NEUTROPHILS IN ISCHEMIA REPERFUSION INJURY IN SHOCK

很大一部分损伤发生在缺血和 再灌流已被确定为发生在再灌注期。 这种再灌注损伤是由白细胞介导的，并阻断了白细胞。 对单抗的坚持提供了显著的保护作用 远离伤病。同样，轻度低体温症(1-4摄氏度)在 缺血和再灌流可显著减轻损伤。 缺血再灌注可见于多种临床疾病。 包括心肌梗塞，中风，器官移植， 失血性休克和创伤性损伤。显然，这一广谱 可能有再灌流损伤成分的临床疾病 使其成为美国死亡和发病的主要原因 各州。在这些疾病中，长期的缺血会导致显著的 由于组织缺氧和快速恢复灌注造成的损伤将会减少 这种类型的伤害。然而，先前缺血的组织的再灌流 可以引发炎症反应，导致显著的 额外的组织损伤。阻断白细胞黏附已被证明 实验上显著减少了部分再灌流 受伤。同样，亚低温已被证明可以减少损伤。 这发生在缺血和再灌流中。这个项目的假设 主要有：1)白细胞黏附分子的表达和/或激活 内皮细胞或中性粒细胞(PMN)依赖于小分子 温度变化(1到4摄氏度)。黏附分子功能 在亚低温期间减少，从而提供保护 缺血再灌注损伤。2)显著的组织保护 再灌注后即刻亚低温的结果 时段(4-8小时)。因此，维持患者的轻微体温 在此期间可提供免受伤害的保护，并 显著的治疗效益。具体目标是：目标1 A：至 检测黏附分子CD62P、CD62E、 人脐静脉内皮细胞CD106、CD54及其mRNA的表达 33℃、35℃、37℃和39℃时的EL ISA和Northern印迹分析 1B：通过了解低温介导的抑制机制 在内皮细胞中的基因表达的改变，将有可能逆转 或激活体内低温的影响-目标2：测量 刺激后中性粒细胞功能和激活的指标 含PMA的白细胞、热致死细菌和C5a。气温将会是 设置为33摄氏度、35摄氏度、37摄氏度和39摄氏度瞄准 3.观察亚低温对小鼠白细胞滚动和运动的影响。 在33、35、37和39℃的流动条件下的粘附性 度C.目标4：确定表达的数量变化 兔耳缺血后CD62P、CD62E、CD54及其mRNA的表达 在22摄氏度和24摄氏度的环境温度下再灌流。 目的5：确定有效的低温治疗时机。 预防缺血再灌流后的组织损伤。目标6：达到 确定以下降温措施的保护效果 失血性休克和复苏。该项目的实验 将使用细胞和分子生物学技术，并将于 采用体内和体外实验。

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.