Cristina R. Bosoi

Identifying the direct effects of ammonia on the brain

Elevated concentrations of ammonia in the brain as a result of hyperammonemia leads to cerebral dysfunction involving a spectrum of neuropsychiatric and neurological symptoms (impaired memory, shortened attention span, sleep-wake inversions, brain edema, intracranial hypertension, seizures, ataxia and coma). Many studies have demonstrated ammonia as a major player involved in the neuropathophysiology associated with liver failure and inherited urea cycle enzyme disorders. Ammonia in solution is composed of a gas (NH3) and an ionic (NH4+) component which are both capable of crossing plasma membranes through diffusion, channels and transport mechanisms and as a result have a direct effect on pH. Furthermore, NH4+ has similar properties as K+ and, therefore, competes with K+ on K+ transporters and channels resulting in a direct effect on membrane potential. Ammonia is also a product as well as a substrate for many different biochemical reactions and consequently, an increase in brain ammonia accompanies disturbances in cerebral metabolism. These direct effects of elevated ammonia concentrations on the brain will lead to a cascade of secondary effects and encephalopathy.

AST-120 (spherical carbon adsorbent) lowers ammonia levels and attenuates brain edema in bile duct-ligated rats.

The pathogenesis of hepatic encephalopathy is multifactorial, involving gut‐derived toxins such as ammonia, which has been demonstrated to induce oxidative stress. Therefore, a primary hepatic encephalopathy treatment target is reducing ammonia production in the gastrointestinal tract. AST‐120, an oral adsorbent of engineered activated carbon microspheres with surface areas exceeding 1600 m2/g, acts as a sink for neurotoxins and hepatotoxins present in the gut. We evaluated the capacity of AST‐120 to adsorb ammonia in vitro and to lower blood ammonia, oxidative stress and brain edema in cirrhotic rats. Cirrhosis was induced in rats by bile duct ligation for 6 weeks. AST‐120 was administered by gavage preventively for 6 weeks (0.1, 1, and 4 g/kg/day). In addition, AST‐120 was evaluated as a short‐term treatment for 2 weeks and 3 days (1 g/kg/day) and as a sink to adsorb intravenously infused ammonium acetate. In vitro, AST‐120 efficiently adsorbed ammonia. Ammonia levels significantly decreased in a dose‐dependent manner for all AST‐120–treated bile duct‐ligated rats (nontreated: 177.3 ± 30.8 μM; AST‐120, 0.1 g/kg/day: 121.9 ± 13.8 μM; AST‐120, 1 g/kg/day: 80.9 ± 30.0 μM; AST‐120, 4 g/kg/day: 48.8 ± 19.6 μM) and significantly correlated with doses of AST‐120 (r = −0.6603). Brain water content and locomotor activity normalized after AST‐120 treatments, whereas arterial reactive oxygen species levels remained unchanged. Furthermore, AST‐120 significantly attenuated a rise in arterial ammonia after ammonium acetate administration (intravenously). Conclusion:AST‐120 treatment decreased arterial ammonia levels, normalized brain water content and locomotor activity but did not demonstrate an effect on systemic oxidative stress. Also, AST‐120 acts as an ammonia sink, efficiently removing blood‐derived ammonia. Additional studies are warranted to evaluate the effects of AST‐120 on hepatic encephalopathy in patients with advanced liver disease. (HEPATOLOGY 2011;)

Increased brain lactate is central to the development of brain edema in rats with chronic liver disease

BACKGROUND & AIMS The pathogenesis of brain edema in patients with chronic liver disease (CLD) and minimal hepatic encephalopathy (HE) remains undefined. This study evaluated the role of brain lactate, glutamine and organic osmolytes, including myo-inositol and taurine, in the development of brain edema in a rat model of cirrhosis. METHODS Six-week bile-duct ligated (BDL) rats were injected with (13)C-glucose and de novo synthesis of lactate, and glutamine in the brain was quantified using (13)C nuclear magnetic resonance spectroscopy (NMR). Total brain lactate, glutamine, and osmolytes were measured using (1)H NMR or high performance liquid chromatography. To further define the interplay between lactate, glutamine and brain edema, BDL rats were treated with AST-120 (engineered activated carbon microspheres) and dichloroacetate (DCA: lactate synthesis inhibitor). RESULTS Significant increases in de novo synthesis of lactate (1.6-fold, p<0.001) and glutamine (2.2-fold, p<0.01) were demonstrated in the brains of BDL rats vs. SHAM-operated controls. Moreover, a decrease in cerebral myo-inositol (p<0.001), with no change in taurine, was found in the presence of brain edema in BDL rats vs. controls. BDL rats treated with either AST-120 or DCA showed attenuation in brain edema and brain lactate. These two treatments did not lead to similar reductions in brain glutamine. CONCLUSIONS Increased brain lactate, and not glutamine, is a primary player in the pathogenesis of brain edema in CLD. In addition, alterations in the osmoregulatory response may also be contributing factors. Our results suggest that inhibiting lactate synthesis is a new potential target for the treatment of HE.

Brain edema in acute liver failure and chronic liver disease: Similarities and differences.

Hepatic encephalopathy (HE) is a complex neuropsychiatric syndrome that typically develops as a result of acute liver failure or chronic liver disease. Brain edema is a common feature associated with HE. In acute liver failure, brain edema contributes to an increase in intracranial pressure, which can fatally lead to brain stem herniation. In chronic liver disease, intracranial hypertension is rarely observed, even though brain edema may be present. This discrepancy in the development of intracranial hypertension in acute liver failure versus chronic liver disease suggests that brain edema plays a different role in relation to the onset of HE. Furthermore, the pathophysiological mechanisms involved in the development of brain edema in acute liver failure and chronic liver disease are dissimilar. This review explores the types of brain edema, the cells, and pathogenic factors involved in its development, while emphasizing the differences in acute liver failure versus chronic liver disease. The implications of brain edema developing as a neuropathological consequence of HE, or as a cause of HE, are also discussed.

Elevated cerebral lactate: Implications in the pathogenesis of hepatic encephalopathy

Hepatic encephalopathy (HE), a complex neuropsychiatric syndrome, is a frequent complication of liver failure/disease. Increased concentrations of lactate are commonly observed in HE patients, in the systemic circulation, but also in the brain. Traditionally, increased cerebral lactate is considered a marker of energy failure/impairment however alterations in lactate homeostasis may also lead to a rise in brain lactate and result in neuronal dysfunction. The latter may involve the development of brain edema. This review will target the significance of increased cerebral lactate in the pathogenesis of HE.

Portacaval anastomosis-induced hyperammonemia does not lead to oxidative stress

Ammonia is neurotoxic and believed to play a major role in the pathogenesis of hepatic encephalopathy (HE). It has been demonstrated, in vitro and in vivo, that acute and high ammonia treatment induces oxidative stress. Reactive oxygen species (ROS) are highly reactive and can lead to oxidization of proteins resulting in protein damage. The present study was aimed to assess oxidative status of proteins in plasma and brain (frontal cortex) of rats with 4-week portacaval anastomosis (PCA). Markers of oxidative stress, 4-hydroxy-2-nonenal (HNE) and carbonylation were evaluated by immunoblotting in plasma and frontal cortex. Western blot analysis did not demonstrate a significant difference in either HNE-linked or carbonyl derivatives on proteins between PCA and sham-operated control rats in both plasma and frontal cortex. The present study suggests PCA-induced hyperammonemia does not lead to systemic or central oxidative stress.

Induction of systemic oxidative stress leads to brain oedema in portacaval shunted rats

The pathogenesis of hepatic encephalopathy (HE) is multifactorial and often associated with the development of brain oedema. In addition to ammonia playing a central role, systemic oxidative stress is believed to aggravate the neuropsychological effects of ammonia in patients with chronic liver disease (CLD). The aim of this study was to (i) induce systemic oxidative stress in hyperammonaemic portacaval anastomosed (PCA) rats by inhibiting the antioxidant glutathione using Dimethyl maleate (DEM) and (ii) investigate whether a synergistic relationship between ammonia and oxidative stress contributes to the pathogenesis of brain oedema in CLD.

Enoxaparin does not ameliorate liver fibrosis or portal hypertension in rats with advanced cirrhosis

Recent studies suggest that heparins reduce liver fibrosis and the risk of decompensation of liver disease. Here, we evaluated the effects of enoxaparin in several experimental models of advanced cirrhosis.

The bile duct ligated rat: A relevant model to study muscle mass loss in cirrhosis

Muscle mass loss and hepatic encephalopathy (complex neuropsychiatric disorder) are serious complications of chronic liver disease (cirrhosis) which impact negatively on clinical outcome and quality of life and increase mortality. Liver disease leads to hyperammonemia and ammonia toxicity is believed to play a major role in the pathogenesis of hepatic encephalopathy. However, the effects of ammonia are not brain-specific and therefore may also affect other organs and tissues including muscle. The precise pathophysiological mechanisms underlying muscle wasting in chronic liver disease remains to be elucidated. In the present study, we characterized body composition as well as muscle protein synthesis in cirrhotic rats with hepatic encephalopathy using the 6-week bile duct ligation (BDL) model which recapitulates the main features of cirrhosis. Compared to sham-operated control animals, BDL rats display significant decreased gain in body weight, altered body composition, decreased gastrocnemius muscle mass and circumference as well as altered muscle morphology. Muscle protein synthesis was also significantly reduced in BDL rats compared to control animals. These findings demonstrate that the 6-week BDL experimental rat is a relevant model to study liver disease-induced muscle mass loss.

573 ATTENUATION OF OXIDATIVE STRESS PROTECTS THE BRAIN IN RATS WITH “ACUTE-ON-CHRONIC” LIVER FAILURE

related to hyperammonemia is based on studies of cell cultures, animal models and clinical studies where the actual tissue or plasma concentrations of ammonium range from clinical relevant levels in the micromolar range to more than 5 mM. To assess the significance of the ammonium concentration we studied the extracellular release of lactate, glutamate, and lactate in cerebral cortex of rat brain slices in a dose–response study. Methods: We applied concentrations of ammonium from 0.15 mM to 10 mM to 29 brain slices in a perfusion chamber with exposure times up to 90 minutes. We measured the extracellular changes in lactate, adenosine and glutamate by the use of enzymatic biosensors inserted into cerebral cortex. Results:We found a consistent reduction in the extracellular lactate concentration ranging from 4 to 400 micromolar independent of the ammonium concentration (R=0.006, ammonium vs. lactate). The reduction in lactate was not affected by inhibition of the neuronal lactate transporter MCT-2 by adding alpha-cyano-4hydroxycinnamic acid. We found a positive correlation between the ammonium concentration and the glutamate increase (R=0.43, p < 0.05) with a marked release of glutamate (up to 55 micromolar) with exposure to 10 mM of ammonium. We also observed a positive correlation between the ammonium level and the change in adenosine (R=0.68, p < 0.05) where ammonium levels above 500 mM were associated with adenosine release up to 18 micromolar.. The peak in glutamate preceded the peak in adenosine release by 18±7 minutes (p < 0.05). Conclusion: Cortical tissue exposed ammonium displayed a linear dose–response-like relationship between ammonium concentration and the changes in glutamate and adenosine. Interestingly, we found that ammonium induced a reduction in the extracellular lactate concentration independent of the ammonium concentration within the studied range. The reduction appeared not to be related to increased neuronal uptake of lactate.