| Hepatic encephalopathy (HE) is one of the most common causes for the complications and deaths of hepatic patients. Though there exist many theories explaining its pathogenesis mechanism, it has not yet been fully elucidated. There is no specific therapy, which makes the hepatic encephalopathy researches more important and urgent. Studies in recent years reveal that an important inhibitory neurotransmitter, Gamma-amino butyric acid (GABA), in the central nervous system is obviously related to the cerebral cell's dysfunction of hepatic encephalopathy disease. KCC2/NKCC1 is a pair of chloride ion transmitters which adjusts the GABA status. So researches on GABA's plastic changes under HE can start from here and HE's central mechanism could be illuminated from an new angle.Aim:1. To observe the colocalization of KCC2 or NKCC1 with GFP-positive neurons within the substantia nigra pars reticulata (SNr), the glutamate decarboxylase 67-green fluorescence protein (GAD67-GFP) knock-in mice were used in the present study.2. To establish SYBR Green I real-time fluorescence quantitative PCR method to test normal mice's KCC2 and NKCC1 expressions and hepatic encephalopathy mice's expressions at different disease stages in substantia nigra reticulate, and also to discuss its function in the pathogenesis of hepatic encephalopathy.Methods:1. Double-1abeled techniques were used by in situ hybridization combined with immunohistochemistry for GFP and double immunofluorescence histochemistry for GFP and KCC2 or NKCC1. The stained sections were observed under light microscope and a confocal laser-scanning microscope.2. The internationally recognized TAA intraperitoneal hepatic encephalopathy animal injection model and SYBR real-time fluorescence quantitative PCR were used to test normal mice's KCC2 and NKCC1 expressions and hepatic encephalopathy mice's expressions at different disease stages in substantia nigra reticulate.Results:1. The results showed that over 90% of GFP-positive neuronal cell bodies in the SNr showed hybridization signals for KCC2 mRNA, and that almost 50% of neuronal cell bodies with KCC2 mRNA signals were GFP-positive. At the same time, about 80% of GFP-positive neuronal cell bodies in the SNr showed hybridization signals for NKCC1 mRNA, and 35% of neuronal cell bodies with NKCC1 mRNA signals were GFP-positive. In addition, we found that about 50.5% of KCC2-positive neurons and 90.0% of GFP-positive neurons were KCC2/GFP double-labeled neurons in the SNr. Similiarly, about 42.5% of NKCC1-positive neurons and 80.5% of GFP-positive neurons were NKCC1/GFP double-labeled neurons in the SNr.2. Compared with normal mice, KCC2 expression in substantia nigra reticulate of hepatic encephalopathy ones reduced (P<0.05) on the first day after the model was established, and NKCC1 increased (P<0.05); then KCC2 was increasing and NKCC1 was falling down gradually; in the fourth week, compared with the normal ones, KCC2 of the hepatic encephalopathy mice still reduced (P<0.05) and NKCC1 still increased (P<0.05); these expressions did not have obvious difference between these two kinds of mice in the eighth week.Conclusions:1. The present results indicate that majority of GABAergic neurons within the SNr of GAD67-GFP knock-in mice, which are GFP-positive, are colocalizaed with KCC2 or NKCC1, suggesting an important role of the cation-chloride cotransporters in the regulation of GABAergic transmission in substantia nigra.2. The changes of the expressions of KCC2 and NKCC1 are related to the acute pathogenesis of hepatic encephalopathy. With the development of the disease, the body would make compensatory adjustments, which made the expressions of KCC2 and NKCC1 return to the normal level. The changes of the expressions of KCC2 and NKCC1 and also the change of GABA's status caused by them, might be one of the pathogenesis mechanisms of hepatic encephalopathy. |
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