Note:

A melatonina é uma indolamina sintetizada principalmente pela glândula pineal, cuja função está associada à marcação do escuro. Além deste papel cronobiótico, a melatonina também tem papel na defesa e é sintetizada por outros sítios podendo exercer ação parácrina e autócrina, como em células imunocompetentes. Concentrações substancialmente elevadas de melatonina são encontradas no liquido cefalorraquidiano (LCR) que tem sido vinculada à síntese de melatonina por células do sistema nervoso central (SNC), como as microglias. Sabendo-se que estas células são os macrófagos residentes no SNC e que a síntese de melatonina por estes fagócitos já é comprovada, nosso trabalho teve por objetivo avaliar se microglias cerebelares sintetizam essa indolamina e se esta atua potencializando a fagocitose destas células. Nossos resultados mostram que o bloqueio dos receptores de melatonina com o antagonista luzindol, diminuiu tanto a fagocitose induzida por melatonina exógena, quanto à fagocitose basal, indicando que há síntese de melatonina por microglias cerebelares que, por sua vez, age na fagocitose. Esses resultados são relevantes e indicam que a melatonina sintetizada pela microglia, pode estar relacionada com a homeostase do ambiente neural. Sendo assim, nossos dados podem contribuir com estudos que estabeleçam novas estratégias terapêuticas para doenças neuroinflamatórias. / Melatonin is a indolamine synthesized primarily by the pineal gland, whose function is associated with the marking of the dark phase. Beyond this chronobiotic function, melatonin also plays a role in defense and is synthesized by other sites. It may exert paracrine and autocrine action, like in immunocompetent cells. High substantially concentrations of melatonin are found in the cerebrospinal fluid (CSF) that has been linked to the synthesis of melatonin by the central nervous system cells (CNS), such as microglia. Knowing that these cells are the resident macrophages in the CNS and that melatonin synthesis by these phagocytes is proven, our study aims to assess whether cerebellar microglia synthesize this indolamine and whther this acts enhancing phagocytosis of these cells. Our results show that blocking the melatonin receptors with the antagonist, luzindole, both the exogenous melatonin-induced phagocytosis and the basal phagocytosis decreased, indicating that there is melatonin synthesis by cerebellar microglia which acts on phagocytosis. These results are significant and indicate that melatonin synthesized by microglia may be related to the neural environment homeostasis. In this way, our data can contribute, for example, in studies to establish new therapeutic strategies for neuroinflammatory diseases.

A melatonina é uma indolamina sintetizada principalmente pela glândula pineal, cuja função está associada à marcação do escuro. Além deste papel cronobiótico, a melatonina também tem papel na defesa e é sintetizada por outros sítios podendo exercer ação parácrina e autócrina, como em células imunocompetentes. Concentrações substancialmente elevadas de melatonina são encontradas no liquido cefalorraquidiano (LCR) que tem sido vinculada à síntese de melatonina por células do sistema nervoso central (SNC), como as microglias. Sabendo-se que estas células são os macrófagos residentes no SNC e que a síntese de melatonina por estes fagócitos já é comprovada, nosso trabalho teve por objetivo avaliar se microglias cerebelares sintetizam essa indolamina e se esta atua potencializando a fagocitose destas células. Nossos resultados mostram que o bloqueio dos receptores de melatonina com o antagonista luzindol, diminuiu tanto a fagocitose induzida por melatonina exógena, quanto à fagocitose basal, indicando que há síntese de melatonina por microglias cerebelares que, por sua vez, age na fagocitose. Esses resultados são relevantes e indicam que a melatonina sintetizada pela microglia, pode estar relacionada com a homeostase do ambiente neural. Sendo assim, nossos dados podem contribuir com estudos que estabeleçam novas estratégias terapêuticas para doenças neuroinflamatórias. / Melatonin is a indolamine synthesized primarily by the pineal gland, whose function is associated with the marking of the dark phase. Beyond this chronobiotic function, melatonin also plays a role in defense and is synthesized by other sites. It may exert paracrine and autocrine action, like in immunocompetent cells. High substantially concentrations of melatonin are found in the cerebrospinal fluid (CSF) that has been linked to the synthesis of melatonin by the central nervous system cells (CNS), such as microglia. Knowing that these cells are the resident macrophages in the CNS and that melatonin synthesis by these phagocytes is proven, our study aims to assess whether cerebellar microglia synthesize this indolamine and whther this acts enhancing phagocytosis of these cells. Our results show that blocking the melatonin receptors with the antagonist, luzindole, both the exogenous melatonin-induced phagocytosis and the basal phagocytosis decreased, indicating that there is melatonin synthesis by cerebellar microglia which acts on phagocytosis. These results are significant and indicate that melatonin synthesized by microglia may be related to the neural environment homeostasis. In this way, our data can contribute, for example, in studies to establish new therapeutic strategies for neuroinflammatory diseases.

Microglia are immunocompetent cells in the central nervous system that have many similarities with macrophages of peripheral tissues. Their activation protects local cells from foreign microbial infection in the CNS. However, “over-activated“ microglia become a “Double-edged sword” which show neuronal toxicity and are implicated in a variety of neurodegenerative diseases. Previous studies have suggested that potassium channels play a role in regulating microglial activation, migration and proliferation. However what kinds of potassium channel subunits are expressed in microglia, whether their expression changes after microglial activation and the functional role of most potassium channels expressed in microglia are still not fully characterized. To address these questions, we used the N9 mouse microglial cell line as a cell model for experiments in vitro. We first optimized the cell culture and lipopolysaccharide (LPS), the endotoxin of gram-negative bacteria, mediated stimulation of microglial activation that results in subsequent nitric oxide (NO) release. Using qRT-PCR, we analyzed mRNA expression of >80 potassium channel pore-forming subunits and their regulatory subunits in both LPS-treated (1μg/ml, 24hr) and untreated microglia. The subunits which displayed the highest mRNA expression in resting N9 cells included Kcnma1 (KCa1.1), Kcnk6 (K2p6.1), Kcnc3 (Kv3.3) and Abcc8 (SUR1). In addition, N9 cells also expressed the mRNAs for other channel subunits previously reported in microglia such as Kcnn4 (KCa3.1), Kcna3 (Kv1.3) and Kcna5 (Kv1.5) subunits. Of these channel subunits LPS had no significant effect on mRNA expression except for Kcnk6 which was significantly reduced. We then examined whether pharmacological manipulation of these channels controlled LPS-induced NO release. It was found out that the KCa3.1 selective blocker Tram34 and the Kv1.5 inhibitor propafenone (PPF) significantly decreased LPS-induced NO in agreement with data in primary microglia. Ba2+ that inhibits inwardly rectifying potassium channels as well as K2p6.1 also significantly attenuated LPS-induced microglial activation. Inhibition or activation of KCa1.1 channels by paxilline and NS1619 respectively had no significant effect. However, paxilline significantly attenuated the effect of Tram34, PPF and Ba2+ to control LPSinduced NO release while NS1619 significantly facilitated the effect of Tram34 and PPF. To investigate the major ionic currents expressed in N9 microglia with and without LPS application, we examined whole-cell ionic currents using the patch-clamp technique. Resting N9 cells display a small outward current at positive potentials but a large inwardly rectifying component at negative potentials in physiological potassium gradients. The outward current was dramatically increased by LPS application that was dependent upon the intracellular free calcium concentration. Paxilline or Tram34 was then applied to acutely block this apparent outward KCa current. The result indicated that the LPS triggered KCa current was mainly paxilline sensitive supporting a role for an LPS-induced increase in KCa1.1 channel current. In addition, by using current clamp the mean resting membrane potential of N9 cells was -50.6±6.6mV (N=7) determined in the presence of 1μM [Ca2+]i and -59.4±8.5mV (N=10) with 10nM [Ca2+]i. N9 cells did not display any spontaneous action potentials and the resting membrane potential was not significantly affected by LPS. To conclude, the work presented in this thesis extends the current knowledge regarding potassium channel mRNA expression in microglia and their function in microglial NO release. What is more, it was found that KCa1.1 current expression was increased in LPS-activated N9 cells and revealed KCa1.1 channels as a modulator of NO release by activated microglia.

Approximately 7 – 9 % of the population is living with some form of diabetes. When poorly controlled (which is often the case), this disease is associated with cerebrovascular pathology such as microbleeds and impairments in cognitive function. The presence and burden of microbleeds in the brain has been strongly linked with cognitive decline and increased risk of dementia. Microglia, the resident immune cells of the central nervous system, dynamically respond to vascular insults by extending their processes to the site of injury. The rapid actions of microglia are thought to play a beneficial role in vascular repair since inhibiting these responses can exacerbate injury. Here, we hypothesized that diabetes, especially if not well controlled with insulin, will disrupt microglia responses to damaged microvessels in the brain which will lead to increased plasma leakage from damaged microvessels. Using two-photon in vivo imaging, we show that chronic hyperglycemia in the streptozotocin model of type one diabetes leads to decreased microglial process accumulation around the site of microvascular injury and increased permeability of fluorescent dyes from the damaged vessel 30 minutes after induction of the bleed. Importantly, this impaired microglial and vascular response could be partially mitigated with tight control of blood glucose levels with insulin. These results indicate that chronic hyperglycemia disrupts microglial based repair of damaged microvessels, which may help explain why poorly controlled diabetes is associated with greater a risk of cerebrovascular dysfunction and cognitive decline. / Graduate / 2018-01-12

Microglia are the resident macrophages of the central nervous system (CNS) and, as key immune effector cells, form the first line of defence. Microglial cells also provide support for maintaining neuronal homeostasis and more generally normal brain physiology and cognitive function. It has been speculated that in order to support homeostasis, microglia adapt to a variety of brain microenvironments leading to regional phenotypic heterogeneity. To date this hypothesis lacks convincing empirical evidence, yet is critical to better understand microglial function in health and age-related neurodegenerative disease. In 2010 it was estimated that in the UK approximately 10 million people are over the age of 65, which is expected to double by 2050. Ageing is one of the strongest risk factors for neurodegenerative diseases such as Alzheimer’s and Parkinson‘s disease and growing evidence implicates neuroinflammatory mechansims that may involve microglial dysfunction in disease aetiology. The majority of age-related neurodegenerative diseases develop in a region-specific manner but the reasons are poorly understood. Accordingly, the work described in this thesis sought to determine the extent and nature of regional transcriptional heterogeneity of microglia and how this is affected by ageing. To examine the function and phenotype of these cells a technique for isolating pure microglia from the adult mouse brain was established. Microglia were consistently extracted by density-gradient and immunomagnetic cell separation. In vitro assays showed purified microglia retained key functional properties including phagocytosis, polarisation and production of pro-inflammatory cytokines in response to exogenous stimulation. Thus, freshly isolated microglia are not altered or dysfunctional during the extraction process and are likely to adequately represent the 'real' in vivo state. Genome-wide transcriptional network analysis of young adult mouse microglia from four discrete regions of the brain (cerebellum, cerebral cortex, hippocampus and striatum) uncovered regional heterogeneity in the microglial transcriptome driven particularly by bioenergetic and immunoregulatory functions. Transcriptional profiles of cerebellar and hippocampal microglia suggest a higher immune vigilance and alertness, which was supported by functional differences in the capability of microglia to phagocytose and control replication of bacteria. Region-dependent heterogeneity of microglia was largely consistent throughout the ageing process; however the region-specific phenotypes were more pronounced as age increased indicating region-dependent kinetics of microglial ageing. Collectively, the outcome of this study implies that microglia adapt to region-specific demands of brain tissue under steady-state conditions and are susceptible to ageing. Region was found to have a greater impact on microglial diversity than age. Overall, these findings will generate a substantial advance in our understanding of microglial function in the healthy brain and in age-related neurodegeneration.

Excessive tissue-damaging inflammation can exacerbate acute brain injury, and non-resolving inflammation is implicated in chronic neurodegeneration. Understanding the mechanisms that resolve deleterious inflammation in the brain is imperative to develop new therapeutic strategies. However current knowledge is limited, partly due to a lack of tractable models. Studies in extra-cerebral tissues have shown that myeloid cells are central to the inflammatory response. The aim of this thesis was to develop a model of self-limiting acute brain inflammation that is optimised to address mechanisms controlling resolution. The model was used to define the temporal profile of myeloid cell accumulation in the brain and establish the precise identities, origin and functional contribution of cell subsets in the resolution of the inflammatory response in the brain. Cerebral inflammation was induced by stereotaxic injection of inflammatory stimuli (LPS, HMGB1, MSU); LPS produced a robust inflammatory response and neutrophil influx and loss defined clear phases of initiation and resolution. Cellular changes (e.g. glial activation, endothelial activation and leukocyte influx) in response to LPS were characteristic of acute inflammation. Bone marrow chimaeric (Csf1r-EGFPC57Bl/6J) and monocyte reporter (Ccr2+/RFP) mice were used to distinguish between infiltrating macrophages and resident microglia. Analysis over 28 d showed the temporal profile of myeloid cells during brain inflammation, and monocyte accumulation contributed to expansion of the total mononuclear phagocyte population. Ccr2RFP/RFP knock-in mice showed that monocyte recruitment and resolution were independent of CCR2, and selective depletion of Ly6Clo monocytes with an anti- CSF1R antibody did not affect macrophage recruitment. Monocyte depletion using clodronate failed to deplete the Ly6Cint population and monocytes were still recruited into the brain. Together these results suggest multiple monocyte subsets could be involved in the inflammatory response in the brain. These data show that myeloid cell subsets of distinct origins accumulate in the inflamed brain. This work establishes a model system to identify endogenous mechanisms of resolution in cerebral inflammation and provides a platform to test CNS-targeted pro-resolution agents.

Neural stem cell (NSC) transplantation strategy offers great potential to treat spinal cord injury (SCI). NSCs may replace lost neurons or oligodendrocytes, and act as a source of neurotrophic factors to support the survival of remaining cells. Their efficiency was limited by poor survival after transplantation, and they had more tendencies to differentiate into astrocytes, but not neurons and oligodendrocytes. This project investigated whether activated microglia is a factor that contributes to this phenomenon, and studied the potential role of minocycline, a widely used antibiotic drug, to modify the negative effects of microglia on NSCs. In the first part of this study, we used organotypic spinal cord slice (SCS) culture to mimic in vivo local environment after SCI, and NSCs were grafted on their surface or shared culture medium with them. After specific depletion of microglia with clodronate loaded liposome, more grafted NSCs survived, and in the co-culture system, the NSC neuronal differentiation rate increased while glial differentiation rate decreased, the apoptosis rate also decreased. This suggested that activated microglia may impair NSC survival, and neuronal differentiation, but improve glial differentiation. In the second part of this study, we first tested the direct effects of minocycline on NSC apoptosis, proliferation and differentiation in vitro, to test whether minocycline has any side effect on NSCs. The results showed that at the concentration 10μg/ml or lower, minocycline did not affect NSC survival and proliferation, but impaired neuronal differentiation. Then we treated primary microglia culture with LPS or LPS plus minocycline, and collected the conditioned mediums (CM-LPS and CM-LPSMC) to test their effects on NSC　apoptosis and differentiation. The results showed that compared with CM-LPS, CM-LPSMC resulted in a significantly lower apoptotic rate of NSCs, also allowed NSC neuronal differentiation. This suggested that minocycline may impair the pro-apoptotic effect of activated microglia on NSCs. In conclusion, our study showed that activated microglia may impair NSC survival and neuronal differentiation. This indicated that in NSC transplantation strategy for SCI, microglia would be a target to be manipulated to improve graft survival and neuronal differentiation. Although minocycline may suppress NSC differentiation towards neurons, it has the potential to protect NSCs from the toxic effects of activated microglia. This showed the therapeutic potential of minocycline in NSC transplantation strategies for SCI. / published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

No description available.

Thesis (Ph. D.)--Virginia Commonwealth University, 2009. / Prepared for: Dept. of Microbiology and Immunology. Bibliography: leaves 69-85. Also available online via the Internet.