Study On The Relationship Between PI-3K/Akt Signaling Pathway And Cerebral Ischemic Tolerance In Gerbils

The phenomenon that sublethal cerebral ischemic preconditioning could prevent neurons from the following severe ischemic insult, now known as cerebral ischemic tolerance, provides us an opportunity to study the neuroprotective mechanism of ischemic preconditioning. However, the definite mechanism of cerebral ischemic tolerance remains unclear.It has been reported that ischemic tolerance (IT) might be related to excitatory amino acid(EAA), adenosine, immediate early gene(IEG), heat shock protein(HSP) and oncogene B cell lymphoma-2(Bcl-2), etc.The serine-threonine kinase Akt, also known as protein kinase B(PKB), is mainly activated by phosphatidylinositide 3'-OH kinase (PI-3K), another serine-threonine kinase that can be completely inhibited by Wortmannin. It has been established that activation of Akt plays an important role in celluar survival by phosphorylating its substrates at serine or threonine residues. Thus, the PI-3K/Akt kinase cascade has generally been accepted as a vital pathway for trophic factors to promote cellular survival. But it remains to be determined if Akt is implicated in cerebral ischemic tolerance.Here we aimed at investigating the relationship between PI-3K/Akt signaling pathway and cerebral ischemic tolerance in gerbils, that is, themolecular mechanism of cerebral ischemic tolerance. In this study, cerebral ischemic tolerance was induced by 2 minutes of ischemic preconditioning, and intracerebroventricular administration with a microdose ofWortmannin was performed with a stereotactic operation frame at 30 minutes before ischemic preconditioning. The expression of Akt and I kB and the apoptosis of pyramidal neurons in hippocampal CA1 region were examined by immunohistochemistric staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), respectively. The changes of ultrastructure in hippocampal neurons were detected by transmission electron microscope.There are four sections in this project: 1.The role of ischemic preconditioning in inducing the expression of Akt in hippocampal CA1 region in gerbils. 2. The expression of Akt and I k B in hippocampal CA1 region in cerebral ischemic tolerance in gerbils. 3. The relationship between PI-3K/Akt signaling pathway and hippocampal ischemic tolerance in gerbils. 4. The changes of ultrastructure in hippocampal pyramidal neurons in cerebral ischemic tolerance in gerbils.The results showed that 2 minutes of ischemic preconditioning could induce the up-regulation of Akt expression, and its expression level was dependent upon the duration of reperfusion. It was found that the Akt expression in hippocampal CA1 region started at 24h, reached the peak at 48h and returned to the normal level at 7d after reperfusion. 2 minutes ofischemic preconditioning could also inhibit the apoptosis of pyramidal neurons so as to protect the brain from following 5 minutesof ischemic insult. That is, ischemic preconditioning could induce cerebral ischemic tolerance in gerbils. It revealed that ischemic preconditioning could block the up-regulation of I K B expression induced by 5 minutes of severe ischemia. Intracerebroventricular administration with Wortmannin alone, a specific inhibitor of PI-3K, resulted in rare apoptotic neurons and Akt-positive cells in non-ischemia group. By contrast to intracerebroventricular administration with phosphate buffer saline, however, intracerebroventricular administration with Wortmannin at 30 minutes before ischemic preconditioning showed fewer Akt-positive cells but more apoptotic neurons. Transmission electron microscope saw swelling of mitochondria, pyknosis and malformation of nucleus when gerbils were subjected to 5 minutes of ischemia. Given 2 minutes of ischemic preconditioning at 48h before 5 minutes of severe ischemia, however, gerbils showed slight ultrastructure damage in hippocampal neurons.From the above results, we reckon here that cerebral ischemic tolerance may...