Endogenous glutathione protects cerebral endothelial cells from traumatic injury

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.