Longitudinal neurometabolic changes in the hippocampus of a rat model of chronic hepatic encephalopathy

•In hepatic encephalopathy, early neurometabolic changes occur 2–4 weeks post-bile-duct ligation.•In hepatic encephalopathy, brain glutamine increases, but creatine and ascorbate decrease.•Changes in astrocyte morphology are observed 4 weeks after bile-duct ligation.•Early changes in hepatic encephalopathy are suggestive of osmotic/oxidative stress.•Early changes in hepatic encephalopathy are accentuated 6–8 weeks after bile-duct ligation. Background & AimsThe sequence of events in hepatic encephalopathy (HE) remains unclear. Using the advantages of in vivo 1H-MRS (9.4T) we aimed to analyse the time-course of disease in an established model of type C HE by analysing the longitudinal changes in a large number of brain metabolites together with biochemical, histological and behavioural assessment. We hypothesized that neurometabolic changes are detectable very early, and that these early changes will offer insight into the primary events underpinning HE.MethodsWistar rats underwent bile-duct ligation (BDL) and were studied before BDL and at post-operative weeks 2, 4, 6 and 8 (n = 26). In vivo short echo-time 1H-MRS (9.4T) of the hippocampus was performed in a longitudinal manner, as were biochemical (plasma), histological and behavioural tests.ResultsPlasma ammonium increased early after BDL and remained high during the study. Brain glutamine increased (+47%) as early as 2–4 weeks post-BDL while creatine (−8%) and ascorbate (−12%) decreased. Brain glutamine and ascorbate correlated closely with rising plasma ammonium, while brain creatine correlated with brain glutamine. The increases in brain glutamine and plasma ammonium were correlated, while plasma ammonium correlated negatively with distance moved. Changes in astrocyte morphology were observed at 4 weeks. These early changes were further accentuated at 6–8 weeks post-BDL, concurrently with the known decreases in brain organic osmolytes.ConclusionUsing a multimodal, in vivo and longitudinal approach we have shown that neurometabolic changes are already noticeable 2 weeks after BDL. These early changes are suggestive of osmotic/oxidative stress and are likely the premise of some later changes. Early decreases in cerebral creatine and ascorbate are novel findings offering new avenues to explore neuroprotective strategies for HE treatment.Lay summaryThe sequence of events in chronic hepatic encephalopathy (HE) remains unclear, therefore using the advantages of in vivo proton magnetic resonance spectroscopy at 9.4T we aimed to test the hypothesis that neurometabolic changes are detectable very early in an established model of type C HE, offering insight into the primary events underpinning HE, before advanced liver disease confounds the findings. These early, previously unreported neurometabolic changes occurred as early as 2 to 4 weeks after bile-duct ligation, namely an increase in plasma ammonium and brain glutamine, a decrease in brain creatine and ascorbate together with behavioural and astrocyte morphology changes, and continued to progress throughout the 8-week course of the disease. The sequence of events in hepatic encephalopathy (HE) remains unclear. Using the advantages of in vivo 1H-MRS (9.4T) we aimed to analyse the time-course of disease in an established model of type C HE by analysing the longitudinal changes in a large number of brain metabolites together with biochemical, histological and behavioural assessment. We hypothesized that neurometabolic changes are detectable very early, and that these early changes will offer insight into the primary events underpinning HE. Wistar rats underwent bile-duct ligation (BDL) and were studied before BDL and at post-operative weeks 2, 4, 6 and 8 (n = 26). In vivo short echo-time 1H-MRS (9.4T) of the hippocampus was performed in a longitudinal manner, as were biochemical (plasma), histological and behavioural tests. Plasma ammonium increased early after BDL and remained high during the study. Brain glutamine increased (+47%) as early as 2–4 weeks post-BDL while creatine (−8%) and ascorbate (−12%) decreased. Brain glutamine and ascorbate correlated closely with rising plasma ammonium, while brain creatine correlated with brain glutamine. The increases in brain glutamine and plasma ammonium were correlated, while plasma ammonium correlated negatively with distance moved. Changes in astrocyte morphology were observed at 4 weeks. These early changes were further accentuated at 6–8 weeks post-BDL, concurrently with the known decreases in brain organic osmolytes. Using a multimodal, in vivo and longitudinal approach we have shown that neurometabolic changes are already noticeable 2 weeks after BDL. These early changes are suggestive of osmotic/oxidative stress and are likely the premise of some later changes. Early decreases in cerebral creatine and ascorbate are novel findings offering new avenues to explore neuroprotective strategies for HE treatment.