胆红素脑病SD乳鼠模型中脑水通道蛋白-4表达发生变化

Expression and Significance of Brain AQP4 in the Neonatal SD Rat Model of Bilirubin Encephalopathy

神经细胞水肿是胆红素脑病(bilirubin encephalopathy,BE)发生发展过程中的重要病理变化。水通道蛋白-4(aquaporin-4,AQP4)的表达及分布异常与多种疾病所致细胞毒性脑水肿的发生发展具有密切联系。但胆红素脑病中AQP4的表达变化规律及其在病理进展中的作用尚不清楚。采用7日龄SD大鼠小脑延髓池注射胆红素溶液的方法,建立新生大鼠胆红素脑病模型。胆红素脑病模型根据胆红素作用时间的不同,分为12 h、24 h、48 h、72 h和7 d组。采用HE及尼氏染色,检测各新生大鼠脑组织的病理改变;应用透射电镜(TEM),检测胆红素作用24 h后,鼠脑组织超微结构的变化;应用免疫荧光及Western印迹,检测AQP4在脑组织中的表达变化。通过上述实验,以探讨AQP4的表达变化与胆红素所致脑损伤的关系。HE及尼氏染色结果显示,随着胆红素沉积时间的延长,神经细胞逐渐肿胀,细胞间隙增大,尼氏小体数量逐渐减少;电镜结果显示,胆红素脑病24 h后神经细胞线粒体出现肿胀;免疫荧光染色显示,24 h组AQP4的表达范围明显增加,其后表达范围逐渐减少,表达强度也随之减弱;Western印迹结果显示,AQP4表达在不同时间点呈现先增高后降低的趋势,在24 h达到峰值(24 h组138±011vs对照组087±021,P<005),在之后的各时间点上,AQP4的表达呈现下降趋势,而72 h组与7 d组AQP4表达均低于48 h组(P<005),基本恢复到对照组的表达水平(P>005)。上述结果提示,胆红素脑病中胆红素的毒性作用将引起AQP4表达量的改变,AQP4的表达变化与胆红素脑病中细胞毒性脑水肿的发生相关,并且可能在胆红素脑病脑损伤的进展中发挥作用。

Edema of neurocytes is an important pathological change in the occurrence and development of bilirubin encephalopathy(B E).Abnormal expression and distribution of aquaporin-4(AQP4)are closely related to the occurrence and development of cytotoxic brain edema caused by various diseases.However,the expression of AQP4 in BE and its role in the development of BE are still unclear.The BE model of neonatal SD rats was established by injecting bilirubin solutions into the cerebellomedullary cistern of 7-day-old SD rats.The BE model was divided into 12 h,24 h,48 h,72 h and 7 d groups according to the different acting intervals of bilirubin.HE and Nissl staining were used to detect the pathological changes of each brain tissue of neonatal rats;transmission electron microscopy(TEM)was used to detect the ultrastmctural changes of brain tissues after 24 h of bilirubin treatment;immunofluorescence and Western blotting were used to detect the changes of AQP4 expression in brain tissues.The temporal and spatial changes of AQP4 expression during the development of BE was studied by the above-mentioned experiments,and the relationship between the changes of AQP4 expression and brain injury was explored.The results of HE and Nissl staining showed that with the prolongation of bilirubin deposition duration,neurocytes become swollen and intercellular space is increased,with the number of Nissl bodies decreased gradually.TEM showed that the mitochondria of neurocytes are swollen after 24 hours of bilirubin treatment.Immunofluorescence staining showed that the expression range of AQP4 increased significantly in the 24 h group,and then subsequently decreased gradually;Western blotting results showed that the expression of AQP4 increased firstly and then decreased at different durations.At the 24 h group,the expression of AQP4 reached its peak(1.38±0.11 in 24 hours仍0.87±0.21 in the control,P<0.05).Subsequently,the expression of AQP4 decreased,while the expression of AQP4 in the 72 h group and 7 day group was lower than that in the 48 h group(P<0.05),and it is basically restored to the level of the control group(P>0.05).These results suggest that the toxic effect of bilirubin in BE will cause the change of AQP4 expression,which may be related to neuronal edema in BE and play a role in the progression of brain injury in BE.