The Change And The Role Of Heme Oxygenase-1/Carbon Monoxide In Hypoxic-Ischemic Brain Damage Of The Neonatal Rats

ObjectiveIn order to observe the change of Heme Oxygenase-1/Carbon Monoxide (HO-1/CO) in the neonatal rats with hypoxic-ischemic brain damage(HIBD) and investigate the role of HO-1/CO in the mechanism of the HIBD.Our research may provide a new idea for clinical diagnosis and therapy of neonatal HIE.Methods1.Establishment of HIBD model:1.1 42 seven-day-old Wistar rats were randomly divided into sham-operation control group(Sham),HIBD 1h,6h,12h,24h,48h and 72h groups(n=6 per group).The Sham group were sacrificed 24h after operation.The HIBD groups were sacrificed 1h,6h,12h,24h,48h and 72h after operation respectively.1.2 48 seven-day-old Wistar rats were randomly assigned to sham operation group(sham),normal saline group(NS),Znpp group(Znpp) and Hemin group(Hemin). 50mg/kg NS,Znpp and Hemin were intraperitoneally injected to NS,Znpp and Hemin group after HI respectively.Rats were sacrificed 24h after operation.1.3 The rats were subjuected to a middle anterior neck incision.Isolated and ligated the left common carotid artery,they were exposed to hypoxic environment (8%O₂,92%N₂) for 2h.Sham group were only isolated the left common carotid artery.2.Observation behavior of rats:The turnover ability and levorotary ability when their tails were gripped were observed before and after HI respectively.3.Examination the appearance of the brain.4.The activity of SOD,the content of MDA,the cellular apoptosis ratio,the expression of HO-1 gene in brain and the concentration of CO in blood:The activity of SOD,the content of MDA,the cellular apoptosis ratio,the expression of HO-1 gene in the brain were tested by the method of Xanthine Oxidase,Thibabituric Acid(TAB),the flow cytometry with FITC-AnnexinV/PI and the Real- Time fluorescent quantitation PCR(RT-Q PCR) respectively.The concentration of CO in blood was examined by the absorbance of 420nm and 432nm.Results1.Behavior change of the rats:Before the experiment all rats were healthy.After experiment sham group were still healthy.While out of 72 rats with HI,30 rats couldn't turn themselves over,28 rats became levorotary when their tails were gripped,another14 rats exhibited both behavior problems.10-15min after HI,the rats were agitated;20-30min later,they were cyanosis,pallor,tachypnea,and then in suppression.Some rats appeared convulsion,lethargy or coma;lh after in normal atmosphere,the rats became more active;4h Later,the abnormal signs disappeared.2.General examination of the brain:2.1 The two brain hemispheres of sham group showed no pathological change. The ligated brain hemisphere of HIBD group showed pallor at 6h,obvious pallor and edema at 12-24h.The volume was larger than the opposite side.Some brain hemorrhaged or cerebral ventricles hematoceled.The edema of the ligated brain hemisphere lessened at 72h,but the frontal and parietal lobe were still pallor.2.2 The ligated brain hemisphere of NS group showed pallor and edema,but that of Znpp group obviously pallor and edema,even showed liquefactive necrosis lesion,while the edema of the Hemin group rats' brain alleviated.3.Expression of HO-1 gene in brain and the concentration of CO in blood of the rats with HIBD:The HO-1 gene expression and the concentration of CO were extremely low in sham group.In HIBD groups,They all began increasing at 1h,and reached peak at 12h.Compared to other groups,the differences were statistically significant,(p＜0.01).Then they gradually decreased,while still higher than that in Sham group at 72h,the difference was still statistically significant(p＜0.01).The changing tendency of expression of HO-1 gene and that of the concentration of CO were similar.They were positive correlated(r=0.914,P＜0.01).4.The activity of SOD,the content of MDA,the cellular apoptosis ratio,the expression of HO-1 gene in brain and the concentration of CO in blood of the rats of Sham group,NS group,Znpp group and Heroin group:The HO-1 gene expression and the concentration of CO in NS group,Znpp group and Heroin group were highter than that in Sham group(P＜0.05).However they were lower in Znpp group but highter in Hemin group than that in NS group(P＜0.05).The content of MDA and the cellular apoptosis ratio of rat brain in NS group,Znpp group and Heroin group were highter than that in Sham group,while the activity of SOD was lower than that in Sham group(P＜0.05).However the content of MDA and the cellular apoptosis ratio were much higher in Znpp group,while they lower in Hemin group than that in NS group(P＜0.05).The activity of SOD was lower in Znpp group but higher in Hemin group than that in NS group(P＜0.05).Conclusions1.The HIBD model was successfully established.2.After HIBD,the content of MDA increased and the activity of SOD decreased, that indicated there was the oxygen free radical injury after HI.The cellular apoptosis ratio increased,that showed there was the apoptosis after HI.3.The expression of HO-1 gene and the concentration of CO were increased quickly after HI,and reached peak after HI 12h.The expression changes of HO-1 gene and CO after HI in neonatal rats may be significant to the pathogenesis of HIBD.4.HO-1/CO system might protect the brain tissue after HI through inhibiting the expression of MDA,protecting SOD and decreasing the apoptosis of neuron.Our conclusion might provide new direction for the mechamism of HIBD.5.The expression of HO system changed through HO-1 inhibitor or HO-1 inducer might aggravate or release disease,So our conclusion might be useful for the clinical diagnosis and treatment of neonatal HIBD.