Protective effect of hypothermia on ammonia toxicity and energetic disturbances in astrocytes

Introduction: Ammonia is a key factor in the pathogenesis of hepatic encephalopathy (HE) due to acute liver failure (ALF). Acute ammonia treatment causes cell swelling and energy failure of astrocytes in HE, which are able to compensate partly by increased anaerobic metabolism and lactate synthesis. On the other hand, astrocytes selectively express glutamine synthetase (GS), which is responsible for ammonia detoxification. It has been the prevailing hypothesis that the osmotic disturbance induced by glutamine leads to astrocyte swelling and consequently brain edema in HE. However, recent studies point to a limited capacity of GS and to direct effects of ammonia on mitochondrial energy metabolism. Mild hypothermia is known to offer protection from severe encephalopathy and lactate accumulation in human ALF. However, the mechanisms are still unknown. Aims: Since astrocytes are the primary pathological target in HE, our aim was to investigate if hypothermia protects against ammonia-induced energy failure in cultured astrocytes. Multinuclear NMR spectroscopy was used to evaluate possible underlying mechanisms in this protection. Methods: Primary cultures of cortical astrocytes were prepared from 1-2 day old Sprague-Dawley rats and cultivated for four weeks. Then, confluent astrocyte cultures were incubated for 12 hours with DMEM containing 5 mM [1-C]glucose in the presence or absence of ammonia (5 mM NH4Cl) under normothermic (37 °C), mild hypothermic (33°C) or moderate hypothermic (27°C) conditions. After removal of the incubation media, the cells were extracted with perchloric acid (PCA) and lyophilized. The lyophilized samples were redissolved in 0.5 ml D2O and centrifuged. Prior to the NMR analysis, the samples were neutralized with D2O and NaOH to allow unique chemical shift assignments and to prevent glutamine-carbamate formation in the media. After recording of Hand C-NMR spectra, samples were treated with EDTA (to form divalent cation complexes) to perform proton-decoupled P-NMR measurements. H, Cand P-NMR spectra were recorded on a Bruker WB360 spectrometer. Results: Cellular energy state. P-NMR spectra of PCA extracts depict high-energy phosphates such as nucleoside diand triphosphates (NDP’s, NTP’s), in particular ADP and ATP (adenosine diand triphosphate), and phosphocreatine (PCr). The analysis of these spectra revealed that 12 hour treatment of astrocytes with 5 mM NH4Cl resulted in decreased ATPand PCr concentrations to <50% of control values (Fig. 1). The levels of creatine (Cr) (calculated from H-NMR spectra, Fig. 2) concomitantly increased to 150% of control, resulting in markedly decreased PCr/Cr ratios to 34% of controls. Mild hypothermia (33°C) alone produced no or only slight changes in high-energy phosphates of astrocytes, but significantly attenuated the ammonia-induced depletion of ATP and PCr under normothermic conditions. While a further decrease in the incubation temperature (from 27°C to 33°C) did not cause a further attenuation of ammonia-induced ATP depletion, PCr levels were completely restored (p<0.05). Glutamine and glutamate. 12 h treatment of astrocytes with NH4Cl resulted in increased glutamine levels to 159±7.5% of controls. Mild and moderate hypothermia in these cells attenuated the increase in glutamine levels slightly by 6% (ns) and 29% (p<0.05), respectively. Its de novo synthesis after glucose entry into the TCA cycle by pyruvate carboxylase (PC) (glutamine labelled at C-2) increased to 220±13.4% after treatment with NH4Cl, leading to an increased percentage C enrichment to 139±15% of controls. Mild and