Sequential and spatial profiles of apoptosis in ischemic penumbra after two-vein occlusion in rats

Abstract

Object The two-vein occlusion model is known to be useful for ischemic penumbra studies in vivo. It was applied here to examine sequential changes in the expression of Bax and Bcl-2 proteins and in apoptotic cells to assess the relationship between penumbra and apoptosis. Methods Two cortical veins were occluded photochemically by using rose bengal dye in 27 Wistar rats. The animals were killed with perfusion fixation at the following intervals: 4, 12, 24, 48, 96, and 168 hours after vein occlusion (four at each interval; three additional rats were sham-treated). Immunohistochemical analysis for the Bcl-2 family of proteins was performed along with the terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay to examine the relationship to single-cell death. Cells positive for antiapoptotic proteins began to appear in the TUNEL assay for animals killed 24 hours after vein occlusion, with a peak at 48 hours. These cells were localized in the core of infarction. Immunohistochemical staining for Bax protein showed an increased presence around ischemic lesions at 4 hours after vein occlusion, and the amounts continued to rise until 24 hours, when the localization was diffuse around the core of infarction. Negative findings on immunohistochemical studies for Bcl-2 protein were seen at the early phase after two-vein occlusion. Conclusions After vein occlusion, apoptosis appeared sequentially and widely in cortical lesions considered to be the penumbra. Therefore, control of apoptosis would be expected to offer a therapeutic window for treatment of venous infarction.