PRECONDITIONING: PMN ADHESION AND MICROVASCULAR INJURY

描述：（逐字从应用程序）：最近的一些结果 研究表明，短暂的局部缺血会增加 骨骼肌和其他组织的有害影响， 暴露于缺血和再灌注（IR）24小时后，出现一种称为 延迟或晚相缺血预适应（延迟IPC）。虽然 预处理减少缺血后组织损伤的机制是 不清楚，我们实验室的初步数据表明，延迟IPC 通过抑制白细胞粘附防止由IR诱导的肌肉坏死， 第二次缺血损伤后再灌注期间的迁移。但 在这一期间发起的活动之间的联系机制 缺血预处理对缺血后微血管的影响 功能障碍和肌细胞坏死尚不清楚。因此， 本申请中概述的项目旨在确定 延迟IPC可抑制氧化剂产生、P-选择素表达、白细胞 粘附和迁移穿过毛细血管后微静脉、微血管屏障 骨骼肌破裂、毛细血管无复流和肌细胞坏死 随后在24小时后暴露于延长的缺血和再灌注（IR）。 我们假设一氧化氮（NO）在延迟IPC中起关键作用， 最初作为触发器，然后随后作为 保护为解决这个问题，我们建议确定：（1）否 在预适应期间， 缺血并触发延迟IPC的保护作用;（2）eNOS活性 是由在重复的剪切过程中发生的增加的剪切应力刺激的。 在每个IPC周期后释放闭塞时发生的充血或 IPC诱导的缓激肽释放;（3） 预处理缺血通过A启动延迟IPC的保护作用 涉及黄嘌呤氧化酶衍生氧化剂生成的机制 （4）蛋白激酶C（P KC）有助于 延迟预处理mg，如果是这样，如果亚型特异性PKC易位是 （5）缺血预处理循环中NO的生成对心肌细胞的保护作用; 预处理骨骼肌再灌注过程中NO的产生有助于 延迟IPC的保护作用通过iNOS依赖机制。到 为了实现这些目标，我们将利用活体显微镜方法， 定量氧化剂产生、白细胞粘附和迁移、微血管 野生型小鼠提睾肌蛋白渗漏和毛细血管无复流 对照小鼠（C57 BL 16）和缺乏eNOS、iNOS或nNOS的转基因小鼠。的 延迟IPC对黄嘌呤氧化酶活性及IR诱导P-选择素的影响 表达和肌细胞坏死。亚型特异性PKC 易位，NOS mRNA水平，亚型表达和活动将被 在IPC和HR期间进行检查。拟议的研究不仅应实质上 提高我们对延迟IPC降低 骨骼肌微血管功能障碍和肌细胞坏死 随后的长时间缺血和再灌注，但也应 为开发药理学方法提供了理论基础， 复制了它非常强大的保护作用。

DESCRIPTION: (Verbatim from the application): The results of a number of recent studies indicate that brief episodes of ischemia increase the tolerance of skeletal muscle and other tissues to deleterious effects of a more prolonged exposure to ischemia and reperfusion (IR) 24 hours later, a phenomenon referred to as delayed or late phase ischemic preconditioning (delayed IPC). Although the mechanisms whereby preconditioning reduces postischemic tissue injury are not clear, preliminary data from our laboratory indicates that delayed IPC prevents muscle necrosis induced by IR by inhibiting leukocyte adherence and emigration during reperfusion after the second ischemic insult. However, the mechanisms linking events that are initiated during the period of preconditioning ischemia to the reduction in postischemic microvascular dysfunction and myocyte necrosis are unclear. Thus, the overall goal of the projects outlined in this application is to determine the mechanisms by which delayed IPC attenuates oxidant production, P-selectin expression, leukocyte adhesion to and emigration across postcapillary venules, microvascular barrier disruption, capillary no-reflow, and myocyte necrosis in skeletal muscles subsequently exposed to prolonged ischemia and reperfusion (IR) 24 hours later. We hypothesize that nitric oxide (NO) plays a critical role in delayed IPC, acting initially as a trigger and then subsequently as the mediator of the protection. To address this issue, we propose to determine whether: (1) NO derived from endothelial NOS is produced during the period of preconditioning ischemia and triggers the protective actions of delayed IPC; (2) eNOS activity is stimulated by the increased shear stress that occurs during the repeated hyperemias that occur on release of the occlusion after each cycle of IPC or by IPC-induced bradykinin release; (3) NO produced during the period of preconditioning ischemia initiates the protective effects of delayed IPC by a mechanism that involves the generation of xanthine oxidase derived oxidant species; (4) protein kinase C (P KC) contributes to the beneficial actions of delayed precondition mg and, if so, if isoform-specific PKC translocation is induced by the NO formed during the cycles of preconditioning ischemia; and (5) NO production during reperfusion of preconditioned skeletal muscles contributes to the protective actions of delayed IPC by an iNOS dependent mechanism. To accomplish these aims, we will utilize intravital microscopic approaches to quantify oxidant production, leukocyte adhesion and emigration, microvascular protein leakage, and capillary no-reflow in cremaster muscles in wild-type control mice (C57BLl6) and in transgenic mice lacking eNOS, iNOS or nNOS. The influence of delayed IPC on xanthine oxidase activity and IR induced P-selectin expression and myocyte necrosis will also be investigated. Isoform-specific PKC translocation, NOS mRNA levels, isoform expression, and activities will be examined during IPC and hR. The proposed studies should not only substantially improve our understanding of the mechanisms whereby delayed IPC reduces microvascular dysfunction and myocyte necrosis in skeletal muscles subjected to subsequent prolonged periods of ischemia and reperfusion but should also provide a rationale for the development of pharmacologic approaches that duplicate its remarkably powerful protective effects.