M-Type K+ Channels: In Vivo Neuroprotective Role during Cerebrovascular Stro

描述（由申请人提供）：中风是美国人类死亡的第三大原因，并且仍然是定义导致组织损伤的病理生理机制的重点。脑卒中后许多脑区发生神经元损伤和细胞损失，因此为研究提供了高度相关的背景。本项目通过“m通道”研究缺血性中风期间的神经保护作用，这是一种关键的K+通道，通过平衡兴奋性阳离子电流的去极化效应来稳定静息电位。电压门控M通道由KCNQ2-5亚基组合产生，在控制神经元兴奋性和动作电位放电中起关键作用。我们之前已经表明，大多数m型通道被活性氧（ROS）上调，活性氧通常在缺血性脑血管中风期间和之后产生。本研究假设M电流介导的神经元沉默通过增加KCNQ通道的开放概率，从而降低神经元活性，延长细胞级联的激活，从而导致细胞死亡，从而具有神经保护作用。使用了两种体内小鼠模型，第一种模型涉及由激光控制的光血栓形成产生的顶叶皮层内的脑梗死。当光敏染料“孟加拉玫瑰”被激光照射时，自由基的形成导致内皮损伤，血管内容物渗漏到薄壁组织和血管血栓形成。第二种体内模型是大脑中动脉闭塞（MCAo），在这种模型中，一根细丝通过颈动脉无创地进入大脑中动脉。MCAo模型在同侧半球产生灾难性的中风，相应的明显的大脑缺陷。在两种小鼠模型中，使用m通道打开剂瑞加滨（RTG）和氟吡汀，以及阻滞剂XE991，在中风后的不同时间点给药，m电流活性将发生药理学改变。此外，显性阴性KCNQ3敲入小鼠将用于两种模型，以确认特定的m通道机制。最后，设计用于区分损伤程度和神经功能的行为分析将用于描述由于通道激活或阻塞而导致的功能的微小变化。运动和协调缺陷将通过平衡木、梯子灵活性、露天运动和加速旋转测试进行分析。学习缺陷将使用Morris水迷宫进行分析。因此，本研究使用了两种强而兴奋的中风模型，这可能为通过调节m型K+通道的活性来减少常见的缺血性发作期间引起的神经元损伤提供新的治疗干预模式。

DESCRIPTION (provided by applicant): Stroke is the third leading cause of human death in the USA and remains the focus of efforts to define the pathophysiologic mechanisms leading to tissue damage. Neuronal damage and cell loss occurs in numerous brain regions after strokes, thus providing a highly-relevant context for study. This project investigates neuroprotection during ischemic stroke through "M-channels" which are critical K+ channels that stabilize resting potentials by counterbalancing the depolarizing effects of excitatory cation currents. Produced by combinations of KCNQ2-5 subunits, voltage-gated M channels play critical roles in control of neuronal excitability, and action potential firing. We have previously shown that most M-type channels to be up- regulated by reactive oxygen species (ROS), molecules commonly produced during and after ischemic cerebrovascular stroke. This study hypothesizes that M current-mediated neuronal silencing has a neuroprotective role by increasing the open-probability of KCNQ channels, thus decreasing neuronal activity and prolonging activation of cellular cascades resulting in cell death. Two in vivo mouse models are used and the first involves a cerebral infarct within the parietal cortex produced by laser-controlled photothrombosis. When the photo-sensitizing dye, Rose Bengal, is irradiated by laser light, free-radical formation occurs causing endothelium damage, leakage of vascular contents into the parenchyma and vascular thrombosis. The second in vivo model is the middle cerebral artery occlusion (MCAo), in which a filament is introduced non-invasively into the MCA via the carotid artery. The MCAo model produces a catastrophic stroke in the ipsilateral hemisphere, with correspondingly pronounced cerebral deficits. The M-current activity will be pharmacologically altered in both mouse models using the M-channel openers, retigabine (RTG) and flupirtine, and the blocker, XE991, with drug application at various time points post-stroke. Additionally, dominant- negative KCNQ3 knock-in mice will be used in both models to confirm a specific M-channel mechanism. Finally, behavioral assays designed to distinguish degrees of injury and neural function will be used to delineate minute changes in function due to channel activation or blockage. Motor and co-ordination deficits will be assayed with the balance beam, ladder dexterity, open-field locomotion and accelerating rotarod tests. Learning deficits will be assayed using the Morris water maze. Thus, this study uses two strong and exciting stroke models, which may provide novel modes of therapeutic intervention for reducing neuronal damage caused during commonly-occurring ischemic attacks, by modulating the activity of M-type K+ channels.