大鼠脑冲击伤早期启动神经元焦亡的发生及可能机制

Incidence and possible mechanism of neuronal pyrolysis at the early stage of blast-induced traumatic brain injury in rats

目的探讨大鼠脑冲击伤(bTBI)早期是否启动神经元焦亡的发生及可能机制。方法选取雄性SD大鼠52只,按随机数字表法分为空白对照组和bTBI组,bTBI组又根据观察时间分为1,6,12 h三个亚组,各组13只大鼠。bTBI组麻醉后将颈部以下保护,用BST-Ⅰ型激波管致头部冲击伤。空白对照组除不致伤外,其他操作与bTBI组一致。伤后6,12 h进行改良神经功能严重程度评分(mNSS)检测,观察大鼠神经功能障碍程度。伤后1,6,12 h行MRI T2WI扫描观察异常信号影;解剖大鼠头部进行大体观察;HE染色观察脑组织病理改变;电镜下观察神经元超微结构改变;ELISA检测炎症因子[白细胞介素(IL)-1β、IL-18]的表达水平;免疫荧光共染检测蛋白[凋亡相关微粒蛋白(ASC)、核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)]在神经元上的表达情况。结果(1)bTBI 6 h组mNSS[3(3.0,3.0)分]高于空白对照组[0(0,0)分](P<0.05),bTBI 12 h组mNSS[2(2.0,2.0)分]高于空白对照组[0(0,0)分](P<0.05);bTBI 6 h组mNSS与bTBI 12 h组比较,差异无统计学意义(P>0.05)。(2)与空白对照组比较,bTBI组MRI T2WI序列均发现脑组织体积增大,整体T2信号影增高,扣带回增宽变扁,大脑皮质、第三脑室较周围存在高信号影,但各bTBI亚组间无明显差异。(3)bTBI组大体观察发现脑组织出现不同程度的水肿和充血;HE染色显示各bTBI亚组脑组织皮质区部分细胞出现肿胀变形和血管管腔收缩,血管周围间隙增宽。(4)电镜下发现bTBI组神经元肿胀主要发生在线粒体,且在伤后12 h内肿胀程度逐步加深。(5)bTBI 1,6,12 h组IL-18水平分别为(4.12±0.42)pg/ml、(5.20±0.29)pg/ml、(6.82±0.61)pg/ml,较空白对照组的(2.94±0.49)pg/ml均明显升高(P均<0.01);bTBI 1,6,12 h组IL-1β水平分别为(2.48±0.15)pg/ml、(4.10±0.38)pg/ml、(5.04±0.28)pg/ml,较空白对照组的(1.86±0.32)pg/ml均明显升高(P均<0.01);各bTBI亚组血清中IL-18及IL-1β蛋白在伤后12 h内均呈不同程度的升高(P均<0.05)。(6)ASC和NLRP3蛋白在脑皮质神经元阳性细胞的胞质上表达,且随时间延长表达均呈上升趋势。结论大鼠在bTBI早期启动脑皮质神经元发生焦亡。bTBI大鼠神经功能的缺失可能是冲击波作用于头部导致神经元焦亡引起。

Objective To investigate activation of neuronal pyrophosis at the early stage in blast-induced traumatic brain injury(bTBI)in rats and its possible mechanism.Methods Fifty-two adult male SD rats were divided into blank control group and bTBI group according to the random number table method.The bTBI group was subdivided into 1-hour,6-hour and 12-hour groups according to the observation time.Each group contained 13 rats.In bTBI group,the rats were protected below the neck to inflict head impact injuries with a BST-I surge tube after anesthetization.In blank control group,the rats underwent the same procedures except for the free of injury.A modified neurological severity score(mNSS)was performed at 6 hours and 12 hours post-injury to observe the degree of neurological deficits.After 1 hour,6 hours and 12 hours of injury,MRI T2WI scans were performed to observe abnormal signal shadows,brains were dissected for gross observation,HE staining was performed to observe histopathological changes in the brain,neuronal ultrastructural changes were observed under electron microscopy,ELISA method was performed to detect expression levels of inflammatory factors including interleukin(IL)-18 and IL-1β,and immunofluorescence co-staining was used to detect expression levels of proteins on neurons,including apoptosis-associated microprotein(ASC)and nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3).Results(1)The mNSS in bTBI 6-hour group[3(3.0,3.0)points]and in bTBI 12-hour group[3(2.0,2.0)points]were higher than that in blank control group[0(0,0)points](both P<0.05).The mNSS score had no statistical significance between bTBI 6-hour group and 12-hour group(P>0.05).(2)Compared with blank control group,the MRI T2WI sequence in bTBI group found that the brain volume was increased,overall T2 signal shadow increased,cingulate gyrus widened and flattened;the cerebral cortex and third ventricle had high signal shadows but there was no statistical significance among bTBI subgroups.(3)The gross observation in bTBI group showed varying degrees of edema and congestion in brains.HE staining showed swelling and deformation of some cells in the cortical area of brain tissues,constriction of the vascular lumen and widening of the perivascular gap in each bTBI subgroup.(4)Electron microscopy revealed that the swelling of neurons in bTBI group occurred mainly in the mitochondria,with gradually increased degree of swelling within 12 hours after injury.(5)Levels of IL-18 in 1-hour,6-hour and 12-hour groups were(4.12±0.42)pg/ml,(5.20±0.29)pg/ml and(6.82±0.61)pg/ml,respectively,significantly higher than those in blank control group(all P<0.01).Levels of IL-1βin bTBI 1-hour,6-hour and 12-hour groups were(2.48±0.15)pg/ml,(4.10±0.38)pg/ml and(5.04±0.28)pg/ml,respectively,significantly higher than those in blank control group[(1.86±0.32)pg/ml](all P<0.01).Serum levels of IL-18 and IL-1βin each bTBI subgroup showed different degrees of elevation at 12 hours post-injury(all P<0.05).(6)ASC and NLRP3 proteins were expressed on the cytoplasm of brain cortical neuron-positive cells,with tendency increase over time.Conclusions Neuronal pyroptosis is initiated at the early stage of bTBI in rats.Neurological dysfunction in bTBI rats may be associated with neuronal pyrolysis generated by shock wave acting on the head.