Hippocampal apoptosis and mitochondrial dysfunction in a low grade hepatic encephalopathy model.

TALLIS, S.; LORES-ARNAIZ, S.; ROSELLO, D.; LAGO, N.; LEMBERG, A.; BOVERIS, A.; PERAZZO, J.C.; BUSTAMANTE, J.

Buenos Aires, Argentina

Simposio; Advances in Biomedical Sciences, International Symposium of the International Master in Biomedical Sciences University of Buenos Aires (Argentina) and Albert Ludwigs University of Freiburg (Germany) “Bright minds for a better world”.; 2008

Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires

Introduction Portal Hypertension (PH) and Hyperammonemia, a cytotoxic of the Central Nervous System, are major features in several human liver pathologies, as cirrhosis, and could lead to different grades of Hepatic Encephalopathy. Prehepatic PH could be regarded as a valid experimental model of Low Grade Hepatic Encephalopathy (LGHE) specially focusing in the Hippocampal (H) brain area damage. Different mechanisms lead to neuronal death. One of them is the apoptotic pathway that could be triggered by extrinsic and/or intrinsic pathways, the last involve the mitochondrial participation. The aim of the present work was to study if in experimental LGHE due to PH Apoptosis (A) could be present and related to the mitochondrial H changes previously described. LGHE was induced through portal vein stenosis in rats. To evaluate the possible presence of A, TUNEL assay and protein Bax expression were used. Respiratory mitochondrial parameters and mtNOS activity and expression were used as mitochondrial functionality markers.   Methods Animal model: Adult male Wistar Kyoto rats divided into 2 groups: LGEH and Sham operated rats were used. LGHE was induced performing a calibrated stenosis of the portal vein. Ten days after surgery rats were anesthetized, portal pressure recorded and plasma ammonia concentrations determinate. Isolation of mitochondria: H pool from four rats was used for the isolation of mitochondrial fraction by differential centrifugation. Further mitochondrial purification was performed by Ficoll gradient. Mitochondrial respiration: A two-channel respirometer for high-resolution respirometry was used. Mitochondrial respiratory rates were measured using malate-glutamate as substrate to measure state 4 respiration rate and adding ADP to measure state 3 respiration rate for the determination of respiratory controls, determinated as the ratio of oxygen uptake in state 3/state 4. Mitochondrial nitric oxide synthase activity: NO production was measured in submitochondrial membranes using a spectrophotometric method by following the oxidation of oxyhemoglobin to methemoglobin. Mitochondrial nitric oxide synthase expression (Western blot): Submitochondrial membranes were separated by SDS-PAGE. After transference, membranes were probed with a polyclonal antibody against the neuronal isoform of nitric oxide synthase. The nitrocellulose membrane was subsequently incubated with a secondary antibody conjugated with peroxidase and revealed by chemiluminescence. Hippocampal Apoptosis: At the time hippocampus were isolated and in order to identify apoptosis, a nuclear DNA fragmentation was determinate by the in situ nick end labeling (TUNEL) assay in thin H tissue sections and then analyzed under fluorescence microscopy. Bax expression (Western blot): Submitochondrial membranes were separated by SDS-PAGE. After transference, membranes were probed with a polyclonal antibody against Bax protein. A secondary antibody conjugated with peroxidase and revealed by chemiluminescence was used.   Discussion This study describes for the first time Hippocampal Apoptosis in Central Nervous System in a LGHE model induced by prehepatic PH. This could be regarded as a probable pathway in the onset of LGHE.