Chronic imaging of cellular dynamics after cortical microhemorrhage

描述(申请人提供)：脑微出血与认知能力下降和痴呆症风险增加有关，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成像了这些损伤的影响。令人惊讶的是，我们没有发现神经细胞死亡或树突病理变化的证据，这两者都是在小动脉闭塞后观察到的。相反，我们观察到了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导致轴突营养不良而导致认知功能障碍，而是以更微妙的方式导致Impac神经功能。最近的研究表明，在发育和成年期，小胶质细胞在突触的监视和修剪中发挥着积极的作用。此外，这些细胞的激活与突触脊椎更新率的升高有关，但对整体脊柱密度没有影响。在这里，我们建议检验这一假设，即微出血导致脊柱翻转速度的异常增加，并且这种增加是由激活的炎症细胞与突触的相互作用引起的。突触更新率的升高与可塑性有关，可塑性有助于脑损伤(如中风)后的功能恢复。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连接都可能导致功能障碍。在目标1中，使用微出血后的慢性双光子成像来区分脑内驻留的小胶质细胞和血液来源的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创造的嵌合动物来实现对小胶质细胞或巨噬细胞的特定标记。然后我们测试阻断小胶质细胞激活(抑制P2Y受体)和巨噬细胞募集(阻断白细胞黏附)的方法。这项工作将量化微出血后脑部炎症的空间尺度、时间动力学和细胞角色，并建立工具来调节这种炎症反应。在目标2中，我们量化了与对照组相比，脊柱周转率和微出血附近兴奋性和抑制性神经元密度的变化。我们的初步数据显示，离职率上升了两倍，并持续了两周以上。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将脊柱周转率改变到基线水平。如果是真的，这将支持我们的假设，即 微出血后的炎细胞增加了脊柱的周转率。最后，我们试行实验，直接成像微出血后炎症细胞过程与突触棘突相互作用的变化。脑部炎症发生在许多神经退行性疾病中，如阿尔茨海默病，这里研究的炎症细胞介导的突触更新增加也可能有助于这些疾病的认知影响。

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.