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REACTIVE OXYGEN SPECIES LIKELY INDUCE PRO INFLA MMATORY GENE TRANSCRIPTION AND p 53 ACTI VITY FOLLOWING OXYGEN GLUCOSE DEPRIVATION IN CULTURED MICROGLIA By David Hartmann A Thesis Submitted to the Division of Natural Sciences New College of Florida In partial fulfillment of the requirements for the degree of Bachelor of Arts in Chemistry and Neurobiology Under the sponsorship of Dr. Alfred Beulig Sarasota, Florida May 2012
ii Acknowledgments I would like to thank a host of family, friends, professors, and lab mentors, who have all been there to elevate or alleviate my levels of oxidative stress both types of regulation have served to formulate a wonderful and edifying collegiate experience. Sincerest thanks to the professors of New College. In particul ar, Drs. Beulig, Scudder, Walstrom, and Demski opened my mind up to the many intersections of chemistry and neurobiology th at are waiting to be explored. Excellent teaching aside, I greatly appreciate the mentorship and support of Dr. Scudder as my academi c advisor and Dr. Beulig as my thesis sponsor. Thanks as well to Dr. Shipman for devoting so much of his time and energy to helping his students. This would not have been possible without Dr. Gwenn Garden and Stephanie Hopkins at the University of Washin gton I thank them profusely for accepting me into their laboratory, and for genuinely listening to the ideas of an undergraduate from across a leave of absence in order to conduct this research during fall semester, and to the Council of Academic Affairs for helping in funding my thesis travels Finally, thank you to both of my parents and all of my siblings. I have felt your support throughout these years, even though I know very little of what I have said about my studies has made much sense. I could not have done this without the friendships of Erica, Roz, Mark, Andrew, and Nathan.
iii Table of Contents Acknowledgments ................................ ................................ ....................... ii Table of Contents ................................ ................................ ....................... iii List of Figures ................................ ................................ ............................ vi Abstract ................................ ................................ ................................ ..... vii Abbreviations ................................ ................................ ............................. ix Chapter 1: Introduction ................................ ................................ ............ 1 1.1 Microglia ................................ ................................ ............................. 1 1.1.1 Glia More than Brain Glue ................................ ..................... 1 1.1.2 Microglia: Sentinels of the CNS ................................ ................ 2 1.1.3 Reactive Oxygen Species and p53: Putative Promot ers of a Pro Inflammatory Response ................ 4 1.1.4 Thesis Goals ................................ ................................ ............... 7 1.1.5 Downstream of Microglial Activation: Blades of the Double Edged Sword ................................ ........... 7 1.1.6 p53 in Microglial Activation ................................ .................... 10 1.2 p53 ................................ ................................ ................................ .... 11 1.2.1 p53: A Complex Regulator of Cell Death & Growth Arrest ... 1 2 1.2.2 p53 and Reactive Oxygen Species (ROS) ............................... 1 4 1.2.3 p53 Relevance in the CNS ................................ ....................... 1 6 1.2.4 p53 in Ischemic Brain Injury ................................ ................... 16 1.3 Ischemia Reperfusion Brain Injury ................................ ................... 1 9 1.3.1 Stroke Has Many Untreatable Stages of Cell Death ................ 1 9 1.3.2 Injury in the Ischemic Core ................................ ...................... 2 1 1.3.3 Injury in the Ischemic Penumbra ................................ ............. 25 1.3.4 Inflammation in Ischemia: Role of Microglia ......................... 26 1.3.5 Microglia an d ROS in the Ischemic Context ........................... 2 9 1.3.6 Unanswered Questions about ROS, Microglia, and Inflammation in Ischemia Paradigms ................................ ................................ ................. 31 1.4 Research in the Garden Laboratory ................................ .................. 32 1.4.1 Context of Thesis Research ................................ ..................... 32
iv Chapter 2: Experimental ................................ ................................ ......... 36 2.1 Acquiring an d Maintaining Cells ................................ ................... 36 2.1.1 Cell Culture Media and Chemical Solutions .............................. 36 2.1. 2 Mice ................................ ................................ ......................... 36 2.1.3 Making Primary Microglial Cultures ................................ ....... 37 2.1.4 Maintaining Mixed Cultures and Harvesting Microglia .......... 37 2.1.5 BV 2 Cell Cultures ................................ ................................ .. 38 2.2 Detecting ROS Produ ction in Cells ................................ ............... 38 2.2.1 Detecting ROS Stimulated by Hydrogen Peroxide .................. 38 2.2.2 Detecting ROS Stimulated by Oxygen Glucose Deprivation (OGD) Reperfusion ................................ ................................ .......................... 39 2.2.3 Theory behind DCFH 2 DA ROS Detection ............................. 40 2.3 Gene Transcript Experiments ................................ ........................ 43 2.3.1 Extraction of RNA and Reverse Transcriptase Reaction ........ 43 2.3.2 Quantitative Polymerase Chain Reaction (qPCR) ................... 44 2.4 Justifying the OGD Reperfusion Paradigm ................................ ... 45 2.5 Statistics ................................ ................................ ......................... 46 Chapter 3: Results ................................ ................................ .................... 47 3.1 Characterizing the ROS Detection and Inhibition Paradigm ......... 47 3.2 Oxygen Glucose Deprivation Reperfusion Experiments .............. 51 3.2.1 BV 2 Cells ................................ ................................ .............. 51 3.2.2 Primary Microglia ................................ ................................ ... 5 2 3.3. Effects of ROS on Transcription of IL 1Beta and MARCO ....... 5 7 3.3.1 Qualitative Observations of Cells Prior to RNA Extraction ... 5 7 3.3.2 qPCR Reactions ................................ ................................ ...... 57 Chapter 4: Discussion ................................ ................................ .............. 61 4.1 Restatement of Goals and Findings ................................ ................ 61 4.2 Direct Evidence of ROS Production in Microglia Following OGD Reperfusion ................................ ......................... 61 4.3 ROS Influenced Gene Transcription Greatly ................................ .. 63 4.4 Attempting to Localize the Source of Detected ROS ..................... 65
v 4.5 Qualitative Observations about OGD Reperfusion Effects on Morphology ................................ ................................ .. 67 4. 6 Support for ROS as Initiators of a Pro Inflammatory Response .... 68 4.7 Quantitative Differences between In Vitro and In Vivo ................. 69 4.8 Equivocal Results about MARCO Transcription ........................... 70 4.9 Implications for p53 ................................ ................................ ........ 71 4.10 Conclusion ................................ ................................ .................... 74 References ................................ ................................ ................................ 76 Appendix ................................ ................................ ................................ ... 9 6
vi List of Figures Figure 1: Light micrographs displaying characteristic microglial morphologies ............. 5 Figure 2: Overview of microglial activation ................................ ................................ ..... 7 Figure 3 : Responses of p53 and subsequent effects on the cell ................................ ....... 1 3 Figure 4 : Visual representation of the ischemic core and penumbra ............................... 20 Figure 5 : Timeline of chemical events in the ischemic core that lead to neuronal death 22 Figure 6 : Reperfusion following ischemia is associated with a burst of ROS ................. 25 Figure 7 : Overview of inflammatory processes that are initiated by oxidative stress ...... 32 Figure 8 : p53 transcriptional activity was increased following OGD reperfusion ........... 34 Figure 9 : Schem atic of experimental procedures ................................ .............................. 40 Figure 10 : General mechanism for the oxidation of DCFH 2 ................................ ........... 42 Figure 1 1 : Confirming the requirement for both ROS and DCFH 2 in producing DCF .... 48 Figure 1 2 : Increase in fluorescence over time, BV 2 cells treated with H 2 O 2 ................. 49 Figure 1 3 : Primary microglia treated with H 2 O 2 and NAC ................................ .............. 50 Figure 1 4 : OGD reperfusion results in BV 2 cells ................................ ........................... 52 Figure 1 5 : OGD reperfusion results in wild typ e primary microglia ............................... 53 Figure 1 6 : Comparing ROS production of p53 knockout and wild type microglia ......... 54 Figure 1 7 : Bar graph comparing all groups at 90 minutes after reperfusion .................... 55 Figure 1 8 : Testing DCFH 2 saturation after OGD reperfusion with H 2 O 2 ................................ ... 56 Figure 1 9 : The qPCR results for serial dilutions of the positive control .......................... 58 Figure 20 : IL 1Beta transcription, expressed as fold above control ................................ 59 Figure 2 1 : MARCO transcription, expressed as fold above control ................................ 60
vii REACTIVE OXYGEN SPECIES LIKELY INDUCE PRO INFLA MMATORY GENE TRANSCRIPTION AND p 53 ACTI VITY FOLLOWING OXYGEN GLUCOSE DEPRIVATION IN CULTURED MICROGLIA David Hartmann New College of Florida, May 2012 ABSTRACT The pro inflammatory microglial response is widely believed to exacerbate brain injury following ischemia reperfusion, whereas the anti inflammatory microglial response is reported to improve neurological outcome. Because brain levels of reactive oxygen sp ecies (ROS) and the transcription factor p53 are known to increase following ischemia reperfusion, and these molecules have been shown to bias microglia towards the pro inflammatory response following other insults, ROS and p53 are prime candidates in the search for molecules that promote transcription of pro inflammatory factors such as interleukin 1Beta (IL 1Beta) in response to ischemia reperfusion. Importantly, ROS are known to activate p53 and are known to result from p53 activation in various cell typ es, but the existence of this positive feedback loop and its influence on microglial transcription have not been examined. By utilizing the redox dichlorodihydrofluorescein, this thesis demonstrates that murine and BV 2 cell line microg lia subjected to oxygen glucose deprivation (OGD) exhibit ~30% greater ROS production upon reperfusion as compared to microglia maintained in normoxic normoglycemic media. The same OGD reperfusion paradigm elicited an increase in p53 activity in earlier st udies, m eaning that i ncreased ROS product ion possibly contributed to the previously observed increase in p53 activity. No significant differences in ROS production were observed in microglia harvested from p53 knockout mice, suggesting
viii that ROS produce d upon reperfusion are not a result of p53 activity Pre treating cells with the radical scavenger N acetylcysteine (NAC) abrogated ROS production in all conditions. The amount of IL 1Beta transcript measured by quantitative PCR was approximately 1500 fold higher in microglia subjected to OGD reperfusion when compared to controls, an effect that was greatly inhibited by NAC, indicating that ROS are largely responsible for the observed increase in IL 1Beta transcription after OGD reperfusion. Transcription of the p ro inflammatory marker macrophage receptor with collagenous structure ( MARCO ) showed similar trends, but with much less robust differences between groups. These findings extend the role of ROS in activating the pro inflammatory microglial phenotype to the context of ischemia reperfusion. Future studies are needed to confirm the role of ROS in activating p53, and to examine if p53 activity mediates the ROS induced increases in pro inflammatory transcription. Dr. Alfred Beulig
ix Abbreviations: ABeta amyloid beta AD ATP a denosine tri phosphate BDNF brain derived neurotrophic fa ctor cDNA complementary DNA CNS c entral nervous system DAMP d amage associated molecular pattern GDNF glia derived neurotrophic factor HAND HIV associated neurocognitive disorder ICAM 1 intercellular adhesion molecule 1 IFN gamma interferon gamma IGF 1 insulin growth factor 1 IL 1Beta interleukin 1 beta, a pro inflammatory cytokine IL 4 interleukin 4, anti inflammatory cytokine IL 6 interleukin 6, pro inflammatory cytokine iNOS inducible nitric oxide synthase normally at very low levels but can be upregulated in response to certain stressors LPS lipopolysaccharide, a cell wall component of gram negative bacteria. Triggers extensi ve M1 activation in microglia. M1 classical, pro inflammatory activation profile of microglia M2 alternative, anti inflammatory profile of microglia MARCO macrophage receptor with collagenous structure MCAO middle cerebral artery occlusion MCP 1 monocyte chemoattractant protein 1 MDM2 murine double minute 2, an enzyme that links ubiquitin to p53, promoting p53 degradation NCX3 sodium calcium exchanger isoform 3 NGF nerve growth factor NMDA N methyl D aspartate, an agonist of a particular glutamate recepto r
x on neurons -this receptor was then named after this specific agonist nNOS neuron specific nitric oxide synthase NO nitric oxide NOS nitric oxide synthase OGD oxygen glucose deprivation (defined here as 1% O 2 instead of the ambient 22%, and 250uM glucose in OGD instead of 25mM glucose in controls) p53KO p53 knockout (or p53 / ) PAMP Pathogen associated molecular pattern PHOX NADPH oxidase enzyme primarily present in phagocytic cells PUMA p53 upregulated mediator of apoptosis ROS reactive oxygen species (pr imarily H 2 O 2 superoxide, peroxynitrite, hydroxyl radical) SOD superoxide dismutase
1 Chapter 1: Introduction 1.1: Microglia 1.1.1: Glia -More Than Brain Glue Homeostasis and maturation of the central nervous system (CNS) requires synergistic and delicately balanced chemical and electrical dialogue between myriad cell types. Although inter neuronal communication has been established as the biological substrate for memory and other mental processes, communication between neuronal and non neuronal cells greatly contributes to the development and function of the CNS. Chief among these vital non neuronal cells is a class of cells known as glia, which were recently reported to be approximately equal in number to neurons in the primate brain (Azevedo, et al. 2009). Because glia are unable to generate the electrical impulses observed in neurons, it was initially believed that glial cells serve a passive, structural literature has emerged that supports an active and essential role of glia in the function, and dysfuncti on, of nervous systems in organisms ranging from Caenorhabditis elegans to humans (Oikonomou & Shaham 2010). Of the four known types of glia -polydendrocytes, oligodendrocytes, microglia, and astrocytes -microglia are unique in that their differentiation a nd development occur outside of the neuroectoderm, the embryonic cell layer that develops into the central nervous system (Saijo & Glass 2011). Microglia are instead derived from hematopoietic stem cells in the yolk sac that surrounds the embryo. Early in development, these microglial precursors then migrate from the yolk sac into the CNS, where they fully differentiate into microglia (Ginhoux, et al. 2010) and subsequently proliferate to become
2 approximately 12% of the total number of cells in the mature b rain (del Rio Hortega 1932). Many of the transcription factors and cell surface receptors that are involved in differentiation and proliferation of microglia play similar roles in the development of tissue macrophages -peripheral cells that are also derive d from hematopoietic stem cells and are responsible for the phagocytosis of pathogens, and the activation of the immune response (Saijo & Glass 2011; Unanue & Allen 1987). Thus, when looking at embryonic lineage and the molecules involved in development, w e see that microglia and tissue macrophages are very similar, while neurons and other types of glia share little besides residency in the CNS with microglia. Although the other glial cell types are critical for CNS function, this thesis is focused on micro glia and their impact on neuronal environment. 1.1.2: Microglia: Sentinels of the CNS The functional roles of mature microglia are logical extensions from their developmental similarities to macrophages and their anatomical proximity to neurons and astroc ytes: microglia function as the primary phagocytic immune cells of the CNS. As such, microglia detect and respond to pathological events in the brain. Detection of insults occurs via various pattern recognition receptors on the cell surface which enable mi croglia to identify pathogen associated molecular patterns (PAMPs) like bacterial cell wall components or viral DNA/RNA (Block, et al 2007). There are also pattern recognition receptors capable of identifying damage associated molecular patterns (DAMPs) su ch as adenosine tri phosphate (ATP), amyloid beta fibrils (ABeta), heat shock proteins (HSPs), and other molecular indicators of stress or damage to nearby host cell
3 populations (Brown & Neher 2010). Real time in vivo imaging revealed that microglia active ly scan their environment for signs of stress by extending and retracting their outermost branches, though it was previously believed that microglia detected insults by passively awaiting the arrival of signaling factors to their microenvironment (Nimmerja hn, et al. 2005; Davalos, et al. 2005). Recent evidence even suggests that surveying microglia frequently make physical contact with synapses, and the extent of this interaction is increased in response to neuronal activity as well as ischemic insult -furt her evidence that microglia actively seek and detect changes in their environment (Wake, et al. 2009). The stereotypical microglial response following detection of CNS insult is the branched with slend er cell body) transformation that can be completed in a little as two hours (Stence, et al. 2001). This stimulus induced morphological transition represents an abrupt chang e in functional state, as microglia switch roles from surveillants to modulators of the cellular environment. An investigation of ~19,000 gene transcripts indicated that microglia activated by lipopolysaccharide (LPS), a component of gram negative bacteria l cell walls, actually bear more transcriptional resemblance to peripheral macrophages than to quiescent microglia, exemplifying the drastic chemical changes underlying microglial activation (Schmid, et al. 2009). The transcriptional profile resulting from microglial activation is highly dependent upon the activating stimulus. The changes observed in gene expression after ABeta activation (Walker, et al. 2006) were markedly different than those seen following LPS treatment (Schmid, et al. 2009; Walker, et a l. 1995), or spinal
4 cord injury (Byrnes, et al. 2006). Although the expression levels of specific genes depend upon the type and severity of CNS insult, all genes transcribed by activated microglia typically serve one of two functional purposes: pro inflam matory (resulting in the recruitment of local and peripheral immune cells to a site of injury; associated with tissue damage), or anti inflammatory (resulting in reduction of the immune defense response and the enhancement of the growth of host cells; asso ciated with tissue regeneration). Numerous injuries to the CNS, such as ischemic stroke, elicit simultaneous activation of pro and anti response to injury (Wang, et al. 2007). These di fferent phenotypes of microglia elicit vastly different effects on neighboring cells, and much research is currently being done to find therapies that harness the anti inflammatory, neuroprotec tive capacities of microglia which suppress the neurodegenerative pro inflammatory tendencies (Block, et al. 2007). 1.1.3: Reactive Oxygen Species and p53: Putative Promoters of a Pro Inflammatory Response Evidence is emerging that reactive oxygen species (ROS) are initiators of the pro inflammatory microglial respons e (Block & Hong, 2005). Microglia produce ROS and transcribe pro inflammatory proteins after treatment with LPS: if ROS are inhibited, then so too are the pro inflammatory transcriptional changes, suggesting that ROS transmit an early pro inflammatory sign al to the nucleus (Pawate, et al. 2004). Considering that the pro inflammatory microglial reaction to ischemic stroke is widely believed to exacerbate cell death in the brain (Dirnagl, et al. 1999), and that ROS production is increased following an ischemi c stroke (Peters, et al. 1998), one would anticipate that ROS
5 morphology showing a thicker cel l body and processes, with fewer branches on stout processes. Taken from Ayoub and Salm (2003). signaling contributes to pro inflammatory transcriptional changes in microglia following ischemic stroke. Indeed, based on results from whole brain studies using ischemia models, it is often proposed that microglia produce ROS, and th ese ROS then promote pro inflammatory transcription in microglia (Chen, et al. 2011; Genovese, et al 2011). However, there is little cell type specific evidence to show that microglia produce ROS
6 following ischemia, o r that these microglia specific ROS promote pro inflammatory signaling. Like ROS, the transcription factor p53 has also been shown to pus h microglia towards pro inflammatory transcription following several different stimuli (Garden, et al. 2004; Jayadev, et al. 2011). Interestingly, p53 knock out (p53KO) microglia not only show a suppressed pro inflammatory response to insults, but they al so show a boosted anti inflammatory response that is neuroprotective (Garden, et al. 2004; Jayadev, et al. 2011). This makes microglial p53 an interesting therapeutic target for the treatment of post ischemic inflammation. There is currently only indirect evidence suggesting that microglial p53 contributes to inflammation following ischemia: p53KO mice show significantly less severe injury following stroke (Crumrine, et al. 1994; Leker, et al. 2004; Enzo, et al. 2006; Culmsee, et al. 2001) p53KO mice showe d a larger amount of microglia with anti inflammatory markers following stroke (Jayadev, et al. 2011), and there are unpublished results showing that oxygen glucose deprivation (OGD) followed by reperfusion activates p53 transcription in microglial culture s (Gwenn Garden, personal communication) However, the mechanisms underlying this observed p53 activation, or the downstream effects of p53 activation, are unknown. ROS are well known activators of p53 (Achanta & Huang 2004), and ROS are known to transmit pro inflammatory signals in microglia ( Block and Hong, 2005). If microglia do indeed produce ROS following ischemia reperfusion as is often proposed, it would therefore be plausible that ROS were the cause for the OGD reperfusion induced p53 activation, which would consequently promote transcri ption of pro inflammatory genes. If this were true, this
7 would provide a mechanism by which ROS production can elicit pro inflammatory transcription. 1.1.4: Thesis Goals The goal of this thesis is to determine if ROS contribute to pro inflammatory transcri ptional changes in microglia subjected to oxygen glucose deprivation (OGD) with reperfusion. A component of this goal is to discern if ROS are possibly upstream and/or downstream of the activation of p53 that was previously observed ( unpublished) in OGD re perfusion (Gwenn Garden, personal communication). Figure 2: Visual overview of the process of microglial activation, showing the involvement of p53 in promoting the pro inflammatory response. Please s ee A bbreviations page for c larification. 1.1.5: Downstream of Microglial Activation: Blades of th e Double Edged Sword Critical among the myriad transcriptional changes in microglial activation is the upregulation of cytokines (cell signaling molecules, usually proteins, that are released from glia and immune cells), and cytokine receptors (Lee, et al. 2002; Hanish 2002). Via
8 the production and release of cytokines, microglia can communicate the perturbations detected in their environment, and effect a response from nearby cells. Specific CNS perturbations yield specific cytokine signaling. For example, microglia were shown to release neurotrophic cytokines following exposure to mild, moderate, and severe hypoxic neuronal injury, but released the pro inflammatory cytokines interleukin 1Beta (IL 1Beta) and tumor necrosis factor alpha (TNF alpha) only foll owing mild neuronal injury (Lai & Todd 2008). A tightly regulated microglial response such as this is essential for physiological function: it was shown that low doses of TNF alpha were neuroprotective against excitotoxic injury, while larger doses of TNF alpha potentiated excitotoxic neuron death; the converse was true for IL 1Beta (Benardino, et al. 2005). The net effect of cytokines on cells in the CNS is thus dependent upon the identity of the signaling molecule (Allan & Rothwell 2001), its concentratio n (Benardino, et al. 2005; Li, et al. 2007), and the presence of other molecules in the environment that may have synergistic or inhibitory contributions (He, et al. 2002). Considering that microglia are one of the main sources of pro inflammatory cytokine s in the brain (Hanish 2002), and that there are so many factors deciding between a protective or deleterious outcome of cytokine signaling, tight regulation of the microglial response to perturbations in the CNS is vital. Indeed, dysregulation of microgli al output --is characterized by excessive production of pro inflammatory cytokines and other potentially cytotoxic molecules, and is now known to contribute to neurodegeneration in many types of CNS injury (Giulian, et al. 1995). The inju rious role of microglia was proposed after high numbers of activated microglia were seen localized to sites of injury in the brains of patients afflicted with ischemic stroke, as well as neurodegenerative diseases such as
9 Prion Protein disorders, Parkinson Disease, and HIV associated neurocognitive disorder (HAND) (reviewed in Block, et al. 2007). Further investigation revealed that extensive microglial activation and proliferation is more than just a conseq uence of neuronal injury in these pathologies. It has been convincingly demonstrated that excessive microglial release of pro inflammatory molecules such as IL 1Beta, interleukin 6 (IL 6), TNF alpha, interferon gamma (IFN gamma), and reactive oxygen specie s (ROS) contribute to CNS injury in these and many other neuropathologies (e.g.: Lull & Block 2005; Lorton 1997; Combs, et al. 1999; Kaushal & Schlichter 2008). Although the pro inflammatory response of microglia can contribute to neuropathogenesis, micro glia are capable of promoting a regenerative, anti inflammatory environment in the CNS as well. Microglial phagocytosis of apoptotic neurons decreases the production of pro inflammatory molecules like TNF alpha and nitric oxide synthase (NOS) (Takahashi, e t al. 2005), and microglial phagocytosis of harmful plaques in Prion protein disorders and AD (Kranich, et al. 2010; El Khoury, et al. 2007) has been associated with positive behavioral outcomes in mice, in part due to clearance of debris and the release o f trophic factors like nerve growth factor (NGF) (Heese, et al. 1998) and insulin growth factor 1 (IGF 1) (Lalancette Hebert, et al. 2007). Proliferation of microglia, a hallmark of the activation cascade, was important in the recovery from cerebral ischem ia in mice: ablating the capacity of microglia to proliferate resulted in nearly 3 fold higher cell death than controls in an in vivo model of stroke (Lalancette Hebert, et al. 2007). Therefore, activated microglia are capable of creating an environment th at promotes recovery and/or degeneration in the CNS. This two faced
10 nature of microglial activation has driven efforts to develop therapies that simultaneously the benefic ial anti 2010; Lakhan, et al. 2009). Certain stimuli such as interleukin 4 (IL 4) or apoptotic host cells reliably induce M2 activation, whereas M1 activation consistently follows micro glial detection of IFN gamma or pathogen associated molecules like bacterial cell wall components (Neumann, et al. 2009). A primary goal of modern translational microglia research is to promote M2 but suppress M1 microglial activation. This goal has been m et with limited success (Ohtaki, et al. 2008; Yrjanheikki, et al. 1999 ). We will now discuss p53 in more detail, a transcription factor that was recently discovered to regulate the microglial activation phenotype. This discovery has opened new therapeutic avenues, but more work must be done before its therapeutic potential can be realized. 1.1.6: p53 in Microglial Activation While the stimuli and effects of M1 and M2 activation profiles have been well classified, the transcription factors that mediate a sh ift towards M1 or M2 are largely unknown. The transcription factor p53, widely known for its role as a tumor suppressor in cancer research, has emerged as one important contributor to the microglial activation phenotype. Microglia harvested from p53 knock out (p53KO) mice show greater expression of M2 associated gene transcripts following treatment with the HIV envelope protein gp120 when compared to p53 + / + microglia in a model of HAND (Jayadev, et al. 2011). Interestingly, gp120 induced neuronal death onl y occurred when p53 + / + neurons were co cultured with p53 + / + microglia; p53KO microglia treated with gp120 actually
11 brought apoptosis of p53 + / + neurons to below baseline levels, suggesting that stimulation of p53KO microglia actually induces a neuroprotecti ve M2 activation response (Garden, et al. 2004). When looking at whole brain, p53 has been implicated in cell death following a number of CNS insults, with cerebral ischemia being among the most studied. In general, inhibiting or deleting p53 is associated with less cell death in experimental models of stroke (Leker, et al. 2004; Endo, et al. 2006; Culmsee, et al. 2001; Crumrine, et al. 1994; but see Maeda, et al. 2001). It was found that p53KO microglia, significantly more than wild type, expressed molecul ar markers of M2 activation in a middle cerebral artery occlusion (MCAO) mouse model of stroke (Jayadev, et al. 2011), possibly one reason why p53KO mice show less cell death in the brain following ischemic insult (Crumrine, et al. 1994; Yonekura, et al. 2006). Although the presence of p53 was shown to affect microglial phenotype following MCAO, it is not known if microglial p53 is activated in ischemia, or how p53 affects the microglial inflammatory response to ischemia. It would be of great clinical inte rest to ascertain ways to manipulate p53 in hopes of tipping the microglial response towards the beneficial M2 phenotype. However, the mechanisms of p53 activation in microglia, in any pathological context, are unknown. We will now look into previous resea rch on p53 in other cell types to attain clues on how p53 may be regulated in microglia, and how it might affect transcription of inflammatory factors. Additionally, an investigation of p53 action in other cells will help to parse out the reasons why p53 i nhibition is beneficial following stroke (e.g: Leker, et al. 2004). 1.2: p53
12 1.2.1: p53: A Complex Regulator of Cell Death & Growth Arrest In its role as a tumor suppressor, p53 responds to cellular stress, particularly DNA damage, by arresting cell growt h and division, initiating DNA repair, or promoting apoptosis of damaged cells to avoid aberrant growth of potentially mutated cells (reviewed in Levine & Oren, 2009). The extent of cell damage, the type of cellular insult, the cellular environment, and th e cell type are the principal factors that decide the fate of p53 activity. For instance, thymocytes exposed to ionizing radiation tend to undergo p53 dependent apoptosis whereas fibroblasts exposed to a similar dose of radiation undergo p53 dependent grow th arrest (reviewed in Amundson, et al. 1998). Following radiation exposure, a murine leukemia cell line will undergo p53 dependent cell cycle arrest in the presence of the cytokine interleukin 3, but p53 dependent apoptosis in the absence of interleukin 3 illustrating the influence of cellular environment on p53 function (Amundson, et al. 1998). Specificity in the p53 mediated effects on the cell are generated via differential activation of particular molecular cascades, or activation of identical cascade s to a different extent (Vousden & Lane 2007). In general, p53 responds to insult in a graded fashion -the amount of p53 activity, and the resultant effects on the cell, depend on the degree of cell stress (see Fig. 3 ; Vousden & Lane 2007). In the absence of cellular stress, p53 is present at low levels in the cell because of post translational degradation mechanisms. A substantial percentage of the small amount of p53 that is present in basal con ditions is bound, via a deep hydrophobic pocket, to the murine double minute 2 (MDM2) protein (Vassilev 2007). The MDM2 p53 binding site overlaps with the transactivation domain of p53, prohibiting transcriptional activity of
13 Figure 3 : Th e graded response of p53, with the possible effects on the cell, are displayed. The result of p53 activation is dependent upon the severity of insult, cell type, and cellular environment. Different molecular cascades are initiated to produce a specific eff ect on the cell. From Vousden & Lane (2007). p53. MDM2 also ubiquitinates p 53 at several amino acid residues, setting p53 up for degradation by the proteasome (Kubbutat, et al. 1997). Stabilization, and therefore accumulation and activation of p53 occurs when the p53 MDM2 association is inhibited. The mechanism of p53 MDM2 bindin g inhibition is specific to the type of cellular stress, but phosphorylation of p53 is a very common mechanism for the liberation of p53. As an example, pharmacological DNA damaging agents caused the phosphorylation of Ser15 and Ser20, whereas a hypoxia mi micking molecule elicited only phosphorylation at Ser15 (Ashcroft, et al. 2000). The mechanisms allowing for the activation of p53 in microglia have yet to be determined.
14 The consequences of p53 accumulation stem largely from the transcription of p53 resp onsive genes, though p53 can effect apoptosis via transcription independent mechanisms as well (Amundson, et al. 1998; Culmsee & Mattson 2005; Vousden & Lane 2007). Microglia in HAND affected human brains showed a ccumulated p53 with increased levels of p2 1 and Bax, suggesting that pro apoptotic and growth arrest genes are transcribed by p53 in microglia (Jayadev, et al. 2007). The roles of these transcribed genes in promoting a pro inflammatory response are undetermined. 1.2.2: p53 and Reactive Oxygen Spe cies (ROS) High concentrations of reactive oxygen species, small unstable molecules with high energy electrons localized to an oxygen atom, lead to the accumulation and/or activation of p53. After adding exogenous H 2 O 2 to glioma cells (Datta, et al. 2002), or inhibiting the endogenous ROS scavenger superoxide dismutase (SOD) in colon carcinoma cells, p53 protein levels increased (Achanta & Huang 2004). During hypoxia in a breast cancer cell line, increased production of ROS by mitochondria was required for the accumulation of p53 (Chandel, et al. 2000). In response to transient global cerebral ischemia, a mouse model of stroke that is known to elicit increased levels of ROS, p53 transcriptional activity was increased, as evidenced by high levels of PUMA prot ein (Niizuma, et al. 2009). The p53 transcriptional activity in the aforementioned study was inhibited when SOD was overexpressed, confirming that ROS production was upstream of p53 transcriptional activity. The mechanisms by which p53 is stabilized were n ot determined in the above studies, but one investigation showed that Ser15 of p53 was phosphorylated by Jun kinase 1 or 2 as a result of glutamate induced ROS production in
15 neurons (Choi, et al. 2011). Many propose that ROS induce DNA damage, which is det ected by proteins like Jun kinase 1, and is then signalled to p53 by way of phosphorylation or other protein modifications (Culmsee & Mattson 2005). Many studies show that ROS are also downstream of p53, and that ROS are an essential effector of p53 growt h arrest or apoptosis. In carcinoma cell lines, higher levels of p53 protein were associated with higher levels of intracellular ROS in a PUMA and Bax dependent manner, suggesting that p53 effected the production of ROS by mechanisms at the mitochondria a n important locus for cellular ROS production (Macip, et al. 2003). Bax, PUMA, and two other p53 induced genes named PIG3 and PIG6, increase ROS by permeabilizing the mitochondrial membrane and disrupting the electron transport chain (Liu, et al. 2008, Cul msee & Mattson 2005). Studies are scarce on ROS downstream of p53 activity in brain cell types. One investigation showed that ROS were upstream and downstream of p53 in the synaptosome (the proteins that comprise a synapse) when treated with glutamate or D NA damaging molecules (Gilman, et al. 2003). In this study, blocking p53 with pifithrin alpha or using synaptosomes from p53KO mice reduced the amount of ROS after administration of the stimulus. Mitochondrial membrane disruption was essential in generatin g ROS downstream of p53 (Gilman, et al. 2003). With ROS being upstream and downstream of p53, a positive feedback loop is plausible, in which ROS produce p53, and p53 produce ROS. This positive feedback loop has not been investigated, but would be extreme ly harmful if it existed in microglia, considering that higher amounts of ROS correspond to greater pro inflammatory gene transcription (Block, et al. 2007).
16 1.2.3: p53 Relevance in the CNS In replicating cells, functional p53 is essential in cancer preve ntion, but in non replicating cell types such as mature neurons, activation of p53 can lead to apoptosis and a permanent decrease in cell number. Thus, loss of p53 function is a major risk factor for uncontrolled cell growth and tumorigenesis, but in the C NS it is the overactivation of p53 that is more of a concern: p53 can directly lead to neuronal death via activation of p53 mediated apoptotic mechanisms (see Culmsee & Mattson 2005 for review), and p53 can kill neurons indirectly by promoting microglial p roduction of harmful cytokines (Jayadev, et al. 2011; Garden, et al. 2004). Indeed, p53 protein appears to promote cell death in models of neurodegenerative disorders such as AD (de la Monte, et al. 1997; et al. 2005; Bernstein, et al. 2011), ischemic stroke (see below), HAND (Garden, et al. 2004), and others. Additionally, it is possible that sub lethal activation of p53 can instigate degeneration of neurites by p53 mediated destruction of synaptic mitocho ndria (Gilman, et al. 2003). Inhibiting p53 or genetically removing the p53 gene is typically found to reduce CNS cell damage in the aforementioned pathological contexts, supporting the hypothesis that p53 activation in the CNS is deleterious (reviewed in Culmsee & Mattson 2005). 1.2. 4 : p53 in Ischemic Brain Injury P53 was first implicated as a contributor to ischemic brain injury for the following reasons (Crumrine, et al. 1994): protein synthesis dependent programmed cell death (apoptosis) was observed following transient cerebral ischemia (Shigeno, et al. 1990); p53 was identified as a transcriptional mediator of apoptosis in multiple cell types
17 (Yonish rouach, et al. 1991); levels of p53 protein and mRNA increased following transient cerebral ischemia in rats, and only in the hemisphere that was ipsilateral to artery occlusion (Li, et al. 1994). Further investigations confirmed the involvement of p53 in ischemic injury: upregulation of p53 protein occurred specifically within apoptotic neurons following ischemic insult (Li, et al. 1997), and blocking the upregulation of p53 via antisense oligonucleotides prevented hypoxia induced neuronal apoptosis in culture (Banasiak & Haddad 1998). It was recently shown that the extent of hippocampal cell death was ab out six fold greater in p53 + / + mice subjected to transient global ischemia, when compared to p53KO hippocampi, validating the older studies that hinted towards the importance of p53 in ischemic injury (Yonekura, et al. 2006). Even when administered 1 6h af ter the ischemic insult, pharmacological inhibition of p53 also resulted in reduced cell death (Leker, et al. 2004; Endo, et al. 2006; Culmsee, et al. 2001). Inhibition of cell death during this protracted time course supports the hypothesis that p53 contr ibutes to apoptosis, a type of cell death that requires several hours to complete after ischemia. Another study found that administering a p53 inhibitor 6 9 days after ischemia improved neurological outcome and increased maturation of newborn neurons (Luo, et al. 2009). Because p53 deficiency leads to alternative activation in microglia, and alternatively activated microglia promote neurogenesis, an attractive hypothesis would be that the neurological benefits reaped by the p53 inhibitor are mediated by mic roglia (Jayadev, et al. 2011; Ekdahl, et al. 2009; Luo, et al. 2009). Activation of p53 in ischemic injury is possibly mediated by upstream ROS. Hypoxia, especially when coupled with hypoglycemia, is well known to induce intracellular ROS generation in man y cells types, including primary astrocytes and
18 neurons (Abramov, et al. 2007), and immortal cell lines of microglia (Hur, et al. 2010). Mice subjected to global cerebral ischemia showed an increase in PUMA, which was blocked by SOD overexpression (Niizuma et al. 2007), evidence that p53 is activated by ischemia induced ROS. Sources of increased ROS in ischemia are numerous, with xanthine oxidase, NADPH oxidase, and mitochondria (Brennan, et al. 2009; Piantadosi & Zhang 1996) being prominent sources. With mitochondria being a target of p53 and a well known contributor to ROS after ischemia (Piantadosi & Zhang 1996), it is quite possible that stabilization of p53 plays a role in ROS production following ischemia. In support of p53 dependent ROS production fo llowing ischemia, ROS were shown to be downstream of p53 stabilization in synaptosomes in response to oxidative stress; p53 mitochondrion interactions were cited as the source of this increased ROS (Gilman, et al. 2003). Downstream production of ROS follow ing p53 upregulation has not yet been investigated in any CNS related paradigm except for synaptosomes, which are extracted and homogenized components of the neuronal synapse. It would be of particular interest to investigate the feedback between ROS and p 53 in microglia in ischemia, given that ROS production by microglia is deleterious to surrounding neurons (discussed in later sections), and p53 promotes a pro inflammatory microglial response. Thus, p53 may lead to neuronal damage via two mechanisms in mi croglia -induction of ROS and of the pro inflammatory response. With known roles in mitochondrial viability, ROS production, neuronal apoptosis, and microglial inflammation, p53 has the potential to influence almost every phase of ischemic brain injury. To look into the potential ways in which p53 becomes activated
19 and the role of p53 in ischemic injury, one must consider some of the established mechanisms of brain ischemia reperfusion injury. 1.3: Ischemia Reperfusion Brain Injury 1.3.1: Stroke Has Many Untreatable Stages of Cell Death Stroke is the third leading cause of death in industrialized countries, and there are around four million stroke survivors in the United States coping with its debilitating effects (Dirnagl, et al. 1999). The pathological e ffects are a result of extensive neuronal death, though glia are also affected and can contribute to neurological deficits (Nedergaard & Dirnagl 2005). In middle cerebral artery occlusion (MCAO), a common type of stroke in humans and a very widely used exp erimental model of focal stroke, blockage of blood flow to the brain depletes oxygen and glucose in specific regions, triggering multiple cytotoxic cascades that can lead to the formation of dead brain tissue n surrounding the artery blockage that receives less than 15% of normal b lood flow; in MCAO, the core mostly comprises t he lateral striatum and parts of the overlying parietal and somatosensory cortices (Lipton 1999). Peripheral to the core, more distal to the blockage, is an area named the entorhinal cortex, and other parts of neocortex generally classify as penumbral tissue in MCAO. The penumbra is classified as an area of b rain that can make a full recovery, but is at risk of dying and adding to the infarct volume if pharmacological intervention or resumption of blood flow do not occur. The size of the core or penumbra depends largely on the intensity of the injury. Longer, more intense ischemic insults produce a larger core
20 and smaller penumbra, while the opposite is true of brief insults. Because the extent of oxygen and glucose deprivation is different between core and penumbra, the cell death mechanisms differ between the two regions (see Fig. 4 ) (Lipton 1999). Necrotic (or passive) cell death predominates in the core, whereas apoptosis (programmed cell death) predominates in the penumbra (Doyle, et al. 2008; Charriaut Marlangue, et al. 1996). There is overlap in cell deat h mechanisms between the two regions, however. Microglial inflammation is most evident and plays the most substantial role in t he ischemic penumbra (see Fig. 4 ; Dirngal, et al. 1999), and we will therefore primarily focus on the penumbra. Figure 4 : This is a visual representation of the ischemic core and penumbra following middle cerebral artery occlusion (MCAO).There is a spectrum of oxygen and glucose deprivation; it is most severe in the ischemic core (red), and becomes less severe as one goes far ther from the artery blockage towards the penumbra (blue through yellow). Accompanying the differential severity of ischemic insult in the various brain regions are different cell morphologies and biochemical events.
21 1.3.2: Injury in the ischemic core Tho ugh cell death in core regions has been shown to continue for weeks after ischemic insult (Lipton 1999), the ischemic core suffers extensive metabolic stress, leading to a loss of ion homeostasis and necrotic or excitotoxic death that can occur within min utes (Doyle, et al. 2008). With low levels of oxygen and glucose, cells are unable to generate ATP via oxidative phosphorylation. Brain tissue is then quickly depleted of its ATP stores, rendering neurons unable to maintain activity of the ATPase Na + / K + io nic pump, creating an increase in extracellular K + A higher extracellular concentration of K + causes an increase in resting potential of the neuron, which facilitates release of Ca 2+ from intracellular stores, and can open voltage sensitive ion channels, allowing the neuron to become more depolarized. The intracellular Ca 2+ increase leads to spontaneous vesicular release of glutamate from excitatory neurons. With a more positive resting potential, and an increased concentration of excitatory neurotransmitt ers in the noxic 4 5 ). Typically, rapid depolarization occurs 3 5 min after onset of ischemia, and large changes in the composition of the ionic envi ronment ensue: Na + Cl and Ca 2+ rush into cells, while K + efflux continues. The cell cannot re polarize because of the lack of ATP necessary to pump ions out of the cell, against their electrochem i c al gradient. With an overall increase in intracellular i on concentration, water enters the cell because of an osmotic obligation, and the plasma membrane can rupture and cause edematous cell death (Lipton 1999). If the cell survives beyond the influx of water, the influx of Ca 2+ triggers extensive glutamate rel ease, leading to a 10 60 fold increase in extracellular gl utamate concentration (see Fig 5 ). Binding of glutamate to
22 Figure 5 : A timeline of the chemical events that induce cell death, predominately in the ischemic core. Briefly after oxygen and/or glucose deprivation, the ATPase Na + /K + can no longer pump ions due to insufficient ATP. Changes in ion concent rations across cell membranes partially depolarize neurons, which then release small amounts of glutamate into the extracellular milieu. This will prompt the neuron to fully depolarize, releasing large amounts of glutamate and skewing the ionic concentrati ons greatly. If ATP is not restored, the cell will not restore ion concentrations, setting the stage for cell death. From Martin, et al. 1994. the neuronal NMDA receptor then allows further intracellular Ca 2+ increase. Meanwhile, the altered ionic concentration gradients disrupt as trocytic glutamate and K + clearance, thus increasing the rapid depolarization of neighboring neurons and the release of
23 glutamate, creating a positive feedback loop that culminates in a severe imbalance in ion and glutamate concentrations (reviewed in Mart in, et al. 1994). Once intracellular Ca 2+ levels are increased, a number of mechanisms promoting necrosis can be initiated. Increases in Ca 2+ promote the activation of phospholipase A2, an enzyme that catalyzes the hydrolysis of phospholipids, and is assoc iated with membrane rupture and cell death following ischemia (Bonventre, et al. 1997). Calpains, a family of calcium respo nsive proteases, also become activated during ischemia and proteolyze proteins such as the cytoskeletal protein spectrin, which leads to loss of cell membrane integrity (Bevers & Neumar 2008). Calpains also cleave isoform 3 of the intramembrane sodium calc ium exhanger channel (NCX3), thereby reducing neuronal ability to rid the cell of excess Ca 2+ (Bano & Nicotera 2007). Increased neuronal Ca 2+ can activate the neuron specific subtype of nitric oxide synthase (nNOS) only minutes after onset of ischemia, cau sing production of nitric oxide (NO), a free radical that has been shown to inhibit the electron transport chain and cause DNA damage (Matsui, et al. 1999). Importantly, Ca 2+ can be taken up by the negatively charged matrix of mitochondria via transfer thr ough the energy independent uniporter channel. Because Ca 2+ entry into the mitochondrion generates a more positively charged matrix, protons that rest outside of the matrix experience an attenuated electrochemical drive to enter the matrix. Under physiolog ical conditions, mitochondria utilize this electrochemical gradient to generate ATP via the FoF1ATP synthase enzyme (Dong, et al. 2006). Thus, Ca 2+ entry into the mitochondrial matrix leads to depolarization of mitochondria (Lipton 1999), and a decrease in ATP production because of the reduced protonmotive force across the inner mitochondrial membrane.
24 The excessive production of reactive oxygen species (ROS) is widely believed to be a major contributor to ischemic injury. Under physiological conditions, fr ee radical scavengers such as glutathione, superoxide dismutase (SOD), and catalase work to neutralize these highly reactive molecules and prevent them from chemically modifying membrane lipids or cellular proteins. During ischemia, and especially during r eperfusion when O 2 is available for superoxide production (Br oughton, et al. 2009, see Fig. 6 ), levels of ROS are elevated as a result of several purported mechanisms. Calcium influx through NMDA receptors, as occurs extensively in ischemia, leads to the a ctivation of NADPH oxidase, an enzyme that transfers an electron from NADPH to O 2 yielding superoxide (Abramov, et al. 2007; Brennan, et al. 2009). Phospholipase A2, an enzyme activated during ischemia induced Ca 2+ increases, cleaves membrane lipids to yi eld arachidonic acid, which is then metabolized by cyclooxygenase 2 to give superoxide as a byproduct (Bonventre, et al. 1997). Meanwhile, there also exist Ca 2+ independent sources of ROS such as xanthine oxidase (Abramov, et al. 2007). While the sources o f ROS and the reasons for their overproduction during ischemia are debated, it is known that the downstream actions of ROS serve to exacerbate ischemic brain injury. One particularly harmful product of ROS generation is peroxynitrite (ONOO ), a molecule th at is formed in a reaction between NO and superoxide, and is known to damage membranes and alter protein function by nitrosylating tyrosine residues (Eliasson, et al. 1999). Neurons release glutamate and ATP in the ischemic core, both of which have been re cently shown to induce the microglial production of ROS (Mead, et al. 2012), TNFalpha, IL 1Beta, and a number of other cytokines that exacerbate ongoing changes in cells in the core (Kaushal & Schlichter 2008; Melani, et al. 2006). IL 1Beta, for instance,
25 can increase the amount of Ca 2+ influx through NMDA receptors, promoting cell death by the necrotic mechanisms discussed above (Huang, et al. 2011). Figure 6 : Reactive oxygen species (ROS) are slightly increased throughout this 3 h middle cerebral artery occlusion (MCAO). When the artery was re opened at 60 min after MCAO onset, a substantial increase in ROS is evident. This is in vivo evidence for a burst of ROS upon reperfusion. From Peters, et al. (1998). 1.3.3: Injury in the ischemic penumbra In the ischemic core, ATP levels quickly drop to ~25% of normal, whereas the ATP levels in the penumbra range from ~50 70% of normal during ischemia (Lipton 1999). Upon reperfusion, Ca 2+ distribution returns to pre ischemic levels in the penumbra, but ATP levels are permanently decreased (~70% of normal) even after
26 reperfusion (Sims & Anderson 2002). As a result of these differences between the core and the penumbra, the penumbra generally does not undergo immediate necrotic death due to metabolic failure. Instead cells in the penumbra continue to slowly die off, up to weeks after the insult, due to cell death resulting from inflammation and apoptosis (Dirnagl, et al. 1999). Because the penumbra comprises cells that are not immediately committed to cell death, the re is a chance for recovery, and it is thus extensively studied as a therapeutic target. Microglia are considered an especially important aspect of progression of penumbral damage, because they release potentially harmful cytokines onto a population of vul nerable neurons (Lipton 1999). The therapeutic potential of rescuing cells in the penumbra is supported by the positive correlation between the volume of salvaged penumbra and the neurological improvement following stroke in humans (Furlan, et al. 1996). 1.3.4: Inflammation in Ischemia: Role of Microglia Within 30 min after MCAO, microglia in the penumbra extended their processes around pre synaptic boutons for a much longer duration of time than in non ischemia controls in vivo (Wake, et al. 2009). With s uch extensive exposure to neurons, toxic or beneficial factors released by microglia can have a major impact on ischemic brain injury. Microglia are less likely to die during the ischemic insult than are neurons because of higher levels of antioxidant mole cules such as glutathione (Hirrlinger, et al. 2000), but some death of microglia is observed in the ischemic core (Kato, et al. 1996). Because microglia are capable of proliferating, it is not the death of microglia that is of therapeutic concern, but rath er neurodegenerative microglial M1 activation that occurs in response to
27 ischemic injury. Indeed, activated microglia appear in the penumbra prior to neuronal death, implicating them in the progression of penumbral cell death (Rupalla, et al. 1998). Glutam ate (Kaushal & Schlichter 2008), ATP (Melani, et al. 2006), K + (Abraham, et al. 2001), and ROS are all known to induce M1 activation in microglia (Roy, et al. 2008). These activators diffuse from the ischemic core to the penumbra, where microglial activat ion appears to be the strongest (Zhang, et al. 1997) and the earliest, occurring in as little as 30 min after MCAO (Rupalla, et al. 1998). Upon activation, microglia proliferate and change morphology and gene expression patterns drastically. The number of microglia doubles in the brain hemisphere that is ipsilateral to an ischemic insult, peaking at 24h following 1h of MCAO, and slowly decreasing over the course of weeks (Gelderblom, et al. 2009). Microglia mostly accumulate in areas of ischemia induced neu ronal damage, suggesting that microglia migrate to areas of injury (Morioka, et al. 1993). A high density of microglia in injured areas does not mean that microglia necessarily induce damage; some regions show microglia accumulation but not neuronal death (Abraham, et al. 2000). Indeed, some studies have even shown that a higher number of microglia results in better neurological outcome: injecting cell culture derived microglia into rats 48h after transient MCAO decreased infarct volume when infarct was mea sured 1 week after artery occlusion (Narantuya, et al. 2010). Nonetheless, there is substantial evidence suggesting that microglia exacerbate neuronal injury following ischemia by adopting an M1 phenotype and promoting inflammation. Indeed, therapies that reduce microglial activation confer neuroprotection following transient MCAO ( Yrjanheikki, et al. 1999 ).
28 Microglia are the first immune cells to arrive at the site of injury. They accumulate prior to the infiltration of peripheral immune cell types, such as dendritic cells, neutrophils, macrophages, and lymphocytes (Gelderblom, et al. 2009). Neurons subjected to ischemia release complement proteins C3 and C9, as well as chemokines such as monocyte chemoattractant protein 1(MCP 1) that cooperate to recruit microglia to sites of neuronal damage (Lai & Todd 2006). Once recruited and activated following ischemia, microglia release cytokines and chemokines that assist in breaking down the blood brain barrier and recruiting cells from the periphery. While accumu lation of microglia appears to have beneficial as well as detrimental effects, the accumulation of peripheral immune cells, which occurs around 3d after stroke, is widely believed to be entirely detrimental (Weinstein, et al. 2010). IL 1beta is produced al most exclusively by microglia after ischemia and disrupts the blood brain barrier, recruits peripheral neutrophils into the brain, and stimulates production of other pro inflammatory cytokines (Bhat, et al. 1996 and discussed therein). IL 1alpha and IL 1be ta double knockout mouse models showed a 70% reduction in infarct volume following 30 min MCAO and reperfusion (Boutin, et al. 2001). This reduction in infarct volume in IL 1KO mice is likely due in part to the decreased recruitment of peripheral immune ce lls (Huang, et al. 2006). New evidence shows that IL 1Beta treatment potentiates Ca 2+ influx through NMDA receptors in neurons, meaning that direct action of IL 1Beta on neurons could also contribute to the deleterious effects of IL 1Beta after ischemia (H uang, et al. 2011). I n vivo and in vitro experiments have therefore revealed a predominately harmful rol e of IL 1Beta production following stroke.
29 1.3.5: Mic roglia and ROS in the Ischemic C ontext The release of ROS from activated microglia, particularly f rom different forms of NADPH oxidase, is believed to be a major source of neuronal injury following ischemia and reperfusion. However, very few studies have shown ROS production in microglia by direct methods such as spin trapping or fluorescent probes. St ill, experiments in cell culture provide mechanisms of microglial NADPH oxidase activation that might occur in ischemia in vivo. Administration of glutamate, ATP, or GABA triggered superoxide production by stimulating microglial neurotransmitter receptors and subsequently activating microglial NADPH oxidase; glutamate induced NADPH oxidase activation was found to be particularly neurotoxic (Mead, et al. 2012). Hypoxia without reoxygenation did not increase ROS production in cultured microglia, while hypoxia with reoxygenation showed a marked increase in NADPH oxidase derived extracellular ROS (Spranger, et al. 1998). Such an increase in extracellular ROS has been shown to be harmful in in vivo studies of ischemia. Microglia possess a form of NADPH oxidase th at exists predominately in phagocytes (PHOX) that produces extracellular superoxide designed to neutralize invading pathogens. Mice lacking the gene encoding a necessary subunit of PHOX, gp91 phox showed a decrease in infarct volume after transient cerebr al artery occlusion (Walder, et al. 1997). In this study, blocking microglial PHOX was necessary but not sufficient for neuroprotection; PHOX from both invading peripheral immune cells and microglia had to be eliminated in order for protective benefits to be observed (Walder, et al. 1997). Another study illustrates the benefits of PHOX inhibition in an ischemic context. After administration of ischemia mimicking drugs, PHOX became expressed in microglia, ROS production was increased, and
3 0 neurons exposed to microglia conditioned medium underwent apoptosis. Inhibiting the expression of PHOX by siRNA abrogated ROS production in microglia, and neurons exposed to the media from these siRNA treated microglia did not undergo apoptosis (Hur, et al. 2010). New resea rch indicates that protective effects of PHOX inhibition are not only a result of reduced extracellular ROS. Intracellular ROS signalling, mediated by PHOX and other types of NADPH oxidase enzymes, has recently emerged as an important component of the pro inflammatory microglial phenotype. The activation of PHOX, followed by the conversion of superoxide to H 2 O 2 has been shown to induce transcription of pro inflammatory genes in microglia (Block, et al. 2007). The transcription of TNFalpha, iNOS, IL 1Beta a nd IL 6 in response to the bacterial cell wall component lipopolysaccharide (LPS), or the cytokine interferon gamma, was significantly reduced when PHOX was inhibited (Pawate, et al. 2004). Importantly, there exists a positive feedback loop in microglia be tween pro inflammatory cytokine production and ROS generation; IL 1Beta and TNF alpha were shown to activate microglial NADPH oxidase in cell culture, yielding hydrogen peroxide (Mander, et al. 2006). Thus, IL1 Beta and TNFalpha induce ROS, and ROS can upr egulate these and other cytokines. Interestingly, the NADPH oxidase derived H2 O 2 was required for the proliferation of microglial cells in response to IL 1Beta and TNF alpha administration (Mander, et al. 2006). This shows that IL 1Beta and TNF alpha promo te ROS formation via NADPH oxidase in cultured microglia, and that this increase in ROS is largely responsible for the proliferation of microglia. When combining this finding with others showing that ROS upregulate IL 1Beta, one can see how ROS production in microglia is
31 a central component of the microglial pro inflammatory response. Indeed, although the source of ROS was uncertain, scavenging intracellular ROS with the antioxidant N acetylcysteine was shown to prevent microglia from expressing a marker of M1 activation (Roy, et al. 2008). 1.3.6: Unanswered Questions about ROS, Microglia, and Inflammation in Ischemia Paradigms Unfortunately, the importance of R OS mediated intracellular signa ling in eliciting the microglial pro inflammatory response has be en little studied in the context of ischemia. Two studies found that inhibiting or genetically knocking out PHOX reduced whole brain transcription of IL 1Beta following ischemia, but the microglia specific contribution to these effects is uncertain (Chen, et al. 2011, Genovese, et al. 2011). Other potential sources of ROS, such as mitochondria, have not been investigated as mediators of pro inflammatory transcription, though suppressing ROS by overexpressing SO D1 has been shown to reduce post ischemia i nfla mmation (Chen, et al. 2009), and mitochondria produce ROS following ischemia reperfusion (Piantadosi & Zhang 1996). Finally, because p53 is known to be induced by ROS (Niizuma, et al. 2009), and is known to regulate transcription of pro inflammatory genes (Jayadev, et al. 2011), it would be interesting to see if activation of p53 is a connection between intracellular ROS and transcription of pro inflammatory genes. Thesis work in the Garden laboratory helps elucidate the role of ROS signalling in the transc ription of pro inflammatory genes in an ischemia reperfusion context, with consideration of the contributions that p53 can make to this ROS signaling.
32 Figure 7 : An overview of the inflammatory processes that are initiated by oxidative stress and/or exc itotoxicity following stroke. Microglia and astrocytes transduce the stroke insult into chemical signals that exert many different effects: breakdown of the blood brain barrier, recruitment of neutrophils, and direct damage to neurons. Astrocytes play a mu ch less pronounced role in the formulation of an immune response, but they too generate cytokines and control extracelluar glutamate concentrations, which will both modulate the microglial response. 1.4: Research in the Garden Laboratory 1.4.1: Context o f Thesis Research p53 has emerged as an important contributor to neuronal injury in several neuropathologies, ischemic stroke important among them (Culmsee & Mattson 2005).
33 Many studies have shown that inhibition or absence of p53 is neuroprotective follow ing experimental models of stroke, making p53 a promising therapeutic target. However, one study has also shown that the absence of p53 exacerbates brain damage following transient ischemia (Maeda, et al. 2001). Therefore, the mechanisms by which p53 aggra vates or prevents ischemia induced brain injury must be elucidated before therapies can be developed. Microglia, via the production of pro inflammatory molecules, are believed to play a critical role in brain injury following an ischemic insult (Lakhan, e t al. 2009). Because p53 activation has been shown to bias microglia towards the inflammatory phenotype (Garden, et al. 2004; Jayadev, et al. 2011), perhaps the action of p53 within microglia contributes to ischemic injury. Indirect evidence that inhibitin g p53 in microglia is neuroprotective showed that microglia from p53KO mice were much more likely to express markers of anti inflammatory microglia (Jayadev, et al. 2011). Thus, p53 deficiency promotes alternative activation following transient MCAO. But, conversely, does the activation of p53 promote pro inflammatory microglial activation in ischemia? Unpublished research from the Garden lab attempts to answer this question. The first step was to show that p53 is indeed activated in an ischemia paradigm. The Garden lab showed that p53 transcription is activated in microglial cultures in response to an in vitro paradigm of ischemia reperfusion called oxygen glucose deprivation reperfusi on (OGD reperfusion) (see Fig. 8 ). Interestingly, the reperfusion step w as essential; p53 was not activated in response to OGD alone (data not shown). Because previous studies have shown that ROS are generated in microglia only if this reperfusion step occurs (Spranger, et al. 1998), it was proposed that OGD reperfusion
34 Figure 8 : A luciferase reporter assay showed increased transcription of p53 response elements upon subject ing microglia to an in vitro ischemia and reperfusion paradigm. Nutlin is an MDM2 blocker that causes accumulation of p53. Reprinted with permission of Dr. Gwenn Garden. leads to ROS production, which then activates p53. This proposal is in accordance with a study in neurons that showed ROS to be an upstream requirement of p53 accumulation (Choi, et al. 2011). However, it was not known if this OGD reperfusion paradigm in microgl ia caused an increase in ROS production, nor was it confirmed that microglia adopt a pro inflammatory phenotype when subjected to OGD reperfusion. The aim of this thesis is to show that ROS are indeed increased following OGD reperfus ion in p53KO and wild type microglia, giving credence t o the hypothesis that ROS contribute to the induction of p53 shown in Fig. 8 Additionally, I aim to show that
35 ROS production following OGD reperfusion is an important step in mounting a pro infl ammatory response in microglia. This research can provide important links between microglial ROS production, p53, and pro inflammatory gene transcription, which can help to guide the design of anti inflammatory therapies in ischemic stroke.
36 Chapter 2: Experimental 2.1: Acquiring and Maintaining Cells 2.1.1 : Cell Culture Media and Chemical Solutions Normoglycemic, serum free medium was made by adding, to high glucose DMEM, insulin transferrin selenium (1% final concentration) and 1mg/mL Albu max. Hypoglycemic, serum free medium was created by adding, to glucose free DMEM, insulin transferrin selenium (1% final concentration), 1mg/mL Albumax, 250M glucose, and 25mM endotoxin dichlorodihydrofluorescein diacetate (DCFH 2 DA) (Cayman Chemical) was dissolved in dimethylformamide (Acros Organics) to achieve a 500M stock solution, which was stored wrapped in aluminum foil at 20 o C to inhibit photoxidation and/or hydrolysis. Solid N acetylcysteine (NAC) (Sigma) was dissolved in distilled water (0.5M stock), sterilized with a syringe operated filter (0.2M mesh), and stored at 20 o C until use. See Appendix for other cell culture media. 2.1.2: Mice Wild type and p53KO mice of the C57/ 36 BL6 strain were maintained in a pathogen f ree environment under the guidance of the University of Washington Institutional Animal Care and Use Committee (IACUC) standards. p53KO mice were developmentally normal, but were prone to develop tumors, as initially characterized by Donehower, et al. (199 2). Mouse pups at postnatal day 3 or 4, determined by their appearance, were collected for the production of microglial cultures.
37 2.1.3: Making Primary Microglial Cultures Mixed glial cultures (astrocytes and microglia) were made from postnatal day 3 or 4, according to established protocols (as in Garden, et al. 2004). The making of cultures is reviewed in the Appendix 2.1.4: Maintaining Mixed Cultures and Harvesting Microglia Culture medium, with 20% L929 conditioned medium in D10C in order to provid e essential growth factors was changed once every 6 8 days for up to one month, after which time the culture flask was discarded. Over the course of 6 8 d, mature microglia would detach from the cell culture flask and become suspended, leaving astrocytes adhered to the flask. These mature microglia were harvested by gently shaking cell culture flasks, and collecting the supernatant. The supernatant was centrifuged at 1100 RPM for 5 min. The resulting pellet was resuspended in D10C so as to achieve a cell d ensity of 50,000 to 200,000 cells/mL. To determine cell density of the resuspension, a random sample of the resuspension was added to a hemacytometer (Bright Line by American Optical) that provided a regimented, reproducible method of hand counting cells. Microglia on the hemacytometer were viewed and counted under a 100X light microscope. Less than 5% of cells viewed at this magnification were a different cell type than microglia, indicating that the cells used in experiments were almost exclusively microg lia.
38 2.1.5: BV 2 Cell C ultures BV 2 cells, an immortal cell line of microglia, were grown in BV 2 medium. Because BV 2 clones proliferate rapidly, cell s were harvested every 2 5 days. See Appendix for details on maintaining and harvesting BV 2 cells 2. 2: Detecting ROS Production in Cells 2.2.1: Detecting ROS Stimulated by Hydrogen Peroxide At a density of 10,000 per well, primary microglia were added to a poly D lysine coated (to make cells adhere) 96 well plate at the time of harvesting. The day after cells were plated, the culture medium was changed to warm macrophage serum free medium (MSFM, Invitrogen). In selected wells, NAC stock was added at this stage (5mM final concentration). Twenty three hours afterwards, DCFH 2 DA stock solution was added to a chieve a final concentration of 50M in each well, unless noted otherwise in Results. Cells were incubated 60 min with DCFH 2 DA, and media were removed from each well. Wells were rinsed once with 50L PBS, and 50L of MSFM was added to each well. Approxima tely one minute following the application of fresh MSFM, cells were imaged in the chamber of an automated plate reader (Packard Fusion Universal Microplate Analyzer) that was heated to 37 o C. DCFH 2 DA fluorescence was measured at 485 + 10 nm excitation and 535 + 13 nm emission for all experiments hereafter. Following this baseline reading, selected cells were stimulated with hydrogen peroxide (500M unless noted otherwise), and the fluorescence was measured periodically afterwards, for a minimum of 90 min.
39 Hydrogen peroxide stimulated ROS in BV 2 cells were detected in a similar manner with the following modifications. BV 2 were plated a t a density of only 5,000 cells per well, because of the rapid proliferation that occurs in these cells within two days. Th e 96 well plates used for BV 2 cells were not coated with poly D lysine. 2.2.2: Detecting ROS Stimulated by Oxygen Glucose Deprivation (OGD) Reperfusion At a density of 10,000 per well, primary microglia were added to two poly D lysine coated, transparen t 96 well plates at the time of harvesting. The day after cells were plated, cell media of both plates were replaced with normoglycemic, serum free media (see Fig. 9 ) In order to pre equilibrate the gas mixture within cell media, an aliquot of fresh normo glycemic medium was placed into a normoxic cell culture chamber for at least 24 h. Likewise, an aliquot of hypoglycemic serum free medium was placed into a hypoxia incubator (ThermoScientific) that automatically injects nitrogen to attain a 1% oxygen, 5% C O 2 and 94% nitrogen environment. Following 24h of serum starvation in normoglycemic media, media in the control plate were replaced with pre equilibrated normoglycemic serum free media. Hypoxic, hypoglycemic serum free media were added to wells in the pla te that will undergo OGD reperfusion. At this point, a subset of the wells from each plate was treated with NA C (5mM). After 23h of normoxia / OGD p lates were incubated for 60 min each with DCFH 2 DA in their respective oxygen environments. T he media were r emoved and the c ells were washed with 50L PBS, then 50L of normoxic, normoglycemic, serum free media were added to the rinsed wells luorescence was recorded by the automated plate reader at 10 minute intervals for 6 0 min and then every 30 minutes thereafter (>120 min
40 total). For the most part, cells were maintained at 37 o C and were protected from ambient light. After detecting fluorescence for at least two hours, half of the wells from each condition were treated wi th H 2 O 2 (500M) under ambient light to test if all of the DCFH 2 DA had been oxidized. Fluorescence was read for at least 1 h following treatment with H 2 O 2 Once all fluorescence imaging was complete, cells were viewed under the light microscope to observe cell morphology. OGD reperfusion stimulated ROS in BV 2 cells were detected in a similar manner with the following modifications. BV 2 were plated a density of only 5,000 cells per well, and the 96 well plates used for BV 2 cells were not coated with poly D lysine. Figure 9 : A schematic of the experimental paradigm that applies to primary microglia and BV 2 cells. The red bar indicates the fluorescence of the 96 well plates is being detected. Not depicted is the induction of ROS by hydrogen peroxide aft er the 2h fluorescence detection period. NAC = N acetylcysteine, DCFH2 dichlorodihydrofluorescein diacetate. 2.2.3: Theory behind DCFH 2 DA ROS Detection: DCFH 2 DA enters the cell via passive transport because it is relatively membrane soluble w ith its acetate moieties. Once inside the cell, DCFH 2 DA has its
41 acetate groups cleaved by intracellular esterases, leaving DCFH 2 with fairly acidic phenolic groups (pKa 7.9 and 9.2) that can become phenolates inside the pH neutral cell environment (Wardma n 2007). Because the DCFH 2 molecule now has a net charge with hydrophilic phenolate groups, it cannot leave the cell via passive transport, and DCFH 2 rem ains inside the cell (see Fig. 10 ) (Halliwell & Whiteman 2004). The applied concentration of DCFH 2 DA wa s 50M in this study, but when this concentration was added to a well without cells, the fluorescence detector was saturated. Therefore, the amount of DCFH 2 that permeated cells is far less than 50M, as DCF fluorescence in cell based experiments, followin g a change of media, was ~5% the fluorescence saturation point. Although thousands of studies have used DCFH 2 DA to detect intracellular reactive oxygen species, it is not certain which ROS oxidize this probe in times of cellular oxidative stress. It is un reactive to superoxide, and is only reactive to hydrogen peroxide in the presence of Fe 2+ by way of the Fenton reaction : Fe (II) + H 2 O 2 OH + OH + Fe (III) Recent research suggests that DCFH 2 oxidation to DCF during cellular stress is a result of tr anslocation of Fe 2+ ions from mitochondria or lysosomes into the cytosol, where Fe 2+ can react with H 2 O 2 (Karlsson, et al. 2010). Studies have also shown that DCFH 2 can be oxidized by heme proteins without the need for reactive oxygen species providing an other mechanism by which Fe 2+ translocation could oxidize DCFH 2 (Ohashi, et al. 2002). H ydroxyl radical s react with DCFH 2 at the rate of diffusion, but this is true for the rea ction of OH with many aromatic molecules, and scavenging OH only decreased DC F fluorescen ce slightly in a purely chemical array (Wardman 2007, LeBel et al. 1992).
42 Figure 10 : A proposed mechanism showing the critical steps that lead to formation dichlorodihydrofluores cein (DCFH 2 DA). ROS = reactive oxygen species, RNS= reactive nitrogen species. Adapted from Halliwell & Whiteman, 2004. In the pr oposed mechanism shown in Fig. 10 a reactive species such as OH would abstract the H at the sp 3 carbon, producing a resonan ce stabilized radical with a singly occupied p orbital. Another species, likely oxygen (Wardman 2007), would oxidize the intermediate radical, allowing DCFH to become DCF. The oxidation of the DCFH radical by O 2 would thus create sup eroxide, which could then be dismutated into H 2 O 2 two of the very same species that DCFH 2 is purported to be detecting (Bonini, et al. 2006). Additionally, wavelengths of light greater than 300nm have been shown to initiate the reduction of DCF back to DCF which can then promote oxidation of DCFH 2
43 (Bilski, et al. 2002). Experimental a rtifacts such as these, which would overestimate DCF fluorescence are expected to occur equally in all conditions of this study. This assumption is valid because the detecti on of DCF fluorescence is occurring in the same media, with equal levels of O 2 and the exposure to light is equal across cell treatment groups. Therefore, comparing amounts of DCF fluorescence between groups can provide a valid measure of relative ROS pro duction, despite the aforementioned artifacts. Figure 10 shows that it is possible that the free radical scavenger used in this study NAC, is scavenging the DCFH radical rather than physiologically relevant radicals and that this is responsible for the i nhibition of fluorescence produced by NAC. However, data show that NAC does not inhibit H2 O 2 stimulated increases in DCF fluorescence (Fig. 1 3 ), suggesting that NAC does not scavenge the DCF radical in this paradigm. 2.3: Gene Transcript Experiments 2.3. 1:Extraction of RNA and Reverse Transcriptase Reaction Primary microglia were plated at a density of 500,000 cells per poly D lysine coated 35 mm culture dish. From one microglia harvest, four such dishes were plated for the following conditions: normoxia normoglycemia, normoxia normoglycemia with 5mM NAC, OGD reperfusion, OGD reperfusion with 5mM NAC. Cells in culture dishes und erwent the same OGD or normoxia normoglycemia paradigm as discussed above for cells in 96 well plates. Reperfusion, however, was d ifferent. Normoglycemic media was added to all dishe s after 24 h of OGD or normoxia normoglycemia. All dishes were then
44 reperfusion period, all cells were lysed. Total RNA w as extracted from all cells using the RNAse free environment. Following isolation, RNA was kept at 80 o C. The concentration of RNA for all four conditions was determined with a range of 10 22 ng/L. The same mass of RNA underwent reverse transcription for each sample. DNA complementary to the isolated RNA was created using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer Bio Rad thermal cycler. This kit utilized random primers for complementary DNA (cDNA) synthesis. 2.3.2: Quantitative Polymerase Chain Reaction (qPCR) The cDNA produced by reverse transcription was used in a quantitative PCR (qPCR) reacti on that probed the expression of mouse genes for interleukin 1Beta (IL 1Beta) and macrophage receptor with collagenous structure (MARCO), with TATA binding protein (TBP) serving as the housekeeping gene. All reactions were loaded in triplicate. The qPCR re action was run using a StepOne qPCR machine and the accompanying software (Applied Biosystems) for 40 cycles. The relative quantity of gene transcripts (RQ) was quantified using the comparative threshold cycle ( C T ) method established by Pfaffl (2001) in St epOne software. R esults are expressed as a fold difference in gen e expression above the normoxia normoglycemia condition. See Appendix for details on reagents and reaction setup. A positive control was used to ensure that the qPCR construct effectively det ermined differences in transcription of TBP, IL 1Beta, and MARCO. Our positive
45 control was cDNA from BV 2 cells treated with lipopolysaccharide (LPS) for 24 h. This treatment is known to induce pro inflammatory microglial activation and transcription of IL 1Beta and MARCO. LPS treated BV 2 cDNA was serially diluted 1:10 to provide concentrations of 1:1, 1:10, 1:100, 1:1000, and 1:10,000. Each dilution was run in duplicate. With the average C T values for each dilution, the efficiency of each qPCR reaction wa s calculated by StepOne software and was used to calculate the RQ value as in Pfaffl (2001). 2.4: Justifying the OGD Reperfusion Paradigm The oxygen glucose deprivation paradigm was previously confirmed by Weinstein, et al. (2010) to upregulate hypoxia in ducible factor 1 (HIF1), a protein that accumulates only in hypoxic conditions. The Weinstein study established that 250M glucose and 1% oxygen for 24 h did not elicit widespread death of primary microglia, but did initiate a molecular response that is kn own to accompany ischemic cell stress. Weinstein, et al. also observed transcriptional changes induced by our paradigm of OGD with reperfusion, many of which involved upregulation of pro inflammatory cytokines (2010). Thus, our paradigm, adopted from Weins tein, is known to activate ischemia associated cell signaling and has been shown to induce pro inflammatory transcriptional changes in microglia. This is particularly interesting when considering that the same paradigm sh owed an induction of p53 (Fig. 8 ), opening the possibility that p53 is responsible for transcription of pro inflammatory genes. The paradigm used by Weinstein was followed exactly in the investigation of gene transcripts, but modifications had to be made for imaging ROS production. This is because a burst of ROS is associated with the
46 initial stages of reperfusion (Fabian, et al. 1995; Yamaguchi, et al. 2002; Watanabe 1998; Peters, et al. 1998), so that ROS had to be imaged at the start of reperfusion, rather than at the end of 24 h of reper fusion. 2.5: Statistics A two way analysis of variance (ANOVA) with repeated measures test was performed in time course studies (fluorescence x time) with a Bonferroni post test. When the fluorescence at one time point was examined, a one way ANOVA with a Tukey post test was utilized. GraphPad Prism software was used for statistical analysis and the plotting of graphs using raw fluorescence data, and Microsoft Excel was used to plot graphs for which data were expressed in percentages or increases. Because no replicates were performed for the gene expression experiment, statistical tests cannot be performed. To compare the results as best we could, the maximum and minimum possible fold changes (range), given the acquired Ct values, were calculated with Step One software (Applied Biosystems), and plotted with Microsoft Excel.
47 Chapter 3: Results 3.1: Characterizing the ROS Detection and Inhibition P aradigm To confirm that the amount of oxidized DCF was proportional to the amount of fluorescence detected by the plate reader, the concentration of incubated DCF was varied from 0 100M and the fluorescence was observed after treatment with 500M H 2 O 2 As the incubation concentration of DCFH 2 was increased in BV 2 cells, the amount of fluorescence above baseli ne increased linearly, as indicated by an R 2 value of 0.9914 (Fig. 1 1 ). To establish that DCF fluorescence was proportional to the concentration of ROS, the amount of H 2 O 2 added to cells was varied from 0 1000M. DCF fluorescence was linearly (R 2 =0.9516) d ependent upon the dose of H 2 O 2 (Fig. 1 1 ). Note that cells not treated with DCFH 2 or H 2 O 2 showed very minimal fluorescence increase over time; both were required for a substantial increase in fluorescence to be detected. These linear relationships recapitul ate characteristics of DCFH 2 in previous studies (LeBel, et al. 1992). These data support the reaction mechanism for the oxidation of DCFH 2 (Fig. 10 ) is valid, as the reaction between ROS and DCFH 2 is first order with respect to each molecule. To investiga te the lag time between the introduction of ROS and the detection of ROS by DCFH 2 fluorescence readings were taken periodically following treatment with H 2 O 2 Fluorescence values plateau at 90 min, ind icating that production of ROS has leveled off by this time (Fig, 1 2 ). Cells n ot treated with DCFH 2 DA fluoresced at the same levels as cell media alone (~4000 RFU, Fig. 12). This is likely due to phenol red or other components in the cell m edia that may exhibit fluorescence.
48 Figure 1 1 : BV 2 cells, plated at a density of 5000/well, were grown for two days, and then their response to H 2 O 2 was investigated in the presence or absence of DCFH 2 Average fluorescence values before H 2 O 2 treatment were subtracted from a verage fluorescence values 90 min after H 2 O 2 treatment (n=6 wells/condition). Top: BV 2 cells treated with 0, 10, 50, or 100 M DCFH 2 for 60 min were exposed to 500 M H 2 O 2 Bottom: BV 2 cells treated with 50 M DCFH 2 were exposed to 0, 250, 500, or 1000 M of H 2 O 2 R = 0.9914 0 1000 2000 3000 4000 5000 6000 7000 0 10 20 30 40 50 60 70 80 90 100 Average fluorescence increase over baseline after 90min H2O2 (RFU) [DCF] in uM [DCFH 2 ] Varied, 500 M H 2 O 2 90 min [DCF] Varied, n=6 wells each R = 0.9516 0 1000 2000 3000 4000 5000 6000 0 250 500 750 1000 Average fluorescence increase over baseline after 90min H2O2 (RFU) [H 2 O 2 ] in uM [H 2 O 2 ] Varied, with 50 M DCFH 2, 90 min [H2O2] Varied, n=6 wells each
49 Figure 1 2 : Time course of fluorescence increase. Average raw fluorescence values (n=6/condition) of BV 2 cells were plotted over time, with the baseline reading being 0 min. Because DCF has been reported to be mildly cytotoxic, most studies use DCFH 2 concentrations below 100M (Wardman 2007). As seen in Figs. 1 1 and 1 2 incubation with 50M, but not 10M of DCFH 2 detected a substantial increase in DCF fluorescence upon treatment with H 2 O 2 Therefore, cells were incubated in 50M DCFH 2 for th e remaining experiments. To confirm that our ROS detection assay was effective in primary microglia, harvested microglia were subjected to DCFH 2 incubation and H 2 O 2 treatment. We also wanted to test the ability of N acetylcysteine (NAC) to block the H 2 O 2 i nduced increase in DCF fluorescence. NAC is reported to scavenge ROS itself (Benrahmoune, et al. 2000), and is reported to be metabolized over time to the endogenous antioxidant glutathione, which will also scavenge ROS (Banks & Stipanuk 1994). However, NA C did not significantly inhibit H 2 O 2 induced increase in fluorescence 0 2000 4000 6000 8000 10000 12000 14000 0 50 100 150 200 Fluorescence (RFU) Time (min. after H 2 O 2 addition) Increase in Fluorescence Over Time 100uM DCF, 500uM H2O2 50uM DCF, 500uM H2O2 50uM DCF, 1000uM H2O2 50uM DCF, no H2O2 10uM DCF, 500uM H2O2 MSFM only Cells with no DCF, 1000uM H2O2
50 in primary microglia when incubated for 1h or 24h prior to H 2 O 2 treatment (Fig. 1 3 ). This could be because the H 2 O 2 insult overpowered NAC Lower doses of H 2 O 2 were not robust enough to induce a significant change in fluorescence, and 1 0mM doses of NAC were harmful to cells. Experiments were thus m oved t o OGD reperfusion with the hope that NAC could inhibit production of a milder, intracellular form of ROS. Figure 1 3 : Hydrogen peroxide (500M) treated cells showed a significantly higher DCF fluorescen ce when compared to cells treated with DCF and not H 2 O 2 ( indicates significant difference from cells treated with only DCF p<0.05). NAC ( 5mM, 1 or 24 h incubation) did not make a significant difference when compared to H 2 O 2 treatment without NAC. The flu orescence of each well at 90 min after treatment was divided by the baseline value for that same well, giving each individual well a percentage of baseline. The percent of baseline values of the same treatment condition were averaged together and the avera ges are shown above (n=6 wells/condition). Error bars are + S.D. 0 20 40 60 80 100 120 140 160 180 200 Fluorescence 90 min after H2O2 Treatment/Baseline (%) H 2 O 2 Induced DCF Fluorescence in Primary Microglia No Treatment DCF DCF + H2O2 DCF + H2O2 + NAC 1h DCF + H2O2 + NAC 24h
51 3.2: Oxygen Glucose Deprivation Reperfusion Experiments 3.2.1: BV 2 Cells A fter confirming that the experimental paradigm c an detect ROS BV 2 cells were subjected to 24 h oxygen glucose deprivation (OGD) a nd their production of ROS was detected via DCF fluorescence for the first 3 h of reperfusion. Cells subjected to OGD reperfusion showed significantly (p<0.05) higher DCF fluorescence than normoxia normoglycemia (control) cells, starting at 10 min after re perfusion (Fig. 1 4 ). NAC treatment (5mM) for 1 h or 24 h abrogated DCF fluorescence for both conditions, confirming that ROS were the cause of the increase in fluorescence that occurred, and showing that NAC is an effective inhibitor of endogenous ROS prod uction. The kinetics of DCF fluorescence increase appear very similar to what was seen with acute H 2 O 2 application (as in Fig. 1 2 ). The amount of DCF fluorescence is dependent upon the number of cells within each well. Therefore, following reperfusion, cel ls were observed under light microscope to examine differences in cell density. When viewing the cells subjected to OGD reperfusion, there were substantially fewer cells than seen in the control conditions. No differences in cell density were observed with in the conditions of the OGD reperfusion or control plates. Even with much fewer cells, the OGD reperfusion condition produced more ROS than normoxia normoglycemia controls. A high amount of cells along with the volatile, constit utively activated nature of the BV 2 cell line likely contributed to the high amount of ROS production that is seen in d ata collected from the Control condition.
52 Figure 1 4 : BV 2 cells were subjected to OGD reperfus ion (labeled OGD) or equivalent periods of normoxia normoglycemia (labeled control) DCFH 2 was oxidized to DCF to a significantly greater extent in the OGD reperfusion condition, suggesting higher amounts of ROS are produced in these cells. No significant differences between any NAC treated conditions. Error bars are + S.D. (n= 6 wells/condition). 3.2.2: Primary Microglia The results from BV 2 cells were recapitulated in primar y microglia. Cells subjected to OGD reperfusion showed a significantly higher amount of ROS production during reperfusion, beginning a t 20 min of r eperfusion (Fig. 1 5 ). All ROS were inhibited
53 Figure 1 5 : In primary microglia, OGD reperfusion elicits a significantly higher amount of ROS production than normoxia nor moglycemia, beginning at 20 min of reperfusion (p< 0.05). NAC treatment for 24 h abrogates ROS production; there is no significant difference between OGD reperfusion and control conditions treated with NAC. N=6 wells/condition. Error bars + S.D. by 24 h NAC treatment. Because ROS are known to be downstream of p53, we investigated if the presence of p53 influenced the amount of ROS production. Wild type and p53KO cells showed no significant differences in ROS production for OGD reperfusion or control conditions, and the effects of NAC were not genotype dependent (Fig. 1 6 ). Cells treated with NAC and DCFH 2 showed fluorescence values very similar to cells not exposed to DCFH 2 (data not sho wn for clarity), representing suppression of DCFH 2 oxidation and presumably ROS production -by NAC.
54 F igure 1 6 : Deficiency in p53 does not affect the production of ROS following OG D reperfusion (top) or normoxia normoglycemia reperfusion (bottom), nor does it affect the ability of NAC to inhibit ROS (n=6 wells/condition, p> 0.05).
55 Figure 1 7 : The data shown in Fig 1 5 and 1 6 are shown here at 90 min after reperfusion to highlight differences between groups. There is a statistically significant difference between OGD and normoxia normoglycemia treated cells (marked by #). Pre treatment with 5mM NAC for 2 4h significantly suppressed DCF fluorescence in all conditions (p < 0.05). p53 knockout (p53KO) cells showed no significant differences from their wild type counterparts. N=6 wells/condition, error bars are + S.D. In order to investigate why DCF fluorescence stopped increasing at approximately 60 min, one half of the wells for each c ondition was treated with 500 M H 2 O 2 after 180 min of reperfusion. After 1h, H 2 O 2 treated cells showed a noticeable but statistically insignificant increase in fluorescence while n ontreated cells did not (Fig. 1 8 ). This
56 suggests that the cessation of DCF fluorescence increase occurred because ROS are no longer being produced, and not that all DCFH 2 had been oxidized to DCF. Following reperfusion and treatment wit h H 2 O 2 cells were viewed under the light microscope. Unlike in BV 2 cells, there were no observable differences in cell density between any of the conditions. B esides o bvious H 2 O 2 induced damage to cells there were no other detectable differenc es in morphology between any of the groups. Figure 1 8 : Following 150 min of reperfusion, one h alf of the wells shown in Fig 1 5 1 7 were treated with 500 M H 2 O 2 There is a visible, but statistically insignificant increase in fluorescence only i n cells treated with H 2 O 2 (n=3, p> 0.05). Error bars + S.D.
57 3.3: Effects of ROS on Transcription of IL 1Beta and MARCO 3.3.1: Qualitative Observations of Cells Prior to RNA Extraction The OGD reperfusion experimental paradigm utilized in this study had be en previously shown to elicit increases in transcription of pro inflammatory cytokines (Weinstein, et al. 2010), but the role of ROS was not investigated. Therefore, we next investigated the effects that ROS generated in our OGD reperfusion paradigm might have on pro inflammatory gene transcription. Cells were subjected to the same OGD paradigm as in DCF assays, but with a 24 h reperfusion period. When viewed under the light microscope, the normoxia normoglycemia condition showed ramified, elongated process es throughout the entire experiment. The OGD cells were elongated and ramified after OGD and before reperfusion, indicating that OGD did not elicit microglial activation. However, after 24h reperfusion, cells adopted an amoeboid phenotype with stout proce sses and enlarged cell bodies, indicating activation of these microglia. Fewer cells were adhered to the culture dish th an cells in the normoxia normoglycemia condition, and OGD reperfusion dishes had clusters of unidentified floating detritus. In contrast OGD reperfusion cells treated with NAC showed the same morphology as controls. Thus, in these culture dishes, the amount of ROS exposure affected the morphology of the microglia. 3.3.2: qPCR Reactions To validate t he qPCR construct, cDNA from a positiv e control, BV 2 cells treated with L PS for 24 h, was reacted with probes for TATA binding protein (TBP) the housekeeping gene and our genes of interest (IL 1Bet a and MARCO). These genes were
58 chosen because they are well known markers of M1 activation and have been shown to be transcribed in a p53 dependent man ner in microglia exposed to LPS and IFN gamma which will be relevant for future studies (Jayadev, et al. 2011). As seen in Fig. 1 9 the threshold cycle (C T ) value was responsive t o the amount of cDNA present in the reaction well (R 2 >0.99). Our qPCR construct was able to effectively discern differences in gene expression levels for all of our genes studied, and differences in gene expression did not appear to be due to artifacts of the qPCR reaction. Figure 1 9 : The average threshold cycle (C T ) value (n=2 per dilution) for each dilution of positive control cDNA was plotted against the log 10 of the BV 2 cDNA dilution factor. These data show that for each gene of interest, the qPCR output is lo garithmically proportional to the amount of gene transcript present in the reaction well, as is expected for qPCR reactions. Please s ee Appendix for slopes The increases in gene expr ession relative to the normoxia normoglycemia condition (control) show that OGD reperfusion induced a 1521 fold average increase in IL1 Beta transcript, with a range of 244 fold (control C T =28.2) When treated with 5mM R = 0.9967 R = 0.9995 R = 0.9999 0 5 10 15 20 25 30 35 40 -5 -4 -3 -2 -1 0 Threshold Cycle log of BV 2 cDNA dilution qPCR of Positive Control cDNA TBP MARCO IL-1Beta
59 NAC just p rior to OGD, IL 1Beta transcript was markedly red uced to 6.4 fold above normoxia normoglycemia controls ( range = 3.0 fold). The normoxia normoglycemia condition treated with NAC showed an average 1.6 fold increase over control (range= 0.3 fold). All OGD reperfusion conditions therefore showed an increase in IL 1Beta gene transcription that extended beyond the statistical range of the contro l (0.5 fold) but the range of the normoxia normoglycemia with NAC condition overlapped with the range of the control indicating that NAC did not itself have an effec t on gene transcription (Fig. 20 ). Figure 20 : IL 1Beta transcript averages (n=3) relativ e to normoxia normoglycemia control. The OGD reperfusion without NAC condition is not shown here because its transcript levels were at approximately 1500 fold above control. The top of the error bar represents the maximum fold change e xpected using the e xperimental C T values, and the bottom of the error bar represents the minimum e xpected value. Average gene expression differences of another M1 activation marker, macrophage receptor with collagenous structure (MARCO), followed the same trends as 0 1 2 3 4 5 6 7 8 9 Fold Difference Above Control IL 1Beta Gene Expression Control Normoxia/Normoglycemia + NAC OGD-Reperfusion + NAC
60 IL 1Beta, but with much less robust differences between conditions. Additionally, the C T values obtained were fairly scattered, leading to the large ran ges seen in Figure 2 1 Because gene expression values are relative to the control, perhaps the large range of the control value s overshadowed a more substantial difference that might exist between cells subjected to OGD reperfusion with and without NAC. Re lative to OGD reperfusion with NAC, OGD reperfusion without NAC showed a fold increase average of 2.7 with a range of 0.8 to 5.5, indicating that the variability of C T values is too high to declare that NAC inhibits transcription of MARCO. Figure 2 1 : MA RCO transcript aver ages (n=3) relative to normoxia normoglycemia control MARCO expression. The top of the error bar represents the maximum fold change calculable using the experimental C T values, and the bottom of the error bar represents the minimum value The ranges overlap when comparing the control to all conditions.The averages do show the same trends as what was observed for IL 1Beta, however. 0 50 100 150 200 250 300 350 400 450 Fold Difference Above Control MARCO Gene Expression Control Normoxia/Normoglycemia + NAC OGD-Reperfusion OGD-Reperfusion + NAC
61 Chapter 4: Discussion 4.1: Restatement of Goals and Findings The goal of this thesis was to determine i f ROS contribute to pro inflammatory transcriptional changes in microglia subjected to oxygen glucose deprivation (OGD) with reperfusion. A component of this goal was to discern if ROS are possibly upstream and/or downstream of the activation of p53 that w as previously observed in an identic al experimental paradigm (Fig. 8 ). The results of this study show that OGD with reperfusion increased DCF fluoresce nce ~30% more than did normoxia normoglycemia controls in BV 2 cells (Fig. 1 4 ) and primary microglia (Fig 1 7 ). These increases in DCF fluorescence were abrogated by incubation of cells with NAC, an established ROS scavenger, confirming that production of ROS was reponsible for increases in DCF fluorescence. p53KO and wild type microglia showed no significant difference in ROS production in response to OGD reperfusion, indicating that ROS are likely not downstream of p53 action in this paradigm. Finally, it was shown that ROS induced by OGD reperfusion were largely responsible for a substantial (~1500 fold) in crease in transcription of the pro inflamm atory cytokine IL 1Beta (Fig. 20 ). These findings give credence to the hypothesis that ROS generated by OGD reperfusion activate p53, which then promotes transcription of pro inflammatory genes in microglia. 4.2: Direct Evidence of ROS Production in Microglia Following OGD Reperfusion Our finding that OGD reperfusion in microglia elicits a burst of ROS is in accordance with a corpus of literature that has established ischemia reperfusion as an inducer of a temporar y burst of ROS in the brain (Peters, et al. 1998). Spranger, et al.
62 (1998) incubated microglia in hypoxia for 24 h without hypoglycemia, and found that reperfusion was necessary for a burst of ROS from cultured microglia. However, in their paradigm, micro glia in all conditions were stimulated with phorbol 12 myristate 13 acetate (PMA), an activator of the superoxide generating enzyme NADPH oxidase (Spranger, et al. 1998). Their results do not show the effects of hypoxia reoxygenation without PMA stimulatio n. Additionally, hypoglycemia in combination with hypoxia has vastly different effects on microglia than hypoxia alone (Lyons & Kettenmann, 1998, described below). Thus, our finding that OGD reperfusion produces ROS is corroborated by findings from Sprange r, et al. (1998), but consideration must be given to the differences in experimental paradigms. There is other evidence that hypoxia reoxygenation induces ROS production in microglia, though ROS were never directly detected in these studies. Following 4h hypoxia and 24 h reoxygenation, Lai and Todd (2006) observed substantial increases in microglial activation and production of inducible nitric oxide synthase (iNOS) and IL 1Beta. There are two lines of evidence that implicate ROS in this observed response: iNOS and IL 1Beta are known to be downstream of ROS (Roy, et al. 2008; Cheret, et al. 2008), and the application of phenolic radical scavengers abrogated the increases in expression that were induced by hypoxia reoxygenation (Lai & Todd 2006; Kraus, et al. 2005). In another study, upregulation of FasL, ligand to the Fas death receptor, was observed following both hypoxia reoxygenation and application of H 2 O 2 implicating ROS as the mediating factor in the induction of FasL by hypoxia reoxygenation (Vogt, e t al. 1998). It is unknown if the combination of hypoglycemia and hypoxia will affect ROS differently than hypoxia alone. This uncertainty again makes it difficult to interpret the
63 results of this thesis in terms of previous work, especially considering th e vastly different effects between hypoxia alone and hypoxia with hypoglycemia. One study observed little cell death in microglia exposed to 6h or 42h hypoxia (0.3%) with reperfusion, but over 80% of microglia died when glucose was replaced by 2 deoxygluco se during 6h of hypoxia (Lyons & Kettenmann, 1998). Interestingly, when mannitol was used as a replacement for glucose rather than 2 deoxyglucose, approximately only 50% of cells died during this insult (Lyons & Kettenmann, 1998). The authors attribute thi s increased survival to the radical scavenging ability of mannitol, suggesting that the combination of hypoxia with aglycemia is more likely to produce cytotoxic ROS than hypoxia alone (Lyons & Kettenmann, 1998). Our paradigm did not produce cell death dur ing hypoxia and hypoglycemia, possibly because both the hypoxic and hypoglycemic insults were less severe in our experimental paradigm as compared to that of Lyons and Kettenmann (1998). However, we did observe that OGD with reperfusion elicited cell death in culture dishes, and that this was inhibitable by NAC, recapitulating aspects of the Lyons and Kettenmann study (1998). 4.3: ROS Influenced Gene Transcription Greatly The increase in ROS production that we observed following OGD reperfusion was follow ed by an approximately 1500 fold increase in the transcription of IL 1Beta, which was greatly inhibited by incubation with NAC. This result suggests that ROS are vital in the upregulation of pro inflammatory factors in ischemia reperfusion. Our results the refore highlight the two pronged mechanism by which microglial ROS can be harmful to surrounding cells: ROS generated by microglia can directly damage nearby cells via
64 peroxidation of lipid membranes or other mechanisms, and ROS can promote transcription o f pro inflammatory cytokines. The direct cytotoxic action of ROS is illustrated by the lipid peroxidation and oxidative DNA damage that were observed following ischemia reperfusion, both of which were prevented with a phagocytic NADPH oxidase (PHOX) inhibi tor (Wang, et al. 2006; Chen, et al. 2009). Indirect cytotoxicity of ROS is exemplified by PHOX dependent upregulation of IL 1Beta and a number of other pro inflammatory cytokines and chemokines such as intercellular adhesion molecule 1 (Genovese, et al. 2 011; Chen, et al. 2011; Chen, et al. 2009). Because PHOX is more highly expressed in microglia than other brain cell types (Kahles, et al. 2010), it can be inferred that it is microglial PHOX, and therefore microglial ROS, that are predominately responsibl e for the increase in oxidative damage and pro inflammatory transcription. However, some of the effects of PHOX inhibition or gene knockout could be due to events occurring in astrocytes or peripheral immune cells that infiltrated the brain (Green, et al. 2001). A study in BV 2 cultures provides microglia specific data that also link ROS, pro inflammatory cytokine transcription, and cytotoxicity. Hur, et al. (2010) found that chemical simulation of ischemia elicited increases in ROS, iNOS, and IL 1Beta pro duction in BV 2 cells. Extensive neuron death was observed when cultured neurons were incubated with the media in which the BV 2 cells underwent ischemia (Hur, et al. 2010). Small inhibitory RNA (siRNA) knockdown of PHOX abrogated the neurotoxicity of the microglia conditioned media (Hur, et al. 2010). This result suggests that PHOX induced ROS during ischemia were responsible for the neurotoxicity of ischemia treated microglia.
65 However, Hur, et al. (2010) did not prove that ROS or cytokine production were reduced following siRNA knockdown. This thesis contributes to the literature described above by directly showing for the first time that primary microglia produce ROS in a model of ischemia reperfusion, and that this ROS production can be critical for tra nscription of pro inflammatory genes. This study therefore strengthens the possibility of a two pronged mechanism by which ROS can contribute to cytotoxic actions of microglia in ischemia reperfusion. However, this study does not provide evidence that ROS or ROS induced changes in transcription that we observed following OGD reperfusion are cytotoxic to neighboring cells. It would be of great interest to investigate the effects of NAC pre treatment on the neurotoxicity of the media in which microglia were s ubjected to OGD reperfusion. 4.4: Attempting to Localize the Source of Detected ROS The production of both intracellular and extracellular ROS is central to the idea of a two pronged mechanism of microglia mediated neurotoxicity. Extracellular ROS would directly damage nearby cells, while intracellular ROS signaling would promote transcription of pro inflammatory factors (Chen, et al. 2011). Our ROS detection paradigm operates on intracellular DCF, but this does not ensure that ROS is produced intracellul arly. Indeed, Fig. 1 1 shows that application of H 2 O 2 elicited an increase in DCF fluorescence, indicating that extracellular sources of ROS will be detected by this intracellular probe. Although DCF fluorescence was not specific to extra or intra cellular sources of ROS, the antioxidant NAC provides evidence that OGD reperfusion elicited intracellular ROS. Incubation of NAC for 24 h did not reduce fluorescence induced by
66 exogenous H 2 O 2 suggesting that NAC incubation does not reduce DCF fluorescence caused by extracellular ROS. Incubation with NAC, which must be intracellular because of cell media changes, did reduce fluorescence induced by OGD reperfusion, implicating an intracellular mechanism of ROS production. However, several other studies have investi gated ROS production with intracellular probes like DCF in activated microglia, and have found that application of extracellular ROS scavengers greatly reduced the downstream effects of ROS (Roy, et al. 2008; Pawate, et al. 2004). Our data support intracel luar ROS production, but there is only suggestive evidence to exclude extracellular sources. Mitochondria are widely implicated as an intracellular source of ROS following ischemia reperfusion (Piantadosi & Zhang 1996; Chan 2001). One study showed that tr eating microglia with the hypoximimetic drug sodium azide increased mitochondrial ROS production, but literature is otherwise scarce (Park, et al. 1999). Given that drastic changes in intracellular Ca 2+ occur in microglia following hypoxia reoxygenation (S pranger, et al. 1998), and that Ca 2+ initiates mitochondrial ROS in ischemia of other cell types (Dong, et al. 2006), one might expect mitochondrial ROS to contribute to the intracellular ROS that we observed following OGD reperfusion. Our finding that p53 KO mice elicited similar amounts of ROS as did wild type cells somewhat discourages the possibility that the ROS we observed are derived from mitochondria. P53 negatively regulates mitochondrial SOD (Holley, et al. 2010), promotes ROS formation by protein protein interactions at the mitochondrial membrane (Endo, et al. 2006), and transcribes proteins that promote ROS at the mitochondrial membrane (Culmsee & Mattson, 2005). In light of our findings in p53KO microglia, and the numerous studies that implicate
67 different forms of NADPH oxidase in ischemia induced ROS and inflammation (Spranger, et al. 1998; Chen, et al. 2009, 2011; Hur, et al. 2010), it seems most plausible that the ROS we observed were derived from the NADPH oxidase enzyme. Studies using NADPH o xidase or mitochondria specific inhibitors would greatly help in elucidating the source of ROS in microglia following OGD reperfusion. Knowing the source of ROS in microglia would enable therapies to be designed that could abrogate the harmful direct and i ndirect effects of ROS signaling in microglia. This is clinically critical because NADPH oxidase is a critical mediator of vasodilation in cerebral arteries, and a non cell specific inhibition of NADPH oxidase could have detrimental effects on stroke outco me (Paravicini, et al. 2004). 4.5: Qualitative Observations about OGD Reperfusion Effects on Morphology Qualitative observations are in support of our quantiative results. Incubating cells with NAC during OGD prevented the widespread activation of microgl ia that occurred after reperfusion, suggesting that it is the formation of ROS that leads to microglial activation in this paradigm. Changes in microglial activation were only observable in response to OGD reperfusion when cells were cultured in a 35 mm cu lture dish, and not in a 96 well plate. This is possibly because cells in the 35 mm dish were plated at 1.6x higher density than in the 96 well plate (for the sake of extracting sufficient amounts of RNA), and the effects of pro inflammatory intercellular signalling were magnified as a result. The higher cell density in 35 mm culture dishes as compared to 96 well plates may also help to explain how a seemingly minor ~ 30% increase in ROS production following OGD reperfusion elicited a ~1500 fold increase in IL 1Beta gene transcription in the
68 present study. In light of previous studies, it is very plausible that an increase in cell density is responsible for amplifying microglial activation. Through cytokine signaling and the release of ROS, activated microgl ia are known to promote activation of nearby microglia, causing a positive feedback loop that amounts to widespread activation (Block and Hong, 2005). Results in our study provide evidence of ROS and IL 1Beta production in microglia exposed to OGD reperfus ion, supporting the possibility that a positive feedback loop of microglial activation exists in our cell cultures. With a higher density of cells, the effects of such a pro inflammatory cascade are expected to be amplified because this will equate to a hi gher concentration of activating signals such as ROS or IL 1Beta. Thus, differences in cell density might explain how a minor increase in ROS production could plausibly lead to a 1500 fold increase in transcription of IL 1Beta. Cell density differences may also explain why very distinct, NAC inhibitable morphological differences were only observed in microglia subjected to OGD reperfusion in 35 mm dishes, and not in 96 well plates. 4.6: Support for ROS as Initiators of a Pro Inflammatory Response The dras tic effects of NAC on morphology and IL 1Beta transcription fo llowing OGD reperfu sion (Fig. 20 ) are in accordance with the hypothesis that ROS are initiators of pro inflammatory microglial activation (Block and Hong, 2005; Block, et al. 2007). There is a cadre of supporting evidence for this hypothesis in other stimulation paradigms. The production of ROS was the first detectable event after stimulating dependent transcription of pro inflammatory cyto kines and subsequent neurotoxicity (Qin, et al. 2004; Gao, et al.
69 2002; Pawate, et al. 2004; Kang, et al. 2001). By inhibiting ROS in these studies, microglial activation was greatly suppressed, as was transcription of pro inflammatory cytokines such as IL 1Beta and TNF Considering that these cytokines are important signals for activating nearby microglia, one could assert that inhibiting ROS will prevent the positive feedback loop of microglial activation from ev er occurring, and a severe reduction in pro inflammatory activity is expected. The drastic effects we observed following incubation with NAC corroborate these previous studies and extend the role of ROS as an initiator of microglial activation to the conte xt of isc h emia reperfusion. 4.7: Quantitative Differences between In Vitro and In Vivo Results of ischemia reperfusion in vivo are qualitatively similar to those of this thesis, but the studies presented here are quantitatively very different. After 60 mi n MCAO, and 1 d of reperfusion, IL 1Beta transcription was increased by 20 fold in MCA territory; PHOX KO mice showed an 8 fold increase (Chen, et al. 2011). Our study showed an approximately 1500 fold increase in IL 1Beta transcription, brought to about 6 fold when cells were incubated with NAC (Fig. 20 ). Firstly, more biological replicates are needed to confirm these drastic changes, but even the lowest of three values we attained showed over a 1200 fold increase in transcription. If this finding is indee d replicated in our paradigm, then the greater severity of the insult in our paradigm may be responsible for the large differences between our results and those found in vivo MCAO was found to bring O 2 in the ischemic core down to about 4 % of pre ischemi c values, rising to near pre ischemic levels upon reperfusion (Liu, et al. 2004). Our paradigm
70 utilized 1% partial pressure O 2 for hypoxia, which is also about 4% of the pre ischemic value (21%, atmospheric). Glucose levels have been shown to decrease by a factor of 10 during ischemia in vivo (Langemann, et al. 1995), but our paradigm reduced glucose levels by a factor of 100. The greater effects of NAC on IL 1Beta transcription in our paradigm, as compared to those seen by knocking out PHOX, are likely a r esult of the total abrogation of ROS by NAC that we observed, whereas knocking out PHOX allows ROS production from other sources (Chen, et al. 2011). Another likely contributor to the inflated effects of OGD reperfusion that we observed is the greater dens ity of microglia in culture than what is seen in vivo (Gwenn Garden, personal communication). Additionally, our cultures did not contain neurons or astrocytes, cell types that are known to inhibit pro inflammatory signaling (Block, et al. 2007). Thus, our in vitro paradigm is somewhat of clinical relevance in that it simulates the lack of oxygen and glucose that would be experienced by microglia in the ischemic core in vivo Future attempts should be made to model the penumbra in culture, where microglia mo stly accumulate in stroke patients (Price, et al. 2006). The lack of other cell types in culture, the high microglial density, and the severe insult likely conspire to amplify effects that are qualitatively similar in vivo (Chen, et al. 2011). 4.8: Equiv ocal Results about MARCO Transcription Although we saw an unequivocal increase in the transcription of IL 1Beta, our qPCR results were less definitive about MARCO (Fig. 2 1 ). MARCO is an inducible cell surface scavenger receptor that detects invading pathog ens (Milne, et al. 2005). MARCO initiates differentiation of microglia into antigen presenting cells and is therefore a
71 marker of a pro inflammatory response (Milne, et al. 2005). Interestingly, although qPCR detected that MARCO mRNA is upregulated in resp onse to 24 h permanent MCAO, transient MCAO of shorter duration followed by reperfusion showed no induction of MARCO mRNA (Milne, et al. 2005). In our study, qPCR reactions conducted in triplicate showed increases in average MARCO gene expression following OGD reperfusion and OGD reperfusion with NAC as compared to normoxia normoglycemia controls (Fig. 2 2 ). This shows the same trend as IL 1Beta, and is in accordance with previous findings showing that ischemia induces MARCO mRNA production (Milne, et al. 20 05). However, the threshold cycle values produced by the qPCR reactions were inconsistent, and the validity of these results is questionable. The poor precision suggests experimental errors rather than a lack of MARCO induction following OGD reperfusion. 4.9: Implications for p53 Garden, et al. showed that transcriptional activity of p53 is increased following OGD reperf usion, but not OGD alone (Fig. 8 unpublished results). The work of this thesis showed that ROS are produced following OGD reperfusion, a nd that p53 is not required for this inc rease in ROS production (Fig. 1 7 ). These findings together support the hypothesis that ROS are the cause of the observed p53 induction. However, Hur, et al. (2010) found an increase in ROS production during chemicall y simulated ischemia in microglia, without the requirement for reperfusion. If the increase in p53 activity is to be attributed to microglial ROS, it is a wonder why p53 activity was not also increased during OGD, given the findings by Hur, et al (2010). O ther studies in whole brain have also found increased ROS production during ischemia without reperfusion (Peters, et al.
72 1998; Fabian, et al. 1995; Solenski, et al. 1997). Additionally, hypoxia without reoxygenation has been shown to elicit p53 accumulatio n in neuron cultures (Banasiak & Haddad, 1998), and MCAO without reperfusion revealed a p53 dependent mode of cell death (Crumrine, et al., 1994). Because p53 activity is highly specific to the extent of oxidative stress, it is possible that, in our paradi gm, a threshold of oxidative stress was not reached until after reperfusion. In response to mild oxidative stress, p53 will transcribe stress response genes that assist in protecting against damage; severe insults instead elicit transcription of pro oxidan ts Bax and PUMA (Sablina, et al. 2005). The reporter assay that revealed p53 transactivation following our OGD reperfusion paradigm utilizes a promoter for PUMA to drive transcription of luciferase, indicating that the assay is only capable of detecting p5 3 transcription of high stress genes. However, if p53 transcribed antioxidant enzymes as in Sablina, et al. (2005), one would expect an increase in ROS production in p53KO cells which we did not find (Fig. 1 7 ). Thus, because of the pro or anti oxidant r oles that p53 plays in controlling oxidative stress, one might have expected either higher or lower ROS produced by p53KO cells if p53 had accumulated in wild type cells by the end of OGD. Our finding that ROS production did not depend on the presence of p 53 supports the idea that p53 is not present at significant levels during OGD, and that reperfusion is required for its activation. Because p53 plays a significant role in classical microglial activation and therefore neurotoxicity (Garden, et al. 2004; Ja yadev, et al. 2011), future studies should be conducted to elucidate the mechanisms responsible for p53 activation in microglia. In proving that ROS activate p53 in microglia, it would be helpful to show that our paradigm of OGD alone does not produce as m uch ROS as OGD reperfusion, but for our assay this would require a
73 hypoxic environment in the fluorescence microplate reader. Future studies should also investigate ROS levels after reperfusion, when p53 is known to have accumulated in microglia (Fig. 8) to test i f ROS are downstream of p53 activity. It would be interesting to see if p53 generate ROS that serve to sustain p53 activation and pro inflammatory action. The increase in ROS, p53 transcriptional activity, and pro inflammatory transcription following OGD r eperfusion lead to the attractive hypothesis that p53 is involved in the neurotoxic inflammatory response commonly observed following stroke. This hypothesis is strengthened by previous studies. It was found that p53 was required for the upregulation of IL MCAO (Jayadev, et al. 2011). Another study showed significant neurological improvements in rats treated with a p53 specific inhibitor 6 9 days after the start of reperfusion (Luo, et al. 2009). The authors attributed this improvement to an increase in neurogenesis in the p53 inhibited condition (Luo, et al. 2009). Considering that neurogenesis is believed to be hindere d by classically activated microglia, but promoted by alternatively activated microglia (Ekdahl, et al. 2009), the inhibition of p53 may have inhibited pro inflammatory microglial activity, decreasing inflammation and offering a facilitative environment fo r neurogenesis (Luo, et al. 2009). Many other studies have found protective benefits of p53 inhibition or knockout, but none have investigated the contribution made by microglial p53 to ischemia induced damage (Culmsee, et al. 2001; Crumrine, et al. 1994; Endo, et al. 2006; Leker, et al. 2004). These studies typically show a reduction in neuronal apoptosis resulting from p53 inhibition, but inflammatory
74 cytokines like IL 1Beta have been shown to promote apoptosis in neurons (Holmin & Mathiesen, 2000). This means that the inhibition of p53 during stroke can reduce extrinsic apoptosis by reducing inflammation and activation of death receptors, and p53 inhibition can inhibit intrinsic apoptosis because of the actions of p53 at the mitochondrial membrane. The w ork presented in this thesis is therefore consistent with findings from previous studies that show p53 exacerbates ischemia reperfusion injury, though the role of p53 in microglia in ischemia reperfusion has not been specifically investigated. Results pres ented here provide clues for a mechanism of microglia mediated neuroprotection following p53 inhibition, but it is still unclear exactly how to connect the increases in ROS, p53 activity, and transcription of pro inflammatory genes that we observed. 4.10: Conclusion Although many studies reference microglial capacity for ROS production in the context of ischemia reperfusion brain injury, there has been very little direct evidence that ischemia reperfusion causes an increase in ROS production in microglia. This thesis directly showed that microglia do indeed increase their production of ROS following an in vitro paradigm of ischemia reperfusion named oxygen glucose deprivation reperfusion (Fig. 1 7 ). The detected ROS were intracellular, but the sources of ROS remain to be elucidated. These intracellular ROS had downstream effects on pro inflammatory gene transcription, eliciting an approximately 15 00 fold increase above normoxia normoglycemia controls in IL 1Beta gene expression, as determined by qPCR. Inhibit ing ROS with the radical scavenger NAC reduced IL 1Beta transcription to approximately 6
75 fold, providing further evidence that ROS were responsible for the increase in IL 1Beta transcript observed. Gene expression of MARCO showed similar trends, but these experiments must be replicated before making conclusions on the effects of OGD reperfusion on MA RCO transcription (Fig. 2 1 ). Thus, this study provides evidence that the ROS produced by OGD reperfusion can create a cytotoxic environment through direct actio n as highly reactive oxidants, and through indirect action as inducers of pro inflammatory transcription. P53 is a critical mediator of the pro inflammatory phenotype in microglia (Garden, et al. 2004; Jayadev, et al. 2011). It was recently found that repe rfusion was required for an increase in transcriptional activity following OGD, suggesting ROS to be important in microglial p53 activation (Garden, et al. unpublished results). This thesis showed that the same paradigm of OGD reperfusion elicited an incr ease in ROS, providing support for the possibility that ROS activated p53. Additionally, p53KO microglia produced similar ROS to wild type microglia, somewhat supporting the claim that p53 is silen t shortly following OGD (Fig. 1 7 ). Our qPCR results also su pport the hypothesis that ROS are responsible for the increase in p53 activity. The upregulation of IL 1Beta and MARCO transcripts in microglia is known to require p53 in response to the hypothesis that ROS upregulate p53 following OGD reperfusion, pushing microglia towards a pro inflammatory phenotype. To prove the inv olvement of ROS in microglial p53 activity would be of great clinical relevance for ischemia induced brain injury, which is known to have an important inflammatory component (Dirnagl, et al. 1999).
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96 Appendix 2.1.1 : Cell Culture Media and Chemical Solutions All culture medium products are purchased from Invitrogen. Stored at 4 o C in sterile conditions unless otherwise noted. Media for BV ium (DMEM) supplemented with 25 U/mL penicillin, 2 5 mg/mL streptomycin, 10% Fe tal Bovine Serum (FBS), and 0.2 mM L glutamine. To make D10C media, DMEM was supplemented with 10% heat inactivated horse serum, 10% Ham's F 12 medium, 2 mM L glutamine, 10mM% HEPES, 2 5 U/mL penicillin, and 25mg/mL streptomycin. L929 murine fibrobl ast cell lines, growing on a 17 5 cm 2 tissue culture flask, were incubated in D10C, and the supernatant was extracted and frozen at 20 o C weekly. This medium provides macrophage colony stimulating factor (MCSF), a cytokine that is import ant for culture microglia viability. 2.1.3: Making Primary Microglial Cultures Under sterile conditions, the cortices from mouse pups were separated from the cerebellum and hippocampus and were then placed into cold Neurobasal A (Invitrogen) buffered with HEPES to pH 7.4. The meninges were removed. Cortices, grouped into a centrifuge tube by genotype, were homogenized in a solution of ~0.6% trypsin 2+ or Ca 2+ (Invitrogen). DNAse (at 0.008% v/v, Invitro gen) was then added to prevent clumping. Trypsin inhibitor was Neurobasal A buffered with HEPES and was then pipetted numerous times until a homogeneous suspension was achieved. The suspension volume was about 6mL for less than 8 homogenized brains. The centrifuge tube was centrifuged at 1100 RPM for 5 min,
97 and the supernatant was removed. Pellet was rinsed and resuspended in D10C. The resuspension was added to poly D lysine coated plastic tissue culture flasks that contained enough warm D10C to amply cover the bottom of the flask. After 1 3 days of incubation at 37 o C, 5% C O 2 cell medium w as exchanged for fresh D10C. Once cells were confluent, L929 conditioned medium was added to achieve 20% of the total volume. It is important to note that the cells within the same flask were of the same p53 genotype, but were taken from any available litt er of mouse pups, ensuring high genetic variability from one cell to the next within the same flask. Adding to the variability between cells used for experiments, cells from different flasks were typically combined prior to plating cells for experimentation. 2.1.5: BV 2 cell cultur es BV 2 were harvested by first aspirating the supernatant, and rinsing cells in sterile phosphate buffered saline (PBS). A small volume of 0.125% trypsin was added, and the flask was manipulated until most cells no longer adhered to the culture flask. Fre sh BV 2 medium was added to the cell trypsin mixture, and then was collected and centrifuged for 5 min at 1100 RPM. The supernatant was aspirated, and cells were resuspended and counted in the same way as primary microglia. To propagate the cell line, appr oximately 1 mL of the resuspension was added to a new flask, not coated with poly D lysine, containing warm BV 2 media. 2.3.2: Quantitative Polymerase Chain Reaction (qPCR) Each qPCR reaction that occurred in one well of a 96 well reaction plate used 10L Taqman Universal PCR Master Mix (Applied Biosystems), 4L distilled water, 5L experimental cDNA, and 1L of a DNA primer/Taqman probe mixture. A
98 primer/Taqman probe mixture kit was purchased for the three genes being investigated (gene catalog no.: TBP Mm01277042_m1, IL 1Beta Mm00434228_m1, MARCO Mm00440265_m1, Applied Biosystems). 3.3.2: qPCR Reactions y = 3.4814x + 21.205 R = 0.9967 0 5 10 15 20 25 30 35 40 -6 -4 -2 0 Threshold Cycle TBP in BV 2 Positive Control TBP in BV-2 y = 3.5318x + 18.452 R = 0.9995 0 5 10 15 20 25 30 35 -6 -4 -2 0 Threshold Cycle MARCO in BV 2 Positive Control MARCO in BV-2 Linear (MARCO in BV-2)
99 Raw qPCR Data: TBP MARCO IL1Beta Efficiency (%) 93.7532578 91.933052 89.16782 CT value CT value CT value Normo 22.7751637 28.930744 28.16246 22.6322517 20.703897 28.02245 22.4001236 21.140728 27.92674 AVG 22.602513 23.59179 28.03721 SD 0.18928036 4.628826 0.118555 Normo NAC 22.5608749 22.45705 27.36386 22.6003036 22.432314 27.29004 22.7330132 22.982143 27.3902 AVG 22.6313972 22.623836 27.34804 SD 0.09018315 0.3105498 0.051921 OGD 22.6139278 17.320274 16.55221 22.5509224 15.594255 16.55869 22.6996136 15.412047 16.57715 AVG 22.6214879 16.108859 16.56269 SD 0.07463332 1.0530647 0.012942 OGD NAC 22.8095226 17.621782 25.55166 23.1171627 18.448118 25.61892 23.223505 18.190357 25.6027 AVG 23.0500635 18.086753 25.59109 SD 0.21499323 0.4227981 0.035098 y = 3.6121x + 22.134 R = 0.9999 0 10 20 30 40 -6 -4 -2 0 Threshold Cycle IL 1Beta in BV 2 Positive Control IL-1Beta in BV-2