A scalable optogenetic system of focal stroke induction in zebrafish for testing stroke disparity genes

项目摘要/摘要 中风是美国主要的死亡原因之一，也是成人严重残疾的主要原因 社会负担。缺血性中风，由脑动脉血栓栓塞性闭塞引起，构成 大多数中风发生，其余的是血管破裂的出血性中风。在美国， 中风发病率在非裔美国人和高加索人之间存在明显差异，发病率要高得多 (约240%)在黑人中。尽管密集寻找治疗方法，重组组织型纤溶酶原激活剂(RtPA) 仍然是FDA批准的唯一疗效有限的中风后药物。在这令人悲哀的不足的背后 中风疗法的缺乏在于难以产生足够大量的中风诱发疾病 用于有效药物筛选的动物，作为一种高度劳动密集型的外科程序(大脑中动脉 闭塞)仍然是诱发模型动物缺血性卒中的首选方法。 光血栓形成是一种迅速被采用的新方法来诱导啮齿类动物的缺血性卒中，有许多 具有高度可重复性的梗塞面积和位置，死亡率最低等优点。在这个过程中，焦点 限定波长的光照射暴露的头骨可激活静脉注射的光敏化学物质 (例如，在血液中，导致内皮细胞损伤和局部血小板聚集，导致 与受影响的血管堵塞有关。然而，即使在这种情况下，劳动密集型程序也不能 必须使用不易放大的方法来创建笔划模型。最近，光凝血性缺血性卒中 也成功地在成年斑马鱼的大脑中通过手动注射后的焦点照明诱导 从而为中风研究提供了一个额外的脊椎动物模型系统。在这方面， 一种令人兴奋的新的遗传方法，它消除了手动注射光敏化学物质， 最近成功制造和测试了选择性地通过光照诱导成人细胞凋亡的方法 斑马鱼的心脏。 在这里，我们建议创建一种易于扩展的、光遗传诱导的中风系统，该系统使用 转基因斑马鱼，开发行为测试系统来识别新的治疗化学物质， 并测试人类APOL1的GWAS(基因组范围关联研究)确定的中风风险变体 基因，在非洲血统的人中也被称为突出的肾脏疾病风险因素。

PROJECT SUMMARY/ABSTRACT Stroke is one of the leading causes of death in the U.S. and also a leading cause of adult disability with severe societal burdens. Ischemic strokes, caused by the thromboembolic occlusion of cerebral arteries, constitute the majority of stroke incidences and the rest are hemorrhagic strokes with ruptured blood vessels. In the U.S., stroke incidence shows clear disparity between African Americans and Caucasians with much higher frequency (~240%) in blacks. Despite intensive search for treatment, recombinant tissue plasminogen activator (rtPA) remains the only FDA-approved post-stroke medicine with limited effectiveness. Behind this woefully inadequate dearth of stroke therapeutics lies the difficulties in generating a sufficiently large number of stroke-induced animals for effective drug screening, as a highly labor-intensive surgical procedure (middle cerebral artery occlusion) is still the method of choice to induce ischemic strokes in model animals. Photothrombosis is one of rapidly adopted new methods to induce ischemic strokes in rodents, with a number of advantages such as highly reproducible infarct size and location with minimal mortality. In this procedure, focal illumination of defined wavelength light on the exposed skull activates an IV-injected photosensitive chemical (eg. Rose-Bengal) in the bloodstream, causing injuries in endothelial cells and local platelet aggregation, leading to the clogging of the affected blood vessel. However, even in this case labor-intensive procedures that cannot not easily be scaled up must be utilized to create the stroke model. Recently, photothrombic ischemic stroke has also been successfully induced in the adult zebrafish brain by focal illumination following a manual injection of Rose-Bengal, thus providing an additional vertebrate animal model system for stroke research. In this regard, an exciting novel genetic approach, which eliminates the manual injection of photosensitive chemicals, was recently created and tested successfully to selectively induce apoptotic cell death by light illumination in the adult zebrafish heart. Here we propose a creation of a readily scalable, optogenetically induced stroke system using transgenic zebrafish, development of a behavioral test system to identify novel therapeutic chemicals, and to test GWAS (genome wide association study)-identified stroke risk variants of the human APOL1 gene, also known as prominent kidney disorder risk factors in people of African ancestry.