摘要目的:探讨胆道闭锁(biliary atresia，BA)小鼠生存晚期的脑损伤情况及N-乙酰半胱氨酸(N-acetylcysteine，NAC)对BA小鼠脑损伤的治疗作用。方法:将160只新生BALB/c小鼠按随机数字表法平均分为疾病组、空白对照组、治疗组及药物对照组，每组各40只。疾病组出生后第3天腹腔注射30 μl恒河猴轮状病毒(rhesus rotavirus，RRV)诱导BA小鼠模型；治疗组出生后第3天注射等量RRV后每日腹腔注射10 mg/kg的NAC治疗至第12天；空白对照组出生后第3天予腹腔注射30 μl未扩增RRV的MA104细胞培养上清；药物对照组出生后第3天起每日腹腔注射10 mg/kg的NAC至第12天。取第19天小鼠进行神经功能评分，检测血浆脑损伤标志物神经元特异性烯醇化酶(neuron specific enolase，NSE)和中枢神经特异蛋白S100β水平。评估小鼠海马锥体细胞层病理切片苏木精-伊红(hematoxylin and eosin，HE)染色、尼氏染色及Iba1免疫组织化学染色情况。评估小鼠肝门管区HE染色、细胞角蛋白19(cytokeratin 19，CK19)免疫组织化学染色及天狼猩红染色情况。组间样本神经功能评分、血浆脑损伤标志物水平比较采用 t检验，组间样本海马锥体细胞层HE染色评分、尼氏染色评分、Iba1免疫组织化学染色评分比较采用Wilcoxon秩和检验。 结果:生后第19天疾病组小鼠神经功能评分中一般评分及局灶评分分别为(10.24±1.27)分、(6.84±1.05)分，与空白对照组小鼠均为0分比较，一般评分( t＝－39.90， P<0.001)、局灶评分( t＝－32.03， P<0.001)差异均有统计学意义。出生后第19天治疗组小鼠神经功能评分中一般评分及局灶评分分别为(4.84±0.92)分、(1.88±0.59)分，与疾病组小鼠比较，一般评分( t＝16.81， P<0.001)、局灶评分( t＝20.25， P<0.001)差异均有统计学意义。出生后第19天药物对照组小鼠神经功能评分一般评分及局灶评分均为0分。疾病组与空白对照组小鼠血浆NSE水平分别为(27.79±2.50)ng/ml、(18.93±2.78)ng/ml，差异有统计学意义( t＝－10.63， P<0.001)，治疗组血浆NSE水平为(23.08±1.96)ng/ml，与疾病组比较，差异有统计学意义( t＝6.64， P<0.001)。药物对照组血浆NSE水平为(18.95±2.79)ng/ml，与空白对照组比较差异无统计学意义( t＝－0.02， P＝0.983)。疾病组与空白对照组血浆S100β水平分别为(285.01±27.84)pg/ml、(180.77±35.52)pg/ml，差异有统计学意义( t＝－10.33， P<0.001)，治疗组血浆S100β水平为(218.29±31.02)pg/ml，与疾病组比较，差异有统计学意义( t＝7.16， P<0.001)。药物对照组血浆S100β水平为(188.11±46.29)pg/ml，与空白对照组比较差异无统计学意义( t＝－0.57， P＝0.571)。疾病组HE染色评分0、1、2分视野数量为8、17、5个，空白对照组HE染色评分均为0分，差异有统计学意义( Z＝－5.74， P<0.001)。治疗组HE染色评分0、1、2分视野数量为17、13、0个，与疾病组比较，差异有统计学意义( Z＝－2.77， P＝0.006)。药物对照组所有视野HE染色评分均为0分。疾病组尼氏染色评分0、1、2分视野数量为6、19、5个，空白对照组尼氏染色评分均为0分，差异有统计学意义( Z＝－6.14， P<0.001)。治疗组尼氏染色评分0、1、2分视野数量为22、8、0个，与疾病组比较，差异有统计学意义( Z＝－4.28， P<0.001)。药物对照组所有视野尼氏染色评分均为0分。疾病组Iba1免疫组织化学染色阴性、弱阳性、阳性、强阳性视野数量分别为1、0、14、0个，空白对照组阴性、弱阳性、阳性、强阳性视野数量分别为13、1、1、0个，差异有统计学意义( Z＝－4.57， P<0.001)。治疗组小鼠Iba1免疫组织化学染色阴性、弱阳性、阳性、强阳性视野数量分别为10、3、2、0个，与疾病组相比，差异有统计学意义( Z＝－4.11， P<0.001)。第19天药物对照组小鼠Iba1染色阴性、弱阳性、阳性、强阳性视野数量分别为13、2、0、0个，与空白对照组相比，差异无统计学意义( Z＝－0.07， P＝0.944)。肝门管区HE染色见疾病组炎症细胞浸润，治疗组炎症细胞浸润减少。肝门管区CK19免疫组织化学染色可见疾病组胆管缺如，治疗组可见少量发育不全的胆管。肝门管区天狼猩红染色可见疾病组大量肝组织纤维化，治疗组肝组织纤维化减少。 结论:BA小鼠生存晚期存在脑损伤，早期使用NAC治疗可以减轻其脑损伤。

abstractsObjective:To explore the status of brain injury in late survival mice with biliary atresia (BA) and to examine the therapeutic efficacy of N-acetylcysteine (NAC) for brain injury in BA mice.Methods:A total of 160 neonatal Balb/c mice were randomized into four groups of blank control, disease, treatment and drug control (n=40 each). Treatment protocols of each group: Disease group adopted the BA murine model of chronic liver injury induced by an intraperitoneal injection of 30 μl rhesus rotavirus (RRV) at Day 3 post-birth; Treatment group received a daily intraperitoneal injection of 10 mg/kg of NAC after an injection of 30 μl RRV until Day 12 post-birth.Blank control group received 30 μl MA104 cell culture supernatant without RRV amplification at Day 3 post-birth.Drug control group received a daily intraperitoneal injection of 10 mg/kg NAC from Day 3-12 post-birth.Twenty-five mice at Day 19 in each group were selected for neurological function score.Plasma levels of neuron-specific enolase (NSE) and central nervous system specific protein (S100β), markers of brain injury, were assayed in 21 subjects from each group.Hematoxylin and eosin (HE) and Nissl stains of pathological sections were evaluated with 5 mice in each group and 6 40× visual fields were randomly selected for each mouse.Three mice in each group were selected and 5 40× visual fields in each mouse were randomized for evaluating ionized calcium binding adapter molecule 1 (Iba1) in pathological sections.HE, cytokeratin 19 (CK19) immunohistochemical and Sirius scarlet stains were evaluated in 3 mice from each group.Student t-test was employed for comparing neurological function scores and plasma markers of brain injury between two groups.Wilcoxon rank-sum test was utilized for comparing HE scores, Nissl and Iba1 immunohistochemical stain scores in hippocampal pyramidal cell layer between two groups.Results:At Day 19 post-birth, general and focal scores of disease group were (10.24±1.27) and (6.84±1.05) points respectively.As compared with blank control group with 0 points, the differences of general score ( t=-39.90, P<0.001) and focal score ( t=-32.03, P<0.001) were statistically significant.General and focal scores were (4.84±0.92) and (1.88±0.59) points in treatment group and the differences in general score ( t=16.81, P<0.001) and focal score ( t=20.25, P<0.001) were statistically significant as compared with those in disease group.Neurological function score of drug control group was 0.The levels of plasma NSE were (27.79±2.50) and (18.93±2.78) ng/ml in disease and blank control groups and the differences were statistically significant ( t=-10.63, P<0.001). Plasma NSE level was (23.08±1.96) ng/ml in treatment group and the difference was statistically significant as compared with disease group ( t=6.64, P<0.001). The plasma NSE level of drug control group was (18.95±2.79) ng/ml.There was no significant difference with blank control group ( t=-0.02, P=0.983). The plasma levels of S100β in disease and blank control groups were (285.01±27.84) and (180.77±35.52) pg/ml respectively.And the difference was statistically significant ( t=-10.33, P<0.001). Plasma S100β level was (218.29±31.02) pg/ml in treatment group and the difference was statistically significant as compared with disease group ( t=7.16, P<0.001). Plasma S100β level of drug control group was (188.11±46.29) pg/ml and no significant inter-group difference existed ( t=-0.57, P=0.571). Counts of visual fields with HE stain scores of 0, 1 and 2 were 8, 17 and 5 in disease group while those of blank control group were all 0.And the difference was statistically significant ( Z=-5.74, P<0.001). The number of visual fields with HE staining scores of 0, 1 and 2 in treatment group was 17, 13 and 0, and the difference was statistically significant compared with disease group ( Z=-2.77, P=0.006). HE stain score of all visual fields was 0 in drug control group.Counts of visual fields with Nissl stain scores 0, 1, and 2 in disease group were 6, 19 and 5 while Nissl stain scores were all 0 in blank control group and the difference was statistically significant ( Z=-6.14, P<0.001). The number of visual fields with Nissl scores of 0, 1 and 2 was 22, 8 and 0 in treatment group and the difference was statistically significant as compared with disease group ( Z=-4.28, P<0.001). Nissl stain score of all visual fields was 0 in drug control group.The number of negative, weak positive, positive and strong positive visual fields of Iba1 immunohistochemical stain was 1, 0, 14 and 0 in disease group respectively, while counts of negative, weak positive, positive and strong positive visual fields in blank control group was 13, 1, 1, 0, respectively, with statistical significance ( Z=-4.57, P<0.001). Counts of Iba1 immunohistochemical stain negative, weak positive, positive and strong positive visual fields were 10, 3, 2 and 0 in treatment group and the difference was statistically significant as compared with disease group ( Z=-4.11, P<0.001). At Day 19, counts of Iba1 negative, weak positive, positive and strong positive visual fields were 13, 2, 0 and 0 in drug control group and there was no significant difference as compared with blank control group ( Z=-0.07, P=0.944). HE staining of hepatic portal area revealed an infiltration of inflammatory cells in disease group and diminished in treatment group.CK19 immunohistochemical stain of hepatic portal area hinted at absence of bile ducts in disease group and few dysplastic bile ducts were seen in treatment group.Sirius scarlet stain of hepatic portal area indicated massive liver tissue fibrosis in disease group and mild liver tissue fibrosis in treatment group. Conclusions:BA mice develops brain injury in late survival and early NAC treatment can significantly lessen brain injury.