Experimental study of N-acetylcysteine in the treatment of brain injury in mice with biliary atresia

Abstract

Objective 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.