Neuronal and astroglial injury and recovery from stroke-induced depolarizations

描述（由申请人提供）：在缺血性卒中中，血流停止导致局部脑能量储备耗尽。在严重代谢受损的区域，ATP减少导致钠-钾泵失效，导致缺氧去极化（AD）从缺血核心传播。延长的AD引起神经元的急性损伤，表现为细胞肿胀沿着树突珠状和棘丢失，在没有再灌注的情况下变成终末损伤。持续时间较短（<5 min）的复发性梗死周围去极化（PID）始于核心边缘，并在卒中后数小时至数天内对缺血半暗带造成损害。AD和PID是常见的扩散去极化过程的变体，其取决于局部代谢损害的程度（AD在血流量<10%的核心中，PID在血流量为20-40%的半影中）。尽管星形胶质细胞对维持离子平衡、防止神经毒性和为神经元提供营养支持很重要，但对这些扩散性去极化导致的缺血性损伤的早期阶段的动态贡献尚不清楚。拟议的研究将明确定义AD和PID后神经元和星形胶质细胞损伤的程度以及再灌注期间的恢复。实验方法将涉及实时在体内2-光子激光扫描显微镜在单个神经元和星形胶质细胞的水平，同时与AD和PID的电生理记录在两个急性小鼠中风模型。具体目标是：1）验证缺血性损伤的缺氧去极化成分同时影响星形胶质细胞和神经元的假设。2)检验PID急性损伤半暗带中的星形胶质细胞和神经元，导致缺血核心扩张的假设。在目标1中，短暂性颈总动脉闭塞将用于评估神经元和星形胶质细胞损伤和恢复，同时伴有皮质直流（DC）电位（表示AD）和血流的变化。我推测，AD急性损伤星形胶质细胞除了神经元在缺血的早期阶段。在再灌注期间，需要健康的星形胶质细胞来恢复允许神经元再灌注的离子梯度。在目标2中将使用光血栓微循环闭塞，以在半暗带样“风险区域”周围创建缺血性病变。当PID在半影内传播时，该区域中的星形胶质细胞和神经元将被成像，从而在血流和代谢需求之间产生进一步的不匹配。我假设，只要有足够的血流，反复发作的PID损伤的神经元和星形胶质细胞就能够恢复。随着时间的推移，复极化的能量需求不再被减少的能量供应所满足，导致终末损伤和在细胞水平的分辨率上检测到的梗死核心的半影募集。这项工作将星形胶质细胞作为临床中风治疗的新靶点。局部麻醉剂地布卡因将被用于在实验子集中试图抑制PID引起的损伤，从而可能增加中风后的治疗窗口。 公共卫生相关性：作为这项工作的结果，我期望提供新的信息，星形胶质细胞和神经元之间的动态关系，在中风的早期事件。通过星形胶质细胞的恢复促进神经元网络的恢复可能为临床中风治疗提供新的途径。

DESCRIPTION (provided by applicant): In ischemic stroke, cessation of blood flow results in depletion of local brain energy reserves. In areas of severe metabolic compromise decreased ATP leads to failure of the sodium-potassium pump, resulting in anoxic depolarization (AD) propagating from the ischemic core. Prolonged AD causes acute injury to neurons manifested by cellular swelling along with dendritic beading and spine loss, becoming terminal injury in the absence of reperfusion. Shorter duration (<5 min) recurring peri-infarct depolarizations (PIDs) initiate at the edge of the core and cause damage in the ischemic penumbra for hours to days after stroke. AD and PIDs are variations of a common spreading depolarizing process that depends upon the degree of local metabolic compromise (AD in the core where blood flow is <10%, PID in the penumbra where blood flow is 20-40%). Though important for maintaining ionic balance, preventing neurotoxicity, and providing trophic support for neurons, the dynamic contributions of astrocytes to the early stages of ischemic injury resulting from these spreading depolarizations are not well understood. The proposed research will clearly define the extent of neuronal and astroglial injury after AD and PIDs and recovery during reperfusion. The experimental approach will involve real-time in vivo 2-photon laser scanning microscopy at the level of single neurons and astrocytes simultaneously with electrophysiological recordings of AD and PIDs in two acute mouse models of stroke. The specific aims are: 1) To test the hypothesis that the anoxic depolarization component of ischemic injury affects astrocytes concurrently with neurons. 2) To test the hypothesis that PIDs acutely damage astrocytes and neurons in the penumbra, resulting in expansion of the ischemic core. In Aim 1, transient common carotid artery occlusion will be used to assess neuronal and astroglial injury and recovery concurrently with changes in the cortical direct current (DC) potential (which signifies AD) and blood flow. I hypothesize that AD acutely injures astrocytes in addition to neurons during the early stages of ischemia. During reperfusion, healthy astrocytes are needed to restore the ionic gradients that allow neurons to repolarize. Photothrombotic microcirculatory occlusion will be used in Aim 2 to create an ischemic lesion surrounding a penumbra-like "area at risk". Astrocytes and neurons in this area will be imaged as PIDs propagate within the penumbra, creating further mismatch between blood flow and metabolic demand. I hypothesize that neurons and astrocytes damaged by recurring PIDs are able to recover as long as adequate blood flow is available. Over time, energy needs for repolarization are no longer met by the diminishing energy supply, resulting in terminal injury and penumbral recruitment into the infarct core detected at the cellular level of resolution. The proposed work should implicate astrocytes as a novel target for clinical stroke therapy. The local anesthetic dibucaine will be used in an attempt to inhibit damage caused by PIDs in a subset of experiments, potentially increasing the therapeutic window after stroke. PUBLIC HEALTH RELEVANCE: As a result of this work, I anticipate to provide novel information about the dynamic relationship between astrocytes and neurons during the early events of stroke. Restoration of neuronal networks facilitated by astroglial recovery may provide a novel avenue for stroke therapy in the clinic.