Unhealthy ageing and memory loss associated with vascular insufficiency

血管周围星形胶质细胞在大脑中丰富，在神经元和血管之间形成物理桥梁。此外，星形胶质细胞控制神经血管耦合，使局部脑血流量与区域神经元能量使用相匹配，确保大脑的正常功能。血管功能不全发生数年，无明显临床症状，可因环境压力或生活方式的选择而加重（Ritchie et al. 2001）。星形胶质细胞对血管功能不全引起的内皮细胞激活做出反应，随着年龄的增长，这些过程对大脑功能有巨大的影响。最近，我们已经证明，当星形胶质细胞对病理变化做出反应时，它们的氧化和一氧化氮通路失调，这阻碍了星形胶质细胞相应地调节神经血管偶联（Sarmiento et al. 2020）。因此，我们寻求进一步研究受损能量资源与血管周围脑细胞之间的联系，以更好地了解神经血管耦合如何受到影响，以及在不健康衰老过程中大脑损伤和功能降低的倾向。1)您将接受培训，以优化产后和成年小鼠的脑细胞和/或脑切片的培养。这被称为原代培养，这是一项必要的实验室技术，需要高水平的技能。随后，你们将接受培训，调查氧-葡萄糖剥夺（OGD）的影响。这是一个环境压力的实验模型，它模拟了营养不足时大脑中的情况，营养不足可能是由于与年龄相关的血管功能不全造成的，也可能与生活方式有关。您将接受培训，评估星形胶质细胞和内皮细胞（即血管）的反应性和健康使用特定的细胞标志物（如GFAP， STAT3，血管生成素-2，CD-31），以及信号通路相关的神经血管耦合在相同的细胞（血管活性分子，如COX-1， iNOS, 20-HETE）。此外，内皮和星形细胞功能的电生理测量与细胞内钙（Fura 2）和一氧化氮（DAF-FM）的测量将与相同环境应激下细胞标志物和信号通路的变化相关。2)在描述星形细胞对ODG的反应后，您将使用优化的体外方案培养血管周围细胞和/或来自小鼠模型的脑切片，该模型概括了血管功能不全的特征，如内皮激活、神经炎症和记忆缺陷（Boehm-Sturm et al. 2017，卒中）。这是一个外科模型，如果你愿意，你可以接受动物处理和外科手术方面的培训。脑区域将被分离，用于培养原代细胞和/或切片，以检查参与血管功能不全反应的分子通路的变化，作为翻译管道的下一步。我们假设星形胶质细胞将作为手术的结果而被激活为反应性表型，这将使我们能够研究支撑这一现象的分子机制，并可能适用于治疗。我们的团队一直在研究几种影响大脑氧化应激途径的化合物，这些化合物将在培养的细胞中进行检测，可能会与使用神经保护药物对OGD进行进一步的阈值操作相结合。3)最终，对组织培养有益的化合物可以在同一手术小鼠模型中进行测试。功能结果将通过磁共振成像（MRI）（可能包括计算图像分析的训练）或行为测试（取决于资金和学生的偏好）在整个生物体水平上进行评估。然而，对化合物功效的机制洞察将通过组织学和分子生物学技术得到证实。

Perivascular astrocytes are abundant in the brain and form a physical bridge between neurons and blood vessels. In addition, astrocytes control neurovascular coupling that serves to match local cerebral blood flow to regional neuronal energy use and ensures normal functioning of the brain. Vascular insufficiency takes place over several years without any obvious clinical symptoms and could be exacerbated by environmental stress or lifestyle choices (Ritchie et al. 2001). Astrocytes in particular respond to endothelial cell activation that arises from vascular insufficiency, and collectively these processes have a huge impact on brain function as we age. Recently, we have shown that when astrocytes react to pathological changes, they have dysregulated oxidative and nitric oxide pathways and this prevents astrocytes to regulate neurovascular coupling accordingly (Sarmiento et al. 2020). Therefore, we seek to further investigate the link between compromised energy resources and perivascular brain cells to better understand how neurovascular coupling may be affected and predispose the brain to damage and reduced function during unhealthy ageing. 1) You will be trained to optimise the culture of brain cells and/or brain slices from postnatal and adult mice. This is called primary culture, and it is an essential laboratory technique that requires a high level of skill. Subsequently, you will be trained to investigate the effects of oxygen-glucose deprivation (OGD). This is an experimental model of environmental stress that mimics what happens in the brain when there are insufficient nutrients, which may occur as a result of vascular insufficiency associated with age and may be compounded by lifestyle. You will be trained to assess the reactivity and health of astrocytes and endothelial cells (i.e., blood vessels) using specific cell markers (e.g., GFAP, STAT3, angiopoietin-2, CD-31), as well as signalling pathways associated with neurovascular coupling in the same cells (vasoactive molecules such as COX-1, iNOS, 20-HETE). In addition, electrophysiological measurements of endothelial and astrocytic functions in conjunction with measurements of intracellular calcium (Fura 2) and nitric oxide (DAF-FM) will be correlated to changes in cell markers and signalling pathways in the same environment stress.2) Following characterisation of astrocytic responses to ODG, you will use the optimised in vitro protocols to culture perivascular cells and/or brain slices from a mouse model that recapitulates features of vascular insufficiency such as endothelial activation, neuroinflammation and memory deficits (Boehm-Sturm et al. 2017, Stroke). This is a surgical model, and the possibility exists to be trained in animal handling and surgical procedures if you desire. Brain regions will be isolated for the culture of primary cells and/or slices to examine changes in the molecular pathways involved in response to vascular insufficiency as the next step in the translational pipeline. We hypothesise that the astrocytes will be primed towards a reactive phenotype as a result of the surgical procedure, and this will allow us to investigate molecular mechanisms that underpin this and may be amenable to treatment. Our groups have been working with several compounds that influence oxidative stress pathways in the brain, and these will be examined in the cultured cells, potentially in conjunction with further threshold manipulations of OGD with neuroprotective drugs. 3) Ultimately, compounds that provide benefit for the tissue culture could be tested in the same surgical mouse model. Functional outcomes will be assessed at the whole organism level using either magnetic resonance imaging (MRI) (which could include training in computational image analysis) or behaviour testing (depending on funding, and preferences of the student). However, mechanistic insight into the compound's efficacy will be confirmed using histology, and molecular biology techniques.