Changes Of Apoptosis And Its Mechanisms In Focal-Focal Type Brain Ischemic Preconditioning In Rats

Background and Purpose: Brain ischemic tolerance (IT) is the phenomenon whereby a preconditioning ischemia of brief duration, inadequate to infarct the brain, protects it from subsequent severe ischemia. However, the molecular and cellular mechanisms underlying IT of brain remain incompletely understood. Neuronal apoptosis plays a critical role in the development of the brain ischemia-reperfusion injury; ischemic preconditioning (IP) may exert protective effects by affecting apoptosis after brain ischemia. The present studies were conducted to characterize the protective effects on brain of the focal-focal type brain IP, to explore the changes of apoptosis, and to evaluate the importance of several candidate proteins associated with apoptosis in the development of brain IT.Materials and Methods: In experiment one, IP was induced, using an intraluminal filament method, by 20 minutes MCAO in rats. The secondary insult of 2 hours MCAO was induced 3 days following preconditioning, 24 hours after reperfusion, we performed brain sections and histology analysis, Infarct size were measured and compared with non-IP rats (n=6). Also, we used TUNEL staining to label the neuronal apoptosis and immunohistochemistry to detect the expression of p53, p21 and Bax protein.In experiment two, temporary MCAO (20 minutes) was used in rats, various periods of reperfusion (i.e., 6 and 12 hours and 1, 2, 3, 5, 7days) were allowed after that and protein expression of Bcl-2 and Bax in brain detected by immunohistochemistry (n=4) were determined.Results: 1. Compared with the non-IP group, hemispheric infarct was significantly (P<0.05) reduced when 20 minutes MCAO was performed 3 days before 2h MCAO (decreased 46%), especially in forebrain cortex. 2. IP also decreased the number of TUNEL positive cells in both ischemic core and penumbra after 2h MCAO (P<0.05). 3. The number of p53 protein positive cells increased in parietal cortex and striatum ischemic penumbra after 2 hours MCAO with 24 hours reperfusion, in the parietal cortex ischemic penumbra, IP lightened this increase (P<0.05), the expression of p21 and Bax protein didn't change in the ischemic penumbra of both IP and non-IP group. 4. After 20 minutes MCAO, the Bcl-2 immunostaining elevated at the 1 st day and peaked at the 3rd day, it kept higher at the 7th day, the changes of the immunostaining of Bax didn't observed at any time point.Conclusions: 1. IP is a powerful inducer of brain IT as reflected by preservation of brain tissue after severe ischemia. 2. IP may protect neurons from ischemia-reperfusion injury through an anti-apoptosis mechanism. 3. IP is also associated with an increased expression of the neuroprotective protein Bcl-2 and a less pronounced postischemia expression of p53; these may contribute to the lessened apoptosis in severe ischemia after IP and induce brain IT. 4. P21 and Bax may not play a key role in brain ischemia-reperfusion injury and the development of brain IT; and it's not through regulating the expression of p21 and Bax that IP protects postischemia brain by inhibiting the activation of p53.