The Involvement Of Chloride Homeostasis Imbalance Within The Central Nervous System In Hepatic Encephalopathy And Its Mechanisms

Hepatic encephalopathy (HE) is defined as a spectrum of neuropsychiatricabnormalities in patients with severe liver dysfunction. It’s one of the mostcommon causes for the complications and deaths of hepatic patients. Thoughthere are many theories explaining its pathogenesis mechanism, it has not yetbeen fully elucidated. There is still no specific therapy.The dyskinesia of HE is closely related to the precise regulation of the basalganglia in the central nervous system (CNS) and the substantia nigra parsreticulate (SNr) plays the central role in the motion regulation in thecomplicated basal ganglia network. SNr is mainly composed of Gamma-aminobutyric acid (GABA)-containing projection neurons. Previous studies haveshown that GABA is closely related to the cerebral cell’s dysfunction of HE, butits mechanism is not clear. The chloride ion (Cl~-) is one of the most important intracellular anions. Inthe nervous system, the balance of chloride ion concentration between the insideand the outside of the neural cells, which is called the chloride homeostasis, isthe internal environmental basis in maintaining the shape and function of bothneurons and local loops. The imbalance of chloride ion can result in the changeof GABA transmission, so the chloride homeostasis and its regulatory factorshave become the potential therapeutic target of many GABA-related neuropathicdiseases. Studies revealed that the chloride homeostasis imbalance in manyplaces of the CNS could lead to different kinds of neurological disorders, but itsfunction in HE is still unknown.Na~+-K~+-Cl~-cotransporter1(NKCC1) and K~+-Cl~-cotransporter2(KCC2)are two chloride ion transporters which are similar in structures but totallyopposite in functions. NKCC1imports Cl into the neurons, while KCC2exports Cl out of neurons. They both play roles in inducing the neuron chloridehomeostasis so that they can work in GABA regulation. Studies showed that theexpressions or the functions of NKCC1and KCC2would change in manyneuropathic diseases and will further affect the neurotransmission of GABA.But whether the two transporters will play roles in the pathogenesis of HE is notyet fully investigated.This study will explore the roles of chloride homeostasis imbalance in SNrduring HE and its potential molecular mechanism, which may provideexperimental evidence for clarifying the pathogenesis of HE from a new viewand searching effective clinical treatment for HE. In the present study, glutamatedecarboxylase67(GAD67)-green fluorescent protein (GFP) knock-in transgenicmice were used to easily detect GABAergic neurons with GFP fluorescencewithin the SNr, because GFP expression was controlled by the promoter of the major GABA synthesizing enzyme, GAD67.Aim:1. We aimed to analyze the involvement and possible mechanisms ofaltered chloride homeostasis of GABAergic neurons within the SNr in the motordeficit in a model of the acute form of HE in acute liver failure usingGAD67-GFP transgenic mice.2. We aimed to detect the expression of NKCC1and KCC2in differentpathological type patients and analyse the relationship between the levels ofthem and liver or nerve function.Methods:1. We set up the HE model by intraperitoneal injection of thioacetamide(TAA). The HE and hypolocomotion of mice were confirmed by serumdetection of hepatic enzymes, blood and brain ammonia estimation, brain watermeasurement, histological staining of liver and neurological evaluations, as wellas open field test. We then incubated the brain slices with the fluorescent dyes incombination with laser confocal microscopy to observe the changes of chlorideion concentration of GABA neurons in the SNr of HE mice. Reversetranscriptase polymerase chain reaction (RT-PCR) and western blot were usedto test the mRNA and protein expressions of NKCC1and KCC2. Doubleimmunofluorescence staining, including detection of the GABA neurons in theSNr by anti-GFP antibody and the dopaminergic neurons in the substantia nigrapars compacta (SNc) by the anti-tyrosine hydroxylase (TH) antibody, was usedto detect the subcellular distribution of KCC2in the neurons. Nissl stain wasdone to evaluate whether the number of GABA neurons in the SNr wouldchange after TAA treatment. We also injected the NKCC1specific inhibitor Bumetanide or KCC2inhibitor di-isooctyladinpate (DIOA) stereotactically tothe substantia nigra to evaluate their effect on serum transaminase, blood andbrain ammonia, brain water content, histological staining of liver and motioncapacity.2. KCC2and NKCC1mRNA levels were detected by real time reversetranscriptase polymerase chain reaction (RT-PCR) in the plasma of36cirrhosispatients with HE of grade III-IV,20cirrhosis patients without HE and15healthy controls. Calculations of expression were made with the2CTmethod.The relations between the expression of KCC2and Child-Pugh scores, MELDscales, Glasgow coma scores and blood ammonia were also analyzed.Results:1. TAA treatment would lead to HE and hypolocomotion. Chlorideimaging indicated an enhanced local intracellular chloride concentration in theGABAergic neurons within the SNr of transgenic mice with HE. In addition, themRNA and protein levels of KCC2were reduced, particularly on neuronal cellmembranes; in contrast, NKCC1did not change significantly. TAA treatmentcould not change the number of GABAergic neurons in the SNr. Injection ofDIOA to the substantia nigra could increase the serum transaminase in thenormal mice. Injection of bumetanide to the substantia nigra could not decreasethe aspartate aminotransferase (AST) and alanine aminotransferase(ALT) in HEmice, though the lactate dehydrogenase (LDH) could be a little better. Livers ofnormal mice received injection of DIOA were histological normal and injectionof bumetanide could not improve the liver necrosis and inflammation of the HEmice. There was no significant difference in the blood and brain ammonia andbrain water content in the HE mice received DIOA or bumetanide injection tothe substantia nigra. The injection of DIOA to the substantia nigra would reduced motor activity in the normal mice and led to deterioratedhypolocomotion in the HE mice. In contrast, the injection of bumetanide to thesubstantia nigra did not normalize motor activity in HE mice.2. KCC2mRNAs in cirrhosis patients with HE showed significantdecreases when compared with those in cirrhosis patients without HE or healthycontrols. However, NKCC1mRNAs in different groups did not change. Inaddition, for cirrhosis patients with HE, there were significant negativecorrelations between KCC2levels with Child-Pugh scores, MELD scales andthe levels of blood ammonia; there was a significant positive correlationbetween KCC2levels with Glasgow coma scores.Conclusion:1. Our data suggest that altered chloride homeostasis might be responsiblefor the pathophysiology of hypolocomotion following HE. Drugs aimed atrestoring normal chloride homeostasis would be a potential treatment for hepaticfailure.2. Our study suggests that imbalance of KCC2and NKCC1may be a signalto predict HE.