This study investigates the activation and cleavage of caspase-3 in apoptosis induced by experimental cerebral ischemia in adult mice. Using immunohistochemistry, immunoblots, and a fluorogenic substrate, the researchers examined caspase-3 expression, activation, and localization in the brain after 2 hours of middle cerebral artery occlusion followed by reperfusion. They found that caspase-3p32, a proform of caspase-3, is constitutively expressed in neurons throughout the brain, particularly in the piriform cortex. Caspase-like enzyme activity increased after reperfusion, peaking within 30-60 minutes. Caspase-3p20, the cleavage product, became prominent in neuronal perikarya in the middle cerebral artery territory at reperfusion and was detected in immunoblots 1-12 hours later. DNA laddering and TUNEL staining were observed 6-24 hours after reperfusion, indicating apoptosis. Caspase-3p20 immunoreactivity was detected in TUNEL-positive cells, suggesting a time-dependent evolution of ischemic injury with caspase-like enzyme activation preceding morphological and biochemical features of apoptosis. The study also shows that caspase-3 is essential for apoptosis in the brain, as caspase-3-deficient mice exhibit abnormal brain development and die early. The findings support the role of caspase-3 in ischemic cell death and suggest that caspase inhibitors may protect against brain injury. The study provides biochemical and immunochemical evidence for caspase-3 activation in ischemic brain and highlights the importance of caspase-3 in apoptotic signal transduction in injured brain.This study investigates the activation and cleavage of caspase-3 in apoptosis induced by experimental cerebral ischemia in adult mice. Using immunohistochemistry, immunoblots, and a fluorogenic substrate, the researchers examined caspase-3 expression, activation, and localization in the brain after 2 hours of middle cerebral artery occlusion followed by reperfusion. They found that caspase-3p32, a proform of caspase-3, is constitutively expressed in neurons throughout the brain, particularly in the piriform cortex. Caspase-like enzyme activity increased after reperfusion, peaking within 30-60 minutes. Caspase-3p20, the cleavage product, became prominent in neuronal perikarya in the middle cerebral artery territory at reperfusion and was detected in immunoblots 1-12 hours later. DNA laddering and TUNEL staining were observed 6-24 hours after reperfusion, indicating apoptosis. Caspase-3p20 immunoreactivity was detected in TUNEL-positive cells, suggesting a time-dependent evolution of ischemic injury with caspase-like enzyme activation preceding morphological and biochemical features of apoptosis. The study also shows that caspase-3 is essential for apoptosis in the brain, as caspase-3-deficient mice exhibit abnormal brain development and die early. The findings support the role of caspase-3 in ischemic cell death and suggest that caspase inhibitors may protect against brain injury. The study provides biochemical and immunochemical evidence for caspase-3 activation in ischemic brain and highlights the importance of caspase-3 in apoptotic signal transduction in injured brain.