鼠早产和低氧浓度对胆红素神经毒性的增强作用

Influences of prematurity,hypoxia and hyperoxia preconditioning on bilirubin neurotoxicity in neonatal rats

目的探讨早产、氧浓度异常对胆红素的神经细胞毒性的影响及其可能的发生机制。方法 7 d龄新生足月和早产大鼠各36只,完全随机分为常氧+生理盐水组、常氧+胆红素组、低氧+生理盐水组、低氧+胆红素组、高氧+生理盐水组、高氧+胆红素组。常氧+生理盐水组,经小脑延髓池注射0.02 ml生理盐水;常氧+胆红素组,经小脑延髓池注射胆红素20μg/g;低氧+生理盐水组,在氧浓度8%的密闭箱中暴露3 h,经小脑延髓池注射0.02 ml生理盐水;高氧+生理盐水组,在氧浓度95%的密闭箱中暴露3 h,经小脑延髓池注射0.02 ml生理盐水;低氧+胆红素组,氧浓度8%的密闭箱中暴露3 h后经小脑延髓池注射胆红素20μg/g;高氧+胆红素组,氧浓度95%的密闭箱中暴露3 h后经小脑延髓池注射胆红素20μg/g。各组均于24 h断头取脑。HE染色及TUNEL显示海马神经细胞凋亡,免疫组织化学染色法检测iNOS和TNF-α表达。结果①注射胆红素的大鼠均出现俯伏、转圈、四肢颤动、翻滚、活动减少等异常神经行为,早产组最明显。②HE染色观察胆红素+低氧组海马神经细胞坏死、凋亡最明显。③TUNEL显示,早产胆红素组海马神经细胞凋亡数(30.17±3.76)较足月组(23.33±4.89)明显升高(P<0.05);足月和早产低氧+胆红素组的海马神经细胞凋亡率[分别为(27.67±3.01)和(34.33±3.01)]较胆红素组[分别为(23.33±4.89)和(30.17±3.76)]明显升高(P<0.05),而高氧+胆红素组海马神经细胞凋亡率与胆红素组相似。④早产胆红素组海马神经细胞TNF-α释放(0.425±0.014)较足月组(0.388±0.020)显著增多(P<0.05),两组iNOS表达水平相似;低氧+胆红素组海马神经细胞TNF-α和iNOS表达[(0.437±0.024)和(0.440±0.001)]较胆红素组[(0.253±0.010)和(0.388±0.020)]显著增加(P<0.05);高氧+胆红素组海马神经细胞TNF-α释放(0.416±0.011)显著增多(P<0.05),iNOS表达无明显升高。结论胆红素可诱导TNF-α等炎症因子释放增多;低氧、早产增加TNF-α和/或iNOS等炎症因子的释放和表达,增强胆红素的神经毒性。减少围产期窒息、早产,控制炎症因子释放,对防治胆红素脑病有重要意义。

Objective To determine the effects of prematurity,hypoxia and hyperoxia preconditioning on bilirubin neurotoxicity.Methods Seventy-two 7-day-old rats in full-term group and premature group（36 in each group）,were randomly divided into 6 subgroups,control group,bilirubin group,hypoxia group,hyperoxia group,hypoxia＋bilirubin group,and hyperoxia＋bilirubin group（n=6）.After the rats were kept in a chamber of 95%O2（for hyperoxia groups） or 8% O2（for hypoxia groups） for 3 h,0.02 ml normal saline or 10 mg/ml bilirubin at 20 μg/g body weight was injected into the cerebellomedullary cistern in control group and bilirubin groups.After behavior evaluation at 24 h after injection,the rats were sacrificed,and their brain tissues were collected.The apoptosis and necrosis of neurocytes,the expressions of iNOS and TNF-α in the hippocampus were detected by HE staining,TUNEL and immunohistochemical staining,respectively.Results The rats in the bilirubin groups appeared to have different degrees of abnormal behaviors,such as proneness,tumble,limbs twitched,rolled and less activity.These behaviors were more obvious in the premature rats.HE staining showed that the apoptosis and necrosis of neurocytes in hypoxia＋bilirubin group were more significant than in the other groups.TUNEL showed that compared with the bilirubin group in full-term mice（23.33±4.89）,the apoptotic rate in the hippocampus of bilirubin group in premature mice（30.17±3.76） was significantly increased（P〈0.05）;Compared with the bilirubin groups of full-term and premature mice（23.33±4.89 and 30.17±3.76）,the apoptosis rates of the corresponding hypoxia＋bilirubin groups（27.67±3.01 and 34.33±3.01） were increased dramatically（P〈0.05）;whereas those of hyperoxia＋bilirubin groups didn＇t not show obviously increase（P〉0.05）.Immunohistochemistry showed,compared with the rats in full-term bilirubin group,TNF-α in the hippocampus of bilirubin premature mice（0.425±0.014） was increased significantly（P〈0.05）,while the expression of iNOS（0.253±0.009） was not significantly increased,and the expression of iNOS（0.437±0.024） and TNF-α（0.440±0.001） in hypoxia＋bilirubin group was increased remarkably（P〈0.05）.In contrast,in hyperoxia＋bilirubin group,the expression of iNOS（0.254±0.010） was not obviously increased（P〉0.05）,whereas the release of TNF-α（0.416±0.011） was not significantly increase（P〈0.05）.Conclusion Bilirubin aggravates the damage of hippocampus neurocytes by inducing cell apoptosis and releasing TNF-α.Hypoxia and prematurity improve the expression and releases of inflammatory factors and deteriorate bilirubin neurotoxicity.To control perinatal asphyxia and prematurity and inhibit bilirubin-induced inflammation play vital role in prophylaxis of bilirubin encephalopathy.