La rigidité artérielle est considérée comme un facteur de risque important pour le développement du déclin cognitif. Toutefois, les effets précis de la rigidité artérielle sur le cerveau sont peu connus et, à ce jour, aucun modèle animal ne permet d’étudier l’effet isolé de ce facteur sur l’homéostasie cérébrale. Dans cette étude, nous avons développé un nouveau modèle de rigidité artérielle qui se base sur la calcification de l’artère carotide chez la souris. Au niveau artériel, ce modèle présente une fragmentation de l’élastine, une augmentation de la distribution du collagène et de l’épaisseur intima-média ainsi qu’une diminution de la compliance et de la distensibilité artérielles démontrant la rigidité artérielle. De plus, le modèle ne présente pas d’augmentation de pression artérielle ni de changement de rayon du lumen indiquant une absence d’hypoperfusion globale et d’anévrisme. Au niveau cérébral, les résultats montrent que la rigidité artérielle induit une augmentation de la pulsatilité du flux sanguin cérébral menant ainsi à une augmentation du stress oxydatif. Ce dernier induit une inflammation cérébrale, détectée par l’activation de la microglie et des astrocytes, induisant ultimement une neurodégénérescence. Ces effets sont surtout observés au niveau de l’hippocampe, la région cruciale pour la mémoire et la cognition. Ainsi, cette étude montre que la rigidité artérielle altère l’homéostasie cérébrale et mérite d’être considérée comme une cible potentielle dans la prévention et le traitement des dysfonctions cognitives chez les personnes âgées. / Arterial stiffness is considered as an important risk factor for the development of cognitive decline in the elderly population. However, its precise effects on the brain are unknown and, to date, no animal model allows to study the precise outcome of arterial stiffness on the brain homeostasis. In this study, we developed a new animal model of arterial stiffness based on the calcification of the carotid artery in mice. On the arterial level, this model shows a fragmentation of elastin, increased collagen distribution and intima-media thickness as well as decreased arterial compliance and distensibility, thus fulfilling the major arterial stiffness properties. In addition, this model does not a show an increase in blood pressure or change in arterial lumen radius indicating a lack of global hypoperfusion and aneurysm. Regarding the brain, the results show that arterial stiffness induces an increase in cerebral blood flow pulsatility leading to increased oxidative stress. Oxidative stress induces brain inflammation, detected by the activation of microglia and astrocytes, ultimately leading to neurodegeneration. These effects are particularly observed in the hippocampus, a crucial area for memory and cognition. Thus, this study shows that arterial stiffness alters brain homeostasis and therefore should be considered as a potential therapeutical target for the prevention and treatment of cognitive dysfunction in the elderly.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed June 6, 2008). Includes bibliographical references.

O diabetes mellitus tipo 1 (DMT1) tem sido associado com complicações a longo prazo no sistema nervoso central, além dos efeitos periféricos comuns relacionados à doença, causando disfunções cognitivas no encéfalo. Por outro lado, o enriquecimento ambiental (EA) induz mecanismos de plasticidade dependentes da experiência, especialmente no hipocampo, melhorando o desempenho dos animais em testes de aprendizado e memória. Assim, nosso objetivo foi avaliar a influência do EA sobre o déficit de memória, a atividade locomotora, os níveis de corticosterona, a imunorreatividade da proteína sinaptofisina, e a densidade e a ativação de astrócitos e microglia no giro denteado (GD) do hipocampo de ratos diabéticos tipo 1. Para isso, ratos Wistar machos com 21 dias de idade, foram expostos ao EA ou mantidos em caixamoradia padrão (controles, C) por 3 meses. Quando adultos, os animais tanto C quanto EA foram randomicamente divididos e induziu-se diabetes através de injeção de estreptozotocina em metade dos animais de cada grupo, sendo mantidas as respectivas condições ambientais para cada um dos grupos. A memória espacial dependente de hipocampo foi avaliada em todos os grupos através do teste de reconhecimento de objeto reposicionado, no 41o dia após a indução do diabetes, bem como a locomoção geral dos animais no campo aberto durante o mesmo teste. Os níveis séricos de corticosterona foram medidos ao final do experimento, a imunorreatividade da sinaptofisina foi avaliada por imunoistoquímica, e a densidade e a ativação de astrócitos e da microglia por imunofluorescência no hilo do GD do hipocampo. Nossos resultados mostraram que o EA foi capaz de prevenir ou atrasar o desenvolvimento do déficit de memória causado pelo diabetes em ratos, porém não reverteu o déficit motor observado nos animais diabéticos. Não houve diferença significativa na imunorreatividade da sinaptofisina entre os grupos. Além disso, embora o EA não tenha modificado a densidade e a ativação dos astrócitos nos animais diabéticos, o enriquecimento atenuou os efeitos prejudiciais da hiperglicemia sobre a ativação microglial, bem como reduziu os níveis séricos de corticosterona nos ratos diabéticos adultos. Assim, o EA ajudou a amenizar as comorbidades cognitivas associadas ao diabetes, possivelmente por atenuar a hiperatividade do eixo HPA e a ativação microglial nos animais diabéticos. / Type 1 diabetes mellitus (T1DM) has been associated with long-term complications in central nervous system, besides peripheral common adverse effects, causing neurocognitive dysfunction in the brain. On the other hand, enriched environment (EE) induces mechanisms of experiencedependent plasticity especially in hippocampus, improving the performance of animals in learning and memory tasks. Thus, our objective was to investigate the influence of the EE on memory deficits, locomotion, corticosterone levels, synaptophysin protein immunoreactivity, and density and activation of astrocytes and microglia in the hippocampal dentate gyrus (DG) of type 1 diabetic rats. For this, male Wistar rats, 21 days old, were exposed to the EE or maintained in standard housing (controls, C) for 3 months. At adulthood, C and EE animals were randomly divided and half of them induced to diabetes by streptozotocin, being maintained the respective environmental conditions for each animal groups. Hippocampus-dependent spatial memory was evaluated in all groups in the novel object-placement recognition task, on 41th day after diabetes induction, as well as the general locomotion in the open field at the same test. Serum corticosterone levels were measured in the end of the experiment, contents of synaptophysin was evaluated by immunohistochemistry, and density and activation of both astrocytes and microglia by immunofluorescence in the hilus of the DG in hippocampus. Our results showed that EE was able to prevent or delay the development of memory deficits caused by diabetes in rats, however did not revert the motor impairment observed in group diabetic. There was no significant difference in synaptophysin immunoreactivity among the groups. Furthermore, although the EE did not modify the density and activation of astrocytes in diabetic animals, it attenuated the injurious effect of hyperglycemia over microglial activation, as well as decreased the serum level of corticosterone in diabetic adult rats. Thus, the EE has helped to ameliorate cognitive comorbidities associated with T1DM, possibly by reducing the hyperactivity of HPA axis and the microglial activation in diabetic animals.

O diabetes mellitus tipo 1 (DMT1) tem sido associado com complicações a longo prazo no sistema nervoso central, além dos efeitos periféricos comuns relacionados à doença, causando disfunções cognitivas no encéfalo. Por outro lado, o enriquecimento ambiental (EA) induz mecanismos de plasticidade dependentes da experiência, especialmente no hipocampo, melhorando o desempenho dos animais em testes de aprendizado e memória. Assim, nosso objetivo foi avaliar a influência do EA sobre o déficit de memória, a atividade locomotora, os níveis de corticosterona, a imunorreatividade da proteína sinaptofisina, e a densidade e a ativação de astrócitos e microglia no giro denteado (GD) do hipocampo de ratos diabéticos tipo 1. Para isso, ratos Wistar machos com 21 dias de idade, foram expostos ao EA ou mantidos em caixamoradia padrão (controles, C) por 3 meses. Quando adultos, os animais tanto C quanto EA foram randomicamente divididos e induziu-se diabetes através de injeção de estreptozotocina em metade dos animais de cada grupo, sendo mantidas as respectivas condições ambientais para cada um dos grupos. A memória espacial dependente de hipocampo foi avaliada em todos os grupos através do teste de reconhecimento de objeto reposicionado, no 41o dia após a indução do diabetes, bem como a locomoção geral dos animais no campo aberto durante o mesmo teste. Os níveis séricos de corticosterona foram medidos ao final do experimento, a imunorreatividade da sinaptofisina foi avaliada por imunoistoquímica, e a densidade e a ativação de astrócitos e da microglia por imunofluorescência no hilo do GD do hipocampo. Nossos resultados mostraram que o EA foi capaz de prevenir ou atrasar o desenvolvimento do déficit de memória causado pelo diabetes em ratos, porém não reverteu o déficit motor observado nos animais diabéticos. Não houve diferença significativa na imunorreatividade da sinaptofisina entre os grupos. Além disso, embora o EA não tenha modificado a densidade e a ativação dos astrócitos nos animais diabéticos, o enriquecimento atenuou os efeitos prejudiciais da hiperglicemia sobre a ativação microglial, bem como reduziu os níveis séricos de corticosterona nos ratos diabéticos adultos. Assim, o EA ajudou a amenizar as comorbidades cognitivas associadas ao diabetes, possivelmente por atenuar a hiperatividade do eixo HPA e a ativação microglial nos animais diabéticos. / Type 1 diabetes mellitus (T1DM) has been associated with long-term complications in central nervous system, besides peripheral common adverse effects, causing neurocognitive dysfunction in the brain. On the other hand, enriched environment (EE) induces mechanisms of experiencedependent plasticity especially in hippocampus, improving the performance of animals in learning and memory tasks. Thus, our objective was to investigate the influence of the EE on memory deficits, locomotion, corticosterone levels, synaptophysin protein immunoreactivity, and density and activation of astrocytes and microglia in the hippocampal dentate gyrus (DG) of type 1 diabetic rats. For this, male Wistar rats, 21 days old, were exposed to the EE or maintained in standard housing (controls, C) for 3 months. At adulthood, C and EE animals were randomly divided and half of them induced to diabetes by streptozotocin, being maintained the respective environmental conditions for each animal groups. Hippocampus-dependent spatial memory was evaluated in all groups in the novel object-placement recognition task, on 41th day after diabetes induction, as well as the general locomotion in the open field at the same test. Serum corticosterone levels were measured in the end of the experiment, contents of synaptophysin was evaluated by immunohistochemistry, and density and activation of both astrocytes and microglia by immunofluorescence in the hilus of the DG in hippocampus. Our results showed that EE was able to prevent or delay the development of memory deficits caused by diabetes in rats, however did not revert the motor impairment observed in group diabetic. There was no significant difference in synaptophysin immunoreactivity among the groups. Furthermore, although the EE did not modify the density and activation of astrocytes in diabetic animals, it attenuated the injurious effect of hyperglycemia over microglial activation, as well as decreased the serum level of corticosterone in diabetic adult rats. Thus, the EE has helped to ameliorate cognitive comorbidities associated with T1DM, possibly by reducing the hyperactivity of HPA axis and the microglial activation in diabetic animals.

O diabetes mellitus tipo 1 (DMT1) tem sido associado com complicações a longo prazo no sistema nervoso central, além dos efeitos periféricos comuns relacionados à doença, causando disfunções cognitivas no encéfalo. Por outro lado, o enriquecimento ambiental (EA) induz mecanismos de plasticidade dependentes da experiência, especialmente no hipocampo, melhorando o desempenho dos animais em testes de aprendizado e memória. Assim, nosso objetivo foi avaliar a influência do EA sobre o déficit de memória, a atividade locomotora, os níveis de corticosterona, a imunorreatividade da proteína sinaptofisina, e a densidade e a ativação de astrócitos e microglia no giro denteado (GD) do hipocampo de ratos diabéticos tipo 1. Para isso, ratos Wistar machos com 21 dias de idade, foram expostos ao EA ou mantidos em caixamoradia padrão (controles, C) por 3 meses. Quando adultos, os animais tanto C quanto EA foram randomicamente divididos e induziu-se diabetes através de injeção de estreptozotocina em metade dos animais de cada grupo, sendo mantidas as respectivas condições ambientais para cada um dos grupos. A memória espacial dependente de hipocampo foi avaliada em todos os grupos através do teste de reconhecimento de objeto reposicionado, no 41o dia após a indução do diabetes, bem como a locomoção geral dos animais no campo aberto durante o mesmo teste. Os níveis séricos de corticosterona foram medidos ao final do experimento, a imunorreatividade da sinaptofisina foi avaliada por imunoistoquímica, e a densidade e a ativação de astrócitos e da microglia por imunofluorescência no hilo do GD do hipocampo. Nossos resultados mostraram que o EA foi capaz de prevenir ou atrasar o desenvolvimento do déficit de memória causado pelo diabetes em ratos, porém não reverteu o déficit motor observado nos animais diabéticos. Não houve diferença significativa na imunorreatividade da sinaptofisina entre os grupos. Além disso, embora o EA não tenha modificado a densidade e a ativação dos astrócitos nos animais diabéticos, o enriquecimento atenuou os efeitos prejudiciais da hiperglicemia sobre a ativação microglial, bem como reduziu os níveis séricos de corticosterona nos ratos diabéticos adultos. Assim, o EA ajudou a amenizar as comorbidades cognitivas associadas ao diabetes, possivelmente por atenuar a hiperatividade do eixo HPA e a ativação microglial nos animais diabéticos. / Type 1 diabetes mellitus (T1DM) has been associated with long-term complications in central nervous system, besides peripheral common adverse effects, causing neurocognitive dysfunction in the brain. On the other hand, enriched environment (EE) induces mechanisms of experiencedependent plasticity especially in hippocampus, improving the performance of animals in learning and memory tasks. Thus, our objective was to investigate the influence of the EE on memory deficits, locomotion, corticosterone levels, synaptophysin protein immunoreactivity, and density and activation of astrocytes and microglia in the hippocampal dentate gyrus (DG) of type 1 diabetic rats. For this, male Wistar rats, 21 days old, were exposed to the EE or maintained in standard housing (controls, C) for 3 months. At adulthood, C and EE animals were randomly divided and half of them induced to diabetes by streptozotocin, being maintained the respective environmental conditions for each animal groups. Hippocampus-dependent spatial memory was evaluated in all groups in the novel object-placement recognition task, on 41th day after diabetes induction, as well as the general locomotion in the open field at the same test. Serum corticosterone levels were measured in the end of the experiment, contents of synaptophysin was evaluated by immunohistochemistry, and density and activation of both astrocytes and microglia by immunofluorescence in the hilus of the DG in hippocampus. Our results showed that EE was able to prevent or delay the development of memory deficits caused by diabetes in rats, however did not revert the motor impairment observed in group diabetic. There was no significant difference in synaptophysin immunoreactivity among the groups. Furthermore, although the EE did not modify the density and activation of astrocytes in diabetic animals, it attenuated the injurious effect of hyperglycemia over microglial activation, as well as decreased the serum level of corticosterone in diabetic adult rats. Thus, the EE has helped to ameliorate cognitive comorbidities associated with T1DM, possibly by reducing the hyperactivity of HPA axis and the microglial activation in diabetic animals.

A micróglia é essencial para a homeostase do Sistema Nervoso Central (SNC), função neuro-imune inata, e exerce papel importante na neurodegeneração, envelhecimento cerebral e tumorigênese. Gliomas difusos são tumores cerebrais primários caracterizados por crescimento infiltrativo e altas taxas de heterogeneidade, o que torna a doença praticamente incurável. Avanços em análises genéticas caracterizaram alterações moleculares relacionadas ao tempo de sobrevida e à resposta clínica desses pacientes, especialmente em glioblastomas (GBM). No entanto, a tumorigenicidade dos gliomas não é controlada unicamente por suas alterações genéticas. As interações entre as células tumorais, a micróglia residente e os macrófagos/monócitos infiltrados desempenham um papel crucial na modulação do crescimento e agressividade do glioma. Neste estudo, analisamos o fenótipo de ativação da micróglia/macrófagos em gliomas, incluindo astrocitomas e oligodendroglimas de diferentes graus de malignidade, apresentamos o perfil de expressão gênica da população pura de micróglia cortical e do tecido cerebral total correspondente. Usando sequenciamento de DNA de alta performance, classificamos as amostras de GBM em Proneural, Clássico e Mesenquimal. Em seguida, avaliamos os status de ativação da micróglia/macrófagos dessas amostras. Apesar do alto grau de heterogeneidade, pudemos observar níveis mais altos dos marcadores mielóides (IBA1, CD11b and CD68) em tumores astrocíticos comparados a tumores de origem oligodendrocítica e ao tecido não-neoplásico. Marcadores de anti-inflamação, como CD163, foram mais abundantes em astrocitomas, bem como em GBMs do subtipo Mesenquimal e Clássico; enquanto que marcadores de pró-inflamação, como IL1-beta, mostraram uma expressão mais heterogênea entre as amostras. Em seguida, micróglia foi isolada de 25 amostras de córtex parietal provenientes de autópsia de indivíduos cognitivamente preservados e foi feito o RNA-seq. Os resultados foram comparados à micróglia de camundongo e a outras células mielóides. Boa parte dos genes expressos pela micróglia humana foram similares àqueles expressos pela micróglia murina, como CX3CR1, P2YR12 e ITGAM. Porém, foram identificados genes de característica imune, abundantemente expressos na micróglia humana e não identificados na micróglia de camundongos, como TLR, Fcy, receptores do tipo SIGLEC, e fatores de transcrição NLRC5 e CIITA. A comparação dos dados de expressão gênica da micróglia com monócitos e macrófagos identificou novos marcadores que distinguem a micróglia humana de outras células mielóides. Nossos dados sobre a micróglia em gliomas sugerem características de imunossupressão e de pró-crescimento em tumores de pior prognóstico, ligado a um fenótipo específico de ativação das células mielóides. Este é o primeiro estudo a identificar o transcriptoma da micróglia humana pura, demonstrando que ela é claramente diferente da micróglia murina e de outras células mielóides. Esses resultados abrem portas para estudos de populações específicas de células mielóides em gliomas / Microglia are essential for central nervous system (CNS) homeostasis and innate neuroimmune function, and play important roles in neurodegeneration, brain aging and tumorigenesis. Diffuse gliomas are primary brain tumors characterized by infiltrative growth and high heterogeneity, which renders the disease mostly incurable. Advances in genetic analysis have characterized molecular alterations leading to impact on patients\' overall survival and clinical outcome, particularly in glioblastoma (GBM). However, glioma tumorigenicity is not controlled uniquely by its genetic alterations. The crosstalk between tumor cells, resident microglia and infiltrating monocytes/macrophages plays a crucial role in modulating glioma growth and aggressiveness. Here, we assess the activation status of microglia/macrophages in gliomas,including astrocytomas and oligodendrogliomas of different grades of malignancy, and present the gene expression profile of pure cortical human microglia and corresponding unsorted brain tissue. Using high-throughput DNA sequencing, we have classified GBM samples in Proneural, classical and mesenchymal. Next, we evaluated the activation status of microglia/macrophages within these samples. Despite the great heterogeneity, we observed higher levels of myeloid markers (IBA1, CD11b and CD68) in astrocytic tumors compared to oligodendrocytic ones and to non-neoplastic (NN) tissue. Anti-inflammation markers, such as CD163, are also more abundant in astrocytomas, as well as in the mesenchymal and classical GBM subtypes, while pro-inflammation markers, such as IL1-beta, show a more widespread expression throughout samples. Next, microglia were isolated from the parietal cortex of 25 autopsy samples of cognitively preserved humans and RNA sequenced. Overall, genes expressed by human microglia are similar to mouse microglia, such as CX3CR1, P2YR12, and ITGAM. Interestingly, a number of immune genes, not identified as mouse microglia signature genes, were abundantly expressed in human microglia, such as TLR, Fcy and SIGLEC receptors and NLRC5 and CIITA transcription factors. Comparison of microglia to monocyte and macrophage expression data underscored the CNS-specific functions of microglia and new markers were identified that distinguish human microglia from other myeloid cells. Our glioma-related data suggests an immune-suppressive and growth supportive characteristic for tumors with worse clinical outcome, linked to an activation profile of myeloid cells. This data is the first comprehensive pure human microglia gene expression profile; human microglia clearly differ from mouse microglia and other myeloid cells. These results will help further studies focusing on pure myeloid cells populations in glioma

Toxoplasma gondii is an obligate intracellular parasite that infects virtually all warm-blooded organisms. Systemic dissemination of T. gondii in the organism can cause life-threatening infection that manifests as Toxoplasma encephalitis in immune-compromised patients. In addition, mounting evidence from epidemiological studies indicates a link between chronic Toxoplasma infection and mental disorders. To better understand the pathogenesis of toxoplasmosis, basic knowledge on the host-parasite interactions and the dissemination mechanisms are essential. Previous findings have established that, upon infection with T. gondii, dendritic cells (DCs) and microglia exhibit enhanced migration, which was termed the hypermigratory phenotype. As a result of this enhanced migration, DCs and microglia are used as vehicle cells for dissemination (‘Trojan horse’) which potentiates dissemination of T. gondii in mice. However, the precise mechanisms behind the hypermigratory phenotype remained unknown. In this thesis, we characterized host-parasite interactions upon infection with T. gondii and investigated the basic mechanisms behind the hypermigratory phenotype of T. gondii-infected DCs and microglia. In paper I, we observed that upon infection with T. gondii, DCs underwent rapid morphological changes such as loss of adhesiveness and podosomes, with integrin redistribution. These rapid morphological changes were linked to hypermotility and were induced by active invasion of T. gondii within minutes. T. gondii-infected DCs exhibited up-regulation of the C-C chemokine receptor CCR7 and chemotaxis towards the CCR7 chemotactic cue, CCL19. In paper II, we developed a 3-dimensional migration assay in a collagen matrix, which allowed us to characterize the hypermigratory phenotype in a more in vivo-like environment. The migration of T. gondii-infected DCs exhibited features consistent with integrin-independent amoeboid type of migration. T. gondii-induced hypermigration of DCs was further potentiated in the presence of CCL19 in a 3D migration assay. In paper III, we identified a parasite effector molecule, a Tg14-3-3 protein derived from parasite secretory organelles. Tg14-3-3 was sufficient to induce the hypermigratory phenotype. Transfection with Tg14-3-3-containing fractions or recombinant Tg14-3-3 protein induced the hypermigratory phenotype in primary DCs and in a microglial cell line. In addition, Tg14-3-3 localized in the parasitophorous vacuolar space and host 14-3-3 proteins were rapidly recruited around the parasitophorous vacuole. In paper IV, we found that mouse DCs dominantly express the L-type voltage-dependent calcium channel, Cav1.3. Cav1.3 was linked to the GABAergic signaling-induced hypermigratory phenotype. Pharmacological inhibition of Cav1.3 and knockdown of Cav1.3 abolished the hypermigratory phenotype in T. gondii infected DCs. Blockade of voltage-dependent calcium channels reduced the dissemination of T. gondii in a mouse model. In paper V, we showed that microglia, resident immune cells in the brain, also exhibited rapid morphological changes and hypermotility upon infection with T. gondii. However, an alternative GABA synthesis pathway was shown to be involved in the hypermigratory phenotype in microglia. In summary, this thesis describes novel host-parasite interactions, including host cell migratory responses and key molecular mechanisms that mediate the hypermigratory phenotype. The findings define a novel motility-related signaling axis in DCs. Thus, T. gondii employs GABAergic non-canonical pathways to hijack host cell migration and facilitate dissemination. We believe that these findings represent a significant step forward towards a better understanding of the pathogenesis of T. gondii infection. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.</p>

No description available.

Microglia and brain-resident macrophages are the sentinel immune cells of the central nervous system (CNS), and are ideally situated to respond to any damage to the brain parenchyma or vasculature. Circulating leukocytes are generally excluded from the CNS environment under homeostatic conditions but can gain access to this region in diseases that disrupt immune system function and blood-brain barrier integrity. Although these diverse immune cells exhibit properties that may engender them to be well-suited to resolve microcirculatory insults, their relative contributions to the recanalization of capillary rupture in the cortex, known as cerebral microbleeds (CMBs), has yet to be described. CMBs are particularly concerning in conditions, such as diabetes mellitus (DM), in which these insults occur more frequently and potentially underlie the onset and progression of cognitive decline. Using in vivo 2-photon microscopy and confocal imaging, here I highlight the compromised repair of CMBs in a mouse model of type 1 DM and characterize the robust, heterogeneous macrophage response to these insults. Specifically, 20% of damaged capillaries were eliminated from the circulation in the diabetic cortex and chronic insulin treatment failed to prevent this microvascular loss. Administration of interferon-α or interferon-γ neutralizing antibodies to dampen inflammatory signalling, or dexamethasone to reduce global inflammation, also failed to improve repair rates of damaged microvessels in diabetic mice. In contrast, CMBs in nondiabetic mice repaired without exception. Interestingly, depletion of CNS macrophages using the colony stimulating factor-1 receptor antagonist PLX5622 resulted in microvascular elimination in nondiabetic mice. Given the robust depletion of brain macrophage populations with this treatment, at first these data suggested that these cells were necessary for microvascular repair since their elimination produced vessel loss. However, by parsing the data I identified that microvessels repaired in all cases where macrophages were not identified at the CMB; when CX3CR1+ aggregate was localized to the injury, ~20% of microvessels were eliminated. These findings show that CNS macrophages are not required for microvascular repair following CMB. Immunofluorescent co-labelling of various microglial and macrophage markers within the diabetic CMB milieu revealed a novel population of Mac2+/TMEM119- cells, distinct from homeostatic TMEM119+ microglia. These cells reliably localized to CMBs that failed to repair and rarely associated with vessels that recanalized; Mac2+/TMEM119- cells were not found within nondiabetic CMBs. Treatment of diabetic mice with clodronate liposomes (CLR) to deplete circulating phagocytic leukocytes prevented aggregation of Mac2+/TMEM119- cells to CMBs and improved capillary repair rates. The efficacy of CLR in excluding these cells from the CMB aggregate, coincident with eradication of monocytes from circulation, indicated that these cells likely arose from the periphery. In vivo 2-photon imaging revealed significant increases in lipofuscin at the site of diabetic CMBs relative to the nondiabetic context; other phagocytic markers including CD68 and TREM2 were also upregulated. Mac2+/TMEM119- cells showed elevated lipofuscin content relative to homeostatic microglia; their association with CMBs may thus signal an increase in phagocytosis that contributes to capillary pruning. Taken together, these data identify a novel Mac2+/TMEM119- macrophage associated with pathological microvascular elimination following CMB in the diabetic neocortex. These findings highlight the diversity of immune cell responses to CNS injury and provide insights into the cellular mechanisms of capillary pruning. Furthermore, these advances in our understanding of the regulation of microvascular elimination in the diabetic brain may have clinical implications for patients with DM as they provide evidence for putative adjuvant anti-inflammatory treatments, such as CLR, in mitigating cerebrovascular pathology. / Graduate / 2022-05-06

Clinical findings have linked multiple risk factors and associated pathologies to Alzheimer\''s disease (AD). Amongst them are vascular risk factors such as hypertension and pathologies such as stroke. Coexistence of AD and these associated pathologies worsenes dementia, the clinical hallmark of the disease, as compared to pure AD. One general common denominator of these associated pathologies is the presence of hypoxic tissue conditions. It was asked the question, whether there exists a mutual, causal interaction between hypoxia and AD pathology, that could explain the clinical observations. Alternatively, the worsened clinical state of multiple brain pathologies could \"simply\" be the consequence of multimorbidity, i.e. accumulated disease load, without any causal interaction between the constituents. To approach this question whether hypoxia influences AD progression, use was made of a murine animal model of AD (transgenic mice: APPswe, PSEN1dE). Animals of two ages (8 and 14 months, \"young\" and \"old\" respectively) and two genotypes (transgenic and wild- type) were either treated under hypoxia or normoxia, corresponding to 8% and 21% oxygen, for 20 consecutive days. The resulting changes in the brain were assessed with a variety of techniques, namely by histology, ELISA, dot and Western blotting. Additional experiments in primary cell cultures were performed. Animals exposed to hypoxia showed an increased hematocrit (HCT), weight loss, reactive angiogenesis, but no infarctions. This illustrates that our hypoxic treatment put significant stress on the animals, without causing major pathologies. A large number of variables exists that could potentially be measured to assess the effect of hypoxia on AD. The focus was put on three of them: First, there is the Abeta1-42- protein, known to be the Abeta- isoform associated with the most detrimental disease progression. In AD, the self-combinatory Amyloid- beta peptide (Abeta) accumulates in the brain in so- called plaques, which is a main histologic finding of the disease. Its quantity was determined through histology and ELISA. Secondly, it was attempted to estimate the structural quality of the Abeta- protein by assessing the amount of A!- oligomers present. Abeta- protein does self- accumulate in various grades of complexity, i.e. as monomer, oligomer or fibril. Since oligomers are known to be the most neurotoxic \"species\" of the Abeta- protein, it was hypothesized that under hypoxic treatment their quantity could increase. And third, the organism\''s response to the Abeta- protein stimulus was investigated. Microglial cells have been described as the first cells to encounter the Abeta- protein \"threat\" in the shape of plaques, i.e. Abeta- protein aggregates. They then try to encapsulate and subsequently degrade them. Therefore, the attention was put on this cellular population. It was asked whether hypoxia could change the Abeta- protein quantity in the brain. This was assessed in two ways: First histologically, by staining for Abeta- protein depositions and quantifying them. Second, an ELISA was performed. Our findings state that hypoxic treatment does not alter the Abeta1-42 protein load in the brain, neither in young nor old animals, as assessed by histology and by total ELISA quantification of Abeta1-42 protein. Since hypoxia did not alter the quantity of the Abeta- protein, it was asked whether it influenced it qualitatively? If hypoxia increased oligomer formation, this change in the spectrum of the Abeta- species could, without any change in total Abeta- protein load, lead to increased neurotoxicity in animals under hypoxia. Initial experiments showed that oligomer formation in the brain seems to increase. However, this was not statistically significant and future experiments are necessary to evaluate this hypothesis further. It was then asked, whether hypoxia alters the cellular response to the protein. The total number of microglia in the hippocampal dentate gyrus, our structure of interest for practical purposes, and, it can be argued, by extension the brain, changes dynamically with various factors. First, transgenic animals present an increase in microglia. Second, microglia increase with age. Third, microglia decrease under hypoxia, but only do so significantly in old animals. Next, a parameter called \"plaque occupancy\" was coined to assess the microglia function to confront Abeta- plaques. Plaque occupancy is defined as the number of microglia in spatial proximity to one square millimeter of Abeta- plaque. This means, that microglia restricting one plaque are counted, and then normalized to this plaque\''s area. It was hypothesized that hypoxia would decrease plaque occupancy. Indeed, plaque occupancy roughly halved under hypoxia. Summarizing, our results demonstrate that long- term exposure to hypoxia significantly reduces the number of microglia. The reduced number results in significantly reduced plaque occupancy and compromizes the function of microglia to confront Abeta- plaques. The Abeta1-42 load, however, is not affected. On the other hand, Abeta shows an increased trend towards oligomer formation. A variety of possible explanations to these phenomena have been presented, that in our opinion deserve further investigation.