Apoptotic and necrotic cell death after photothrombotic ring stroke : characterization of a stroke model and its morphological and molecular consequences

Abstract: Cerebral ischemic cell death is a major cause of disability and death among stroke patients. The brain cell demise can occur through apoptosis or necrosis or as a continuum of both. This study aimed at establishing a dual setup of a photothrombotic ring stroke model and exploring its morphological and molecular consequences.    Photothrombotic ring stroke was induced in adult male Wistar rats by a ring shaped laser irradiation beam (514.5nm, outer diameter 5mm, thickness 0.35 mm) for 120 seconds focused on the exposed intact skull bone with simultaneous intravenous infusion of the photosensitizer erythrosin B (17 mg/kg). By using otherwise identical experimental conditions, high intensity irradiation (1.94 W/cm2) resulted in consistent lack of reperfusion in the region at risk whereas low intensity irradiation (0.90 W/cm2) induced late spontaneous reperfusion. The morphological appearance of apoptotic and necrotic cells was demonstrated by H&E, TUNEL and Hoechst stainings under light microscopy, immunohistochemistry and electron microscopy. This was further confirmed by gel electrophoresis showing DNA laddering that coexisted with DNA smear. Cell counts revealed that apoptotic cells appeared earlier (at 24 h) and remained as long as the necrotic cells, that is up to 72 hours after ischemic onset in regions with severe CBF reduction. After low intensity irradiation, we observed early and widespread increased expression of the anti-apoptotic protein bcl-w and a prolonged elevation of Bcl-2 with unchanged pro-apoptotic Bax in mitochondria. In contrast, decreased bcl-w and Bcl-2 with scattered Bax remained after high intensity irradiation. Correspondingly, the release of the pro-apoptotic factor Smac/DIABLO from the mitochondria to the cytosol was more persistent in high- compared with low-intensity irradiation.       Apoptotic and necrotic cell death coexisted in the same regions at the same time after photothrombotic ring stroke induced by low- or high-intensity irradiation, where spontaneous morphological recovery or pannecrosis were evident in the region at risk. The ratios between Bcl-w, Bcl-2 and Bax may direct the translocation of Smac/DIABLO from the mitochondria to the cytosol and thereby influence cell death or survival after focal cerebral ischemia.