The Expression Of PI3K/Akt Signaling Pathway And PTEN In Hippocampus And The Correlation With Cognitive Impairment Of Rats After Neonatal Hypoxic Ischemic Brain Damage

Part oneEstablishment the model of hypoxia ischemia brain damage in neonatal rats and identificationObjective:To set up a reliably severe hypoxia ischemia brain damage (HIBD) model in neonatal rats and several reasonable methods were used to identify whether it is successful or not.Method:Forty healthy 7-day-old Sprague-Dawley (SD) rats were randomly divided into 2 groups:the sham operation group (n=18) and the HIBD model group (n=22). The HIBD model was produced according to the traditional Rice model. The rats were anesthetized with ethylether. The left common carotid artery was exposed, ligated and cut. Then, they were exposed to hypoxia in a normobaric chamber filled with 8% oxygen and 92% nitrogen for 2 hours. In the sham operation group, the left common carotid artery was exposed but was not ligated and cut or exposed hypoxia. To observe the neuroethology changes of rats in 24 hour after HIBD, and the brains were collected after 72 hour. Behavioral ability and neuropatholoic changes were studied in each group. The Morris water maze test was used for evaluating the learning-memory ability when the rats were 28 days old.Results:Compared with the shame operation group, we found all the HIBD model rats have obvious changes in praxiology, anatomy, Nissl staining, and cognitive impairment were showed from the result of water maze.Conclusion:The model of hypoxia ischemia brain damage in neonatal rats is feasible and provide a reliable model for subsequent study.Part twoThe expression of PI3K/Akt signaling pathway and PTEN in hippocampus of rats after hypoxic ischemic brain damageObjective:To investigate the activation of phosphoinositid 3-kinase/Akt (PI3K/Akt) signaling pathway and the expression of PTEN protein in Hippocampus under hypoxia ischemia condition, and to elucidate the role of PI3K/Akt and PTEN regulation after neonatal hypoxia ischemia brain damage (HIBD).Methods:One hundred and ten SD rats aged 7 days were randomly divided into normal control group (n=39), sham operation group (n=39), HIBD model group (n=32). The HIBD model was produced according to the traditional Rice model, the left common carotid artery was exposed, ligated and cut. In the shame operation group, the left common carotid artery was exposed but was not ligated or cut or exposed hypoxia. In the normal control group, the rats received no further processing. The brain tissues were harvested from the rats at 1,4,12 and 24 hours after hypoxia ischemia for Western blot analysis, but for immunofluorescence method only at 4 hours. The AKT, p-Akt and p-PTEN protein expression were detected and measured.Results:In the HIBD model group, the p-Akt protein was significantly increased at 4 hours after operation and to decrease at 8 hours. The p-PTEN expression was significantly increased at 4 hours, reached the peak level at 12 hours, and began to decrease at 24 hours. However, the expression levels of p-Akt and p-PTEN protein in the shame operation group were extremely low at each time point. So, the expression levels of p-Akt in the HIBD model group at 4 and 12 hour was significantly higher than that in the shame operation group (P<0.05), the expression of p-PTEN protein in the experimental group at 4,12 and 24 hours was significant higher than that in the shame operation group (P<0.05). The change of AKT protein in the HIBD model group was not time-dependent, and no significant difference was evident when compared with that of the shame operation group (P>0.05).Conclusion:These results suggested that the HIBD of neonatal rats may activate PI3K/Akt signaling pathway, and further induce the expression of p-Akt. But PTEN is activated at the same time inhibiting the expression of p-Akt. Hope to looking for treatment of neonatal HIBD through the PI3K/Akt signaling pathway and PTEN.Part threeThe learning and memory capacity effects after neonatal hypoxic ischemic brain damage on PI3K/Akt signaling pathwayObjective:To explore the effects of PI3K/Akt signaling pathway on brain learning and memory cognitive ability of neonatal rats with hypoxic-ischemic brain damage. Methods:Hippocampus injected of Wortmannin 2μl 30 minutes before hypoxic-ischemia with the help of brain solid positioned to set the Wortmannin-treated HIBD group. The DMSO-treated HIBD group, HIBD model group, shame operation group and normal control group also counted at a same time. The brain tissues were harvested from the rats at 4 hours after hypoxia ischemia for Western blot analysis to detect the AKT and p-Akt protein. The Morris water maze test in rats was used to for evaluating learning and memory ability when the rats were 28 days old.Results:Using Wortmannin, the PI3K/Akt specific inhibitor, p-Akt protein expression was significantly decreased when compared with the DMSO-treated HIBD group and HIBD model group (P<0.001). After statistical comparison about the data, there was no significant difference between shame operation group and normal control group (P>0.05), also between DMSO-treated HIBD group and HIBD model group (P>0.05). But Wortmannin-treated HIBD group was lower than DMSO-treated HIBD group and HIBD model group (P<0.05), and significantly lower than shame operation group and normal control group in learning and memory ability (P<0.001).Conclusions:PI3K/Akt signaling pathway is an classic anti-apoptotic> urge-survival signal transduction pathway. It plays important regulation and control roles in neurons apoptosis after HIBD. Application of PI3K/Akt signaling pathway inhibitor Wortmannin can worse neurons damage in neonatal HIBD rats, aggravate cognitive impairment further, thus affecting long-term learning and memory capacity.