Effects Of Caspase-3, Calpain And Their Inhibitors On Ischemic Neuronal Damage In Rats

Stroke, with its high incidence, high mortality and high disability rate, remains the first three leading cause of imperiling the health of human beings, and there is currently no effective therapy available. Brain injury following cerebral ischemia results from the interaction of complex pathophysiological proceeds such as excitotoxicity, peri-infarct depolarizations, inflammation as well as apoptosis, and the molecular event is far from clear. In recent years, attention has focused on the mechanisms other than excitotoxic cascade, and much of researchers have concerned with the contribution of two cysteine proteases, caspase-3 and calpain, to cellular dysfunction and cell death in numerous pathological conditions, including cerebral ischemia. With two extensively used systems, one is in vitro model, to induce primary cultures of perinatal rat hippocampal neurons hypoxia followed by reoxygenation, and the other is in vivo model, to temporarily occlude one of the middle cerebral arteries (MCA) followed by prolonged reperfusion, we studied the deleterious role of caspase-3 and calpain in ischemic neuronal damage, and the neuroprotective effects of their inhibitors. The results in vitro showed that caspase-3 and calpain were conactivated in primary cultures of hippocampal neurons following hypoxia/reoxygenation. Pretreatment with caspase-3 inhibitors and/or calpain inhibitors block not only the proteolytic actions of the enzyme, but also the cell death process after hypoxia followed by reoxygenation in a synergistic manner. In vivo studies, we also found that single and double strand breaks of DNA damage were detected in brain sections obtained from rats subjected to 2 hours of left middle cerebral artery occlusion followed by 0 to 48 hours of reperfusion, and single strand breaks precede that of double strand, indicating that apoptotic mechanisms involved in the ischemic neuronal damage. Both mRNA and active protein expression of caspase-3 and calpain in ipsilateral region were increased after recirculation, pretreatments with caspase-3 inhibitor and/or calpain inhibitor decreased the expression of caspase-3 and calpain, and blocked the cell death process itself in the ipsilateral brain after transient focal ischemia in a synergistic manner. Our present results contribute additional evidence that proteases may play a functional role in apoptotic cell death and extend them to include the possibility that endogenenous proteases are capable of inducing the striking DNA fragmentation. Moreover, the synergistic effect of caspase and calpain inhibitors in protecting neurons from ischemic damage suggests that there is a cross-talk between caspase and calpain during apoptosis. These findings suggested that the use of specific caspase and calpain inhibitors is of potential therapeutic utility in ischemic injuries to the human brain...