| Objective:Perinatal hypoxic-ischemic brain damage(HIBD)is one of the major cause of mortality and severe neurologic impairment in children,which brings heavy burden to family and society.We simulated the common causes of acute intrauterine hypoxia in human newborn,established the perinatal rabbit HIBD model,and further studied whether PI3 K / Akt signal pathway is involved in the pathogenesis of HIBD in newborn rabbits.To provide scientific treatment targets for HIBD.Materials and methods:1.Establishment of HIBD model in perinatal rabbits We used the method of embolization of abdominal aorta to block uterine artery of pregnant rabbits indirectly to establish HIBD model in perinatal rabbits.Healthy New Zealand pregnant rabbits were selected(29 d,about 92.06% of pregnancy).After successful anesthesia,the 4F Fogarty artery catheter was inserted into the artery about10 cm,where was the balloon just between the lower end of the opening of the renal artery and the upper end of the uterine artery.0.3ml normal saline was injected to dilate the saccule.25 minutes later,the catheter was withdrawn.24 hours later,the pregnant received cesarean section.The general condition records and neurobehavioral tests were carried out immediately.The serum NSE level and brain water content were measured.The level of Cleaved caspase-3-positive cells in hippocampal CA1 by immunohistochemistry was observed.2.Based on the successful HIBD model in newborn rabbits,further to study whether PI3K/Akt signaling pathway is involved in the pathogenesis of HIBD.32 pregnant rabbits were randomly divided into four groups,8 in each group,which were SHAM group,HIBD group,DMSO group and LY294002 group.In the SHAM group,only the femoral artery was ligated.In DMSO group and LY294002 group,2ml DMSO solvent and LY294002(6.4 ? g/kg,in DMSO solvent 2ml)were injected respectively through the auricular vein of pregnant rabbit immediately after intrauterine hypoxia.RT-q PCR and Western blot were used to detect the expression of proteins and apoptosis genes related with PI3K/Akt signaling pathway.Spss22.0 software was used for statistical analysis.Results:1.The perinatal rabbit HIBD model was established successfully,which is suitable for the study of the mechanism of HIBD in the early stage.In the newborn rabbits,A.HIBD has lower neurological score than sham group: posture score(1.62±0.51?2.75±0.46,P<0.05),righting reflex score(1.50±0.54?2.75±0.46,P<0.05),deglutition reflex score(1.35±0.71?2.75±0.46,P<0.05).B.HIBD has higher level than sham group in serum NSE(1.28±0.18pg/L?0.32±0.13pg/L,P<0.05)and brain water content(89.52±0.15%?88.18±0.22%,P<0.05)increased.C.Brain histopathology showed that in HIBD group,the number of cells decreased,nuclear staining was uneven,nuclear pyknosis,and the number of caspase-3-positive cells in hippocampal CA1 increased.D.The death rate of newborn rabbits in HIBD group was higher than Sham group within24 hours after birth.The score of posture,the number of righting reflex and righting reflex(2.63±0.50,9.00±1.41,2.63±0.50)were still lower than those in sham group(all P<0.05)on the 3th day.Only the body weight of newborn rabbits in HIBD group was lower than that in sham group(66.27±9.2g?86.00±13.94 g,P<0.05)on the 7th day.Neurobehavioral reflex was restored to normal completely.2.PI3 K / Akt signaling pathway is involved in HIBD and its pathogenesis in newborn rabbits.The results of this study showed that the expression of Bcl-2 m RNA decreased(0.72±0.05pg/L?1.10±0.02pg/L,P<0.05),the expression of Bad and Bax m RNA increased(1.59±0.08pg/L?1.00±0.03pg/L;1.48±0.07pg/L?1.00±0.03pg/L)in HIBD group compared with sham group(both P< 0.05).The protein level of PI3 K in HIBD group was lower than that in sham group(0.68±0.07 pg/L?1.28±0.08pg/L,P<0.05).Akt and Bad phosphorylation decreased(0.50±0.06%?0.85±0.07%,0.42±0.05?0.78±0.06,both P<0.05).3.Regulation of PI3K/Akt signaling pathway could repair HIBD in newborn rabbits.The results were showed : A.Compared with HIBD group,LY294002 group further increased the fetal mortality(35.4%?12.30%,P<0.05).B.Compared with HIBD group,LY294002 reduced the neurobehavioral scores of newborn rabbits after hypoxia,posture scores(0.88±0.24?1.62±0.51,P<0.05),righting reflex scores(0.75±0.07?1.50±0.54,P<0.05),swallowing reflex scores(0.75±0.16?1.35±0.71,P< 0.05)were all decreased.C.Compared with HIBD group,NSE(1.75±0.14pg/L?1.28±0.18 pg/L,P<0.05)and brain water content(89.92±0.28%?88.18±0.22%,P<0.05)were increased in LY294002 group.D.Brain histopathology showed that the damage of hippocampus in LY294002 group was more serious and the number of cells was significantly reduced.LY294002 group could increase cell apoptosis in HIBD.E.Compared with HIBD group,the expression of Bcl-2 m RNA in LY294002 group decreased(0.44±0.03 pg/L?0.72±0.05pg/L,P<0.05),the Bad and Bax m RNA increased(1.92±0.09pg/L?1.59±0.08 pg/L,1.86±0.06pg/L?1.48±0.07pg/L,both P<0.05).The ratio of p-Bad/t-Bad and Bcl-2/Bax in LY294002 group was lower than those in HIBD group(0.28±0.02?0.42±0.05,0.41±0.04?0.74±0.07,P<0.05).Conclusion:1.The HIBD model in perinatal rabbits was successfully.It could be used at the early stage of HIBD study.2.It was also found that hypoxia inhibited the activation of PI3K/Akt signaling pathway,which proved that this pathway was involved in HIBD in newborn rabbits.3.PI3K/Akt signal reduces neurons apoptosis and plays a neuroprotective role of HIBD.It could be used as one of the therapeutic targets for HIBD. |
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