P: 68 Brain Regional Susceptibility to Oxidative Stress in a Rat Model of Chronic Hepatic Encephalopathy: In-Vivo 1H MRS, Ex-Vivo ESR Spectroscopy and Histology Findings

Abstract

BACKGROUND: Oxidative stress (OS) is believed to be an important feature in the pathogenesis of chronic hepatic encephalopathy (CHE).1 In CHE impaired ammonium clearance by the diseased liver leads to brain glutamine accumulation. In-vitro, affected ammonium detoxification together with glutamine induces reactive-oxygen-species (ROS) generation associated with astrocyte impairment.2,3 ROS play an important role in cellular signalling, synaptic-plasticity, learning and memory. When in excess, they cause cellular damage.4,5 Systemic oxidative stress was previously shown in bile-duct-ligated rats (BDL). Using in-vivo-longitudinal 1H-MRS we previously observed the indirect presence of OS as a decrease of brain Asc in the hippocampus and cerebellum of BDL rats (model of CHE).6 We aimed to validate these findings using for the first time ex-vivo electron spin (ESR) spectroscopy and histological measures to assess OS levels. METHODS: In-vivo-1H-MRS: Cerebellum/hippocampus of adult rats were scanned before BDL and after every 2-weeks up to week 6 (n = 18) at 9.4T-MR (Varian/Magnex-Scientific) using SPECIAL-sequence 7 (TE = 2.8 ms). Ex-vivo-ESR: ESP300E (Bruker-BioSpin) was used for intracellular superoxide anion detection. Hippocampus/cerebellum were extracted at 6weeks post-BDL/sham-surgery (n = 9), incubated in medium with 10 mM-CMH-cell-permeable spin-trap (Noxygen GmbH). Immunohistochemistry (ICH): GPX1 (anti-oxidative-enzyme) staining was performed (n = 6). RESULTS: The increase in plasma NH4+ and bilirubin confirmed the presence of chronic liver disease. A ∼80% increase in brain glutamine was measured at week6, while Asc showed a stronger decrease in cerebellum (−32% at week 6, P < 0.001) (Figure 1a). ESR revealed differences in redox-state between the two brain regions (∼31% higher in cerebellum, P < 0.004) (Figure 1b). However, the relative change in both regions between BDL and shams was similar ∼42%. The significant increase of hippocampal/cerebellar OS in BDL (P < 0.01, P < 0.001) corroborate the 1H-MRS findings of decreased Asc concentrations (Figure 1a). ICH with GPX1-enzyme,8,9 revealed an increase activity in Purkinje and granular-cell-layer of BDL cerebellum (Figure 1c). Purkinje-cells also showed shrinking soma (BDL: 8.04 ± 1.8 µm2, sham: 12.5 ± 1.2 µm2, P < 0.00001). Elevated GPX1 in cerebellum suggests that GSH-synthesis may increase (confirmed by increased GSH in 1H MRS, Figure 1a) in response to OS-related injury. Elevated OS might suggest that ROS could lead to brain functions disruption.10–12 CONCLUSIONS: Our results showed for the first time the presence of central OS in BDL rats at 6-weeks post-surgery. Changes varied according to brain region and proved a different susceptibility of cerebellum and hippocampus to CHE, reflected by a relationship between increased OS and interrupted antioxidant-defence-system. OS is involved in the propagation of cellular injury and may be an important factor in the aetiology of the CHE.