| Objective: Neonatal hypoxic-ischemic brain injury(HIBI),which is associated with a morbidity of 2.5/1000 live births(4 to 9 million infants globally),is a perinatal brain injury that occurs during fetus distress in utero,suffocating asphyxia during or after parturition,or intrauterine infection.In addition to the high mortality(23%),HIBI can cause long-term neurological sequelae,such as epilepsy,as well as cognitive and memory impairment.At present,there are no satisfactory therapies for HI-induced cognitive,learning and memory deficits.This investigation focus on the protective effect of sevoflurane post-conditioning on neonatal HIBI aiming to achieve the following protective mechanisms: 1.To investigate the number of neurons,microglia and astrocytes in hippocampus of newborn rats 24 h after ischemia and hypoxia,the changes of autophagy and lysosomal protease levels in various cells at 24 h,and the effects of sevoflurane post-treatment on them;2.To investigate the relationship between autophagy and sevoflurane-regulated lysosomal protease levels in microglia,therefore affecting neuronal mortality;3.To demonstrate that sevoflurane regulates autophagy and lysosomal protease levels in microglia and improves long-term prognosis by regulating Ezh2/Pten/mTOR pathway. By this study,it will be clarified about the molecular mechanism of sevoflurane post-conditioning protective effect on neonatal HIBI,and provide new targets for clinical neonatal HIBI treatment.Methods: 1.Neonatal HIBI model: 7-day-old Sprague-Dawley(SD)rats were ligated to the left carotid artery.After waking,the pups were returned to their mothers for 2 h.The pups were then placed in a chamber with constant gas(8% O2,92% N2)exposure for 2 h.The air temperature was controlled at 37 ℃ by submerging the chamber in a temperature-controlled water bath.2.Study groups and drug administration: Random grouping according to random number table.In the first part of the study,the pups were randomly divided into three groups:sham operation group(sham group),hypoxic-ischemia group(HI group),sevoflurane post-treatment group(HI+S group),14 experimental animals in each group(n=14);In the second part of the study,the pups were randomly divided into 6 groups:sham group,sevoflurane post-treatment sham operation group(sham+S group),HI group,HI+S group,rapamycin group(HI+S+R group,HI+R group)(n=36);In the third part of the study,the pups were randomly divided into 6 groups: sham group,sham+S group,HI group,HI+S group,Ezh2 inhibitor group(HI+S+G group,HI+G group)(n=36).3.24 h after HI suffering: Mortality and rat body weight were measured;Western Blot was used to detect autophagy-related proteins LC3 BII,Beclin1,P62/SQSTM,lysosomal protease CathepsinB,proinflammatory cytokines IL-1β,TNF-α,and Ezh2 regulated Pten/Akt/mTOR expression;immunofluorescence double staining detection of NeuN/LC3 B,GFAP/LC3 B,Iba1/LC3 B,NeuN/cathepsin B,GFAP/cathepsin B,Iba1/cathepsin B expression;4.7 day after HI suffering: Determine the body weight of the rats;take the brain and weigh the left and right brain mass ratio;5.28 day after HI suffering:The body weight of the rats was measured;suspension experiments,Morris Water Maze(MWM)experiments were performed for behavioral analysis;Nissl staining was used to observe the survival of hippocampal neurons,and the density ratio of left and right brain neurons was measured.6.Statistical analysis:All data(e.g.,body weight,brain weight,brain weight ratio,neuronal density ratio,protein expression levels,suspension time,swimming speed,platform quadrant crossing times,time spent in each quadrant)are presented as the mean ± the standard deviation(SD),and were analyzed using one-way ANOVA followed by the Student–Newman–Keuls post-hoc test.The escape latency was tested using a two-way repeated ANOVA(treatment as between-groups and time as repeated measure factors)followed by the Bonferroni post-hoc test.The mortality among the groups was analyzed by Z testing.All statistical analyses were performed using GraphPad Prism Version 6.0(GraphPad Software,USA)and SPSS21.0.A P-value of <0.05 was considered statistically significant.Results: 1.In the first part of the study:24h after HI suffering:Compared with sham group,there was no significant change in the number of neurons and astrocytes in hippocampus of newborn rats 24 h after HI(P>0.05);the number of microglia in HI group increased significantly ompared with sham group(P<0.05),however,sevoflurane post-treatment did not significantly change the number of microglia induced by HI(P>0.05);compared with sham group,LC3 BII expression in hippocampus was significantly increased,and the ratio of NeuN/LC3 B double positive cells to NeuN positive cells,the ratio of GFAP/ LC3 B double positive cells to GFAP positive cells,and the ratio of Iba1/LC3 B double positive cells to Iba1 positive cells were significantly increased P<0.05,in the HI+S group,the above results were reversed(P<0.05 compared with HI group);in the neonatal HIBI model,the lysosomal protease CathepsinB was mainly expressed in microglia;compared with the sham group,the expression of cathepsinB in HI group in the hippocampus was significantly increased(P<0.05),and the ratio of Iba1/cathepsinB double positive cells to Iba1 positive cells was significantly higher(P<0.05).The above results were reversed in the HI+S group(P < 0.05 compared to the HI group).2.In the second part of the study:24h after HI suffering:The results of Western Blot showed that compared with the sham group,the expression of LC3 BII,Beclin1,cathepsinB,IL-1β and TNF-α in the hippocampus of HI group increased significantly,while the expression of P62/SQSTM decreased significantly(P<0.05),and the above results in the HI+S group can be reversed(P<0.05 compared with the HI group);the expression of LC3 BII,Beclin1,cathepsinB,IL-1β,TNF-α in the HI+S+R group were up-regulated,and the degree of down-regulation of P62/SQSTM was significantly increased by compared with HI+S P<0.05;compared with HI+S group,the number of LC3B-positive cells in hippocampus of HI+S+R group increased significantly P<0.05;the ratio of Iba1/LC3 B double positive cells to Iba1 positive cells,the ratio of Iba1/cathepsinB double positive cells to Iba1 positive cells were significantly higher P<0.05.7 day after HI suffering:the ratios of left to right brain weight in the HI group were significantly lower than those of the sham group(P<0.05);in the HI+S group,the ratios of left to right brain weight were increased(compared with the HI group,P< 0.05);the ratios of left to right brain weight in the HI+S+R group were lower(P<0.05 compared with the HI+S group).35day after HI suffering:Nissl staining showed a significant decrease in neuron density in the hippocampus of the HI group compared with the sham group(P<0.05);in the HI+S group,the decrease in neuron density in the hippocampus was attenuated(with compared with HI group,P<0.05);the density of neurons in HI+S+R group decreased(P<0.05 compared with HI+S group).3.In the third part of the study:24h after HI suffering:Western Blot showed that compared with sham group,the expressions of Ezh2,H3K27me3/H3,pAkt/Akt and mTOR in hippocampus of HI group were significantly decreased by P<0.05,Pten expression was significantly increased(P<0.05),in HI+S group.Among them,the above results can be reversed(P<0.05 compared with HI group),and the expression of H3K27me3/H3,pAkt/Akt,mTOR is significantly reduced(with HI+S)in the sevoflurane post-treatment group using Ezh2 inhibitor GSK126.Compared with the group,P<0.05),the expression of Pten,LC3 BII and cathepsinB increased significantly(P<0.05 compared with HI+S group).Compared with HI+S group,LC3 B positive cells in hippocampus of HI+S+G group The number increased significantly,P<0.05.The ratio of Iba1/LC3 B double positive cells to Iba1 positive cells and Iba1/cathepsinB double positive cells and Iba1 positive cells were significantly higher than P<0.05.7 day after HI suffering:the ratios of left to right brain weight in the HI+S+G group were lower(P<0.05 compared with the HI+S group).29-34 day after HI suffering:The MWM behavioral test results showed that the average escape latency was significantly longer in the HI group from the third day compared with the sham group(P<0.05);in the HI+S group,the above results were reversed(compared with the HI group,P <0.05);after the application of GSK126,the average escape latency was prolonged(P<0.05 compared with the HI+S group);the Nissl staining results showed that the hippocampus neurons in the HI+S+G group were significantly reduced in density(with HI+S)compared with the HI+S group(P<0.05),the behavioral results were consistent with the degree of brain injury.Conclusion: 1.There was no significant change in the number of neurons and astrocytes in hippocampus of newborn rats 24 h after HI;the number of microglia increased significantly;the level of autophagy in neurons,astrocytes,and microglia increased 24 h post-HI;the lysosomal protease Cathepsin B was mainly expressed in Microglia,and expression increased after HI;sevoflurane post-treatment can inhibit the increase of autophagy and CathepsinB after HI.2.Sevoflurane post-conditioning inhibits autophagy,and regulates the expression level of microglia lysosomal protein Cathepsin B,therefore reduces neuronal death in hippocampus and alleviates long-term prognosis.3.Sevoflurane post-conditioning,inhibits autophagy,and regulates microglia polarization,therefore reduces long-term neuronal death,and improves the prognosis of brain damage in neonatal rats with hypoxia-ischemia through Ezh2 regulated Pten/Akt/mTOR pathway. |
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