following a crush injury in
the rat facial nucleus .................. .......................... ........ .. ........ .... 36

5-1. Photomicrographs of microglia labeling in the cortex of SOD1 rats revealing no
abnormal microglia mophology or activation ................................ ............... 44

5-2. Photomicrographs representing aberrant microglial activation at the level of the
red nucleus in early onset SOD 1 anim als...................................... ............... 47

5-3. Abnormal morphological changes seen at the level of the facial nucleus in SOD1
rats ...................... .................................... 48

5-4. Pathological changes occurring in early SOD1 symptomatic animals...................49









5-5. Percentage of area covered by OX-6 immunoreactive cells in the ventral horn of
lumbar spinal cord of SOD1 transgenic rats and age-matched control rats from
74 days to 56 days. ...................... .. .... ....................... .. ...... .... ........... 49

5-6. Photomicrographs demonstrating change in OX-6 expression with age...................50

5-7. Photomicrographs demonstrating change in OX-42 expression with age.................51

5-8. Percentage of area covered by OX-42 immunoreactive cells in the ventral horn of
the lumbar spinal cord of SOD1 transgenic rats and age-matched control rats
from 74 days to156 days. ............................................... .............................. 52

5-9. Microglial response and changes in SOD1 animals in the ventral spinal cord .........53

5-10. TUNEL positive cells in the ventral lumbar spinal cord in ALS animals ..............54

5-11. Number of TUNEL positive cells in facial nucleus 14 days post axotomy in
non-symptomatic transgenic animals was significantly less when compared to
and age-matched control anim als. ........................................ ......................... 55

5-12. Apoptotic microglial cells in the facial nucleus following injury.........................58















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

ROLE OF MICROGLIA DURING MOTONEURON REGENERATION AND
DEGENERATION: RELEVANCE FOR PATHOGENESIS AND TREATMENT OF
AMYOTROPHIC LATERAL SCLEROSIS

By

Sarah Emily Fendrick

August 2006

Chair: Wolfgang J. Streit
Major Department: Neuroscience

Recently, microglial activation has been identified as a contributing factor in a

number of neurodegenerative diseases and been targeted for therapeutic treatment.

However, this view fails to consider the neuroprotective role of microglia observed in

injury models where microglial activation accompanies neuronal regeneration. The main

goal of this study was to investigate the role of microglia activation in an injury model as

well as the transgenic SOD1 rat model and characterize the accompanying neuronal

responses. In order to determine if microglial activation in the facial nerve paradigm is

beneficial, minocycline, a drug shown to inhibit microglial activation, was utilized

allowing neuronal regeneration to be assessed in the absence of microglial activation.

Microglial activation was assessed in the SOD1 transgenic rat model to characterize the

role of microglial activation in a neurodegenerative disease.

In this study we investigated the effect of minocycline specifically on microglial

mitotic activity and neuronal regeneration within the facial nucleus following a nerve









crush injury. No significant difference was found between minocycline treated and

control rats when comparing the 3H-thymidine labeled microglial cells or fluorogold

labeled neurons at all post-injury time points investigated.

To assess microglial activation in the ALS rat model, microglial morphology and

activation were assessed in various brain regions at three stages of the disease,

asymptomatic, onset of symptoms and end stage. Microglia were found to have a normal

resting morphology in the motor cortex at all time points assessed. In the ventral spinal

cord and brainstem there were signs of intense microglial activation. In addition,

microglia fusions and multinucleated giant cells were seen dispersed throughout the

brainstem and ventral horn of the lumbar spinal cord. To further assess microglial

response and function in the transgenic model a facial nerve axotomy was performed and

apoptotic microglial were quantified. Transgenic animals were found to have

significantly reduced numbers of apoptotic microglial when compared to the age-matched

controls 14 days post axotomy. The findings in the current study suggest that microglia

may undergo both functional and morphological changes as a result of mutant SOD1

contributing to the disease.














CHAPTER 1
INTRODUCTION AND LITERATURE REVIEW

Microglia: An Overview

Introduction

Microglia, the resident central nervous system (CNS) macrophage, represent about

10% of the adult brain cell population (Lawson et al., 1990). Historically, microglia

research focused on the ontogeny of microglia with two conflicting hypotheses. One

hypothesis states microglia precursors are cells of neuroectodermal origin (Kitamura et

al., 1984; Fedoroff and Hao, 1991; Hao et al., 1991; Fedoroff et al., 1997); the other

proposes they proceed from mesodermal cells and originate outside of the developing

CNS (Perry and Gordon, 1991; Ling and Wong, 1993; Cuadros and Navascues, 1998).

The latter view is currently the most accepted by those in the field who believe that

microglia derive either from monocytes that leave the blood stream and populate the

brain parenchyma or from primitive hemopoietic cells that differentiate as microglial

cells within the CNS. The presence of primitive microglia in the developing CNS can be

detected prior to the appearance of monocytes causing speculation on the theory that

microglia precursors are monocytes (Hurley and Streit, 1996; Alliot et al., 1999). An

alternative precursor for microglia is primitive hemopoietic cells, also called fetal

macrophages (Streit, 2001). Fetal macrophages form in the blood islands of the yolk sac,

enter the CNS via the meninges by transversing the pial surface (Navascues et al., 2000)

and expand until assuming the fully differentiated, ramified adult microglial cells.









In the healthy adult CNS, microglia constitute a stable cell population, which

maintains itself by proliferation of resident microglia or recruitment of bone-derived cells

(Barron, 1995; Kreutzberg, 1996; Simard and Rivest, 2004; Bechmann et al., 2005).

Resident microglia in the healthy brain are termed "resting" however this is far from their

actual dynamic and motile functions performed. Resident microglia undergo constant

structural changes allowing microglia to effectively survey and maintain the CNS

environment by sampling the environment with highly motile protrusions (Nimmerjahn et

al., 2005). Upon injury, microglia undergo morphological and phenotypic changes

specifically cells undergo hypertrophy, proliferate and up regulate surface antigens and

various cytokines to transform into an activated state (Graeber et al., 1988a; Streit et al.,

1989b, 1989a).

Microglia Are Neurosupportive

Many studies have demonstrated the ability of microglia to produce growth factors

(transforming growth factor 11 (TGF-B1), IL-B, and nerve growth factor) (Giulian et al.,

1986; Kreutzberg, 1996; Nakajima et al., 2001) that aid in neuronal survival during

development and following injury. Further evidence for a neurosupportive role of

microglia is seen in vitro studies in which cultured neocortical and mesencephalic

neurons show enhanced survival and neurite outgrowth following treatment with

conditioned microglial medium (Nagata et al., 1993). In vivo, microglial activation in

the facial nerve paradigm accompanies neuronal regeneration following injury whereas a

central axotomy, such as a transaction of the rubrospinal tract in the cervical spinal cord,

does not result in regeneration and elicits only a minimal microglial response (Barron et

al., 1990; Tseng et al., 1996; Streit et al., 2000). These in vivo observations clearly

support that microglial activation is required to facilitate regeneration. More direct









evidence is seen in transplantation studies that show cultured microglial cells engrafted

into the injured spinal cord promote neurite outgrowth (Rabchevsky and Streit, 1997).

Another function of microglia is to maintain the environment in the CNS by

removing cellular debris and dysfunctional cells. In the presence of degenerating neurons

microglia transform into phagocytic cells that remove damaged cells eliminating the

potential for toxic products to be released into the CNS environment.

Facial Nerve Axotomy

The facial nucleus is the largest brainstem motor nucleus in the rat with

approximately 3000-5000 motoneurons innervating muscles controlling facial movement,

including whisker movement. The motoneurons are organized into several muscle and

region specific nuclei located in the ipsilateral brainstem.

Axotomy of the facial nerve is a well-established paradigm for the study of

microglial activation in response to injury. Peripheral injury of cranial nerve VII

generates a response in the CNS while maintaining the integrity of the blood brain barrier

restricting leukocyte infiltration. In addition, this model has proven useful in microglia

research because the injury is reproducible and can be done in the absence of direct brain

manipulation. The microglial response is well-documented following facial nerve injury.

Early activation is seen within 24 hours of injury and is characterized by an increase in

molecules with an immune function as well as up regulation of OX-42 immunoreactivity

(Graeber et al., 1988a; Kreutzberg et al., 1989). The next stage of microglial activation

occurs 2-4 days post injury when microglia proliferate and begin to home and adhere to

the axotomized motoneurons allowing microglial processes to strip away afferent axon

terminals (Blinzinger and Kreutzberg, 1968). Following nerve reinnervation which

occurs 2 weeks post injury in the rat, microglia decline in number and return to a resting









state. It has been proposed that microglia undergo cell death following recovery of

axotomized neurons, possibly through apoptosis (Gehrmann and Banati, 1995; Jones et

al., 1997).





















Figure 1-1. Lateral view of the facial nerve (7N) with its upper (UP) and lower (IP)
peripheral branches that innervate the vibrissae follicular muscles. Arrowhead
identifies the location of crush site. (Kamijo et al., 2003)

Amyotrophic Lateral Sclerosis

Amyotrophic Lateral Sclerosis (ALS) is one of the most common adult-onset

neurodegenerative disease affecting ~5 per 100,000 individuals. First described by

Charcot in 1869, ALS is characterized by the selective loss of upper and lower motor

neurons invariably progressing to paralysis and death over a 1-5 year time course. Its

etiology is still poorly understood; however a major breakthrough in the field occurred

with the discovery that mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene

affect approximately 20% of patients with familial ALS (Rosen, 1993; Siddique and

Deng, 1996). This discovery allowed for generation oftransgenic animal models which









closely resemble the motor weakness and degeneration seen in human disease (Gurney,

1994; Wong and Borchelt, 1995; Bruijn et al., 1997; Nagai et al., 2001; Howland et al.,

2002). Prior to animal models, it was proposed the mutations in SOD1 reduced enzyme

activity causing decreased free radical scavenging activity and an increase in oxidative

stress. Contradicting evidence emerged from transgenic SOD1 mice that developed

progressive motor neuron disease despite possessing two normal mouse SOD1 alleles

(Bruijn et al., 1998; Jaarsma et al., 2001). In addition, SOD1 knockout mice live until

adulthood and do not develop motor neuron disease (Reaume et al., 1996) indicating that

the mutation in SOD1 causes a toxic gain of function rather then a loss of dismutase

activity. The mutant SOD1 may catalyze aberrant biochemical reactions which result in

production of damaging reactive oxygen species (ROS) such as the superoxide anion, the

hydroxyl radical, hydrogen peroxide and peroxynitrite (Cluskey and Ramsden, 2001).

Misfolding of the mutant protein causes copper at the active site to be less tightly bound

increasing the release of copper. The unoccupied active site is more accessible to

abnormal substrates such as peroxynitrite which leads to nitration of tyrosine residues

(Beckman et al., 1993) and hydrogen peroxide which generates hydroxyl radicals that can

damage cellular targets (Wiedau-Pazos et al., 1996). In addition, to aberrant chemical

reactions occurring at the active site, the SOD1 toxic gain of function may be due to its

participation in formation of protein aggregates. Protein aggregates or inclusion bodies

intensely immunoreactive for SOD 1 are found in motor neurons of the mouse model of

ALS (Bruijn et al., 1997). Aggregates may be toxic due to additional proteins

associating with them thereby depleting protein functions that may be essential for

neuronal survival. Another hypothesis is that by repetitively misfolding, mutant proteins









are reducing availability of chaperones for proteins required for normal cell function. A

final hypothesis relating to aggregates and their role in ALS is that SOD1 mutants reduce

proteasome activity needed for normal protein turnover. SOD1 aggregates are very

stable and even with treatment using strong detergents and reducing agents the aggregates

are not easily dissociated. Formation of SOD1 aggregates disrupts normal balance of

protein synthesis and degradation interrupting normal degradation of misfolded proteins

critical to cell survival.

While familial ALS has been attributed to mutations located within the SOD1 gene,

mechanisms underlying onset and progression of sporadic ALS, which accounts for the

largest percentage of cases, are still largely unknown. It has been hypothesized that ALS

is due to excitotoxcity, protein aggregation, mitochondrial dysfunction, and recently it

has been proposed microglial activation is a contributing factor in the disease process.

SOD1 mutation Damage io t
aberrant Cu! astrocytes Missl icing of
mediated \ AAT2 mRNA
chemistry
\ lower glitamat tranlL-por
Aggregation by EAAT2

/ "(' Excitotoxicity ,,

t I, 1 -,", Strangulation of !" '-
axonal transport Initiatin
c-F-v--iInitiation of >
i; cascade of cell "
NF misorganization !< t,. death processes
+ t.l '


Figure 1-2. Proposed mechanisms of ALS. (Cleveland, 1999)









Microglia in ALS

Glial cell involvement in ALS pathology is unknown; however recent studies have

presented strong evidence for non-neuronal cell involvement in ALS. A series of

experiments revealed that SOD1 mutations are not directly toxic to motor neurons, but

rather exert their neurotoxic effects in a non-cell autonomous fashion. Initial experiments

showed expression of mutant SOD1 in neurons (Pramatarova et al., 2001; Lino et al.,

2002) or astrocytes (Gong et al., 2000) alone failed to induce motor neuron degeneration.

Clements et al., further implicated glial cells in ALS pathology by showing neuronal

degeneration is dependent not on the type of cell carrying the mutant SOD1 gene, but

rather the number of cells. This conclusion was reached from an experiment in which

mutant SOD1 expression in individual neurons surrounded by wild type glial cells

allowed neuronal populations to remain healthy whereas when the reverse occurred with

wild type neurons surrounded by mutant SOD1 glial cells the result was neuronal

degeneration (Clement et al., 2003). The role of glial cells in ALS is clearly

demonstrated in experiments manipulating expression of mutant SOD1 in various cell

types however further experimentation has specifically shown a role for microglia in ALS

pathology. Cell-specific knock down of mutant SOD1 in microglia and macrophages in

transgenic mice cause increased life span (Cleveland, 2004). Further evidence is seen in

a study conducted by Weydt where microglia isolated from transgenic mice showed

significantly higher levels of tumor necrosis factor alpha (TNF-a) when compared to age-

matched controls (Weydt et al., 2004). Cellular evidence of microglia involvement can

be seen in histological studies of both human and animal tissue where microglial

activation and proliferation is seen in regions of motor neuron loss such as the spinal

cord, brainstem and primary motor cortex (Kawamata et al., 1992; Hall et al., 1998;









Almer et al., 1999; Alexianu et al., 2001; Henkel et al., 2004). Microglial activation

temporally corresponds to onset of motor weakness and neuronal loss. Accordingly,

increased microglia production of TNF-a, IL-1B, iNOS, and COX-2 all pro-inflammatory

mediators are present in ALS patients suggesting a likely role of neuroinflammation in

ALS etiology (Poloni et al., 2000; Elliott, 2001; Nguyen et al., 2001; Olsen et al., 2001;

Hensley et al., 2002; Yoshihara et al., 2002). In addition to the products listed above,

there are a number of microglial activating factors found to be elevated in ALS patients

providing a plausible source for persistent microglial activation. It is unknown if

microglial activation is triggered as a response to neuronal death or some other manner

however it appears that once activated they are able to self-activate furthering an

inflammatory environment. It should be noted that unlike other neurodegenerative

diseases, influx of peripheral immune cells is a rare occurrence only seen at the end stage

of ALS (Kawamata et al., 1992; Bruijn et al., 2004). Therefore, in ALS inflammatory

reactions are generated and sustained only by CNS cells most likely microglia since they

are the major immunocompetent cells of the CNS. Evidence to date strongly supports

active participation of microglial cells in ALS pathogenesis however, it still remains

unknown in what manner microglia exert their detrimental effects and if microglial

activation is a secondary effect of the disease process or an initial contributing factor.

Neuroinflammation

Classically, inflammation is a complex response which aims to repair tissue

damage and is accompanied by the cardinal points described by Celsus: pain, tumor,

rubor and heat. Under normal conditions inflammation is a tightly controlled process

maintained until the initial stimulus is repaired or eliminated however the inability of the

immune system to clear the foreign target or repair the existing stimuli results in chronic









stimulation of immune cells thereby resulting in damage to tissue (Nathan, 2002).

Chronic inflammation in the CNS, often referred to as neuroinflammation, is defined by

the presence of activated microglia, reactive astrocytes and inflammatory mediators with

activated microglia being the central component. Recently, neuroinflammation has been

implicated in a number of neurodegenerative diseases such as Parkinson's, Alzheimer's

and ALS. Microglial activation and its role in neurodegenerative diseases is a highly

debated topic in that it still remains unclear whether microglial activation is beneficial or

detrimental. In the non-diseased brain microglia are found in a resting ramified state

where upon acute injury microglia proliferate and undergo morphological changes to

attain a state of activation to aid in repair of damaged tissue. Following recovery

microglia return to a resting state however in neurodegenerative diseases microglia are

thought to maintain a persistent state of activation. It is proposed that neuroinflammation

may drive a self-propagating toxic cycle of microglia in which several factors of disease

such as protein aggregates, injured neurons, and AB plaques activate microglia

exacerbating neuronal death through production of pro-inflammatory products which in

turn further increases levels of microglial activation. Evidence supporting pro-

inflammatory properties of microglia is extensive in neurodegenerative diseases however

whether the microglial reaction is a secondary response to neuronal death or a causative

factor still remains unresolved. Nonetheless, therapeutic treatments targeting

inflammation in a number of neurodegenerative diseases are currently underway with

variable results. COX-2 inhibitor, rofecoxib, has been used for treatment of Alzheimer's

and Parkinson's however clinical trials failed to demonstrate a beneficial effect (Aisen et

al., 2003; Przybylkowski et al., 2004; Reines et al., 2004). These findings suggest that









inflammation may be a secondary cause of neurodegenerative diseases due to the failure

of treatment to halt disease progression.

Microglia Dysfunction

An alternative to neuroinflammation is that microglial cells undergo functional

changes in the diseased brain where they acquire toxic functions or become incapable of

performing normal functions in the CNS. The inability of microglia to perform their

normal function may have detrimental consequences for neurons thereby propagating

neuronal degeneration. Microglia dysfunction has been observed in HIV patients, the

normal aging brain and neurodegenerative diseases.

HIV-1 associated dementia (HAD) is a syndrome of motor and cognitive

dysfunction in 10% of patients infected with HIV-1 with acquired immune deficiency

syndrome (McArthur et al., 1993; Sacktor et al., 2001). Neurons are not productively

infected with the virus suggesting infected cells in the CNS, in particular microglia,

release signals that lead to secondary neuronal injury. Microglia can be activated by HIV

infection itself, by interaction with viral proteins or by immune stimulation in response to

factors released from other infected cells (Lipton and Gendelman, 1995). In response to

activation, microglia have increased production and release of neurotoxic

immunomodulatory factors such as pro-inflammatory cytokines, free radicals and

neurotoxic amines (Genis et al., 1992; Achim et al., 1993; Bukrinsky et al., 1995; Giulian

et al., 1996; Zhao et al., 2001). Further evidence of abnormal microglia is the presence of

multinucleated giant cells which are found in close proximity to apoptotic neurons.

Phenotypic changes have been shown in microglia with aging such as up regulated

expression of MHC II (Perry et al., 1993; Ogura et al., 1994; DiPatre and Gelman, 1997;

Streit and Sparks, 1997; Morgan et al., 1999), greater exhibition of phagocytic









morphology and IL-la immunoreactivity (Sheng et al., 1998). In addition, several

studies have reported significant changes in microglia morphology, including cytosolic

inclusions (Peinado et al., 1998), higher incidence of clumping in and around white

matter (Perry et al., 1993) and structural changes. Morphological abnormalities in

microglia have been identified in the Huntington's mouse model (Ma et al., 2003) and

human Alzheimer's tissue. Structural changes included bulbous swellings, long stringy

processes, cytoplasmic fragmentation and deramified processes.

Corresponding to phenotypic changes microglia appear to undergo functional

changes due to aging. In culture studies and in the facial nerve paradigm aged microglia

display an increased proliferative response (Rozovsky et al., 1998; Conde and Streit,

2005). In vitro, microglial proliferation progressively increases with donor age up to

400% greater at 24 months vs. 3 months (Rozovsky et al., 1998). Following a facial

nerve axotomy microglial proliferation in aged rats is significantly higher 4 days

following axotomy (Conde and Streit, 2005). The enhanced proliferation of aged

microglia may be due to a loss of response to regulatory mechanisms which is supported

by culture experiments where aged microglia did not respond to TGF-11 in contrast to

young microglia that showed an inhibition of proliferation following TGF- 11 treatment

(Rozovsky et al., 1998). This loss of sensitivity to TGF- 11 with increased age was also

demonstrated in the regulation of prolactin in rat anterior pituitary cells (Tan et al., 1997).

The desensitization to anti-proliferative properties of TGFB -1 provides a plausible cause

for the increased proliferative response of aged microglia. Further impairment of TGF3-

1 regulatory mechanisms is seen in cultured microglia. Lipopolysaccharide (LPS)

treatment induces NO production in all donor age cultures however in young donor









cultures NO production is inhibited following TGFB-1 but TGFB-1 has no effect on NO

production in aged donor cultures (Rozovsky et al., 1998). Aging-related changes in

microglia appear to affect the regulatory mechanism of microglial activation causing the

homeostasis of the local CNS environment to be disrupted.

Minocycline

Minocycline is a second generation tetracycline with antibiotic activity against a

broad-spectrum of bacterial types including both Gram-positive and Gram-negative

bacteria. Completely separate and distinct from its antimicrobial activity, minocycline

exhibits anti-inflammatory effects that are proven to be neuroprotective in a number of

neurodegenerative diseases and brain ischemia (Yrjanheikki et al., 1998; Yrjanheikki et

al., 1999; Du et al., 2001; Kriz et al., 2002; Wu et al., 2002). Although other

tetracyclines can diffuse across the blood-brain barrier into the CNS in small amounts,

the lipophilicity of minocycline allows it to attain significantly higher levels in the CNS

furthering its therapeutic potential in neurodegenerative diseases (Barza et al., 1975).

One proposed mechanism of minocycline is a direct neuroprotective action in which

caspases are inhibited by preventing release of mitochondrial cytochrome c (Zhu et al.,

2002; Teng et al., 2004). Another proposed action of the drug is deactivation of

microglial cells indirectly accounting for the observed neuroprotection (Yrjanheikki et

al., 1999; Du et al., 2001; He et al., 2001; Kriz et al., 2002; Wu et al., 2002). Microglia

deactivation occurs through inhibition of p38 MAPK which is thought to mediate the

inflammatory process within microglia by inducing transcription factors that positively

regulate inflammatory genes (Tikka et al., 2001; Koistinaho and Koistinaho, 2002).

Minocycline treatment administered to SOD1 mice delays onset of motor neuron

degeneration and increases longevity of SOD1 mice lifespan (Kriz et al., 2002; Van Den









Bosch et al., 2002; Zhu et al., 2002). Currently, clinical trials are underway to determine

the benefits of minocycline treatment in human ALS patients.

Minocycline appeared to hold great therapeutic treatment for neurodegenerative

disease however recent findings showed a deleterious effect in Parkinson's (PD),

Huntington's (HD) and hypoxic-ischemia (HI) animal models (Smith et al., 2003; Yang

et al., 2003; Tsuji et al., 2004). In a PD model minocycline increased 1-methyl-4-phenyl-

1,2,3,6-tetrahydropyridine (MPTP) toxicity and showed no effect in the transgenic mouse

model of HD (Smith et al., 2003; Yang et al., 2003). It was also reported that in two

chronic models: the MPTP-intoxicated non-human primate model of PD and the 3-

nitropropionic acid (3-NP) intoxicated model of HD minocycline treatment resulted in

earlier clinical motor symptoms during toxic treatment, decreased motor performance and

greater neuronal loss when compared to controls (Diguet et al., 2003; Diguet et al., 2004).

In addition, minocycline is proposed to exacerbate hypoxic-ischemia brain injury in the

immature mouse cortex, thalamus and striatum while neuroprotective in the immature rat

brain (Arvin et al., 2002; Tsuji et al., 2004). In models of HI, deleterious effects of

minocycline may be due to the reduction in compensatory angiogenesis after HI by

inhibiting endothelial proliferation.














CHAPTER 2
MINOCYCLINE DOES NOT INHIBIT MICROGLIA PROLIFERATION OR
NEURONAL REGENERATION IN THE FACIAL NUCLEUS FOLLOWING A
FACIAL NERVE CRUSH

Introduction

Minocycline is a second-generation tetracycline reported to have an anti-

inflammatory activity independent of its antimicrobial function (Amin et al., 1996).

Recently, minocycline has been shown to inhibit microglial activation and promote

neuronal survival in animal models of neurodegenerative disease and stroke (Yrjanheikki

et al., 1999; Du et al., 2001; He et al., 2001; Kriz et al., 2002; Wu et al., 2002). It has

been hypothesized that in neurodegenerative disease microglia undergo detrimental

activation characterized by increased production and release of neurotoxins that

contribute to neuronal cell death. Accordingly, minocycline has been proclaimed as a

potential treatment for neurodegenerative diseases such as amyotrophic lateral sclerosis

(ALS) and Parkinson's disease which are thought to have a neuroinflammatory

component in their pathogenesis (Du et al., 2001; Kriz et al., 2002; Wu et al., 2002).

On the other hand, experimental studies after acute brain injury show that microglial

activation is a consequence of neuronal injury rather than the cause of it. In particular,

experimental paradigms involving neuron regeneration such as motoneuron axotomy,

show that microglial activation precedes successful regeneration of severed axons

suggesting that activated microglia are neuroprotective and support motoneuron

regeneration (Streit, 1993, 2002, 2005). Following axotomy, greater numbers of

microglia are generated through local proliferation (Graeber et al., 1988b) and these cells









encircle the injured neurons in a manner that suggests neuroprotection through

displacement of afferent synapses and close glial-neuronal apposition which may allow

for targeted delivery of microglia-derived growth factors, such as TGF-beta (Mallat et al.,

1989; Martinou et al., 1990; Araujo and Cotman, 1992; Elkabes et al., 1996; Lehrmann et

al., 1998). Furthermore, there is little if any neuronal cell death within the facial nucleus

of the rat following axotomy (Johnson and Duberley, 1998) suggesting that microglia are

aiding with recovery of damaged neurons rather than harming them (Lieberman, 1971;

Streit and Kreutzberg, 1988; Streit, 1993; Kuzis et al., 1999).

To directly test the hypothesis that minocycline inhibits microglia activation in

vivo, we have quantified microglial proliferation in the axotomized facial nucleus. To

determine if there are functional consequences for neurons of this potential microglial

inhibition, we have also quantified and compared numbers of regenerating motoneurons

between minocycline treated and non-treated animals.

Materials and Methods

Animals and Diet

Animal use protocols were approved by the University of Florida Institutional Use

and Care of Animals Committee (IUCAC). Young adult male Sprague Dawley rats were

divided into two groups, one receiving a standard rodent diet while the second group was

fed a diet enriched with minocycline (Igram/kilogram) obtained from Harlan Tekland

(Madison, WI). The diets were implemented one week prior to surgery and continued

throughout the remainder of the experiment. Levels of food intake were recorded for

each cage of animals. Both treatment groups underwent a unilateral facial nerve

axotomy.









Table 2-1. Average intake of minocycline during experiments.
Survival Time Point Number of Days on Diet Average Drug
for Proliferation Studies Intake/Animal (grams)
2 days post-axotomy 9 0.19286
3 days post-axotomy 10 0.24166
4 days post-axotomy 11 0.27190
Survival Time point for
Flurogold Studies
7 days post-axotomy 14 0.30158
14 days post-axotomy 21 0.50119
21 days post-axotomy 28 0.63218


Facial Nerve Axotomy

Animals were anesthetized with isoflurane using a precision vapor machine with gas

scavenging system attached. Animals were placed in an inducing box where isoflurane

was administered until the pedal and palpebral reflexes were absent. Animals were

transferred to a nose cone where isoflurane was administered to the animal for the

remainder of surgery. Upon full sedation, a small incision was made directly behind the

right ear. Using a pair of angled scissors, the superficial levels of the muscle tissue were

cut until the facial nerve was exposed. Both branches of the facial nerve were separated

from the surrounding tissue and crushed with a pair of hemostats for 10 sec. The incision

was closed with a surgical staple and animals were removed from isoflurane and closely

monitored until fully recovered. The absence of whisker movement was assessed to

confirm that both nerve branches were completely crushed.

3H-Thymidine Injections and Radioactive Perfusions

Microglial proliferation was assessed at 2, 3 and 4 days after facial nerve crush

since it is known that the burst of mitotic activity occurs during this time period. Each

time point included ten animals, five in each treatment group. Animals were weighed

and given an intraperitoneal (i.p) injection of 3 [iCi per gram body weight of [methyl-3H]









thymidine (Amersham Pharmacia Biotech) two hours prior to perfusion. Animals were

caged in radioactive labeled cages until perfusion.

Two hours following the 3H-Thymidine injection each animal was given a lethal

dose of sodium pentobarbital (150 mg/kg; i.p.). In the absence of pedal and palpebral

reflexes animals were transcardially perfused with phosphate buffered saline (PBS)

followed by 4% paraformaldehyde (in PBS). Following perfusion brains were dissected

out and placed in 4% paraformaldehyde overnight at room temperature. Each animal was

assigned a random number to maintain objectivity in quantitative analysis. All liquid, dry

and animal waste were labeled as radioactive and disposed according to the University of

Florida waste management protocol.

Tissue Processing for Audoradiography

Following overnight fixation, a coronal section of the brainstem containing the

facial nucleus was dissected out and rinsed in PBS. Each section was processed for

paraffin embedding by slowly dehydrating through an ascending series of alcohol

beginning with 70% ethanol for 2h, 45 min for 70%, 90%, 95%, 100% ethanol then

100% ethanol overnight. Following overnight incubation, brain sections were placed in

two changes ofxylenes for 2h and transferred to paraffin cassette holders. Cassette

holders were immersed in 2 changes of Surgipath Formula R paraffin (Surgipath,

Richmond, IL) at 600C for 2h each. Lastly, the tissue was removed from cassette holders

and embedded in Surgipath Tissue Embedding Medium paraffin (Surgipath, Richmond,

IL) and allowed to cool. The facial nucleus was serially cut (7[m sections) from rostral

to caudal on a microtome and collected on Superfrost Plus slides.









Audoradiography

Immediately before beginning the developing process, slides were deparaffinized

and hydrated through a series of descending alcohols then rinsed in PBS for 5min. The

sections were dipped in a 50% solution of NTB-2 emulsion (Eastman Kodak) and

allowed to air dry in a darkroom with a safelight with Kodak #2 filter. Slides were

exposed in light- protected slide boxes with desiccant at 40C for 5 weeks and developed

with 50% Dektol developer for 2.5min (Eastman Kodak), rinsed in ddH20 for 10dips,

fixed in Kodak fixer for 5min, washed, counterstained with 0.5% cresyl violet,

dehydrated through alcohols and xylenes and coverslipped using Permount mounting

medium (Fisher Scientific).

Quantitative Analysis for 3H-thymidine Labeled Microglia

For quantitative analysis of proliferating microglia every fourth section of the facial

nucleus was counted for 3H-thymidine labeled microglial cells. The sections were

viewed using a Zeiss Axiophot microscope with a Sony DXC970 camera attached. In the

selected sections the facial nucleus was outlined and the area measured using MCID 6

software (Imaging Research, St. Catherine's, Ontario). The labeled cells in the outlined

area were manually counted under 40X magnification. Labeled cells were pooled

together for each animal and divided by the pooled area measured for each animal to

determine a population density of proliferating microglia in the facial nucleus. Results

are represented as mean values + SEM. The density of dividing microglia was compared

among minocycline treated and non-treated animals at each time point with a t test using

GraphPad Prism software (GraphPad Software, San Diego, CA). A significance level of

p<0.05 was used.









Fluorogold Labeling

Regeneration of motor neurons following minocycline treatment was investigated

at 7, 14, and 21 days after facial nerve crush. Each time point included ten animals, five

in each treatment group. Three days before perfusion, animals were given two 10 itL

injections of 4% fluorogold (in saline): one into the whisker pad and one directly

underneath the eye.

Perfusion and Tissue Processing for Fluorogold Labeling

The transcardial perfusion was performed as detailed in the previous radioactive

perfusion section, however in the absence of radioactive precautions. Following the

perfusion brains were dissected out and placed in 4% paraformaldehyde until sectioning.

The brainstem containing the facial nucleus was dissected out and mounted onto a cutting

block. The facial nucleus was sectioned caudal to rostral on a vibratome in 50 itm

sections and collected on SuperFrost slides. Following collection the slides were allowed

to air dry for one hour, dehydrated through a series of ascending alcohols and xylenes and

coverslipped with Permount mounting medium (Fischer Scientific).

Quantitative Analysis of Fluorogold Labeling

For quantitative analysis of regenerating neurons the entire facial nucleus was

counted for fluorogold labeled cells. Results are represented as mean values + SEM.

The number of fluorogold labeled cells were compared among minocycline treated and

non-treated animals at each time point with a t test. A significance level of p<0.05 was

used.

Results

Microglia proliferation is not inhibited in minocycline treated animals at 2, 3,

and 4 days post facial nerve axotomy. Our objective was to determine in vivo if









minocycline could attenuate microglial activation, and to this end we decided to measure

cell proliferation, which is a reliable, quantifiable parameter of microglial activation.

Consistent with prior reports, we found 3H-thymidine-labeled cells at all three time points

examined. However no statistically significant difference between the number of 3H-

thymidine cells was found in the injured facial nucleus at any time point when comparing

the control rats and the rats fed a diet enriched with minocycline in the injured facial

nucleus at all three time points. No cells labeled with 3H-thymidine were observed on the

uninjured side of the facial nucleus within either treatment group.





Ti 7-
6 m I Control









S2 3 4

Days Post Facial Crush


Figure 2-1. Microglial proliferation in the facial nucleus. There is no statistically
significant difference in the cell proliferation between the control animals and
the animals fed a diet enriched with minocycline at any post-axotomy time
point. (Difference at day two is 1.14 1.171 (p = 0.3587), at day three is 1.12
0.5775 (p = 0.1207), and difference at day four is 0.29 0.1922 (p =
0.2364)).









r;d^



' '
S :..


C II


B w q
4L


-A.


Figure 2-2. Photomicrographs of 3H-thymidine labeled cells in the injured facial nucleus.
A) Control rat 3 days post-injury. B) Minocycline treated rat 3 days post-
injury. Note that 3H-thymidine labeled microglia are in close proximity to the
regenerating neurons. Magnification 250x.


450-
400-
350-
300-
250-
200-
150-
100-
50-
n


ZE1


W Control
M Minocycline


I 1


14 21
Days Post Crush Injury
Figure 2-3. Motor neuron regeneration in the facial nucleus. There is no statistically
significant difference in the number of fluorogold labeled neurons between the
control animals and the animals fed a diet enriched with minocycline at 14
and 21 days after nerve crush. Differences between treatment groups at day
14 is 6 8.051 (p = 0.4774) and at day 21 is 10.8 32.12 (p = 0.7454)
Neuronal regeneration is not inhibited by minocycline treatment. Since any
attenuation of microglial activation by minocycline could have an effect on neuronal









regenerative ability after nerve crush we performed fluorogold injections 7, 14 and 21

days following a unilateral facial nerve crush to determine the number of neurons that

underwent successful reinnervation. No fluorogold labeled neurons were found on day 7

after nerve crush; they first became apparent at 14 days and became more numerous by

day 21. The results show that successful reinnervation of facial muscles takes place

between 2 and 3 weeks (Soreide, 1981; Fawcett and Keynes, 1990). The counts of

regenerating neurons throughout the facial nucleus show that minocycline did not

influence regeneration of neurons between the control and minocycline treated groups.




















Figure 2-4. Fluorogold labled neurons within the lateral and ventral intermediate sections
of the injured facial nucleus. A) Control rat 21 days post-facial nerve injury.
B) Minocycline treated rat 21 days post-facial nerve injury. Bar = 100m.


Discussion

In this study we report that microglial proliferation in vivo is not inhibited by

minocycline within the facial nucleus following a nerve crush. These results differ from

those reported in previous studies, which found that minocycline significantly inhibits









microglial proliferation in vitro (Tikka et al., 2001; Tikka and Koistinaho, 2001). To

reconcile this apparent contradiction in experimental findings, one needs to consider the

fact that there are fundamental differences between microglia in vitro and in vivo (Streit,

2005). Specifically, with regard to cell division, it is important to note that microglial

cells in culture undergo mitosis constitutively and spontaneously, because they exist in a

permanent state of activation in vitro. This differs from resting microglia in the CNS,

which only become activated and proliferate in the presence of a stimulus, such as acute

CNS injury. This mismatch of activation states between cultured microglia and microglia

in the brain likely accounts for the differences in proliferation dynamics observed after

minocycline treatment in vitro and in vivo. In addition, previous studies conducted in the

SOD1 and Parkinson's mice (LPS and 6-hydroxydopamine induced) classified activated

microglia by the presence of cell surface markers such as OX-6, OX-42 and Mac-2 (He et

al., 2001; Kriz et al., 2002; Tomas-Camardiel et al., 2004). In these studies a decrease in

expression of OX-6, OX-42 or Mac-2 was found following minocycline treatment which,

in turn, led to the conclusion that by decreasing microglial activation life span in the

SOD1 mouse is increased and dopaminergic cells become partially protected in the

Parkinson's mouse. However, the exclusive use of cell surface markers is not a reliable

method for quantification of activated microglia. OX-6 labeling of MHC II expression is

not always indicative of microglial activation considering that not all activated microglia

are MHC II positive and MHC II expression can be found on resting microglial cells in

the normal rodent brain, often in the white matter (Streit et al., 1989a). The decrease in

expression of OX-42 and Mac-2 following minocycline treatment could be a result of a

direct neuroprotective effect of the drug which may allow greater neuronal survival









thereby decreasing the intensity of the reactive microgliosis. It has been shown in vitro

and in vivo that minocycline is able to inhibit cyctochrome c release as well as activation

of downstream caspase dependent and independent cell death pathways (Zhu et al., 2002;

Wang et al., 2003). Presently, clinical trials are underway to test the effectiveness of

minocycline vs. placebo in the treatment of ALS patients. These trials resulted after

minocycline was found to increase life span in animal models representing familial ALS.

It is hypothesized that minocycline increased life span in SOD1 mutant mice by

inhibiting microglial activation through the p38 MAPK pathway. However, as we show

in this study minocycline fails to inhibit microglial proliferation, a key characteristic of

microglial activation. Perhaps, minocycline is effective in the SOD1 mouse by being

directly neuroprotective. In the facial nucleus following injury the number of

regenerating neurons was unchanged between the minocycline treated animals and the

control group as was the number of proliferating microglial. This finding offers further

support for the idea that microglial activation after CNS injury is a neuroprotective

mechanism.














CHAPTER 3
DETERMINATION OF MINOCYCLINE CONCENTRATION IN THE BRAIN
AFTER DRUG ADMINISTRATION IN DIET

Introduction

The semisynthetic second-generation minocycline has neuroprotective effects in

models of cerebral ischemia (Yrjanheikki et al., 1998; Yrjanheikki et al., 1999; Tikka et

al., 2001). Minocycline has also been shown to prevent nigrostriatal dopaminergic

neurodengeneration in mouse models of Parkinson's disease (Du et al., 2001; Wu et al.,

2002), delay disease progression in a transgenic model of Huntington disease (Chen et

al., 2000) and increase life span in a mouse model of ALS (Kriz et al., 2002; Van Den

Bosch et al., 2002; Zhu et al., 2002). While all these studies suggest that minocycline

might offer a useful pharmological approach for treatment of numerous

neurodegenerative disease it is still unknown on the concentrations reached in the brain

and how they relate to blood concentrations in animals. Analyses based on simple

protein precipitation (Birminham et al., 1995), liquid-liquid (Mascher, 1998; Araujo et

al., 2001) or solid-phase extraction procedures (Wrightson et al., 1998; Orti et al., 2000)

followed by high-performance liquid chromatography (HPLC) with ultraviolet detection

have been reported for the determination of minocycline in biological samples.

The current study will investigate mincoycline concentrations in the brain

following drug treatment administered through diet. The absorption of minocycline is

believed to be affected by simultaneous administration with food (Leyden, 1985; Meyer,

1996) therefore, to confirm that the lack of inhibition of microglia proliferation in chapter










2 was not a result of insufficient levels of minocycline in the brain, HPLC MS/MS will

be utilized to measure minocycline concentrations in the brain following drug intake

through diet.

Materials and Methods

Reagents

Minocycline hydrochloride and tetracycline hydrochloride were obtained from

Sigma-Aldrich. Stock solutions were prepared by dissolving tetracyclines in methanol at

a concentration of 1 mg/mL. Working standard solutions were prepared from stock

solutions by dilution with methanol. To prepare standards for the concentration curve,

three concentrations of the analyte were prepared in methanol; 0.05 tg/mL, 0.5 tg/mL

and 2.5 [tg/mL.


Tetracycline


.OH
"0 H
NH,


OH 0 OH 0 0


Minocycline









OH o OH o


Figure 3-1. Chemical structures of internal standard and minocycline.









Animals

Two male Sprague Dawley rats weighing 200-250 g were used, one was fed a diet

enriched with minocycline (1 gram/kilogram) for one week prior to analysis while the

other rat was fed a standard rodent diet. Animals were killed by decapitation under deep

anesthesia, brains were rapidly removed and blotted to remove excess surface blood and

stored at -200C until analysis. Water and food were freely available throughout the study.

Extraction

A 200 mg section of the brainstem containing the facial nucleus was removed from

the whole brain and homogenized in 1 mL of 0.01M phosphate buffer pH 7.4, on ice. To

1 mL of the resulting homogenate 25tL of the internal standard tetracycline (10 tg/mL)

and 20 tL of H3PO4 were added, vortex mixed and centrifuged at 2000rpm at 40C for

30min. The precipitate was re-dissolved in 1 mL of 0.01M phosphate buffer, pH 7.4 and

centrifuged. Following centrifugation, the supernatants were combined.

To clean up supernatants lml/30mg Oasis HLB extraction cartridges (Waters) were

used. Prior to applying the homogenized tissue sample cartridges were preconditioned

with 1 mL of methanol followed by 1 mL of distilled water. Samples were loaded into

columns, the columns were rinsed with 1 mL of 5% methanol solution, and dried for 10

min through suction. The samples were eluted with 1 mL of methanol. A blank sample

was processed in tandem with brain samples to eliminate concern of contamination

during extraction.

Equipment

The apparatus used for High Performance Liquid Chromatography was an Agilent

1100 series binary pump (Palo Alto, CA) equipped with an Agilent 1100 UV/V detector










set at 353 nm. Separation was carried out on a Pehnomenex Synergi 4u Hydro-RP 80A

(Torrace, CA) (2 x 150 mm, 4 um) plus C18 guard column (2 mm x 4 mm).

Mass spectrometric detection was performed using a TehermoFinnigan LCQ (San Jose,

CA) with electrospray ionization (ESI).



100 -












60.
3 \ ., 20




so




8 0-


Figure 3.2. Minocycline concentrations found through HPLC/MS/MS analysis. A) In
the minocycline treated animal there was a peak area showing a presence of
minocycline at a concentration of 0.4968 [tg/mL. B) In the control sample
there was no peak area corresponding to minocycline.

Results

Minocycline was found at a significant concentration in the treated animal. In

order to confirm that minocycline had crossed the blood brain barrier, HPLC MS/MS was

performed on brainstem tissue from a control animal and from an animal fed

minocycline. This showed a peak area for the minocycline compound in the treated

animal but not in the control animal. The extract of the minocycline-treated animal was

analyzed twice. From the concentration in the methanol extract and knowing the

extraction procedure, the concentration of minocycline was determined and was found at









a concentration of 0.4968 [g/mL in the treated animal. Using the area of the standard

tetracycline a calibration curve was created. A linear regression yielded the equation of

the line: y = 9.338x 0.0314 where y = the area ratio Minocycline/Tetracycline and x is

the [Minocycline] in [tg/mL. Rearrangement of this equation yielded the equation to

determine the [M] in the unknown: [M] = {(M/T) + 0.0314} /9.3338.

Discussion

The current study demonstrates that significant levels of minocycline were detected

in the brain following drug administration in rat rodent diet. A study conducted by Du

and colleagues found that minocycline orally administered at a concentration of

120mg/kg in the 1-methyl-4-phenyl-1,2,3,6-tetraydropyridine (MPTP) Parkinson's model

provided neuroprotection to midbrain dopamine neurons from the toxic effects of MPTP.

Minocycline levels were assessed using liquid chromatography and mass spectral

detection and found minocycline concentration in the midbrain at 0.32 tg/g compared to

the 2.484 tg/g found in the rat brains assessed in this study. Accordingly, it appears the

lack of inhibition of microglia proliferation in the study described in chapter 2 is not a

result of insignificant levels in the brain but rather an actual lack of effect of minocycline

on microglial activation.

A number of studies using intraperitoneal (i.p.) injection for drug delivery have

claimed minocycline as a neuroprotective agent in neurodegenerative diseases (Van Den

Bosch et al., 2002; Zhu et al., 2002; Wang et al., 2003). It is difficult to determine the

levels of minocycline in the brain using this route of administration due to the lack of

studies investigating minocycline concentrations in brain tissue following an i.p.

injection. The majority of HPLC studies concentrate on the levels of minocycline in the

plasma rather than actual brain tissue.






30


It can be concluded, that oral administration of minocycline is an effective method

for drug delivery and that failure of minocycline to inhibit microglial activation in the

facial nucleus is not a result of low levels of minocycline in the brain.














CHAPTER 4
TIMELINE OF MICROGLIA PROGRAMMED CELL DEATH IN THE FACIAL
NUCLEUS FOLLOWING INJURY

Introduction

Activation and proliferation of microglia is one of the earliest and most common

glial reactions in the injured brain. Upon injury in the CNS, specifically in the facial

nucleus, microglial cells dramatically increase in cell number (Cammermeyer, 1965;

Graeber et al., 1988b; Raivich et al., 1994) and are recruited to perineuronal sites where it

is suggested they displace afferent synaptic terminals (Blinzinger and Kreutzberg, 1968).

Following the initial microglial response and regeneration of motoneurons, activated

microglia migrate into the nearby parenchyma (Angelov et al., 1995) and decline in

number often reaching baseline levels several weeks following injury (Streit et al., 1988;

Raivich et al., 1993). Initially, the mechanism used to maintain homeostasis of

microglial cell numbers was attributed to migration of activated microglia to blood

vessels where microglia exit through the walls (Del Rio-Hortega, 1932; Cammermeyer,

1965) however, recent studies suggest that microglia regulation is controlled by a form of

programmed cell death (Gehrmann and Banati, 1995; Jones et al., 1997).

Programmed cell death is an important mechanism used to control cell population

during development, growth and in regulation of the immune response (Allen et al., 1993;

Bortner et al., 1995; Majno and Joris, 1995). Apoptosis is the most common type of

programmed cell death investigated in the literature and is characterized by chromatin

condensation, DNA fragmentation, membrane blebbing and caspase induced (Gavrieli et









al., 1992; Bohm and Schild, 2003; Jaattela and Tschopp, 2003). Apoptotic cells are

quickly phagocyctosed without induction of an inflammatory response allowing

homeostatic regulation of the CNS. More recently, a number of reports have described a

caspase-independent form of programmed cell death that display activation of other

proteases and changes in morphology not consistent with classical apoptosis (Jaattela and

Tschopp, 2003; Nagy and Mooney, 2003; Lockshin and Zakeri, 2004).

The main objective of the study in the current chapter is to compare the post-

mitotic turnover of microglia at different post-injury time points using the ApopTag

assay, a kit variation of the terminal deoxynucleotidyl transferase-mediated deoxyuridine

triphosphate nick end labeling method. The ApopTag kit labels DNA fragmentation, a

key component of apopotsis, by detecting DNA strand breaks by enzymatically labeling

the free 3'-OH termini with modified nucleotides. The findings from this study will be

used for further experimentation in ALS animal models to determine key differences in

post-mitotic microglia turnover in diseased and non-diseased animals.

Materials and Methods

Animals and Tissue Processing

Male Sprague Dawley rats weighing 200-250 grams were used. Animals

underwent a unilateral facial nerve crush as described in chapter 2. Four animals per

survival time were euthanized at 10, 14, 17 and 21 days post-injury by a transcardial

perfusion as described in chapter 2 without radioactive precautions. Briefly, animals

were given an overdose of sodium pentobarbital and transcardially perfused with 0.1 M

PBS pH 7.4 followed by 4% paraformaldehyde. Immediately following perfusion, brains

were quickly removed and stored in 4% paraformaldehyde for 2h and then transferred to









PBS. Brainstem containing the facial nucleus was paraffin processed as described in

chapter 2 and 7 [tm sections were cut on a microtome.

TUNEL and DAPI Staining

To assess apoptotic cells, TUNEL labeling was performed on the processed tissue.

Sections were deparaffinized through xylenes (2 changes for 15 min each) and

descending alcohols for 2 min each (100%, 100%, 95%, 90%, 70%, 70%) and rinsed in

PBS for 5 min. The ApopTag Red In Situ detection kit (Serologicals Corporation,

Norcross, GA) was used as described in the manufacturer's protocol however the

pretreatment step was omitted. Negative controls omitted the terminal deoxynucleotidyl

transferase (TdT) enzyme.

Following TUNEL labeling, slides were counterstained with DAPI. Slides were

incubated with DAPI at a concentration of 1:333 for 5 min in a light protected box, rinsed

in PBS and coverslipped.

Quantitative Analysis

Eight sections containing the facial nucleus were used for TUNEL quantification.

Sections were imaged using a Spot RT digital camera (Diagnostic Instruments, Sterling

Heights, MI) attached to a Zeiss Axioskop microscope. All TUNEL positive cells

located in the facial nucleus were counted and pooled together per time point and divided

by the total number of sections counted to give an average. Results are represented as

mean values + SEM.

Results

Differences in density of TUNEL positive cells in the injured facial nucleus

were seen at different post-axotomy time points. TUNEL positive cells were found at










all time-points investigated in the injured facial nucleus. There were differences seen in

the number of TUNEL labeled cells at 10, 14, 17 and 21 days (22 1.194, 51 3.061,

10 + 0.064, and 13 0.832) with the greatest density of TUNEL positive cells present at

14 days post-axotomy (Fig. 4-1). No TUNEL labeled cells were found in the

contralateral, unoperated facial nucleus (Fig. 4-2d).


60


50






3 30


S20






0
10 14 17 21
Days Post Axotomy


Figure 4-1. A time line of TUNEL positive microglia in the facial nucleus following
injury. Results are represented as mean values + SEM. (Day 10, 22 + 1.194,
Day 14, 51 + 3.061, Day 17, 10 + 0.064, and Day 21,13 + 0.832)

TUNEL labeled microglia display abnormal cytoplasmic staining at all time

points post-axotomy. The majority of TUNEL positive microglia showed staining

diffusely dispersed throughout the cytoplasm however, lacked intense nuclear staining

commonly found with classic apoptosis. The cytoplasmic staining revealed ramified

processes and a perineuronal location of the TUNEL positive cells similar to that seen of

microglial cells stained with microglia cell surface markers (Fig. 4-2c). A small









population of TUNEL cells displayed both cytoplasmic and nuclear staining revealing an

overlap between TUNEL and DAPI staining (Fig. 4-2a,b). Cytoplasmic staining did not

appear to be contained in lytic vesicles characteristic of phagocytosed debris making it

unlikely to be degraded DNA from adjacent dying cells.

While no co-labeling was performed to identify the TUNEL labeled cells, previous

studies have identified microglia as the only cell to die during the regenerative process in

the facial nucleus (Gehrmann and Banati, 1995). In addition, the morphology of the

TUNEL cells is identical to microglia morphology.


Figure 4-2. Non-classical TUNEL positive cells were found throughout the neuropil of
the injured facial nucleus 14 days post-injury. A) TUNEL labeling in the
facial nucleus displaying cytoplasmic staining representative of microglial
cells. B) Section of double labeled TUNEL/DAPI cells. The arrows identify
nuclear and cytoplasmic staining of microglia. C) Facial nucleus 14 days post-
axotomy with arrows identifying the perineuronal position of microglia. D)
Contralateral, unoperated facial nucleus. Bar = 80tm









Discussion

Axotomy of the rat facial nucleus leads to mitotic division of microglial cells

leading to an increase in cell number (Graeber et al., 1988b; Gehrmann and Banati,

1995). Upon recovery of motoneurons, microglial are thought to undergo programmed

cell death to return to normal homeostatic levels (Gehrmann and Banati, 1995; Jones et

al., 1997). The current study investigated the time course of microglial turnover in the

facial nucleus following a crush injury. The greatest density of TUNEL positive

microglia was seen at 14 days post axotomy where successful reinnervation of the facial

muscles has occurred (Kamijo et al., 2003). The correlation between regeneration and

microglia turnover further supports the essential role of programmed cell death in

regulating the immune response in the CNS following an injury in order to maintain the

homeostatic CNS environment (Fig 4-3).



Soo
500












Cnrt ld 2d 3d 4d 6d ld 12d 1 d
Days after crush injuries

Figure 4-3. Flourogold labeled neurons at different survival times following a crush
injury in the rat facial nucleus. The regenerative patterns of neurons shows a
positive correlation with microglia turnover (Kamijo et al., 2003).









TUNEL labeling in the facial nucleus demonstrated diffuse cytoplasmic and

nuclear staining of microglial cells, similar to previous studies that found non-typical

cytoplasmic staining of microglial cells following facial nerve injury (Jones et al., 1997).

To dismiss handling artifact, the study conducted by Jones and collages examined

different fixations, pretreatment and staining methods where it was found cytoplasmic

staining was present under all experimental conditions. The authors concluded that the

non-typical TUNEL staining suggested microglial turnover occurred through a non-

classical form of programmed cell death.

As the only cell in the CNS capable of transforming into phagocytic cells,

microglia can engulf extracellular and neuronal debris which may contain degraded

DNA. In vivo it is known that microglia are recruited to neuronal debris and responsible

for their removal (Moller et al., 1996) however, this is an unlikely explanation for the

diffuse cytoplasmic staining seen since there was no evidence of phagosomal structures.

Furthermore, facial nerve injury in the rat does not induce neuronal death eliminating the

phagocytic properties of microglia (Moran and Graeber, 2004). However, ricin/axotomy

model leads to extensive neuronal degeneration followed by microglial phagocytosis but

does not show a significant increase in the number of TUNEL labeled cells (Jones et al.,

1997).














CHAPTER 5
MICROGLIA UNDERGO MORPHOLOGICAL AND FUNCTIONAL
ABNORMALITIES IN THE SUPEROXIDE DISMUTASE 1 RAT

Introduction

Amyotrophic lateral sclerosis (ALS) is an adult onset neurodegenerative disease

characterized by selective loss of upper and lower motor neurons. Loss of motor neurons

results in muscle paralysis and ultimately death due to respiratory failure. 5-10% of ALS

cases are familial, inherited in an autosomal dominant pattern (Mulder et al., 1986; Deng

et al., 1993; Rosen et al., 1993; Siddique and Deng, 1996) Of familial ALS cases, 20%

have been linked to mutations located in the Cu/Zn superoxide dismutase 1 (SOD1) gene

(Rosen et al., 1993; Siddique and Deng, 1996). The identification of SOD1 gene

mutations has provided insight into understanding the molecular pathology of ALS.

Specifically, transgenic rodent models expressing SOD1 mutant G93A have provided a

model that closely resembles the human form of the disease, however to date there is no

single mechanism that can be identified in the etiology of ALS. Recent literature has

focused on non-neuronal cells in the propagation of ALS. Several studies have shown

expression of mutant SOD1 limited to motor neurons is insufficient to cause motor

neuron degeneration supporting glial cell involvement in ALS (Pramatarova et al., 2001;

Lino et al., 2002; Clement et al., 2003). In vitro studies found differences in TNF-a

levels secreted following stimulation with LPS when comparing microglia isolated from

transgenic and wild type mice at day 60 (Weydt et al., 2004). Transgenic microglia

produced higher levels of TNF-a. In addition, increased levels of microglial activation









are readily discernible in affected areas in both human and animal models of the disease

(Kawamata et al., 1992; Hall et al., 1998; Alexianu et al., 2001). Thus, from current

evidence it may be proposed that mutant SOD1 may cause abnormalities in microglial

cells in ALS that alter normal cell function. The abnormal microglia could undergo

functional changes that result in increased levels of cytotoxicity further propagating

disease. Alternatively, microglial may become senescent or dysfunctional due to the

SOD1 mutation thereby reducing the number of functional microglia able to provide

trophic support to motor neurons furthering neuronal cell death.

The aim of the present study is to characterize microglia in the mutant G93A SOD1

transgenic rat, specifically the morphological changes that occur throughout the brain and

spinal cord at specific stages of the disease. In addition, this study will investigate

microglia turnover in the spinal cord and following a facial nerve injury to determine if

mutant SOD1 expression in microglia causes the cells to be more susceptible to

apoptosis causing fewer microglial to be readily available in maintaining a healthy

environment for neurons.

Materials and Methods

Animals and Surgery

Animal use protocols were approved by the University of Florida Institutional Use

and Care of Animals Committee (IUCAC). All transgenic animals used in this study

were male Sprague Dawley NTac:SD-TgN(SOD G93A)L26H rats obtained from

Taconic Farms. Animals were monitored daily to assess muscle weakness and to record

disease progression.

To examine microglial morphology OX-42, OX-6 and TUNEL staining were used

at three stages of the disease: asymptomatic where animals had no visual muscle









weakness, onset of symptoms where animals first showed evidence of weakness in the

hind limb and end stage where animals were no longer able to right themselves after 30 s.

Animals were sacrificed by transcardial perfusion as detailed in Chapter 2 at the specified

stage of the disease. Immediately following perfusion brains and spinal cords were

removed and fixed in 4% paraformaldhyde for 2 h. For OX-42, OX-6 and TUNEL

labeling the tissue was placed in 30% sucrose until the tissue was saturated. All time

points included 4 transgenic animals and 4 age-matched wild type controls.

To assess microglial turnover in the facial nucleus, 6 non-symptomatic SOD1

transgenic and 6 age-matched control animals underwent a facial nerve axotomy where

under isoflurane anesthesia, the right facial nucleus was exposed at the exit from the

stylomastoid foramen and crushed with a hemostat for 10 s. At 14 days post-axotony

animals were sacrificed by transcardial perfusion as described in Chapter 2. The brains

were immediately removed and placed in 4% fixative overnight. Tissue sections

containing the facial nucleus were paraffin embedded as detailed in Chapter 2, cut ona

microtome at 7 am, and mounted on SuperFrost Plus slides. A number of slides from

these animals were labeled with lectin as described in the lectin histochemistry section

found in a later paragraph in the materials and methods section.

Table 5-1. Age and corresponding disease stage for animals used in experiments.
Age of Age of Onset Age of
Asymptomatic Animals Endstage
Animals Animals

OX-42/OX-6 74-84 days 113-117 days 135-140 days
Labeling
TUNEL/Lectin 107-114 N/A N/A
Labeling









OX-42 and OX-6 Immunohistochemistry

Lumbar spinal cord, cortical, and brainstem sections were serially cut at 20 [tm on a

cryostat, mounted on Superfrost Plus slides and air dried for one hour. Sections were

pretreated in 0.5% PBS-Triton for 15 min, blocked in 10% normal goat serum for 30 rin

and incubated overnight at room temperature in the primary antibody diluted in buffer.

The primary antibodies included MRC OX-42 (Serotec, Cambridge, UK) and MRC OX-6

(Serotec, Cambridge, UK) at 1:500. The slides were rinsed in PBS and incu bated in

secondary antibody (1:500) for Ih. Following incubation, slides were rinsed for 9 min

and Horseradish Peroxidase Avidin D was applied (1:500) (Vector, Burlingame,CA) and

incubated for 30 min. Slides were washed and immunoreactivity was visualized with

3,3'-diaminobenzidine (DAB)-H202 substrate. After a brief rinse, slides were dehydrated

in increasing concentrations of ethanols, cleared in xylene, and coverslipped using

Permount mounting medium (Fisher Scientific).

Quantification of Immunohistochemistry Labeling in the Ventral Spinal Cord

OX-42 and OX-6 expression in the ventral spinal cord was quantified using Image

Pro Plus software. The area occupied by labeled cells was highlighted and measured for

each section of spinal cord (6 sections per animal) then expressed as a percentage of total

area of ventral spinal cord. Using GraphPad Prism software (San Diego, CA) a t-test was

performed to determine statistical significance between transgenic SOD 1 and control

animals at each time point. A one-way ANOVA was performed to compare differences

among the transgenic animals followed by a Tukey multiple comparison test. A

significance level of p<0.05 was used.









TUNEL labeling and cell identification in the spinal cord

Lumbar spinal cord sections were serially cut at 20 [tm on a cryostat, mounted on

Superfrost Plus slides and air dried for one hour. The ApopTag Red In Situ Apoptosis

Detection Kit (Serologicals Corporation, Norcross, GA) was used as described in the

manufacturer's protocol omitting the pretreatment step. To identify the cell type of

TUNEL positive cells RIP1 oligodendrocytess), GFAP (astrocytes), OX-42 and OX-6

(microglia) were used. Following the TUNEL procedure, sections were pretreated in

0.5% PBS-Triton for 15 min, blocked in 10% normal goat serum for 30 min and

incubated overnight at room temperature in the primary antibody diluted 1:500 in buffer.

The slides were rinsed in PBS and incubated in secondary antibody (1:500) for lh.

Following incubation, slides were rinsed for 9 min and FITC-Avidin D was applied

(1:500) (Vector, Burlingame,CA) and incubated for 30 min. Slides were rinsed briefly

and coverslipped.

Lectin histochemistry

Prior to lectin staining, sections were deparaffinized through xylenes, graded

alcohols and rinsed in PBS. Next, the slides were trypsin treated (0.1% trypsin, 0.1%

CaC12) for 12 min at 370C. Following a 10 min wash the slides were incubated overnight

at 40C in lectin GSA I-HRP (Sigma Chemical Co.) diluted 1:10 in PBS containing

cations (0.1 mm of CaC12, MgC12 and MnC12) and 0.1% Triton-X100. After overnight

incubation slides were briefly rinsed in PBS and visualized with 3,3'-diabimobenzidine

(DAB)- H202 substrate. Sections were counterstained with cresyl violet, dehydrated

through ascending alcohols, cleared in xylenes and coverslipped with Permount.









TUNEL and Lectin Double Labeling

Microglial cell death was visualized in the facial nucleus using TUNEL on tissue

sections from animals 14 days post-facial nerve axotomy. Sections were deparaffinized,

washed in PBS, trypsin treated (0.1% trypsin, 0.1% CaC12) for 12 min at 370C and rinsed

two times in PBS for 5 min each. The ApopTag Red In Situ Apoptosis Detection Kit

(Serologicals Corporation, Norcross, GA) was then used as described in the

manufacturer's protocol omitting the pretreatment step. Following TUNEL, sections

were stained using GSA I-FITC (Sigma, St.Louis, MO) according to protocol described

in the previous lectin section. Six sections containing the facial nucleus per animal were

manually counted for TUNEL labeled cells and statistically evaluated using an unpaired

t test.

Brown and Brenn Gram Stain

A gram stain was performed on tissue containing the facial nucleus from transgenic

SOD1 animals 14 days post-axotomy. The tissue was handled and processed following

the same protocol detailed for the TUNEL labeling in the facial nucleus. Slides were

deparaffinized and hydrated to distilled water. Next 1 mL of crystal violet solution and 5

drops of 5% sodium bicarbonate solution were added to sections and allowed to sit for

one minute then rinsed in tap water. The slides were decolorized with acetone, rinsed in

water, flooded with basic fuchsin working solution for 1 min, rinsed again and placed in

water. Each slide was individually dipped in acetone to start reaction and immediately

differentiated with picric acid-acetone solution until turning a yellowish pink. Finally

slides were quickly rinsed in acetone, then in acetone-xylene solution, cleared in

2 changes of xylene and mounted with resinous medium.









Results

No change in microglia morphology or activation was seen in the cortex of

SOD1 animals. To investigate changes in microglia morphology in the transgenic SOD1

rat, a number of microglia markers were used for staining at various time points

throughout disease progression. Numerous CNS regions were investigated to determine

if microglia change was limited to areas where extensive neuronal degeneration occurred

or rather was a widespread effect.

The cortex revealed OX-42 positive microglia with a normal resting morphology

distributed evenly throughout the cortex (Fig. 5-la). OX-6 staining labeled a small

number of microglial cells in the gray matter indicating an absence of MHC II positive

microglia in the cortex of ALS rats at all stages of the disease (Fig. 5-1b).











Figure 5-1. Photomicrographs of microglia labeling in the cortex of SOD1 rats. A) OX-
42 labeled cells in animals with early onset of symptoms displaying a ramified
morphology representative of a resting state. B) Cortex of end stage animal
labeled with OX-6 showing that there is a lack of MHCII expression in SOD1
animals. The arrow identifies a labeled microglia confirming that the OX-6
stain worked in the cortex. Bar = 40gm

Abnormal microglial fusion and activation was present at the level of the red

nucleus. The brainstem portrayed drastically different characteristics of microglia

throughout disease progression. Morphological changes of microglial cells were seen

prior to onset of symptoms at the level of the red nucleus (Fig. 5-2a,d) and persisted until









end stage. OX-42 staining revealed intense microglial activation within the red nucleus

at all 3 stages of the disease. A distinguishable border was present between the red

nucleus and the surrounding tissue clearly demonstrating that intense microglial

activation was contained to the red nucleus (Fig. 5-2e). It appears that activation

occurred prior to neuronal degeneration since neuronal populations of the red nucleus

were similar between control and transgenic animals in asymptomatic animals. Further

evidence of microglial changes at the level of the red nucleus were seen with the presence

of microglial fusions (Fig. 5-2f). The majority of fusions were located within the nucleus

and contained a large number of microglial cells clumped together. Upon close

observation, it is evident that the fusions were microglia because the cells were OX-42

positive and displayed a morphology representative of microglia. With further disease

progression the fusions were distributed throughout the entire level of the red nucleus.

In the same sections containing the red nucleus, the oculomotor nucleus and

substania nigra were investigated and found to have no microglial fusions and a

population of evenly dispersed ramified microglial cells which remained throughout

disease progression (Fig. 5-2b,c).

SOD1 animals present abnormal microglia fusions at the level of the facial

nucleus. When looking at the level of the facial nucleus similar changes were seen as in

the red nucleus. Asymptomatic animals showed a small number of fused microglia

however no other abnormalities or activation were seen. With the appearance of muscle

weakness through the end stage of the disease, increasing numbers of microglial fusions

were present with the majority of fusions distributed outside the facial nucleus. The

fusions varied in appearance with some displaying a long string of microglial cells









whereas others were rounded fusions of microglial cells representing multinucleated giant

cells (Fig. 5-3a,b,c). The giant cells had microglial nuclei orientated in a circle and were

intensely stained with both OX-42 and lectin (Fig. 5-3d). Unlike the red nucleus, the

facial nucleus did not have increased levels of microglial activation throughout the

disease process. The microglial population in the nucleus was found to have a ramified

morphology indicative of a resting state. An unexpected finding was seen in one animal

where a nidus of bacillus bacteria was detected within the tissue section (Fig. 5-4d).

However, when trying to confirm the presence bacteria using a gram stain there was no

evidence of bacteria.

In addition to microglial abnormalities there was evidence of pathological changes

of neurons. It appears that neuronal bodies are undergoing degenerative changes as seen

by the separation of dendrites from their neuronal bodies (Fig. 5-4a,b). The majority of

fragmented dendrites were located in vacuoles that become increasingly apparent in the

diseased brainstem (Fig. 5-4c).

Microglial activation and abnormalities were evident in the ventral spinal cord

prior to onset of muscle weakness. Microglial response to disease progression in the

ventral spinal cord was assessed using OX-42 and OX-6 markers. OX-6 labeling

revealed an increase in density of MHC II positive labeling with progression of the

disease and was significantly higher in transgenic animals when compared to age-

matched controls after the occurrence of symptoms (Figs.5-5, 5-6). OX-42 density in the

ventral spinal cord initially increased with the occurrence of symptoms however

decreased in number at the end stage of the disease. Elevated levels of OX-42 expression









were found in the transgenic SOD1 animals at all time points when compared to age-

matched controls (Figs.5-7, 5-8).


Figure 5-2. Photomicrographs representing aberrant microglial activation at the level of
the red nucleus in early onset SOD1 animals. All sections were stained with
OX-42. A) Midbrain section displaying intense microglial activation located
in the red nucleus. B) Oculomotor nucleus showing a lack of aberrant
microglial activation. C) Subtania nigra displaying microglia with a ramified
resting morphology. D) Higher magnification of the microglial response in
the red nucleus. (A-D) Bar = 160tm E) The border separating the microglial
activation in the red nucleus from the surrounding tissue. Bar = 80tm F)
Presence of fused microglia located in the red nucleus intensely stained with
OX-42. Bar = 40 tm




























Figure 5-3. Abnormal morphological changes seen at the level of the facial nucleus in
SOD1 rats. A) Microglial fusion displaying a long rod-like structure in
asymptomatic animals (day 74) labeled with OX-42. Note the individual
microglial cells and the ability to observe the processes of microglia. B)
Microglial fusion displaying a rounded structure in asymptomatic animals
labeled with OX-42. C) Rod-like microglial fusions (lectin and creysl violet
labeled) in day 107 animals displaying hind limb weakness. (A-C)
Magnification 250x. D) Multinucleated giant cell of the Langhans type (lectin
labeled) in animals with hind limb weakness. Magnification 630x.

Microglial fusions as well as multinucleated giant cells were present in the gray

matter of the spinal cord with the majority distributed in the ventral horn (Fig.5-9b). The

fusions were identical to those seen in the brainstem and were in addition to phagocytic

clusters that were present as a direct result of neuronal degeneration (Fig. 5-9a). The two

were distinguishable because the clusters were limited to areas surrounding ventral horn

neurons whereas the fusions were not always in the vicinity of neurons. Only one fusion

was located in the white matter, with the majority of the microglial population appearing

normal. In the end stage animals, OX-42 staining revealed swollen, fragmented, non-

ramified microglial cells dispersed throughout the ventral horn suggesting that cell

integrity was impaired (Fig. 5-9c). OX-6 labeled microglial were often associated with










multinucleated giant cells at the end stage, however also revealed a population of

fragmented swollen microglial cells (Fig. 5-9d).





















Figure 5-4. Pathological changes occurring in early SOD1 symptomatic animals. A)
Neuronal fragmentation due to vacuolization. B) Dendrites separated from
neuronal bodies located in vacuoles. C) Vacuole containing a lectin labeled
cell body. D) Evidence of bacillus bacteria suggesting an impaired immune
response. Bar = 16tm


0.03

, 0.025

" 0.02
ei
S 0.015

S0.01

0.005
ei


**


Stages of Disease


Figure 5-5. Percentage of area covered by OX-6 immunoreactive cells in the ventral horn
of lumbar spinal cord of SOD1 transgenic rats and age-matched control rats
from 74 days to156 days. Columns represent mean + S.E.M of 4 animals for
each time point. **P<.001 with respect to the corresponding age-matched
control rats and #P<.05 with respect to presymptomatic transgenic rats.


~






50






A SOD1 Control

Y A

t ; '






S
throughout the disease progression. OX-6 density increased throughout the










si






"a






C



Figure 5-6. Photomicrographs demonstrating change in OX-6 expression with age. A-C)
OX-6 labeled microglia in the ventral spinal cord of SODI animals
throughout the disease progression. OX-6 density increased throughout the
disease progression. Magnification 60x. D-F) OX-6 labeled microglia in the
ventral spinal cord in age-matched control animals. Magnification 125x.













SOD1


,. .. '






I'.
* '
*. .


. ~ ~ ^ : *^ 1] '




**' 1 ': 1 *'* ; '


Control


E
n
d



g
C



e



Figure 5-7. Photomicrographs demonstrating change in OX-42 expression with age. A-
C) OX-42 labeled microglia in the ventral spinal cord of SOD1 animals
throughout the disease progression. An initial increase of OX-42 density is
seen in with the onset of symptoms that declines in end stage animals. D-F)
OX-42 labeled microglia in the ventral spinal cord in age-matched control
animals. Bar = 160am


A


A

Y
m
P
t
o
m
a
t
i







C



i.W~r

c:~i c~;~~












03


025

02
-e
02


-c




01
C.
0,


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Stages of Disease

Figure 5-8. Percentage of area covered by OX-42 immunoreactive cells in the ventral
horn of the lumbar spinal cord of SOD1 transgenic rats and age-matched
control rats from 74 days to156 days. Columns represent mean + S.E.M of 4
animals for each time point. *P<0.05 and **P<0.001 with respect to
corresponding age-matched control rats and #P<0.05 with respect to
transgenic onset of symptom rats.

TUNEL positive cells were dispersed throughout the lumbar ventral spinal

cord in ALS animals. TUNEL labeling was performed on spinal cord sections to

determine if microglial cells were undergoing apoptosis due to cellular senescence.

TUNEL positive cells were found at all stages of the disease with the majority located in

the ventral area of the spinal cord displaying classic nuclear staining. A few isolated

TUNEL positive cells in asymptomatic and early symptomatic animals portrayed nuclear

and diffuse cytoplasmic staining similar to the TUNEL positive cells that were identified

as microglial cells in the facial nucleus (Fig. 5-10a). Control spinal cord sections had

similar occurrence of cytoplasmic/nuclear stained TUNEL positive cells suggesting that

microglial turnover in the spinal cord regulated by apoptosis is unchanged in transgenic


SOD1 Transgemc Ammals
o Age-Matched Control Ammals




#
T









animals (Fig. 5-10c). End stage animals displayed nuclear staining orientated in a string

of labeled cells not representative of microglial cells (Fig 5-10b). Co-labeling with

markers for each CNS cell type revealed no overlap between cell-specific markers and

TUNEL labeled cells not allowing conclusive identification of apoptotic cells.























Figure 5-9. Microglial response and changes in SOD1 animals in the ventral spinal cord.
A) Microglial activation seen in close proximity to motor neurons located in
the ventral horn of asymptomatic animals (OX-42 labeled). B) Rod-like
microglial fusions stained with OX-42 in asymptomatic animals. The fusions
are similar to those seen in the brainstem in SOD1 animals. C) OX-42 labeled
microglia in end stage animals portraying an abnormal swollen morphology
representative of degenerative changes. D) OX-6 labeling of giant cells in end
stage animals suggesting a relationship between giant cells and MHCII
expression. Magnification A,B,D 125x and C 630x.

TUNEL positive cells were found at significantly lower numbers in the SOD1

facial nucleus 14 days post-axotomy. TUNEL-positive cells in the SOD1 transgenic

and wild type axotomized facial nucleus lacked the classic nuclear staining instead

having cytoplasmic staining similar to previous studies of apoptotic microglia (Gehrmann

and Banati, 1995; Jones et al., 1997). In addition, double-labeling revealed an overlap









between lectin and TUNEL staining further identifying TUNEL-labeled cells as

microglia (Fig. 5-12). TUNEL/lectin positive cells distribution was limited to the facial

nucleus.


Figure 5-10. TUNEL positive cells in the ventral lumbar spinal cord in ALS animals. A)
Diffuse cytoplasmic and nuclear staining in asymptomatic animals (Day 74).
B) End stage TUNEL labeling with exclusive nuclear staining orientated in a
string of positive cells. C) Control TUNEL labeling revealing cytoplasmic
and nuclear staining similar to TUNEL positive cells seen in asymptomatic
and early symptomatic animals. Bar = 80gm.










The number of TUNEL labeled cells in the injured facial nucleus was

significantly lower (p<0.001) in the transgenic animals when compared to age matched

controls ((Fig. 5-11) SOD1 transgenic animals 0.5278 0.1847, control animals 5.357

0.5058)). No TUNEL-positive cells were found in the unoperated facial nucleus in both

animal groups. Qualitative analysis of a nissil stain revealed similar numbers of nuclei in

the transgenic and control animals. In addition, the microglial response 14-days post

axotomy appeared to be similar between control and transgenic animals both displaying

perineruonal microglia and qualitatively revealing a similar density of lectin labeling

within the nucleus.








U SOD1 Transgemc
SAnmmals
S[] Age-matched Control
SAnimals













14 Days
0





0-






Days Post Axotomy


Figure 5-11. Number of TUNEL positive cells in facial nucleus 14 days post axotomy in
non-symptomatic transgenic and age-matched control animals. Columns
represent mean + S.E.M of 6 animals for each time point (**P<0.001).









Discussion

The current study demonstrated for the first time a presence of microglial fusions

and Langhans giant cells in the ALS rat model. Additional pathological changes were

observed at the levels of the red and facial nucleus in the brainstem where aberrant

microglial activation and neuronal fragmentation was seen. Evidence of abnormal

microglial function was found in the TUNEL studies where SOD1 animals had

significantly reduced levels of TUNEL positive cells in the facial nucleus compared to

age matched controls 14 days post-axotomy. The current findings demonstrate that

microglial cells undergo morphological and functional changes during the disease

progression in ALS animals.

The presence of multinucelated giant cells was an unexpected finding since these

cells are often only associated with viral and bacterial infections, which to date have not

been reported in ALS animal models. The current study demonstrated a large number of

multinucleated giant cells of the Langhans type throughout the CNS of the rat transgenic

model. Langhans giant cells are characterized by nuclei orientated around the periphery

of the cell and most often associated with granulomatous reactions, specifically

Mycobacterium tuberculosis. The presence of indigestible particles of an organism

causes macrophages to aggregate at the site and engulf the foreign particle. During the

ensuing days, a cell-mediated immunity to the bacterium develops leading to T

lymphocyte recruitment and release of cytokines some of which cause further recruitment

of macrophages and cell fusion through induction of cell surface adhesion molecules (Lee

et al., 1993). In vitro studies have found that interleukin-3 and interferon-y induced

Langhans multinucleated giant cell formation in the presence of granulocyte macrophage-

colony stimulating factor (GM-CSF) (McNally and Anderson, 1995). The characteristics









of Langhans giant cell formation suggests the presence of bacteria in the ALS rat model

which was supported by the observed nidus of bacillus bacteria in a tissue section of a

transgenic animal. The fact that we detected bacilli and giant cells in animals that

develop neurodegenerative disease leads us to conclude that a common link for the

development of brain infection and neurodegeneration may be found in dysfunction of

microglia that results in impaired immunological defense mechanisms and diminished

neuroprotection. Further evidence of this novel theory is found in human

immunodeficiency virus encephalitis (HIVE) cases where viral infected microglial cells

portray altered cytokine production and formation of mulitnucleated giant cells that

accompany neurodengerative changes (Nottet et al., 1997; Zheng and Gendelman, 1997;

Kaul et al., 2001). In ALS, microglial dysfunction may be a direct result of the SOD1

mutation whereas in HIV encephalopathy microglial dysfunction may be a result of viral

infection both rendering detrimental consequences for the neuronal cell population. An

additional link between ALS and HIV is seen in a number of HIV patients that were

clinically diagnosed with ALS suggesting a related mechanism of disease propagation

(Verma et al., 1990; Huang et al., 1993; Casado et al., 1997; Galassi et al., 1998;

MacGowan et al., 2001; Moulignier et al., 2001; Zoccolella et al., 2002).

In addition to the presence of giant cells, intense microglial activation was seen

throughout the brainstem and in the ventral horn of the lumbar spinal cord. Microglial

activation was seen in the spinal cord corresponding to neuronal degeneration, however

in the brainstem the activation is not a direct result of neuronal degeneration but may be

due to elevated levels of microglial-activating factors identified in cerebral spinal fluid

and serum of ALS patients.



















































Figure 5-12. Apoptotic microglial cells in the facial nucleus following injury. A) Lectin
labeled perineuronal cell. B) TUNEL labeled cell. C) Merged images
confirming TUNEL labeled cells are microglial cells. Bar = 40gtm.









Macrophage colony stimulating factor (M-CSF), monocyte chemoattract protein 1 (MCP-

1), tumor necrosis factor alpha (TNF-a), and transforming growth factor beta 1 (TGF-B1)

have all been found at elevated levels in ALS when compared to controls (Poloni et al.,

2000; Elliott, 2001; Hensley et al., 2002; Ilzecka et al., 2002; Yoshihara et al., 2002;

Hensley et al., 2003; Wilms et al., 2003; Henkel et al., 2004). The mechanism in which

microglia activating factors are produced is unclear, but may be released by glial cells

themselves. The glial cells may be affected by the mutant SOD1 thereby becoming

neurotoxic and increasing production of pro-inflammatory molecules. In addition, to

activating microglia the elevated levels of inflammatory molecules may further affect the

function of microglia propagating the disease. Chronic expression of MCP-1 in the

central nervous system causes impairment of microglia function in mice, specifically the

ability of microglia to respond to environmental stimuli (Huang et al., 2005).

It is unclear the cause for isolated microglia activation in the red nucleus, but

absent in other motor nuclei such as the oculomotor and facial nucleus. The aberrant

microglia activation may be dependent on the projection of cells in the specific nuclei.

Cells in the red nucleus are the only cells that directly project to spinal cord levels

whereas those in the oculomotor, facial and substania nigra have no direct projections to

the spinal cord.

Initial observations in the spinal cord confirmed prior reports of prominent

microglial activation in areas of motor neuron degeneration. Microglial activation was

assessed in the ventral spinal cord using OX-42 and OX-6. A variation in expression

between OX-42 and OX-6 was observed. OX-6 expression transiently increased

throughout the disease whereas OX-42 levels were increased in asymptomatic animals,









continued to increase with the onset of symptoms followed by a decline in density in end

stage animals. OX-6 is often used as a marker of microglial activation which suggests

that microglial activation occurs in response to neuronal degeneration in the spinal cord.

However, MHC II is not always indicative of activation since non-activated ramified

microglia present in young and non-diseased human subjects are positively stained with

MHC II (Streit and Sparks, 1997; Streit et al., 2004). Alternatively, MHC II may be a

marker of microglia maturation and an early stage of cellular senescence which is

supported by the increasing number of MHC II positive microglial cells from infancy to

old age (Streit and Sparks, 1997). Therefore, the progressive increase in OX-6 in SOD1

animals may be a result of the SOD1 mutation negatively effecting microglial cells

causing increased cellular dysfunction and senescence. The conflicting decline of OX-42

density in end stage animals may be attributed to differential regulation of cell surface

markers dependent on the life stage of microglia. OX-6 labeling remains elevated in end

stage animals because the remaining microglial cells are senescent whereas OX-42

density declines as a result of microglial degeneration causing a decrease in the overall

microglia population. Evidence of abnormal microglia structure as seen with fragmented,

swollen OX-42 positive cells in end stage animals is suggestive of microglial

degeneration however, the lack of increased levels of apoptotic microglia observed in the

transgenic spinal cord suggests that if microglial are under going cell death it is through

an alternative pathway.

To investigate microglial function in the SOD1 transgenic animal, this study

utilized the facial nerve injury model. Peripheral nerve lesions like the facial nerve

axotomy maintain the integrity of the blood brain barrier thus allowing resident microglia









to be studied in the absence of infiltrating blood-derived cells. In non-diseased animals

microglia proliferate in response to facial nerve injury (Kreutzberg, 1968; Graeber et al.,

1988; Gehrmann and Banati, 1995) reaching a peak at 3 days and gradually decrease in

number (Streit et al., 1988; Raivich et al., 1993). The decline in microglia has been

attributed to non-classical programmed cell death, a possible mechanism used to regulate

post-mitotic microglia populations through elimination of activated microglia (Gehrmann

and Banati, 1995; Jones et al., 1997). The lack of TUNEL-positive microglia in

transgenic animals may be a mark of abnormalities in microglial regulatory mechanisms.

If microglia fail to undergo programmed cell death the levels of microglia will remain

elevated and may maintain an activated state causing a cytotoxic environment. Evidence

for dysfunctional microglial function in ALS has been seen in the study by Weydt et al.

where microglia isolated from the transgenic SOD1 mouse produced higher levels of

TNF-a. Another explanation for the lack of TUNEL positive cells in the transgenic

animals is that post-mitotic turnover of microglia is controlled by an alternative type of

programmed cell death.

Neuronal abnormalities must also be addressed as a factor in the reduction of

microglia turnover in transgenic animals. The motoneuron population in the facial

nucleus may be affected by the disease process resulting in reduced numbers of

motoneurons thereby upon injury a diminished neuronal response is present to induce

microglia activation requiring less cell turnover in order to maintain a resting CNS

environment. However, qualitative analysis of nissl staining of motoneurons in the facial

nucleus revealed similar numbers between transgenic and control animals, which is

further supported by magnetic imaging and histochemical studies that establish









motoneuron populations in the facial nucleus remain unaffected in mice prior to onset of

symptoms (Nimchinsky et al., 2000; Haenggeli and Kato, 2002; Angenstein et al., 2004).

Although, neuronal numbers remain unchanged, previous findings have shown an

impaired neuronal response to peripheral injury in the transgenic SOD1 mouse (Mariotti

et al., 2002). Following a facial nerve axotomy, levels ofNOS immunoreactivity was

significantly reduced in the axotomized facial nucleus of transgenic animals. The precise

function of NOS induction in the facial nucleus is debated however in cranial

motoneurons it has been identified as an indicator of the cell body response to both lethal

(Wu, 2000) and non-lethal (Mariotti et al., 2001) peripheral injuries. The lack of NOS

induction in the transgenic animals may impair microglia-neuronal signaling thereby

reducing the microglia response following injury diminishing microglial turnover.

Contradicting findings on levels of TUNEL labeled cells were seen in the spinal

cord and facial nucleus. The facial nucleus showed decreased levels of TUNEL labeled

microglia whereas the spinal cord had similar levels of TUNEL labeled microglia as the

controls. The discrepancy may be due to regional differences as well as activation levels

of the microglial population. The spinal cord presents intense neuronal degeneration

accompanied by an intense microglial response whereas the facial nucleus had no

evidence of neuronal death.

The current findings suggest an aberrant microglial response in the SOD 1

transgenic animal which may be a direct result of the SOD1 mutant resulting in impaired

function of microglial cells. Previous studies have found that exclusive expression of

SOD1 mutant in neurons (Pramatarova et al., 2001; Lino et al., 2002) or astrocytes (Gong






63


et al., 2000) is insufficient to cause neuronal degeneration indicating that the mutation

must be affecting both the neuronal and glial population.














CHAPTER 6
CONCLUSION

In spite of the large body of evidence indicating that microglial activation might

influence the pathogenesis of degenerative diseases, there is considerable debate

regarding whether microglial activation is beneficial or harmful. To address this

fundamental question we decided to examine the effect of minocycline on microglial

activation and neuronal regeneration. Initially, it was thought that minocycline would

inhibit microglial activation, as seen in a number of previous studies, allowing neuronal

regeneration to be investigated in the absence of a robust microglial response providing

evidence for or against a pro-regenerative role of microglial activation. However, there

was no difference seen in proliferating microglia in the facial nucleus 2, 3, and 4 days

post-axotomy between minocycline treated and control animals. The unexpected effect

of minocycline did not allow the role of microglial activation in neuronal regeneration to

be addressed. Nonetheless, the results provided important insight to the mechanism in

which minocycline provides neuroprotection in a number of neurodegenerative and injury

models. From this study it appears that minocycline does not provide neuroprotection as

a result of microglial deactivation. Further experiments in a lethal motor neuron injury

model would address if minocycline functions by providing direct neuroprotection to

injured neurons. The conflicting findings of minocycline on microglial activation may be

attributed to the model used as well as the marker used to assess microglial activation.

The facial nerve model may elicit a different microglial response than that in a

neurodegenerative model since motor neurons in the facial regenerate and fully recover









whereas the neurons in a neurodegenerative model degenerate providing a chronic source

for microglial activation.

Future studies need to address microglia cytokine production following

minocycline treatment to assess if microglial activation is truly inhibited, specifically,

pro-inflammatory molecules that have been suggested to be detrimental in the CNS. In

addition, a different route of drug administration should be tested to determine if

minocycline protection is dependent on the route of drug administration. An alternative

to oral drug administration would be an i.p. injection of a corresponding dose of drug and

then assessment of microglial proliferation in the facial nucleus following axotomy. An

i.p. injection would determine if the facial nerve paradigm portrays a different response

than that seen in neurodegenerative models following an i.p. injection of minocycline.

To confirm that the lack of effect of minocycline on microglial activation was not a

result of insufficient drug levels in the brain, we performed HPLC/MS/MS to determine

levels of minocycline in the brain following drug administration through oral diet.

HPLC/MS/MS revealed minocycline levels similar to those found in studies where

microglial activation was significantly inhibited following minocycline treatment.

The second aim of the current study investigated changes in microglia morphology

and activation in the rat transgenic model of ALS to assess the role of microglia in

disease progression. Morphological changes of microglia were observed in the

brainstem and spinal cord of ALS transgenic animals at all stages of the disease.

Included in the morphological changes was the formation of giant cells and microglial

clusters. The giant cells portrayed the classic characteristics of Langhans giant cells

suggesting a bacterial infection accompanying neurodegeneration in the ALS animal









model. This finding was further supported by the presence of bacilli bacteria seen in a

symptomatic animal. However, a gram stain failed to identify bacteria in brain tissues

from ALS animals. The failure of the gram stain to identify bacteria may be due to the

short time frame in which the bacteria are present prior to their clearance or the bacteria

present in the brain are acid-fast thereby causing them to be gram resistant. To further

assess the presence of bacterial infections in ALS transgenic animals, future studies

should directly culture brains to determine the presence of bacteria as well as

performance of a broader range of bacterial stains to include all types of bacteria.

Further aberrant microglial activation was present in the red nucleus where intense

activation was seen at all stages of the disease. However, in the same animals the

oculomotor nucleus remained unaffected. The discrepancy between microglial activation

in the two motor nuclei may be a direct result of the nucleuses cell projections. Cells in

the red nucleus directly project into cervical, thoracic and lumbar levels whereas the

oculomotor has no direct pathways to the spinal cord. Degenerative changes in the spinal

cord affecting the rubrospinal tract may cause neuronal changes in the red nucleus

eliciting a microglial response.

Levels of microglial activation in the ventral spinal cord were investigated using

OX-42 and OX-6 to determine a time line of microglial activation in the spinal cord in

response to disease progression. OX-6 expression was found at increased levels in the

transgenic animal when compared to the age-matched controls and showed a continuous

increase in density that corresponded with disease progression. OX-42 density initially

increased with the onset of hind limb weakness however declined in end stage animals.

The differences in OX-42 and OX-6 expression may be due to the heterogeneity of









microglia cells found in the CNS and the differential regulation of cell surface markers

during microglia development. OX-6 positive microglial cells may be in early stages of

cellular senescence as a direct result of the SOD1 mutation thereby increasing in number

during the disease process. OX-42 labels the majority of microglial cells representing the

overall response of microglial cells which declines through microglia degeneration in end

stage animals. Therefore, MHC II positive cells are indicative of late stages of microglial

life cycle thereby remaining elevated in end stage animals whereas the majority of OX-42

cells have undergone structural changes indicating degenerative changes as a result of

cellular senescence therefore, no longer staining with MHC II.

To determine if microglial cells in the ALS model were more susceptible to

apoptosis due to cellular senescence TUNEL labeling was utilized to assess apoptotic

microglia. In the spinal cord TUNEL labeled cells had a morphology that was

representative of microglia however failed to co-label with any of the microglial makers

used. The failure to co-label may be due to differential expression of cell surface markers

on cells undergoing programmed cell death. Future experiments need to further

investigate the identity of the apoptotic cells with of a wider range of microglial markers.

Rather than using a surface antigen, a nuclear stain allowing identification of fragmented

cells should be tested.

In the facial nucleus, TUNEL labeled microglial were significantly fewer in

number in the transgenic animals 14 days post-axotomy. The findings seem to contradict

the hypothesis of cellular senescence and favor dysfunction of transgenic microglia. In

chapter 5, a number of theories were postulated to explain the findings of fewer apoptotic

microglia in the transgenic animals however until the initial microglial response is further









investigated little speculation can be made on the cause behind the lack of microglial

turnover. In order to investigate the initial microglial response, the microglial response at

4 days post-axotomy in the facial nucleus should be compared between asymptomatic

ALS animals and age-matched controls. This experiment will determine if the lack of

turnover is due to a diminished microglial response or is in fact dysregulation of

deactivating mechanisms in microglia. A follow-up to the above experiment would be to

measure microglia-neuronal signals to determine if the lack of response is due to

neuronal dysfunctions or microglia dysfunctions. The cytokine interleukin-6 (IL-6) has

been suggested to be a potential signaling molecule between neurons and microglia and

fractalkine is a chemokine that is found to be constitutively expressed on CNS neurons

while the corresponding receptor CX3CR1 is found on microglial cells another potential

signal between microglia and neurons (Harrison et al., 1998; Nishiyori et al., 1998). Both

of the potential microglia-neuron signals would be key candidates to investigate

microglia-neuron signaling in the facial nucleus.

In conclusion, the microglial cells in the ALS rat appear to be abnormal and

undergoing functional changes due to the SOD1 mutation. It is still unclear in what

manner the mutation renders the microglia dysfunctional, but may be attributed to

oxidative stress. Oxidative stress has been shown to be a key contributing factor in

familial ALS where the mutation catalyzes aberrant chemical reactions that initiate a

cascade of oxidatively damaged products. Experiments addressing oxidative damage

directly effecting the microglial cell population would provide great insight to the

functional and morphological changes of microglia in the ALS rat. Furthermore, the

dysfunction of microglia appears to compromise the integrity of the immune system in






69


the CNS allowing for a bacterial infection to occur and may contribute to

neurodegeneration due to diminished neuroprotective properties.













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BIOGRAPHICAL SKETCH

Sarah Emily Fendrick was born in Mansfield, Ohio and remained in Ohio until her

sophomore year in high school. She then relocated to Minnesota with her family where

she graduated from high school in 1998. For her undergraduate education she attended

the University of Wisconsin-Madison where she wanted to pursue a career in journalism.

However, when attending orientation she made a last minute decision to follow a path

that would allow her to attend medical school. Her mind changed a number of times until

she finally decided that she would like to pursue her degree in genetics.

While in college, her first experience in the field of research was through

employment in a stem cell lab. Her time spent in the stem cell lab was short due to her

dislike of working in close proximity to monkeys. She then obtained employment in a

functional genomics lab where she spent the two years working on a project that did

functional genomics within the E. coli genome. The summer of her junior year Sarah

was selected for an internship funded through the National Science Foundation at the

Whitney Lab in St. Augustine, Florida. During her internship, Sarah's fondness for the

warm weather and sunny days persuaded her to continue her education in Florida.

In 2002, she began her graduate education at the University of Florida in the

Interdisiplinary Program in Biomeidcal Reserach. Entering into graduate school, her

interest was in neuroimmunlogy which caused her to immediately pursue Jake Streit to be

her mentor. Upon active persuasion, Jake accepted Sarah into his lab where her research

focused on the role of microglial activation in ALS. After completing her Ph.D. Sarah






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would like to pursue a career in patent law related to biomedical research and plans to

begin law school in the Fall of 2006 at The Ohio State University.