Introduction: The pathophysiology of subarachnoid hemorrhage (SAH) is complex and includes disruption of the blood-brain barrier (BBB). We have previously optimized methods to freshly isolate BBB endothelial cells (BECs) and performed genome-wide expression profiling to uncover new therapeutic targets in an unbiased manner. In this study we validated 2 of the most highly upregulated targets, cyclooxygenase-2 (Cox2) and angiopoietin-2 (Angpt2).
Methods: The prechiasmatic blood injection mouse model of SAH was used with experimental end-points at 24h. BBB disruption was assessed using intraperitoneal-injected cadaverine-based fluorescent dye. Global neurobehavioral assessments utilized the modified Garcia score. BECs were purified either by magnetic-based sorting for CD45-CD31+ cells or by fluorescence-activated cell sorting for Tie2+Pdgfrb- cells. Microarray results from RNA extracted from BECs were validated with real-time PCR. Immunofluorescence of coronal brain sections and enzyme-linked immunosorbent assays of brain homogenates were performed to determine protein expression. Intraperitoneal injections of the Cox2 inhibitor celecoxib (10mg/kg) were administered 30min and 12h after SAH induction.
Results: CD45-CD31+ BECs derived from SAH mice demonstrated 6.6-fold and 5.4-fold increase in Cox2 and Angpt2 gene expression, respectively (n=4 per group, p<0.05, p<0.01). Tie2+Pdgfrb- BECs similarly showed 5.8-fold and 5.3-fold increase in Cox2 and Angpt2 gene expression after SAH, respectively (n=4 per group, p<0.05, p<0.05), which demonstrates the robustness of these results. We observed increased Cox2 protein expression in intraparenchymal vessels and increased Angpt2 protein expression in brain homogenates after SAH. Celecoxib treatment resulted in modest reduction in BBB disruption and improved neurobehavioral outcomes. Celecoxib treatment abrogated SAH-induced upregulation of Cox2 expression, but not Angpt2, in BECs.
Conclusions: In summary, we identified Cox2 as a potential therapeutic target in SAH. The observed beneficial effects of celecoxib treatment suggest that Cox2 may be critical to BBB disruption after SAH and its mechanism appears to be independent of Angpt2.