Gidday J M, Beetsch J W, Park T S
Department of Neurosurgery, Washington University School of Medicine and St. Louis Children's Hospital, Missouri 62110, USA.
J Neurotrauma. 1999 Jan;16(1):27-36. doi: 10.1089/neu.1999.16.27.
Blood-brain barrier breakdown and edema, indicative of cerebrovascular injury, are characteristic pathophysiologic outcomes following head trauma. These injuries result from both primary mechanical damage and from secondary events initiated by the traumatic insult. Free radicals are recognized as mediators of secondary injury in a number of trauma models. In this study, we used a novel in vitro model of traumatic microvascular injury to test the hypothesis that endogenous glutathione protects cerebral endothelial cells from secondary autooxidative injury following mechanical trauma. Porcine brain cerebral endothelial cells were grown in tissue culture wells with Silastic membrane bottoms, and cellular injury was induced by displacing the membrane different distances with user-defined pressure pulses from a customized device. The resultant endothelial cell injury 2 h following stretch was determined by measuring lactate dehydrogenase in the culture media. Significant stretch-dependent increases in endothelial injury were elicited that depended in a nonlinear fashion on the degree of membrane displacement. Depletion of intracellular glutathione with buthionine sulfoximine (1 mM) increased the extent of traumatic endothelial cell injury by 17-56%, particularly at low to moderate levels of traumatic injury (30-40% of total endothelial cell LDH release). Conversely, traumatic injury was reduced by 22-45% when endothelial cell glutathione levels were augmented threefold (to 140+/-8 nmol/mg protein) by preincubating cells with 2 mM glutathione; the extent of protection was inversely proportional to the extent of the traumatic stretch. Traumatic endothelial cell injury was also significantly and dose-dependently attenuated (up to 40%) by treatment with the xanthine oxidase inhibitor oxypurinol (50 and 100 microM). These results demonstrate that cerebral endothelial cells are the targets of hydrogen peroxide-mediated injury secondary to trauma-induced superoxide radical formation via the xanthine oxidase pathway. The neutralization of peroxides by the endogenous glutathione redox cycle provides endothelial cells a finite capacity to reduce free radical-mediated traumatic injury; this cycle may be amenable to therapeutic manipulation to mitigate posttraumatic edema and other manifestations of vascular dysfunction.
血脑屏障破坏和水肿是颅脑创伤后脑血管损伤的典型病理生理结果。这些损伤既源于原发性机械损伤，也源于创伤性损伤引发的继发性事件。在许多创伤模型中，自由基被认为是继发性损伤的介质。在本研究中，我们使用了一种新型的创伤性微血管损伤体外模型，以检验内源性谷胱甘肽可保护脑内皮细胞免受机械创伤后继发性自氧化损伤这一假说。猪脑脑内皮细胞在底部带有硅橡胶膜的组织培养孔中生长，通过使用定制设备施加用户定义的压力脉冲使膜移位不同距离来诱导细胞损伤。拉伸后2小时的内皮细胞损伤通过测量培养基中的乳酸脱氢酶来确定。引发了与拉伸相关的内皮损伤显著增加，且这种增加以非线性方式依赖于膜移位程度。用丁硫氨酸亚砜胺（1 mM）耗尽细胞内谷胱甘肽会使创伤性内皮细胞损伤程度增加17% - 56%，尤其是在低至中度创伤水平时（占内皮细胞总乳酸脱氢酶释放量的30% - 40%）。相反，通过用2 mM谷胱甘肽预孵育细胞使内皮细胞谷胱甘肽水平增加三倍（至140±8 nmol/mg蛋白）时，创伤性损伤减少了22% - 45%；保护程度与创伤性拉伸程度成反比。用黄嘌呤氧化酶抑制剂奥昔嘌醇（50和100 microM）处理也显著且剂量依赖性地减轻了创伤性内皮细胞损伤（高达40%）。这些结果表明，脑内皮细胞是创伤诱导的超氧化物自由基通过黄嘌呤氧化酶途径形成后过氧化氢介导的继发性损伤的靶点。内源性谷胱甘肽氧化还原循环对过氧化物的中和为内皮细胞提供了有限的能力来减少自由基介导的创伤性损伤；这个循环可能适合进行治疗性调控，以减轻创伤后水肿和血管功能障碍的其他表现。
