Traumatic brain injury results in focal cell damage in areas of the central nervous system that are relatively remote from the site of trauma. Such secondary focal damage may be a critical determinant in the degree of functional recovery after traumatic brain injury. In preliminary data we demonstrate a close association between subarachnoid hemorrhage (SAH) and focal tissue damage after experimental brain trauma. A similar pattern of focal damage is observed after experimental SAH. Taken together, these data support the hypothesis that SAH may be a critical component in the secondary pathogenesis of traumatic brain injury. In this application we employ a model of experimental SAH to study the mechanisms of focal cellular injury and relate these findings to experimental brain trauma. A central hypothesis is that heme oxygenase-1 (HO-1), the 32kDa stress protein, protects brain cells from hemoglobin-induced oxidative stress. To test this hypothesis, we will determine if 1) experimental SAH or traumatic brain injury in the rat is consistently associated with induction of stress proteins in focal areas of the brain and 2) induction of HO-1 prior to brain trauma provides neuroprotection. The role of oxidative stress in experimental SAH, will be evaluated using transgenic mice that overexpress CuZn superoxide dismutase, and knock-out mice that do not express this enzyme. Outcome measures for these studies include in situ hybridization and quantitative immunocytochemistry to study the response of stress proteins and assessment of cell injury and death using HSP70 as a marker for cell injury and by analysis of DNA fragmentation. After SAH, lysis of erythrocytes exposes the brain to oxyhemoglobin, a known spasmogen. We propose that ischemia, resulting from lysed hemoglobin, contributes to focal cell injury. To test this hypothesis we will determine if focal areas of stress protein induction represent areas of ischemia. Outcome measures include assessment of local cerebral blood flow and cerebral glucose utilization. We have demonstrated the induction of HO-1 in microglia in experimental SAH. We hypothesize that hemoglobin, released from the lysis of blood, diffuses through the extracellular space and is transported into microglia where HO-1 is induced to metabolize the heme and ferritin is induced to bind to the released iron. The extracellular paths for heme will be defined by immunocytochemistry. The induction of HO-1 and ferritin will be assessed by in situ hybridization and immunocytochemistry.
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