Subarachnoid Injections of Lysed Blood Induce the hsp70 Stress Gene and Produce DNA Fragmentation in Focal Areas of the Rat Brain

Abstract

Background and Purpose Most experimental studies of subarachnoid hemorrhage have demonstrated little histological evidence of injury. In the present study we examined both the expression of the hsp70 heat-shock gene, a molecular marker of reversible neuronal injury, and DNA fragmentation, a marker of irreversible cell injury and death. Methods Lysed blood, whole blood, oxyhemoglobin, bovine serum albumin, and saline were injected into the cisterna magna of adult rats. The induction of hsp70 mRNA and HSP70 heat-shock protein was assessed with the use of in situ hybridization and immunocytochemistry, respectively. Fragmentation of genomic DNA was studied by DNA nick end-labeling with the use of terminal deoxynucleotidyl transferase and biotinylated dATP. Results Expression of the hsp70 gene was not induced in the brains of rats injected with whole blood, oxyhemoglobin, bovine serum albumin, or saline. Lysed blood injections, however, induced hsp70 mRNA at 6 and 24 hours in the cerebellar hemispheres and in focal regions of the basal forebrain. HSP70 protein was induced by 24 hours and persisted for at least 4 days in the same regions. HSP70 protein was localized to patches of glial cells and occasional neurons in the forebrain. In the cerebellum HSP70 was localized to Bergmann glial cells, granule cells, molecular layer stellate cells, and occasional Purkinje cells. DNA nick end-labeling showed patches of labeled cells in the basal forebrain that occurred in the same regions that hsp70 mRNA was induced. Conclusions The results demonstrate focal stress gene induction and DNA fragmentation after subarachnoid hemorrhage. It is hypothesized that the focal areas of hsp70 induction may reflect ischemic injury due to vasospasm produced by lysed blood and/or injury mediated by direct toxic effects of the lysed blood. The hsp70 induction and DNA nick end-labeling in the same regions suggests that lysed blood produces a spectrum of injury from HSP70 protein-labeled, reversibly injured cells to dead cells with fragmented DNA. Induction of the hsp70 stress gene and DNA nick end-labeling may be useful for evaluating the causes of injury, the spectrum of injury, and potential pharmacological therapies in experimental models of subarachnoid hemorrhage.