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，目前仍是确定导致组织损伤的病理生理机制的重点。中风后许多脑区会发生神经元损伤和细胞丢失，因此为研究提供了高度相关的背景。本项目通过“M-通道”研究缺血性卒中的神经保护作用。“M-通道”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亚基组成，在控制神经元兴奋性和动作电位放电中起着关键作用。我们之前已经证明，大多数M型通道都受到活性氧簇(ROS)的上调，ROS是缺血性脑血管卒中期间和之后通常产生的分子。本研究假设M电流介导的神经元沉默通过增加KCNQ通道的开放概率而具有神经保护作用，从而降低神经元的活动，延长导致细胞死亡的细胞级联激活。使用了两个活体小鼠模型，第一个模型涉及到由激光控制的光血栓形成引起的顶叶皮质内的脑梗塞。当光敏染料Rose Bengal被激光照射时，会产生自由基，导致内皮损伤、血管内容物渗入实质和血管血栓形成。第二种体内模型是大脑中动脉闭塞(MCAO)，通过颈动脉将细丝非侵入性地引入大脑中动脉。MCAO模型在同侧大脑半球产生了灾难性的中风，并相应地出现了明显的大脑缺陷。在两种小鼠模型中，使用M通道开放剂瑞替加宾(RTG)和氟平汀，以及阻滞剂XE991，在中风后的不同时间点应用药物，都将从药物上改变M电流的活性。此外，在两种模型中都将使用显性阴性的KCNQ3敲入小鼠来确认特定的M-通道机制。最后，旨在区分损伤程度和神经功能的行为分析将被用来描述由于通道激活或堵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、开阔场地运动和加速转杆测试进行分析。学习障碍将使用莫里斯水迷宫进行测试。因此，本研究使用了两种强有力的、令人兴奋的卒中模型，通过调节M型K+通道的活性，为减少常见的脑缺血发作时神经元的损伤提供了新的治疗干预模式。 公共卫生相关性：将调查M型K+通道在限制脑血管中风期间缺血/缺氧性损害中的重要作用。体内研究将为中风提供新颖、创新和开创性的病理生理学见解，并为这种毁灭性的常见疾病提供潜在的新治疗方法。

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. PUBLIC HEALTH RELEVANCE: The vital role of M-type K+ channels in limiting ischemic/hypoxic damage during cerebrovascular stroke will be investigated. In vivo studies will provide novel, innovative and ground-breaking pathophysiologic insights into stroke, as well as provide potential new therapeutic approaches to this devastating, common disorder.