Nicolas Chatauret

Effects of hypothermia on brain glucose metabolism in acute liver failure: a 1H/13C-nuclear magnetic resonance study ☆

BACKGROUND & AIMS Mild hypothermia has a protective effect on brain edema and encephalopathy in both experimental and human acute liver failure. The goals of the present study were to examine the effects of mild hypothermia (35 degrees C) on brain metabolic pathways using combined (1)H and (13)C-Nuclear Magnetic Resonance (NMR) spectroscopy, a technique which allows the study not only of metabolite concentrations but also their de novo synthesis via cell-specific pathways in the brain. METHODS (1)H and (13)C NMR spectroscopy using [1-(13)C] glucose was performed on extracts of frontal cortex obtained from groups of rats with acute liver failure induced by hepatic devascularization whose body temperature was maintained either at 37 degrees C (normothermic) or 35 degrees C (hypothermic), and appropriate sham-operated controls. RESULTS At coma stages of encephalopathy in the normothermic acute liver failure animals, glutamine concentrations in frontal cortex increased 3.5-fold compared to sham-operated controls (P < 0.001). Comparable increases of brain glutamine were observed in hypothermic animals despite the absence of severe encephalopathy (coma). Brain glutamate and aspartate concentrations were respectively decreased to 60.9% +/- 7.7% and 42.2% +/- 5.9% (P < 0.01) in normothermic animals with acute liver failure compared to control and were restored to normal values by mild hypothermia. Concentrations of lactate and alanine in frontal cortex were increased to 169.2% +/- 15.6% and 267.3% +/- 34.0% (P < 0.01) respectively in normothermic rats compared to controls. Furthermore, de novo synthesis of lactate and alanine increased to 446.5% +/- 48.7% and 707.9% +/- 65.7% (P < 0.001), of control respectively, resulting in increased fractional (13)C-enrichments in these cytosolic metabolites. Again, these changes of lactate and alanine concentrations were prevented by mild hypothermia. CONCLUSIONS Mild hypothermia (35 degrees C) prevents the encephalopathy and brain edema resulting from hepatic devascularization, selectively normalizes lactate and alanine synthesis from glucose, and prevents the impairment of oxidative metabolism associated with this model of ALF, but has no significant effect on brain glutamine. These findings suggest that a deficit in brain glucose metabolism rather than glutamine accumulation is the major cause of the cerebral complications of acute liver failure.

Loss of expression of glial fibrillary acidic protein in acute hyperammonemia.

Glial fibrillary acid protein (GFAP) is a major component of the glial filament network and alterations in expression of this protein in cultured astrocytes have been reported in response to acute ammonia exposure in vitro. In order to determine the effects of acute hyperammonemia in vivo on GFAP expression, brain extracts from rats with acute liver failure due to hepatic devascularization (portacaval anastomosis followed 24h later by hepatic artery ligation, HAL) were analyzed for GFAP mRNA using reverse transcription-polymerase chain reaction (RT-PCR) and appropriate oligonucleotide primers. GFAP protein was assayed by immunoblotting using a polyclonal antibody. Hepatic devascularization resulted in a significant 55-68% decrease (P<0.01) of GFAP mRNA and a concomitant loss of GFAP protein at precoma and coma stages of encephalopathy when brain water content was significantly increased and brain ammonia concentrations were in the millimolar range (1-5mM). Expression of a second glial filament protein S-100beta was unaffected by acute hyperammonemia. These findings suggest a role for GFAP in cell volume regulation and that loss of GFAP expression could contribute to the pathogenesis of brain edema in acute hyperammonemic syndromes.

MILD HYPOTHERMIA PREVENTS CEREBRAL EDEMA AND CSF LACTATE ACCUMULATION IN ACUTE LIVER FAILURE

Evidence from both clinical and experimental studies demonstrates that mild hypothermia prevents encephalopathy and brain edema in acute liver failure (ALF). As part of a series of studies to elucidate the mechanism(s) involved in this protective effect, groups of rats with ALF resulting from hepatic devascularization were maintained at either 37°C (normothermic) or 35°C (hypothermic), and neurological status was monitored in relation to cerebrospinal fluid (CSF) concentrations of ammonia and lactate. CSF was removed via implanted cisterna magna catheters. Mild hypothermia resulted in a delay in onset of encephalopathy and prevention of brain edema; CSF concentrations of ammonia and lactate were concomitantly decreased. Blood ammonia concentrations, on the other hand, were not affected by hypothermia in ALF rats. These findings suggest that brain edema and encephalopathy in ALF are the consequence of ammonia-induced impairment of brain energy metabolism and open the way for magnetic resonance spectroscopic monitoring of cerebral function in ALF. Mild hypothermia could be beneficial in the prevention of severe encephalopathy and brain edema in patients with ALF awaiting liver transplantation.

Selective alterations of brain osmolytes in acute liver failure: protective effect of mild hypothermia

The principal cause of mortality in patients with acute liver failure (ALF) is brain herniation resulting from intracranial hypertension caused by a progressive increase of brain water. In the present study, ex vivo high-resolution 1H-NMR spectroscopy was used to investigate the effects of ALF, with or without superimposed hypothermia, on brain organic osmolyte concentrations in relation to the severity of encephalopathy and brain edema in rats with ALF due to hepatic devascularization. In normothermic ALF rats, glutamine concentrations in frontal cortex increased more than fourfold at precoma stages, i.e. prior to the onset of severe encephalopathy, but showed no further increase at coma stages. In parallel with glutamine accumulation, the brain organic osmolytes myo-inositol and taurine were significantly decreased in frontal cortex to 63% and 67% of control values, respectively, at precoma stages (p<0.01), and to 58% and 67%, respectively, at coma stages of encephalopathy (p<0.01). Hypothermia, which prevented brain edema and encephalopathy in ALF rats, significantly attenuated the depletion of myo-inositol and taurine. Brain glutamine concentrations, on the other hand, did not respond to hypothermia. These findings demonstrate that experimental ALF results in selective changes in brain organic osmolytes as a function of the degree of encephalopathy which are associated with brain edema, and provides a further rationale for the continued use of hypothermia in the management of this condition.

Alterations in expression of genes coding for proteins of the neurovascular unit in ischemic liver failure

There is evidence to suggest that integrity of the neurovascular unit may be compromised in acute liver failure (ALF). In order to address this issue from a molecular standpoint, expression of an array of genes coding for key cerebrovascular endothelial cell and tight junction proteins were measured by reverse transcription-polymerase chain reaction in cerebral cortex of rats with ischemic liver failure resulting from hepatic devascularization (portacaval anastomosis followed 24h later by hepatic artery ligation) compared to appropriate sham-operated controls. Expression of P-glycoprotein, endothelin-1, von Willebrand factor, caveolin-1, occludin, and the endothelial nitric oxide synthase isoform (eNOS) were measured in brain extracts from rats with ALF at coma/edema stages of encephalopathy. The effects of mild hypothermia (35 degrees C) sufficient to prevent cerebral edema in ALF animals on the expression of these genes were also studied. Brain edema and hepatic coma in normothermic ALF rats was accompanied by selective increases in expression of eNOS. Expression of occludin and von Willebrand factor mRNAs were decreased at coma/edema stages of encephalopathy in ALF rats whereas, expression of other cerebrovascular endothelial cell markers endothelin-1, P-glycoprotein, and caveolin-1 were unaffected. Mild hypothermia led to normalization of brain water content and of eNOS mRNA. However, the correlation between increased eNOS expression and encephalopathy/edema grade was poor suggesting the existence of additional mechanisms. These findings underscore the multifactorial nature of brain edema/encephalopathy mechanisms in ALF and question the role of BBB breakdown as a major pathogenetic factor.

Selectively increased expression of the astrocytic/endothelial glucose transporter protein GLUT1 in acute liver failure.

Acute liver failure (ALF) is consistently accompanied by alterations in brain energy metabolites and recent nuclear magnetic resonance (NMR) studies suggest disturbances in brain oxidative metabolism in experimental ALF. Glucose transport across the blood‐brain barrier is essential to sustain brain energy metabolism and is accomplished by the facilitative glucose transporter GLUT1. To investigate alterations in brain glucose uptake in acute liver failure further, GLUT1 expression and [14C]‐2‐deoxy‐D‐glucose uptake were measured in the brains of rats with hepatic devascularization. RT‐PCR and Western blot analyses showed significant increases in steady‐state levels of GLUT1 mRNA and protein in frontal cortex as early as 6 h following hepatic devascularization, (prior to the onset of brain edema and encephalopathy) which remained elevated at coma stages of encephalopathy. Expression of the astrocytic (45‐kDa) and endothelial (55‐kDa) forms of GLUT1 was increased as a result of hepatic devascularization. Exposure of cultured astrocytes to pathophysiologically relevant concentrations of ammonia resulted in increased GLUT1 expression, suggesting that elevated ammonia levels are responsible for GLUT1 upregulation in ALF. Increased GLUT1 expression in ALF was selective, since expression of the neuronal glucose transporter GLUT3 and other glucose‐regulated proteins (GRP‐78 and GRP‐94) was unaltered. [14C]‐2‐deoxy‐D‐glucose autoradiography revealed increases in cerebral glucose uptake following the induction of GLUT1 in ALF. These results suggest that ammonia‐induced increases of GLUT1 expression resulting in increased cerebral glucose uptake occur in ALF and could contribute to the pathophysiological mechanisms responsible for the neurological complications of this condition.

Alterations in soluble guanylate cyclase content and modulation by nitric oxide in liver disease.

Hyperammonemia is the main responsible for the neurological alterations in hepatic encephalopathy in patients with liver failure. We studied the function of the glutamate-nitric oxide (NO)-cGMP pathway in brain in animal models of hyperammonemia and liver failure and in patients died with liver cirrhosis. Activation of glutamate receptors increases intracellular calcium that binds to calmodulin and activates neuronal nitric oxide synthase, increasing nitric oxide, which activates soluble guanylate cyclase (sGC), increasing cGMP. This glutamate-NO-cGMP pathway modulates cerebral processes such as circadian rhythms, the sleep-waking cycle, and some forms of learning and memory. These processes are impaired in patients with hepatic encephalopathy. Activation of sGC by NO is significantly increased in cerebral cortex and significantly reduced in cerebellum from cirrhotic patients died in hepatic coma. Portacaval anastomosis in rats, an animal model of liver failure, reproduces the effects of liver failure on modulation of sGC by NO both in cerebral cortex and cerebellum. In vivo brain microdialisis studies showed that sGC activation by NO is also reduced in vivo in cerebellum in hyperammonemic rats with or without liver failure. The content of alpha but not beta subunits of sGC are increased both in frontal cortex and cerebellum from patients died due to liver disease and from rats with portacaval anastomosis. We assessed whether determination of activation of sGC by NO-generating agent SNAP in lymphocytes could serve as a peripheral marker for the impairment of sGC activation by NO in brain. Chronic hyperammonemia and liver failure also alter sGC activation by NO in lymphocytes from rats or patients. These findings show that the content and modulation by NO of sGC are strongly altered in brain of patients with liver disease. These alterations could be responsible for some of the neurological alterations in hepatic encephalopathy such as sleep disturbances and cognitive impairment.

Mild Hypothermia in the Prevention of Brain Edema in Acute Liver Failure: Mechanisms and Clinical Prospects

Mild hypothermia (32°C–35°C) reduces intracranial pressure in patients with acute liver failure and may offer an effective adjunct therapy in the management of these patients. Studies in experimental animals suggest that this beneficial effect of hypothermia is the result of a decrease in blood–brain ammonia transfer resulting in improvement in brain energy metabolism and normalization of glutamatergic synaptic regulation. Improvement in brain energy metabolism by hypothermia may result from a reduction in ammonia-induced decrease of brain glucose (pyruvate) oxidation. Restoration of normal glutamatergic synaptic regulation by hypothermia may be the consequence of the removal of ammonia-induced decreases in expression of astrocytic glutamate transporters resulting in normal glutamate neurotransmitter inactivation in brain. Randomized controlled clinical trials of hypothermia are required to further evaluate its clinical impact.

Reduced expression of astrocytic glycine transporter (Glyt-1) in acute liver failure

A growing body of evidence suggests that alterations in N-methyl-D-asparate NMDA-mediated excitatory neurotransmission may be involved in the pathophysiology of hepatic encephalopathy (HE) in acute liver failure (ALF). The NMDA receptor requires glycine as a positive allosteric modulator. One of the glycine transporters Glyt-1 is expressed primarily in astrocytes of the cerebral cortex in association with regions of high NMDA receptor expression. As astrocytic transporters regulate the amino acid concentrations within excitatory synapses, the expression of Glyt-1 was studied in cortical preparations from rats with ischemic liver failure induced by portacaval anastomosis followed 24 hr later by hepatic artery ligation and from appropriate sham-operated controls. Expression of Glyt-1 mRNA, studied by reverse transcriptase-polymerase chain reaction, was significantly decreased in the brain at coma stages of encephalopathy (to approximately 50% of control) concomitant with a significant threefold increase of extracellular glycine, measured by in vivo cerebral microdialysis. These findings suggest that loss of expression of the Glyt-1 transporter may cause an impairment of regulation of glycine concentration at synaptic level and contribute to an overactivation of the NMDA receptor in ALF. The use of NMDA receptor antagonists, aimed specifically at the glycine modulatory site, could offer novel approaches to the prevention and treatment of HE in ALF.

Effects of liver failure on inter-organ trafficking of ammonia: implications for the treatment of hepatic encephalopathy

Abstract  Hepatic encephalopathy due to acute or chronic liver failure is invariably associated with hyperammonemia. High ammonia concentrations have deleterious effects on brain function by both direct and indirect mechanisms. There is increasing evidence to suggest that hyperammonemia in liver failure results from altered inter‐organ ammonia trafficking. Under normal conditions the gut produces ammonia from glutamine and urea. During liver failure, the contribution of the gut to hyperammonemia is predominantly the consequence of a diminished hepatic elimination rather than increased intestinal production. Normally, the liver removes ammonia by two distinct pathways, namely urea and glutamine synthesis catalyzed by enzymes that are, respectively, localized in the periportal and perivenous hepatocytes. The skeletal muscle relies solely on glutamine synthesis to remove ammonia. During liver failure, muscle glutamine production increases and the muscle becomes the major route for ammonia detoxification. The kidney is capable of both producing and removing ammonia. Under normal conditions, the kidney produces ammonia from glutamine which is mainly excreted into the renal vein, the remainder being excreted into the urine. However, in liver failure the excretion of the ammonia produced by the kidney is increased. Like skeletal muscle the brain relies solely on glutamine synthesis to remove ammonia. But unlike muscle, glutamine synthetase in the brain operates at nearly maximal capacity in normal conditions, and its activity is reduced during chronic liver failure. A better understanding of the alterations of inter‐organ ammonia trafficking could give rise to combined therapies aimed at reducing ammonia production and increasing ammonia removal by target organs.