Дисертації з теми "Chronic neuroinflammation"

Post-menopausal women have an increased incidence of Alzheimer's disease (AD) that may be delayed in onset by estrogen replacement therapy (ERT). Estrogen has many neuroprotective and neurotrophic proclivities; therefore, its decline with menopause may leave the brain vulnerable to toxic insults stemming from disease states. Recent clinical trials investigating ERT as a treatment for AD found beneficial effects following short-term treatment that become attenuated, and possibly reversed, following longer treatment intervals. This doctoral dissertation examined the interaction of two conditions known to exist within the female AD brain: the presence of chronic neuroinflammation and either estrogen deprivation or chronic ERT. As the duration of treatment and regimen of estrogen administration may alter the effectiveness of ERT, chronic and fluctuating administration of estrogen were assessed against the behavioral, biochemical and pathological consequences of short- and long-term neuroinflammation in the female rat brain. Overall, the results suggest a strong interaction between neuroendocrine and autonomic function in the female brain with neuroinflammation. In the presence of chronic neuroinflammation, the brain differentially responds depending on the hormone status of the animal. Cognitive performance is impaired with neuroinflammation or constant estrogen; the combined occurrence of both conditions worsened performance more than either condition presented alone. However, gonadally intact females with neuroinflammation were unimpaired on the task and had approximately half the number of activated microglia. The pattern of activated microglia is unique to the female brain and highlights an interesting distribution not seen in male rats. Specifically, an elegant map of activated microglia emerges of brain areas involved in autonomic control, stress regulation and energy homeostasis. Regions showing the densest distribution of activated microglia are important autonomic relay stations that interconnect various brain regions conveying internal state information. Moreover, these regions have extensive bi-directional communication with both endocrine and immune systems, suggesting an extensive interaction occurring in the female brain capable of influencing multiple systems, including hormone secretion, sympathetic output, immune function and behavioral processes. This dissertation proposes that the interactions between these systems have important consequences for post-menopausal women with AD and are likely to underlie the varying effects seen with ERT.

Neural stem cell (NSC) transplantation induces recovery in animal models of central nervous system (CNS) diseases, in part by modulating adaptive and innate immune responses. Since metabolism governs the phenotype and function of immune cells, the aim of this thesis was to investigate whether NSCs have the ability to regulate the immunometabolic components underpinning neuroinflammation. Herein I have identified a new mechanism by which transplanted somatic and directly-induced NSCs counteract CNS-compartmentalised chronic inflammation in mice. NSC transplantation reduces the immunometabolite succinate in the cerebrospinal fluid, while decreasing the burden of mononuclear phagocyte (MP) infiltration and secondary CNS damage. Mechanistically, the anti-inflammatory activity of NSCs arises in response to succinate released by inflammatory MPs, which activates succinate receptor 1 (SUCNR1)/GPR91 on NSCs, thus initiating prostaglandin E2 secretion and extracellular succinate scavenging. This work uncovers a succinate-SUCNR1 axis in NSCs that clarifies how stem cells respond to inflammatory metabolic signals to inhibit the activation of pro-inflammatory MPs in the chronically inflamed brain.

Alcohol use can negatively impact financial, cognitive, and psychiatric aspects of human life. In the brain, alcohol can have many devastating effects. Alcohol is a well-known cytotoxic agent that can cause specific brain pathology in humans; however, the exact biological mechanisms are not well-elucidated. Animal models are invaluable tools to investigate potential novel treatments in a substance abuse model. Mice studies can be used to screen for negative outcomes prior to human trials. We hypothesize that the C1q tumor necrosis factor-related protein, CTRP3, overexpression in mice reduces neuroinflammation from ethanol consumption that has been coupled with a high fat diet when compared to control mice. The CTRP family of proteins are adipokines and CTRP3 specifically influences cell viability, metabolism, and peripheral inflammation levels. Antibody specific immunoblotting is used to probe protein expression changes in neuroinflammatory markers in mouse cerebellum brain tissue in an overexpression mouse model of CTRP3 when compared to high-fat ethanol exposed mice and baseline control mice. The two proteins examined are MAG and GFAP. Myelin associated glycoprotein, or MAG, is a protein expressed by oligodendrocytes that mediate axonal growth and myelin interactions with neurons in the brain. Oligodendrocytes are extremely sensitive to oxidative stress to which cognitive deficits in ethanol exposure is thought to be attributed. Glial fibrillary acidic protein, or GFAP, is a marker of astrocyte reactivity. Astrocytes are cells in the brain that are responsible for environmental stabilization and actively participate in neurotransmission. Currently, GFAP alterations in ethanol-exposed animals are dose and age dependent. We chose to use young adult mice where GFAP reactiveness is increased during chronic ethanol exposure. The proposed studies are essential in determining CTRP3’s relationship to detrimental neuroinflammatory effects of alcohol and high fat diet in mice. The data obtained from these studies will provide compelling evidence for future clinical trials to investigate CTRP3 as a therapeutic agent in people with a high fat diet that use alcohol chronically.

L’inflammation est un processus essentiel à prendre en compte dans la pratique clinique. Nous avons montré durant cette thèse que le statut (neuro)inflammatoire précédant la survenue d’une pathologie cérébrale est à prendre en compte nécessairement puisqu’il modifie drastiquement la réponse inflammatoire suite à un deuxième stimulus comme la survenue d’un AVC. Il est d’autant plus important que 90% des AVC sont associés à des comorbidités comme l’hypertension artérielle, le diabète ou la consommation chronique d’alcool, qui ont d’ores et déjà été décrites comme des maladies avec une composante inflammatoire. Nous avons caractérisé ce statut neuroinflammatoire silencieux, aussi appelé priming, dans le cadre de la consommation chronique d’alcool et dans le traumatisme crânien léger. De plus, nous avons identifié les macrophages périvasculaires comme participants à l’effet aggravateur du priming inflammatoire sur les lésions ischémiques. Ils semblent alors être une cible thérapeutique de choix et feront l’objet de futures études. Il est donc nécessaire de trouver des techniques d’imagerie non invasives pour détecter le priming. L’autoradiographie ciblant le TSPO nous a permis de révéler le priming inflammatoire dans le cadre du traumatisme crânien léger. Nous proposons, au vu de nos résultats obtenus durant cette thèse, la tomographie par émission de positons pour la détection de la neuroinflammation invisible dans les atteintes cérébrales aigüe(s) et chronique(s)
Inflammation is an essential process to be considered in clinical practice. We have shown during this thesis that the (neuro)inflammatory status preceding the occurrence of a cerebral pathology must necessarily be taken into account since it drastically modifies the inflammatory response following a second stimulus such as stroke. This is even more important given that 90% of strokes are associated with comorbidities such as chronic hypertension, diabetes or chronic alcohol consumption, for which inflammation is an important pathophysiological feature. We have characterized this silent inflammatory status, also called priming, in the context of chronic alcohol consumption and in mild traumatic brain injury. We have identified perivascular macrophages (PVM) as mediators of the aggravating effect of inflammatory priming on ischemic stroke. PVM appear to be potential therapeutic targets and will be the subject of future investigations. It is therefore necessary to find non-invasive imaging techniques to detect inflammatory priming. We show that autoradiography targeting TSPO reveals the inflammatory priming provoked by a single mild traumatic brain injury. We propose, in light of the results obtained during this thesis, the positron emission tomography imaging to detect the invisible neuroinflammation in acute and chronic brain diseases

SARM1 is an injury-induced nicotinamide adenine dinucleotide nucleosidase (NADase) that was previously shown to promote axonal degeneration in response to traumatic, toxic, and excitotoxic stressors. This raises the question of whether a SARM1-dependent program of axonal degeneration is central to a common pathway contributing to disease burden in neurological disorders. The degree to and mechanism by which SARM1 inactivation decreases the pathophysiology of such disorders is of interest to establish the rationale to pursue SARM1 as a therapeutic target. In this study, we compare the course and pathology of experimental autoimmune encephalomyelitis (EAE) in Sarm1-knockout (KO) mice and wild-type littermates to test the contribution of SARM1-dependent axonal degeneration specifically in the context of chronic, immune-mediated central nervous system (CNS) inflammation. The question of whether SARM1 loss in Sarm1-KO mice would inhibit, promote, or have a negligible impact on EAE-induced axonal degeneration and more broadly CNS inflammation was explored using a variety of analyses: quantification of clinical score in a chronic EAE model, CNS immune infiltrate profile, axon initial segment morphology in layer V cortical neurons, axonal transport disruption and transection in the lumbar spinal cord. Additionally, we have proposed a method for detecting SARM1 activation in situusing a novel SARM1-mCitrine bimolecular fluorescence complementation (BiFC) technique. Successful implementation of such a molecular tool would allow for a detailed, mechanistic approach to enhance our understanding of upstream intracellular signals that trigger SARM1 activation.

Biomedical Sciences
Ph.D.
The cannabinoid system exerts functional regulation of neural stem cell (NSC) selfrenewal, proliferation, and differentiation during both homeostatic and pathologic conditions. Recent evidence suggests that cannabinoid signaling is neuroprotective against reduction in NSC proliferation and neurogenesis caused by a multitude of conditions including injury due to HIV-1 associated neurotoxic proteins, neuroinflammation, and stroke. Yet not all effects of cannabinoids or cannabinoid-like compounds on neurogenesis can be attributed to signaling through either of the classical cannabinoid receptors CB1 or CB2. The recently de-orphaned GPR55 is targeted by numerous cannabinoid compounds suggesting GPR55 may be causing these aberrant effects. Activation of GPR55 has shown immune-modulatory effects outside the central nervous system (CNS) and anti-inflammatory actions on microglia, the resident immune cells within the CNS. New evidence has confirmed that both human and murine NSCs express functional levels of GPR55 yet the effects that GPR55 activation has on adult neurogenesis or NSC responses to inflammation has not been elucidated. In the present study we sought to determine the role GPR55 signaling has on NSC proliferation and neurogenesis as well as possible neuroprotective mechanisms within the NSC pool in response to inflammatory insult. Activation of GPR55 increased human NSC proliferation in vitro as assessed by BrdU incorporation and flow cytometry. Neuronal differentiation was also upregulated by signaling through GPR55 under homeostatic conditions in both human and murine NSC samples. Expression of NSC differentiation markers (nestin, sox2, GFAP, S100b, DCX, bIII-tubulin) in vitro was determined by immunohistochemistry, qPCR, and flow cytometry. In vivo, C57BL/6 and GPR55-/- mice were administered the GPR55 agonist O-1602 (4 μg/kg/day) directly into the left hippocampus via stainless steel cannula connected to an osmotic mini-pump for a continuous 14 days. O-1602 treatment increased hippocampal NSC proliferation, survival, and immature neuron formation as compared to vehicle treated animals. These results were determined to be dependent on GPR55 activation as GPR55-/- animals did not show any response to agonist treatment. Interestingly, GPR55-/- mice displayed significantly reduced rates of hippocampal NSC proliferation and neuroblast formation as compared to C57BL/6 animals. Chronic production of inflammatory mediators, such as IL-1b seen in neuroinflammation, to NSCs is known to reduce proliferation rates and attenuate neurogenesis both in vitro and in vivo. Addition of GPR55 agonists to IL-1b (10 ng/mL) treated human and murine NSC samples in vitro protected against reductions in neuron formation as assessed by immunohistochemistry and flow cytometry. Moreover, inflammatory cytokine receptor mRNA expression was down regulated by GPR55 activation in a neuroprotective manner. To determine inflammatory responses in vivo, we treated C57BL/6 and GPR55-/- mice with LPS (0.2 mg/kg/day) continuously for 14 days via osmotic mini-pump. Reductions in NSC survival (as determined by BrdU incorporation), immature neurons, and neuroblast formation due to LPS were attenuated by concurrent direct intrahippocampal administration of the GPR55 agonist, O-1602 (4μg/kg/day) in C57BL/6 mice but not in GPR55-/-mice. Neuroprotection by O-1602 treatment was not found to be microglia dependent as microglia activation was not altered by agonist administration. Molecular analysis of the hippocampal region showed a suppressed ability to regulate immune responses by GPR55-/- animals manifesting in a prolonged inflammatory response (IL-1b, IL-6, TNFa) after chronic, systemic inflammation as compared to C57BL/6 animals. Taken together, these results suggest a neuroprotective role of GPR55 activation on NSCs in vitro and in vivo and that GPR55 provides a novel therapeutic target against negative regulation of hippocampal neurogenesis by inflammatory insult.
Temple University--Theses

Alzheimer's disease (AD) is a disorder of aging that is characterized by progressive loss of cognitive functioning, eventually culminating in complete dementia. Many lines of evidence, i.e., epidemiological, pathological and biochemical, suggest inflammatory processes play a role in neurodegenerative disease processes responsible for AD. Recent reports suggest that patients who chronically take anti-inflammatory drugs to treat the symptoms of inflammatory diseases, such as rheumatoid arthritis, have a reduced risk of developing AD. The inflammation found in the brains of AD patients is characterized by activated microglia, and astrocytes in brain regions that demonstrate extensive neuronal degeneration. In addition, the activated glia are closely associated with amyloid plaques and may contribute to their formation. Various molecules involved in inflammatory reactions, including cytokines, complement, arachidonic acid and prostaglandins are found to be elevated in the brain and CSF of AD patients. These molecules may also contribute to the evolution of the pathology in this disorder. The purpose of the first study was to determine if inflammatory processes are toxic to neurons. The results will show that both acute injections and chronic infusions of the proinflammagen lipopolysaccharide into the basal forebrain of rats was toxic to cholinergic cells. Chronic infusions of LPS decreased the level of activity of the acetylcholine synthetic enzyme choline acetyltransferase (ChAT). In addition, these infusions decreased the number of neurons that demonstrated immunoreactivity for this enzyme. Furthermore, low dose, long-term infusions of LPS produced a greater decline in ChAT activity than a single injection of high dose LPS. These results suggest that the toxic effects of neuroinflammation are more time-dependent than dose-dependent and support the hypothesis that long-term inflammatory processes can impair cholinergic neuronal function. The purpose of the second study was to examine the mechanism by which the chronic LPS infusions were neurotoxic to cholinergic neurons within the basal forebrain of young rats. This study investigated the hypothesis that increased activity of glutamate and prostaglandin neurons might underlie the toxic actions of chronic inflammation induced by LPS. First, this study investigated whether chronic administration of a noncompetitive N-methyl-D-aspartate (NMDA) sensitive receptor antagonist, memantine, could provide neuroprotection for forebrain cholinergic neurons. Second, this study also investigated whether a cyclooxygenase-2 (COX2)/lipoxygenase inhibitor, CI987, could provide significant neuroprotection from the cytotoxic effects of LPS-induced neuroinflammation. Daily peripheral administration of memantine (s.c.) or CI987 (s.c.) significantly attenuated the cytotoxic effects of the chronic inflammatory processes upon cholinergic cells within the basal forebrain. However, only CI987 attenuated the neuroinflammation produced by LPS and the subsequent changes in microglial activation. These results indicate that the cytotoxic effects of chronic neuroinflammation may involve prostanoid synthesis and may operate through NMDA receptors, and that the effects of prostaglandins occur upstream to NMDA receptor activation.

La maladie de Parkinson (MP) est une maladie neurodégénérative caractérisée par l'agrégation de l'α-synucléine (αSYN). Les agrégats αSYN sont essentiels à l'activation de la microglie et aux réponses inflammatoires pouvant contribuer à la pathogenèse. Il est donc important de comprendre les mécanismes qui sous-tendent l'activation, la polarisation et la fonction des cellules microgliales. Dans ce travail, nous avons étudié pour la première fois le potentiel inflammatoire d’assemblages d’αSYN dérivés des patients (FPD) sur la microglie primaire et exploré leur capacité à polariser ces cellules vers un phénotype spécifique en combinaison avec des facteurs inflammatoires présents chez les patients tels que le TNFα et la prostagladine E2 (TPFPD). Nos données montrent que les FPD ont un potentiel inflammatoire plus important que des assemblages recombinants et que la microglie exposée à TPFPD acquiert un phénotype fonctionnel et moléculaire spécifique. En particulier, elle présente une libération réduite de cytokines mais plus élevée de glutamate en lien avec l’expression à la hausse du gène Scl7a11 (cystine-glutamate antiporter xCT) et impliqué dans l'excitotoxicité des neurones dopaminergiques. Ensemble, ces résultats soutiennent la relation structure-fonction des polymorphes d’αSYN et mettent en évidence certaines propriétés de la microglie inflammatoire de type chronique
Parkinson’s disease (PD) is a neurodegenerative disorder characterized by α-synuclein (αSYN) aggregation. Mounting evidence indicates that αSYN aggregates are central to microglia activation and inflammatory responses though to contribute to pathogenesis. Therefore, understanding the mechanisms that drive microglial cell activation, polarization and function is of paramount importance as it might have important therapeutic impact. In this work, we investigated for the first time the inflammatory potential of patient-derived αSYN assemblies (FPD) on primary microglia and explored their capacity to polarize these cells toward a specific phenotype when combined to chronic-type and PD-relevant inflammatory cues including TNFα and prostagladine E2 (PGE2) (referred as TPFPD stimulation). Our data show that FPD hold stronger inflammatory potency than structurally different recombinant αSYN fibrils and that TPFPD-exposed microglia acquire specific functional and molecular phenotypes departing from pro-inflammatory M1 cells. In particular, they show reduced cytokine but higher glutamate release in line with up-regulated Scl7a11 gene (cystine-glutamate antiporter xCT) expression and involved in dopaminergic neuron excitotoxicity. Together, these results support structure-function relationship of αSYN polymorphs and highlight some properties of chronic-type inflammatory microglia

Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2009.
Committee Chair: Bellamkonda, Ravi; Committee Member: Babensee, Julia; Committee Member: Butera, Robert; Committee Member: DeWeerth, Steve; Committee Member: Lee, Robert; Committee Member: McKeon, Robert. Part of the SMARTech Electronic Thesis and Dissertation Collection.

As head injuries and their sequelae have become an increasingly salient matter of public health, experts in the field have made great progress elucidating the biological processes occurring within the brain at the moment of injury and throughout the recovery thereafter. Given the extraordinary rate at which our collective knowledge of neurotrauma has grown, new insights may be revealed by examining the existing literature across disciplines with a new perspective. This article will aim to expand the scope of this rapidly evolving field of research beyond the confines of the central nervous system (CNS). Specifically, we will examine the extent to which the bidirectional influence of the gut-brain axis modulates the complex biological processes occurring at the time of traumatic brain injury (TBI) and over the days, months, and years that follow. In addition to local enteric signals originating in the gut, it is well accepted that gastrointestinal (GI) physiology is highly regulated by innervation from the CNS. Conversely, emerging data suggests that the function and health of the CNS is modulated by the interaction between 1) neurotransmitters, immune signaling, hormones, and neuropeptides produced in the gut, 2) the composition of the gut microbiota, and 3) integrity of the intestinal wall serving as a barrier to the external environment. Specific to TBI, existing pre-clinical data indicates that head injuries can cause structural and functional damage to the GI tract, but research directly investigating the neuronal consequences of this intestinal damage is lacking. Despite this void, the proposed mechanisms emanating from a damaged gut are closely implicated in the inflammatory processes known to promote neuropathology in the brain following TBI, which suggests the gut-brain axis may be a therapeutic target to reduce the risk of Chronic Traumatic Encephalopathy and other neurodegenerative diseases following TBI. To better appreciate how various peripheral influences are implicated in the health of the CNS following TBI, this paper will also review the secondary biological injury mechanisms and the dynamic pathophysiological response to neurotrauma. Together, this review article will attempt to connect the dots to reveal novel insights into the bidirectional influence of the gut-brain axis and propose a conceptual model relevant to the recovery from TBI and subsequent risk for future neurological conditions.

Chronic pain affects 20 % of the global population, causes suffering, is difficult to treat, and constitutes a large economic burden for society. So far, the characterization of molecular mechanisms of chronic pain-like behaviors in animal models has not translated into effective treatments. In this thesis, consisting of five studies, pain patient biofluids were analyzed with modern proteomic methods to identify biomarker candidates that can be used to improve our understanding of the pathophysiology chronic pain and lead to more effective treatments. Paper I is a proof of concept study, where a multiplex solid phase-proximity ligation assay (SP-PLA) was applied to cerebrospinal fluid (CSF) for the first time. CSF reference protein levels and four biomarker candidates for ALS were presented. The investigated proteins were not altered by spinal cord stimulation (SCS) treatment for neuropathic pain. In Paper II, patient CSF was explored by dimethyl and label-free mass spectrometric (MS) proteomic methods. Twelve proteins, known for their roles in neuroprotection, nociceptive signaling, immune regulation, and synaptic plasticity, were identified to be associated with SCS treatment of neuropathic pain. In Paper III, proximity extension assay (PEA) was used to analyze levels of 92 proteins in serum from patients one year after painful disc herniation. Patients with residual pain had significantly higher serum levels of 41 inflammatory proteins. In Paper IV, levels of 55 proteins were analyzed by a 100-plex antibody suspension bead array (ASBA) in CSF samples from two neuropathic pain patient cohorts, one cohort of fibromyalgia patients and two control cohorts. CSF protein profiles consisting of levels of apolipoprotein C1, ectonucleotide pyrophosphatase/phosphodiesterase family member 2, angiotensinogen, prostaglandin-H2 D-isomerase, neurexin-1, superoxide dismutases 1 and 3 were found to be associated with neuropathic pain and fibromyalgia. In Paper V, higher CSF levels of five chemokines and LAPTGF-beta-1were detected in two patient cohorts with neuropathic pain compared with healthy controls. In conclusion, we demonstrate that combining MS proteomic and multiplex antibody-based methods for analysis of patient biofluid samples is a viable approach for discovery of biomarker candidates for the pathophysiology and treatment of chronic pain. Several biomarker candidates possibly reflecting systemic inflammation, lipid metabolism, and neuroinflammation in different pain conditions were identified for further investigation.
Uppsala Berzelii Technology Centre for Neurodiagnostics

Chronic traumatic encephalopathy (CTE) is a neurodegenerative disorder caused by repeated concussive or subconcussive blows to the head. Clinically, this disease is characterized by cognitive dysfunction, short-term memory loss, and motor deficits. Pathologically, deposition of the abnormal protein tau, cerebral atrophy, and white matter degeneration is common. CTE has been categorized into Stages I-IV based on increased severity of protein deposition and cerebral atrophy. Acutely, mild traumatic brain injury (TBI) damages the long white matter tracks in the corpus callosum. In addition, it initiates a neuroinflammatory cascade aimed at protecting healthy tissue by clearing any toxic or damaging debris. This cascade results predominantly from the activation of the resident immune cells of the brain, microglia. Inflammation begins immediately and then subsides weeks or months after injury. However, pathological chronic activation of microglia can occur that can cause cell death and degeneration. Several studies have linked traumatic brain injury as well as chronic neuroinflammation to a variety of neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and chronic traumatic encephalopathy (CTE). The present study quantifies the level of inflammation found in the brains of those diagnosed with varying stages of CTE compared to normal, healthy controls. The thickness of the corpus callosum was measured to investigate the correlation between microglial density and white matter degeneration. Cases were selected from the donated brains of former athletes and military veterans who had a history of repetitive mild TBI. Eleven healthy control cases, ten early stage (Stage I/II), and nine late stage (Stage III/IV) CTE cases were selected for analysis. Tissue sections of the anterior and posterior cingulate of each case were stained for microglia, reactive astrocytes, and macrophages using IBA-1, GFAP, and CD68 markers. The percent area stained of each section was calculated to compare inflammatory cell density across progressive stages of the disease. Analysis showed a significant thinning of the corpus callosum of Stage III/IV CTE cases compared to normal controls. There was a significant decrease in microglia and reactive astrocytes of both the anterior and posterior portions of the corpus callosum in both early and advanced stage CTE cases compared to healthy controls. Corpus callosum thickness was significantly decreased in advanced stage (III-IV), but not early stage (I-II) disease. Overall, this suggests that neuroinflammation is decreased in the corpus callosum in CTE despite marked degeneration. Repetitive mild TBI might impair mechanisms of brain inflammation and repair.

Research Doctorate - Doctor of Philosophy (PhD)
Almost daily the role number of roles that microglia have been identified as playing a role in expands. Once considered to only act as the macrophages of the central nervous system, microglia are now recognised to be ‘master-orchestraters’ of events as diverse as neurogenesis, synaptic pruning, synaptogenesis, vascular remodeling, extracellular matrix remodeling, vascular surveillance and many others. Perhaps one of the most interesting and enigmatic features of microglia is their capacity to engage in rapid and profound structural reorganization. This ability has thus fascinated many researchers, a phenomenon that is only continued to grow in response to recent real time imaging of the cells in action. Most of is known about microglial structure has come from static two-dimensional images. Detailed morphological characterization of these cells in two dimensions, while significantly less glamorous, than real time multiphoton imaging has nevertheless proven to be an extremely reliable vehicle for revealing and characterizing the fundamental properties of state changes of these cells. Under normal conditions microglia are recognised to exist in the brain with relatively small somas and a number of thin tapering branches radiating out from the cell body. Often, although not always, these branches split and often split again in what is frequently referred to as their secondary and tertiary branching structures. Following exposure to severe environmental disturbances, microglia are known to engage in profound structural reorganization. The specific form that this reorganization takes is somewhat interventions specific but in many cases it will involve marked somal enlargement and significant process simplification and shortening. These changes are ultimately considered to be relevant and informative because they are known to be linked with changes in the specific functions engaged in by microglia. Chapter 2: At the outset of my studies there had been no quantitative studies to what represented ‘normal’ microglial morphology within uninjured prefrontal cortex (PFC). The PFC is well documented to play a critical role in mood disturbance and our research group has found evidence that chronic stress induces significant changes in microglial activity in this region. Accordingly, the major aim of Chapter Two was to systematically examine the morphology of microglia (identified by ionized calcium-binding adaptor molecule-1 (Iba-1) labelling). Here I undertook many hundreds of manual reconstructions across the five cortical layers of the rat PFC. The reconstruction analysis was used to determine the morphometric parameters including the convex hull area, cell body perimeter, total length, total volume, number of processes, number of branch points, and the complexity of cell. Our results revealed that microglial morphology was consistent across all layers by demonstrating as ramified microglia, while there was a variation in cell size. I additionally determined the density of microglia and found no difference in anterior-posterior direction of the PFC. These findings confirmed that microglia represent a relatively homogenous within the healthy PFC. Chapter 3: Several previous studies have linked expression of the purine ionotropic receptor P2X7, which is primarily found on microglia, with major depression. As chronic stress is recognised to be a significant risk factor in the emergence of major depression. I decided to identify if chronic restraint stress was involved in changes in the expression of P2X7 within the hippocampus. In this study, I utilized an analysis technique recently developed by our group known as cumulative threshold spectra (CTS) to identify if stress altered P2X7 at any of three different time points: acute stress (1 session), chronic stress (21 sessions) and a chronic stress with 7 days recovery time. The findings of this work suggested that exposure to acute or chronic stress significantly reduced the expression of P2X7 receptor within the hippocampus and that levels remained suppressed in recovery period. In addition, the comparative analysis on normalised data showed that the level of the P2X7 reduction was significantly greater in the chronic stress relative to the acute stress group. Furthermore, I found that P2X7 expression rebounded to baseline in 7 days recovery time. Collectively, these results provide the first evidence that chronic restraint stress produces markedly a reduction of the P2X7 receptor within the hippocampus. Chapter 4: In Chapter Four, I investigated how chronic stress influences the roles of glial cells (microglia and astrocytes) as it related to disturbances of glutamatergic signaling. Using immunohistochemical analysis, the results showed that there was a reduction in glial fibrillary acidic protein (GFAP), a marker of astrocytes, in the hippocampus after exposure to chronic stress, while the expression of Iba-1 was increased. In addition, I examined how these changes were associated with glutamatergic signalling proteins following chronic stress. Specifically I assessed changes in the expression of the astrocyte specific glutamate transporters EAAT1 and EAAT2. The proteins were observed to have opposition changes after stress with a increase in EAAT1 and a decrease in EAAT2 expression. Further, I measured the expression of related proteins such as vesicular glutamate transporter (VGLUT1), which was increased and AMPA and NMDA subunits (GluA1, GluA2, GluN1, GluN2A, GluN2B). The AMPA and NMDA subunits showed a complex pattern of changes, however, when taken together with the changes in VLGUT1 and EAATs 1 and 2 are strongly suggestive of dramatic shift in glutamatergic activity. Together, these findings suggest that chronic stress-induced structural remodelling of astrocytes and microglia may be driven by alterations in glutamatergic neurotransmission. Chapter 5: Besides the roles of microglia in response to chronic stress-induced mood disorder, previous studies have demonstrated that microglia appear to be the most important cell involved in inflammatory related mood disturbances. The administration of lipopolysaccharide (LPS) is well-validated model of immune challenge producing microglial activation and sickness behaviours in rodents and humans. Recently, these behaviours have become the representative indicator of how peripheral disturbances in immune signalling can disturb quite complex behaviours. Therefore, I examined the alterations of microglial structure within the PFC following the administration of LPS (100 µg/kg/i.p.) in adult male rats. Immunohistochemistry was conducted to analyse the changes in Iba-1, a specific microglial marker, in the infralimbic and prelimbic of the PFC using a cumulative threshold spectra (CTS) analysis and digital reconstructions. Our results indicated that the somal area of was significantly enlarged, while cell processes became slightly shortened after LPS injection. This data is the first to demonstrate significant microglial alterations within the PFC following LPS administration. Collectively, the experiments clearly outline the complex nature of the structural changes that microglia can undergo in response to routine, yet severe, environmental challenges.

Stroke is a neurovascular disease that frequently results in decreased motor and cognitive functioning. Obesity is a major risk factor associated with ischemic stroke and is thought to worsen the functional deficits observed after stroke. Previous findings from our laboratory suggest that worse motor deficits in obese animals may be a result from an exacerbated neuroinflammatory response. Most animal studies demonstrate an association between obesity and worse cognitive functioning after stroke. However, the mechanisms are not well studied. This study examines the neuroinflammatory response, ischemic brain tissue damage, and cognitive functioning in diet-induced obese mouse models during the chronic phase after ischemic stroke, defined as weeks after stroke. Our study found an earlier onset of cognitive deficits in obese mice after stroke compared to normal weight mice. We found no differences in the degree of brain damage in obese animals and normal weight animals 11 weeks after stroke, but observed higher levels of microgliosis in obese animals compared to normal weight animals. Due to the limitations of our study, additional studies should be done to assess the severity of cognitive deficits in obese animals compared to normal weight animals in the chronic phase after stroke. Further studies also need to be done to confirm our findings regarding the microglial response and degree of ischemic brain damage during the chronic phase.