NEUTROPHILS IN ISCHEMIA REPERFUSION INJURY IN SHOCK

缺血后发生的损伤的很大一部分， 再灌注已经确定在再灌注期间发生。 这种再灌注损伤是白细胞介导的， 与单克隆抗体的粘附提供了显著的保护 从受伤。 同样，轻度低温（1-4摄氏度）， 局部缺血和再灌注导致损伤的显著减少。 缺血再灌注见于多种临床疾病 包括心肌梗塞、中风、器官移植、 失血性休克和外伤。很明显，这种广泛的 可能有再灌注损伤成分的临床疾病 将其列为美国死亡率和发病率的主要原因 States.这些疾病中的长时间缺血导致显著的 由于组织缺氧和灌注快速恢复而造成的损伤将减少 这种伤。然而，先前缺血组织的再灌注 可以引发炎症反应， 额外的组织损伤。已显示阻断白细胞粘附 通过实验来显着减少这种再灌注的部分 损伤同样，轻度低温已被证明可以减轻伤害 发生在缺血和再灌注时。本项目的假设 1）白细胞粘附分子的表达和/或活化 内皮细胞或中性粒细胞（pMN）依赖于小 温度变化（1至4摄氏度）。粘附分子功能 在轻度体温过低时降低，从而提供保护， 缺血再灌注损伤。2）有效的组织保护 再灌注后即刻轻度低温的结果 时间（4-8小时）。因此，维持患者轻度低温 在此期间，可以提供保护免受伤害， 显著的治疗益处。具体目标如下：目标1A： 测定粘附分子CD 62 P，CD 62 E， CD 106和CD 54的表达，以及使用 在33、35、37和39 ℃下进行ELISA和北方印迹分析。目的 1 B：通过了解脑下垂体介导的抑制机制 基因在内皮细胞中的表达，将有可能逆转 或激活体内低温效应-目的2：测量 刺激后PMN功能和激活的指标 含PMA的白细胞、热灭活细菌和C5 a。温度将 设定在33摄氏度、35摄氏度、37摄氏度和39摄氏度。目的 3：确定亚低温对白细胞滚动的影响， 在33、35、37和39 ℃的流动条件下的粘附性 摄氏度目的4：确定表达的定量变化 CD 62 P、CD 62 E和CD 54及其mRNA在兔耳缺血再灌注损伤中的表达 在22摄氏度和24摄氏度的环境温度下再灌注。 目的5：确定有效的低温治疗时机， 防止缺血和再灌注后的组织损伤。目标6： 确定以下温度降低的保护效果 失血性休克和复苏。本项目的实验 将使用细胞和分子生物学技术， 使用体内和体外实验。

A significant portion of the injury that occurs following ischemia and reperfusion has been established to occur during the reperfusion period. This reperfusion injury is leukocyte mediated and blocking leukocyte adherence with monoclonal antibodies provides significant protection from injury. Likewise, mild hypotheremia (1-4 degrees C) during ischemia and reperfusion results in a significant reduction in injury. Ischemia-reperfusion is seen in a variety of clinical disorders including myocardial infarction, stroke, organ transplantation, hemorrhagic shock and traumatic injuries. Clearly, this broad spectrum of clinical disorders that may have a component of reperfusion injury places it as a major cause of mortality and morbidity in the United States. Prolonged ischemia in these disorders results in significant injury due to tissue hypoxia and rapid return of perfusion will reduce this type of injury. However, reperfusion of previously ischemic tissue can initiate an inflammatory response that results in significant additional tissue injury. Blocking leukocytes adhesion has been shown experimentally to significantly reduce portion of this reperfusion injury. Likewise, mild hypothermia has been shown to reduce the injury that occurs in ischemia and reperfusion. The hypotheses of this project are: 1) The expression and/or activation of leukocyte adhesion molecules on endothelial cells or on neutrophils (pMNs) is dependent on small changes in temperature (1 to 4 degrees C). Adhesion molecule function is reduced during mild hypothermia, thus providing protection from ischemia and reperfusion injury. and 2) Significant tissue protection results from mild hypothermia during the immediate post reperfusion period (4-8 hours). Therefore, maintaining patients slightly hypothermic during this period may provide protection from injury and be of significant therapeutic benefit. The specific aims are: Aim 1 A: To determine the stimulated expression of adhesion molecules CD62P, CD62E, CD106, and CD54 as well as expression of mRNA by cultured HUVECs using ELISA and Northern blot analysis at 33, 35, 37, and 39 degrees C. Aim 1 B: By understanding the mechanism of hypothermia-mediated inhibition of gene expression in endothelial cells, it will be possible to reverse or activate the effects of hypothermia in vivo - Aim 2: To measure indicators of PMN function and activation following stimulation of leukocytes with PMA, heat killed bacteria, and C5a. Temperature will be set at 33 degrees C, 35 degrees C, 37 degrees C and 39 degrees C. Aim 3: To determine the effect of mild hypothermia on leukocyte rolling and adherence under flow conditions at temperatures of 33, 35, 37, and 39 degrees C. Aim 4: To determine the quantitative changes in expression of CD62P, CD62E and CD54 and their mRNA in rabbit ear ischemia- reperfusion at ambient temperatures of 22 degrees C and 24 degrees C. Aim 5: To determine the timing of hypothermia that is effective in preventing tissue injury following ischemia and reperfusion. Aim 6: To determine the protective effects of reduced temperature following hemorrhagic shock and resuscitation. The experiments of this project will use cell and molecular biology techniques and will be completed by using in vivo and in vitro experiments.