Chronic imaging of cellular dynamics after cortical microhemorrhage

描述（由申请人提供）：脑微出血与认知能力下降和痴呆风险增加有关，但小出血影响神经细胞功能的机制尚不清楚，部分原因是缺乏良好的动物模型。在最近的工作中，我们使用了一种新的基于激光的方法来破裂小鼠大脑中的靶向小动脉，并使用双光子显微镜对这些病变的影响进行成像。令人惊讶的是，我们没有发现神经细胞死亡或树突病理改变的证据，这两种情况都是在小动脉闭塞后观察到的。相反，我们观察到炎症细胞的快速启动和局部持续激活。这些数据表明，微出血不会通过杀死脑细胞或驱动神经突营养不良而导致认知功能障碍，而是以更微妙的方式影响神经功能。最近的研究表明，小胶质细胞在发育和成年期突触的监视和修剪中起着积极的作用。此外，这些细胞的激活与突触脊柱更新率升高有关，但总体脊柱密度没有变化。在这里，我们提出验证微出血导致脊柱周转率异常增加的假设，这种增加是由激活的炎症细胞与突触的相互作用引起的。突触转换的升高与脑损伤（如中风）后促进功能恢复的可塑性有关。然而，对于这里研究的微出血，没有神经损伤的迹象，这表明任何神经回路的重新布线都可能导致功能障碍。在Aim 1中，使用微出血后的慢性双光子成像来区分脑内小胶质细胞和血源性巨噬细胞在炎症反应中的作用。这些实验使用通过骨髓移植产生的嵌合动物来实现小胶质细胞或巨噬细胞的特异性标记。然后，我们测试了阻断小胶质细胞激活（抑制P2Y受体）和巨噬细胞募集（阻断白细胞粘附）的方法。这项工作将量化微出血后脑炎症的空间尺度、时间动态和细胞参与者，并建立调节这种炎症反应的工具。在Aim 2中，与对照组相比，我们量化了微出血附近脊柱转换率和兴奋性和抑制性神经元密度的变化。我们的初步数据表明，两周以上的离职率会增加两倍。然后，我们确定抑制小胶质细胞激活和/或巨噬细胞募集是否会使脊柱周转率向基线水平转移。如果这是真的，这将支持我们的假设

DESCRIPTION (provided by applicant): Brain microhemorrhages have been linked to cognitive decline and increased dementia risk, but the mechanisms by which small bleeds affect the function of neural cells remains unclear, in part due to a lack of good animal models. In recent work, we used a novel laser-based approach to rupture targeted arterioles in the brain of mice and imaged the impact of these lesions using two-photon microscopy. Surprisingly, we found no evidence of neural cell death or pathological changes in dendrites, both of which are observed after occlusion of small arterioles. Instead, we observed a rapidly-initiated and locally-sustained activation of inflammatory cells. These data suggest that microhemorrhages do not lead to cognitive dysfunction by killing brain cells or driving neurite dystrophy, but rather impac neural function in more subtle ways. Recent work has revealed that microglia play an active role in surveillance and pruning of synapses in development and adulthood. In addition, activation of these cells has been associated with elevated rates of synaptic spine turnover, but without a change in overall spine density. Here, we propose to test the hypothesis that microhemorrhages cause aberrant increases in the rate of spine turnover and that this increase is caused by the interaction of activated inflammatory cells with synapses. Elevations in synaptic turnover have been associated with plasticity that facilitates functional recovery after brain injury, such as stroke. For the microhemorrhages studied here, however, there is no indication of neural injury, suggesting that any rewiring of neural circuits is likely to cause dysfunction. In Aim 1, chronic two-photon imaging after microhemorrhage is used to distinguish the role of brain-resident microglia from blood-derived macrophages in the inflammatory response. These experiments use chimeric animals created through bone marrow transplants to achieve specific labeling of either microglia or macrophages. We then test approaches to block microglia activation (inhibition of P2Y receptors) and macrophage recruitment (blocking of leukocyte adhesion). This work will quantify the spatial scale, temporal dynamics, and cellular players in brain inflammation after microhemorrhage and establish tools to modulate this inflammatory response. In Aim 2, we quantify changes in the rate of spine turnover and density in excitatory and inhibitory neurons near a microhemorrhage as compared to controls. Our preliminary data suggests a two-fold elevation in turnover rates that is sustained beyond two weeks. We then determine if inhibition of microglial activation and/or macrophage recruitment shifts the spine turnover rate toward baseline levels. If true, this would support our hypothesis that the action of inflammatory cells after a microhemorrhage elevates spine turnover. Finally, we pilot experiments to directly image alterations in the interaction of inflammatory cell processes with synaptic spines after a microhemorrhage. Brain inflammation occurs in many neurodegenerative diseases, such as Alzheimer's disease, and the inflammatory cell-mediated increase in synapse turnover studied here may also contribute to the cognitive impact of these conditions.