Roles Of The Cation-chloride Cotransporters KCC2 And NKCC1 Within Substantia Nigra In Hepatic Encephalopathy-induced Hypolocomotion

Hepatic encephalopathy(HE)is defined as a spectrum of neuropsychiatric abnormalities in patients with severe liver dysfunction, after exclusion of other known brain disease. Symptoms of HE include attentional deficits, alterations of sleep patterns and muscular incoordination progressing to stupor and coma. Despite several decades of intensive scientific research, the precise pathogenesis of HE are still unknown. More attention should be focused on this area.Studies of the pathogenesis of a wide range of human neuropsychiatric disorders have been signi?cantly enhanced by the use of animal models. The rodent with thioacetamide-induced toxic liver injury is well-characterized animal model of HE in acute liver failure (ALF) (Type A HE). However, the total dose of thioacetamide was range from 200 mg/kg to 1200 mg/kg and the exact dose did not be reported in literatures. The relationship between the dose of thioacetamide (TAA) and neuropsychiatric abnormalities has not yet reached a consensus. A review of the recent literature reveals that the laboratory rat and rabbit are the most common species currently used for the studies in HE. There is a need of mouse models of type A HE to facilitate the studies.The mechanisms by which HE alters motor function remain unclear. It has been suggested that liver disease alters the neuronal circuit between basal ganglia and cortex that modulates motor function. The signals originated in the basal ganglia are modulated by the substantia nigra pars reticulate (SNr), which contains mainlyγ-aminobutyric acid (GABA)ergic nerous. In immature neurons, GABA receptor-mediated responses are often depolarizing, which is caused by Cl efflux due to high intracellular Cl concentration maintained in immature cells. In the nervous system, cation–chloride co-transporters (CCCs) especially KCC2 and NKCC1 are considered to play critical roles in the regulation of intracellular chloride concentration, and determine the switch from a hyperpolarizing to a depolarizing effect of GABA. But the role of KCC2 and NKCC1 within substantia nigra in HE-induced hypolocomotion is still unknown.In this study, we aimed to investigate the alternation of liver and brain function in mice with HE, and the appropriate dose of TAA to induce the mice model, and to choose reliable parameters for observation. In addition, we focused on the expression profiles of KCC2 and NKCC1 and intracellular Cl concentration in the substantia nigra of mice after TAA induced HE.Firstly, animal models of HE in C57BL/6 mice were induced by three intraperitoneal injections of TAA 200 mg/(kg?d) (n=21), 250 mg/(kg·d) (n=21), 300 mg/(kg?d) (n=22), 400 mg/(kg?d) (n=19) of body weight respectively at 24 hours intervals. Control mice (n=10) received sterile normal saline solution at the same time. Survival was evaluated per 24 hours after the first injection of TAA. The neurological score, motor activities and anxiety were evaluated on day 4. Serum ammonia, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were evaluated on day 5.Secondly, animal models of HE in C57BL/6 mice were induced by three intraperitoneal injections of the appropriate dose of TAA at 24 hours intervals. Control mice (n=6) received sterile normal saline solution at the same time. The expression of KCC2 mRNA and protein, NKCC1 mRNA and protein in the substantia nigra were evaluated by real-time RT-PCR and Western blot in controls and mice on the day 1 (n=6), day 4 (n=6), day 7 (n=6) after three injections of TAA respectively. The intracellular Cl concentration in the substantia nigra of mice was measured by MQAE chloride imaging at the same time.The results are as follows:1. There were significant differences in survival among each TAA groups and the survival in 400 mg/(kg?d) group was too low (31.6%).2. The neurological score, distance movement, time in central zone and the percentage of open arms duration in four TAA groups were significant decreased than the control group, while the percentage of open arms visits showed no significant differences in different groups.3. The level of serum ammonia, AST and ALT in four TAA groups were lower than the control group and the difference among the TAA groups was significant.4. Real-time RT-PCR and Western blot showed that NKCC1 mRNA and protein in the substantia nigra were up-regulated on the day 1, day 4, day 7 after three intraperitoneal injections of TAA [300mg/(kg?d)], while KCC2 was down-regulated.5. MQAE chloride imaging showed that the intracellular Cl concentration in the substantia nigra of mice with TAA-induced HE was increased than the control mice.This study shows that:1. Three intraperitoneal injections of TAA [300mg/(kg?d)] as 24 hour intervals was ideal method of type A hepatic encephalopathy model in mice.2. The brain dysfunction of this model with less motor activities and anxiety can be evaluated by the neurological score, open field and the elevated plus maze.3. The dose-dependent injury of liver by TAA can be evaluated by the level of serum ammonia, AST and ALT.4. NKCC1 mRNA and protein in the substantia nigra were up-regulated, and KCC2 mRNA and protein were down-regulated in mouse models of TAA induced-HE.5. The expressional changes of NKCC1 and KCC2 break the balance of intracellular chloride concentration which may contribute to the mechanism of hyper excitability leading to hypolocomotion.