Effect Of Maternal Deprivation On Hippocampal Apoptosis In Rats With Hypoxic-ischemic Brain Damage

Objective To investigate the effect of maternal deprivation(MD)on hippocampal apoptosis in rats with hypoxic-ischemic brain damage(HIBD)and study molecular mechanism of the regulation in this process.Materials and Methods HIBD SD male rats were established by the method of Rice and randomly divided into control group and MD group.Besides,a sham-operation group were established as the control to HIBD model group.In MD group,rats were separated from their mothers for 3h per day,from the second day after modeling to postnatal day 21.There were no separation treatment in control group.The rats were sacrificed on postnatal day 14,21 and 28,and the brains were taken out.Neuronal apoptosis in hippocampus was observed by Td T-mediated d UTP nick end labeling(TUNEL),the distribution of p53,NF-?B and Bcl-2 in hippocampus was detected by immunohistochemistry,the expression levels of p53 m RNA,NF-?b m RNA and Bcl-2 m RNA were examined by RT-PCR and the protein expressions of p53,NF-?B,Bcl-2 and Bax were detected by western blot.Results1 The results of TUNEL staining revealed that the number of TUNEL positive cells in hippocampus of control group and MD group were all significantly more than sham-operation group on postnatal day 14,21 and 28(all P<0.05),and the numbers in MD group were significantly increased on postnatal day 21 and 28(all P <0.05),compared to control group.2 Immunohisochenical staining showed that the expression levels of p53,Bcl-2 and NF-?B in hippocampus of control group and MD group were all significantly higher than those in sham-operation group on postnatal day 14(all P<0.05),and the expression level of NF-?B but not p53 or Bcl-2 in rats hippocampus of MD group was higher than that in control group on postnatal day 14(P<0.05).The expression levels of p53,Bcl-2and NF-?B in hippocampus of control group and MD group rats on postnatal day 21 were also all higher than those in sham-operation group(all P<0.05),and compared to control group,the expression levels of p53 and NF-?B in hippocampus of MD rats were increased(all P< 0.05),but there was no significant difference in Bcl-2 expression between the two group.Compared to the sham-operation group rats,the expression level of NF-?B was increased(P<0.05)and the levels of p53 and Bcl-2 were no significant difference in hippocampus of control group rats on postnatal day 28 Additionally,the hippocampal expression levels of p53 and NF-?B in MD group rats on postnatal day 28 were significantly higher than those in control group,while the expression level of Bcl-2 in the former group was decreased compared to the later group.3 RT-PCR results show that,compared with sham-operation group,the m RNA expression levels of p53,NF-?B and Bcl-2 in postnatal day-14 rats hippocampus of control group and MD group were all up-regulated(all P<0.05);compared to control group,the expression levels of gene p53 m RNA and NF-?B m RNA were increased in MD group rats at the same postnatal day(all P<0.05),But the levels of Bcl-2 m RNA was no difference.The m RNA expression levels of p53 and NF-?B but not Bcl-2 in hippocampus of control group and MD group rats on postnatal day 21 were significantly higher than those in sham-operation group rats(all P<0.05),and the levels of p53 and NF-?B m RNA expression were all increased in MD group compared to control group(all P<0.05);there were no significant difference in Bcl-2 m RNA expression between control group rats and MD group rats on the same postnatal day.As for postnatal day-28 rats,the expression levels,both in hippocampus of control group and MD group rats of p53 m RNA and NF-?B m RNA were all increased but of Bcl-2 m RNA were significantly decreased compared to the rats of sham-operation group(all P<0.05).In addition,the expression levels of p53 m RNA and NF-?B m RNA in hippocampus of MD group rats were higher than those in control group rats(all P<0.05),and the expression level of Bcl-2 m RNA in the former group was lower than that in the later group(P<0.05).4 According to the western blot results,in hippocampus of postnatal day-14 rats,the protein expression levels of p53,Bcl-2,Bax and NF-?B in control group and MD group were all higher than those in sham-operation group(all P<0.05);compared with control group,the protein expression levels of p53 and NF-?B but not Bcl-2 and NF-?B in MD group rats were significantly up-regulated(all P<0.05).Compared to the sham-operation group,the expression levels of p53,Bax and NF-?B in hippocamous of control group and MD group rats,at postnatal day 21,were all increased(all P<0.05).Besides,in postnatal day 21 rats,the expression level of Bcl-2 protein in control group but not MD group was higher than that in sham-operation group(P<0.05).Compared to the control group rats,the protein expression levels of p53,Bax and NF-?B in hippocampus of MD group rats on postnatal day 21 were all increased(all P<0.05),while the expression level of Bcl-2 protein was decreased(P<0.05).In postnatal day-28 rats,the protein expression of p53 and NF-?B in hippocampus of control group rats were more than those in sham-operation group(all P<0.05).Compared to sham-operation group and control group,the protein expression levels of p53,Bax and NF-?B were all increased(all P<0.05),while the level of Bcl-2 protein expression were decreased(P<0.05).Additionally,the ratio of Bax/Bcl-2 in MD group rats,both on postnatal day 21 and 28,were dramatically higher than those in control group rats or sham-operation group rats.Conclusion MD promotes the hippocampal apoptosis in rats with HIBD,which probably was related to the up-regulation of p53,Bax and NF-?B and the down-regulation of Bcl-2.