Classically, the following three morphological states of microglia have been defined: ramified, amoeboid and phagocytic. While ramified cells were long regarded as “resting”, amoeboid and phagocytic microglia were viewed as “activated”. In aged human brains, a fourth, morphologically novel state has been described, i.e., dystrophic microglia, which are thought to be senescent cells. Since microglia are not replenished by blood-borne mononuclear cells under physiological circumstances, they seem to have an “expiration date” limiting their capacity to phagocytose and support neurons. Identifying factors that drive microglial aging may thus be helpful to delay the onset of neurodegenerative diseases, such as Alzheimer’s disease (AD). Recent progress in single-cell deep sequencing methods allowed for more refined differentiation and revealed regional-, age- and sex-dependent differences of the microglial population, and a growing number of studies demonstrate various expression profiles defining microglial subpopulations. Given the heterogeneity of pathologic states in the central nervous system, the need for accurately describing microglial morphology and expression patterns becomes increasingly important. Here, we review commonly used microglial markers and their fluctuations in expression in health and disease, with a focus on IBA1 low/negative microglia, which can be found in individuals with liver disease.

Placenta-specific 1 (Plac1) is an X-linked gene that is essential for normal placental development. Previous studies have shown that Plac1 is also expressed in the fetal brain and paternally imprinted. Its expression in the fetal brain is markedly downregulated immediately after birth. Plac1 ablation predisposes Plac1-null males and Xm-X Hets (inactive maternal allele) to an increased risk of developing lethal postnatal hydrocephalus suggesting a functional role for Plac1 during embryonic development. The objective of this study was to characterize the effect of Plac1 on brain development and postnatal function. In order to address this, a mutant Plac1 mouse model, established on the C57BL/6J background, was studied. Formalin-fixed, paraffin-embedded whole mount embryos and brain sections were obtained at various stages of development. Plac1 expression was assessed by qRT-PCR and immunohistochemistry (IHC). Brain structure was assessed by histopathological and magnetic resonance imaging (MRI) analysis. Behavioral analysis was conducted using the PhenoMaster automated cage system and a battery of classical behavioral tests. Our results revealed Plac1 expression throughout the embryonic brain when assessed by qRT-PCR and IHC at E16.5 and E18.5. MRI analysis of an adult Plac1 knockout (KO) brain revealed microcephaly (14%), reduced ventricular volume, and increased heterogeneity of the medulla compared to a WT brain. Consistent with these findings, H&E staining of the KO brain revealed a smaller cortical mantle, a dysmorphic hippocampus, and a dysmorphic cerebellum with reduced folia. IHC analyses of NF-M, NeuN, and Iba1 immunostaining revealed significant reductions in axonal and neuronal development and increased activated microglia in KO brain, but not in Xm-X Hets. Although no structural abnormalities were detected in Xm-X Hets, behavioral analyses did reveal reduced activity and behaviors consistent with increased anxiety. In conclusion, Plac1 is a paternally imprinted, X-linked gene that is associated with abnormal brain development, reduced activity, and specific behavioral abnormalities.

Submitted by Daniella Sodre (daniella.sodre@ufpe.br) on 2015-04-08T18:22:35Z No. of bitstreams: 2 TESE Geórgia de Sousa Ferreira Soares.pdf: 2287209 bytes, checksum: 9762ebb4675d95dd929fcb3db11523e7 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-04-08T18:22:35Z (GMT). No. of bitstreams: 2 TESE Geórgia de Sousa Ferreira Soares.pdf: 2287209 bytes, checksum: 9762ebb4675d95dd929fcb3db11523e7 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2014-02-21 / Capes; CNPq / A lectina Concanavalina A (ConA), obtida das sementes de Canavalia ensiformis, é uma proteína que se liga especificamente à manose e glicose (e estruturas mais complexas, como receptores celulares, que contenham resíduos expostos desses carboidratos). Essa lectina possui várias atividades biológicas, incluindo a modulação de propriedades imunológicas e electrofisiológicos do cérebro. No presente estudo, foi caracterizada a ação da ConA sobre o fenômeno dependente da excitabilidade cerebral conhecido como Depressão Alastrante Cortical (DAC); estudou-se também a imunomarcação da microglia com um anticorpo policlonal contra a proteína Iba1 (Ionized calcium binding adaptor molecule 1). Ratos Wistar machos (n = 89) foram amamentados em condições favoráveis ou desfavoráveis de lactação, representadas respectivamente por ninhadas com 6-7 filhotes (grupo N6) ou 12-14 filhotes (grupos N12). Do 5º ao 24º dia pós-natal, foram tratados por via intraperitoneal com 1 mg/kg ou 10 mg/kg de ConA (grupos L1 e L10, respectivamente), ou com solução salina NaCl 0,9% (grupo Sal), ou sem nenhum tratamento (grupo Ingênuo). Aos 90-120 dias de idade, a DAC foi quimicamente induzida no córtex frontal e registrada em dois pontos da região parietal durante 4 h, e seus parâmetros de velocidade de propagação, amplitude e duração das ondas foram mensurados. Após o registro da DAC, os cérebros foram perfundidos e fixados, seguidos de secções cerebrais para reação com anticorpos anti-Iba1 para quantificação da imunomarcação da microglia. Os grupos N12 apresentaram maior velocidade de propagação da DAC que os grupos N6. Em ambas as condições de lactação, o tratamento sistêmico com a ConA resultou em diminuição significativa, dose-dependente, da velocidade de propagação da DAC (p < 0,05), em comparação aos grupos controle (Salina e Ingênuo). A amplitude e duração do componente negativo da variação lenta de voltagem da DAC permaneceram inalterados. Em um grupo adicional de ratos adultos, a aplicação tópica de ConA à superfície cortical também reduziu reversivelmente a velocidade de propagação da DAC. A imunorreatividade da microglia, avaliada no córtex e hipocampo, foi menor nos grupos tratados com ConA, em comparação com os respectivos controles. O hemisfério cerebral no qual a DAC foi repetidamente induzida apresentou maior imunorreatividade em comparação com o hemisfério oposto, principalmente no córtex. Podemos concluir que a atenuação na propagação da DAC e a diminuição da imunorreatividade na microglia em ratos adultos em decorrência da aplicação da lectina ConA (1 mg/kg e 10 mg/kg) durante o período de lactação (fase de grande plasticidade e neurogênese), indicam que essa lectina pode influenciar o desenvolvimento cerebral com ação protetora duradoura no cérebro de ratos. Essa ação não foi dificultada pela condição desfavorável de lactação. Desse modo, a lectina ConA apresentou-se como uma proteína com potencial ação protetora no cérebro, a julgar pelos efeitos sobre a DAC e sobre a reação microglial.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22324号 / 医博第4565号 / 新制||医||1041(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙折 晃史, 教授 竹内 理, 教授 生田 宏一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Microglia, the brain’s resident immune cells, exhibit constitutive expression of the ionized calcium-binding adaptor molecule 1 (IBA1). They are long-lived cells that exhibit a senescent morphology (dystrophy) with aging. It has been reported that dystrophy of IBA1-positive microglia is exacerbated in obese humans. Furthermore, we detected another microglial abnormality, which is the loss of IBA1 immunoreactivity that can create large areas in the brain seemingly devoid of all microglial cells. Here, we systematically compared microglial appearance in human hippocampi derived from obese individuals compared to controls by morphological and spatial analysis. In both groups, areas that were negative for IBA1 contained P2YR12 and glutathione-peroxidase 1 (GPX)-positive microglia. The number and extent of IBA1-negative regions was increased in obese cases. Since some cases of non-obese individuals also exhibited loss of IBA-1 immunoreactivity, we searched for possible confounders and found that hepatic dysfunction strongly impacts the distribution of microglial cells: a spatial analysis of scanned IBA1-stained sections increased Mean Empty Space distances (p=0.016) and IBA1-negative areas (p=0.090) which were independent of the cause of liver dysfunction, but also from aging, were detected. Thus, we report on a novel type of microglia pathological change, i.e. localized loss of IBA1 that is linked, at least in part, to obesity and hepatic dysfunction.:1 Introduction . . . . .. . . . . . . . .. . . . . . . . . . .. 2 1.1 Formation and morphology. .. . . . . . . . . . 2 1.2 Microglia and aging . . . . . . . . . . . . . . . . . 3 1.3 Microglia and obesity . . . . . . . .. . . . .. . . .5 1.4 Hippocampus . . . . . . . . . . . . . . . . . .. . . . 6 1.5 IBA1 . . . . . . . . . . . . . . . . . . . . . . . . .. . . . 7 1.6 Microglial markers . . . . . . . . . . . . .. . . . . 9 1.7 Hepatic dysfunction. . . . . . . . . . . . . . . . .10 1.8 Hepatic encephalopathy . . . . . . . . . . . . .10 1.9 Microglia in hepatic encephalopathy . . . 11 2 Aims and outlines of the dissertation . . . . .13 3 Manuscript of publication . . . . . . . . . . .. . . 15 4 Abstract . . . . . . . . . . .. . . . . . . . . . .. . . . . .25 5 Bibliography. . . . . . . . . . .. . . . . . . . . . .. . . 29 6 Appendix . . . . . . . . . . .. . . . . . . . . . .. . . . . 38 6.1 Supplemental Material .. . . . . . . . . . . . .. 38 7 Darstellung des eigenen Beitrags . . . . . . . 41 8 Erklärung über die eigenständige Abfassung der Arbeit . . . . . . . . . . .. . . . . .. . 42 10 Publications . . . . . . . . . . .. . . . . . . . . . . . .45 11 Acknowledgements. . . . . . . . . . .. . . . . . . 46

<p>There has been a rapid shift in food environment of Western cultures that has increased consumption of diets high in fat and sugar, which have imparted negative effects on metabolic and neurocognitive health. There is also building evidence that the adverse effects of Western diet</p> <p>(WD) are different in males and females, such that males are impacted more at an earlier age and females are impacted later in life. The underlying biological mechanisms linking WD and neurocognitive health are often associated with energy dysregulation or neuroinflammation. WD</p> <p>disrupts glucose homeostasis and causes low grade inflammation in the body, and these can impact</p> <p>the brain by disrupting the blood-brain barrier (BBB). The BBB is the microvasculature found throughout the entire brain that tightly regulates what compounds get into the brain to ensure optimal neuronal function. WD disrupts the BBB, however, the effects of WD on BBB integrity</p> <p>in females and younger individuals remain largely unknown. Based on the metabolic and behavioral effects of WD, we hypothesized that the effects are age- and sex- specific. To test this, we gave male and female rats access to a WD for 8-10 weeks starting in juvenile period (post-natal</p> <p>day 21) or in adulthood (post-natal day 75), then measured body weight, behavior, glucose tolerance, the density of two different markers of BBB integrity. We also measured density of resident immune cells (microglia) to assess the relationship between inflammation and BBB integrity. First, we focused on the impact of hyperglycemia on the BBB since elevated glucose alters glucose transporter 1 (GLUT1). We found sex- and age- specific decreases in GLUT1 density in the prefrontal cortex and hippocampus—two brain regions commonly associated with neurocognitive impairments associated with WD. Correlational comparisons between WD and chow (CH) animals also found that the typically relationship between glucose tolerance and</p> <p>GLUT1 in the PFC and hippocampus were overall disrupted in WD animals. Second, we measured the leakage of albumin, a blood protein, since WD depletes the tight junctions that would typically prevent albumin from entering the brain and triggering a neuroinflammatory response. We did not find an increase in albumin density in WD animals, however, we found a main effect of age which</p> <p>offers insight to differential susceptibilities to BBB leakage. Third, we focused on inflammation and found that WD did not impact microglia density in our experiments, nor did it correlate with GLUT1, albumin, or behavior. Collectively, our findings support the hypothesis that the impact of</p> <p>WD on the BBB is sex- and age- specific, suggest that WD does not increase leakage of compounds such as albumin, and highlights the nuanced relationships between WD, metabolic disruption, behavioral deficits, and neuroinflammation.  </p>