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Development of an in Vitro Model for Activation of Satellite Glial Cells in The Development of an in vitro Model for Activation of Satellite Glial Cells in the Sympathetic Nervous System Master’s Thesis Presented to The Faculty of the Graduate School of Arts and Sciences Brandeis University Graduate Program in Molecular and Cell Biology Susan Birren, Advisor In Partial Fulfillment Of the Requirements for the Degree Master of Science in Molecular and Cell Biology by Surbhi Sona February 2014 Acknowledgement I would like to thank my advisor Dr. Susan Birren for giving me this learning opportunity in her lab. I thank her for her constant guidance and support and for critiquing my work. I also thank her for her insightful questions and suggestions. I am extremely grateful to Dr. Joana Enes for mentoring my work and training me in the various techniques used in this research. I thank her for her patience and valuable guidance which made this a wonderful learning experience for me. I deeply thank her for providing me with some of the materials used in my experiments - She established the primary rat cultures used in this study. The various HEK cell constructs, used in the neurotrophin experiments, were also developed by her. I also thank all the members of the Birren lab for their support and valuable discussions on my work. ii Abstract Development of an in vitro Model for Activation of Satellite Glial Cells in the Sympathetic Nervous System A thesis presented to the Graduate Program in Molecular and Cell Biology Graduate School of Arts and Sciences Brandeis University Waltham, Massachusetts By Surbhi Sona Glial cells of the central nervous system are activated upon injury and modulate the course of several diseases. The glia population of peripheral nervous system is much less explored in this area. I used satellite glial cells (SGCs) from superior cervical ganglia of P0 to P2 rat pups, to develop an in vitro model to study glia activation in the sympathetic nervous system. Several factors including ATP, endothelin-1, lipopolysaccharides and fractalkine have been reported to activate other glia populations and were tested on SGC cultures. Out of these, LPS showed a relatively better consistency in activating SGCs and was used for most of the experiments. To assess activation of SGCs, the cell lysates were analyzed by western blot to check for GFAP expression, a common glia activation marker. There was only a slight difference in GFAP levels between control and treated cells. Immunocytochemistry was used as an alternative approach to assess SGC activation, comparing cell proliferation between the control and activating conditions. The results suggested cell proliferation to be a better assay than GFAP expression to study SGC activation in cell culture. It is also interesting to look for secretary factors released by SGCs iii and to observe if this expression profile alters in response to activation. Using western blot, I validated production of neurotrophins such as NGF and BDNF in the SGC culture. The expression of these factors altered non-significantly in response to activation. The young and adult animals may have different protein expression profile. So, more SGC-specific activation protocol/modulator; SGC culture from adult animals; and a better activation marker for SGC might be required to develop a robust in vitro model to study significance and implications of activation of SGCs in sympathetic nervous system function. These inferences would be valuable as the sympathetic neurons innervate several vital organs such as heart and activation of glia may contribute in affecting the sympathetic drive to these organs. iv Table of Contents 1. Abstract iii 2. List of Tables vi 3. List of Figures vii 4. List of Abbreviations viii 5. Introduction 1 6. Materials and Methods 5 7. Results 11 8. Discussion 22 9. Bibliography 27 v List of Tables Table 1 Activation factors used on satellite glial cells 7 Table 2 Determining the effective concentration and the treatment 7 duration of the activators for satellite glial cells vi List of Figures Figure 1 Design of the Experiment 6 Figure 2 NGF and BDNF expression in satellite glial cells of superior cervical ganglia 12 dissected from P0 to P2 rats Figure 3 GFAP expression in SGCs in response to the activators – ATP, ET-1 and LPS 14 Figure 4.1 Satellite glial cell proliferation in response to LPS 16 Figure 4.2 Quantification of SGC cell proliferation in response to LPS 17 Figure 5 Expression of NGF and BDNF in SGCs in response to LPS treatment 19 Figure 6 TNF-α levels in SGC culture media in response to short term treatment 21 with LPS and FRK vii List of Abbreviations ATP Adenosine Tri-phosphate BDNF Brain Derived Neurotrophic Factor CNS Central Nervous System DAPI 4’,6-diamidino-2-phenylindole DIV Day(s) in vitro ET-1 Endothelin-1 FRK Fractalkine GFAP Glial Fibrillary Acidic Protein LPS Lipopolysaccharides NGF Nerve Growth Factor PNS Peripheral Nervous System SCG Superior Cervical Ganglia SGCs Satellite Glial Cells SMEM Minimum Essential Media for Suspension culture TNF-α Tumour Necrosis Factor – α viii Introduction First described by Rudolf Virchow in 1856, glia were thought to be accessory cell population that simply supported the neurons [Freeman et al., 2013]. Much later, glial cells received the much deserved attention, when through many independent researches studies, it was proposed and established that glia are both affected by as well as contribute significantly to various neuropathological conditions such as Multiple Sclerosis, Epilepsy, Alzheimer’s Disease and Schizophrenia, Parkinson’s Disease, Huntington’s Disease and Amyotrophic Lateral Sclerosis [Miller, 2005 and Lobseiger and Cleveland, 2007]. The glia population in the central nervous system (CNS) comprises of astrocytes, oligodendrocytes and microglia; while Schwann cells and satellite glial cells (SGCs) are the major glia population in the peripheral nervous system (PNS). Relatively much less is described about glia population in the peripheral nervous system as the initial glia research was focussed on glia population of the central nervous system [Ji et al., 2013]. Sympathetic ganglia of PNS relay inputs from CNS to several visceral organs. These ganglionic structures consist of sympathetic neurons enveloped by satellite glial cells [Hanani, 2010]. SGCs share several similarities with astrocytes such as expression of the same glial markers (GFAP and S100) and formation of gap junctions [Ji et al., 2013]. Thus, 1 astrocyte research may provide useful cues for developing a better understanding of the functional significance of SGCs and their role in human pathophysiology. In this context, it is important to mention that sympathetic dysfunction has been implicated in several diseases such as diabetes, hypertension and chronic heart failure. Brain injury activates astrocytes which is manisfested as increased expression of glial fibrillary acidic protein (GFAP) [McMillan et al., 1994]. SGCs in sensory ganglia [Zhang et al., 2009 and Takeda et al., 2009] and in sympathetic ganglia [Elfvin et al.,1987 and Zigmond et al., 2007] also become reactive post injury or inflammation in vivo. Activated astrocytes express elevated levels of a wide range of molecules – structural proteins such as GFAP; growth factors such as NGF; cytokines such as interleukins and TNF- α; and enzymes such as glutamine synthetase, to name a few [Ridet et al., 1997]. Thus, upregulation of one or more of these molecules serve as activation marker for astrocytes and given the similarities between astrocytes and SGCs, some of these may also serve as activation markers for SGCs of sympathetic system. The glia cells also undergo increased proliferation in response to activation [Jasmin et al.,2010]. LPS, a constituent of cell wall of Gram negative bacteria, is widely used to induce activation in astrocytes and microglia, both in vitro as well as in vivo [Zhong et al., 2012 and Yao et al., 2014]. Similarly, astrocytes have been described to turn reactive in response to purinergic signalling mediated by ATP [Tran and Neary, 2006]. ET-1 has been previously described in 2 relation to reactive gliosis in CNS. It is a vasoconstrictor produced by astrocytes in response to injury and has autocrine effect. A study has previously demonstrated that ET-1 promotes activation of astrocytes [Gadea et al., 2008]. Activation of PNS glia is not as extensively studied. A recent work reported activation of SGCs in sensory ganglia using Fractalkine, which is a chemokine [Souza et al., 2013]. Neurotrophins are target derived factors that promote growth and survival of neurons in the peripheral nervous system. They are also known to be modulators of synaptic properties [Poo 2001]. Under pathological conditions, the expression of these factors is altered, for example, in the heart [Govoni et al., 2011]. Apart from the peripheral organs, SGCs of sensory ganglia also produce neurotrophins [Gosselin et al., 2010]. Since glia get activated by injury and other pathological conditions, it would be interesting to explore if that activation has implications on neurotrophin expression by SGCs which in turn would modulate synaptic properties. Since Sympathetic neurons innervate vital organs and such a study could provide useful insights on role of glia in disease pathogenesis and progression. An in vitro model for SGC activation in sympathetic system could help identify factors that mediate such activation. Because in vitro models eliminate complexities of an in vivo process, the SGC culture model could help characterize response of SGC to activation. The mechanisms involved can also be characterized in such models. This would further aid in understanding satellite glial cell 3 biology and the implications of their activation in response to injury, inflammation and other conditions that activate glial cells. 4 Materials and Methods Cell Culture Superior cervical ganglia were dissected from postnatal rats (P0 to P2). The ganglia were incubated 1 hour at 370C in SMEM (GIBCO) supplemented with 350U/mL of collagenase type I (CLS-1, Worthington) and 7.5 U/mL of dipase (Gibco).
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