Helen Marin

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.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).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.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.

Aims: Brain edema is a common occurrence in patients with hepatic encephalopathy (HE) due to chronic liver disease however the underlying pathophysiological mechanisms are weakly described. Hyperammonemia is considered the central component, but levels of plasma ammonia and severity of HE poorly correlate. Oxidative stress and impaired lactate metabolism are pathogenetic factors believed to be involved. This study investigates the temporal resolution of increases in ammonia, oxidative stress and lactate in relation to the development of brain edema in bile-duct ligated rats (BDL), a widely used rat model of chronic liver disease and HE. Methods: Cirrhosis was induced in rats through ligation of the bile duct. Ammonia and reactive oxygen species (ROS) were assessed in arterial plasma using a commercially available kit and a DCFDA-fluorescent technique respectively. Brain water content was measured in the frontal cortex using the specific gravimetric technique and lactate by Amplex Red fluorescence. Measurements were performed in BDL rats vs sham operated controls at 2, 4 and 6 weeks after surgical intervention in plasma and cerebrospinal fluid or brain tissue. Results: See table. Conclusions: Following 2 weeks of BDL, an increase in plasma ROS is observed with no increase in the other factors. Following 4 weeks of BDL, in addition to an increase in systemic ROS, an increase in plasma and brain ammonia and brain (not plasma) lactate is demonstrated however no evidence of brain edema. Brain edema appears at 6 weeks, along with an increase in all 3 factors. The further significant increase in brain lactate and ammonia at 6 weeks compared to 4 weeks, superimposed on systemic oxidative stress, may be responsible the of development of brain edema in BDL rats. Our results support the multifactorial pathogenesis of brain edema in HE and suggests systemic oxidative stress might be an important “first hit”, followed by increases in ammonia and lactate, in the pathogenesis of brain edema in chronic liver failure. SHAMⱡBDL 2 weeksBDL 4 weeksBDL 6 weeks PLASMA AMMONIA(μM)37.24±12.5433.24±16.7371.32±12.03*118.50±10.78* ROS(RFU)0.08±0.032.94±0.39***2.05±0.59***3.68±0.86*** LACTATE(mM)1.40±0.191.22±0.181.21±0.131.75±0.35 CEREBROSPINAL FLUID AMMONIA(μM)36.12±13.2335.94±9.6389.38±13.07*128.4±36.66*** ROS(RFU)26.11±9.9423.95±12.2613.61±1.6325.97±8.21 BRAIN TISSUE LACTATE(μM/ 100 g proteins)88.51±5.2698.06±7.78190.26±26.14*254.60±10.12*** BRAIN EDEMA (%)78.05±0.1477.73 ±0.0777.69±0.0779.03±0.31* ⱡ mean value of SHAM-operated rats 2, 4 and 6 weeks *p<0.05 vs SHAM ***p<0.01 vs SHAM $p<0.05 vs BDL 4 weeks

Background: Liver disease leads to hyperammonemia, a central component in the pathogenesis of hepatic encephalopathy (HE). There is increasing evidence systemic oxidative stress may exacerbate the neuropsychological effects of hyperammonemia in patients with liver disease. With new highly sensitive imaging techniques, brain edema is frequently being observed in patients cirrhosis and HE. Six-week cirrhotic rats develop brain edema, hyperammonemia and also an increase in plasma reactive oxygen species (ROS) along with an increase in xanthine oxidase activity. In order to better understand the relationship between oxidative stress and ammonia and their role in the pathogenesis of brain edema, we evaluated the effect of allopurinol, a xanthine oxidase inhibitor, in rats with cirrhosis. Methods: Secondary biliary cirrhosis was induced in rats by bile duct ligation (BDL) for 6 weeks. BDL and SHAM-operated rats received 100 mg/kg/day of allopurinol for 10 days. A group of BDL and SHAM-operated rats received saline. AST and ALT were measured at the end of the treatment period to assess liver function. Ammonia and ROS were assessed in arterial plasma using a commercially available kit and a DCFDA-fluorescent technique respectively. Brain water content was measured in the frontal cortex using the specific gravimetric technique. Results: Arterial ROS and ammonia levels were significantly increased in BDL vs SHAM-operated rats (p<0.001). BDL rats treated with allopurinol demonstrated a significant decrease in systemic ROS as well as ammonia levels vs non-treated BDL rats (1.70 ± 0.53RFU vs 4.97 ± 1.38RFU, p<0.05, 37.9 ± 13.8µM vs 101.1 ± 10.1µM, p<0.001, respectively), reaching values similar to those seen in SHAM-operated rats. Brain water content increased in BDL vs SHAM-operated rats and normalised in allopurinol treated BDL rats (77.20 ± 0.08% vs BDL: 78.46 ± 0.28%, p<0.05). Liver function markers, AST and ALT, increased in BDL rats compared to their respective SHAM-operated controls (p<0.001) and were not attenuated following allopurinol treatment. Conclusions: Allopurinol treatment decreased systemic ROS and attenuated brain edema as well as arterial ammonia levels. The effect of allopurinol demonstrates oxidative stress plays a role in ammonia metabolism and in the pathogenesis of brain edema. Our results suggest antioxidant treatment directed towards inhibiting ROS production could be beneficial in lowering ammonia and treating HE. Additional studies are warranted to evaluate the implication of oxidative stress in ammonia metabolism.

Aims: Brain edema represents a constant finding in patients with hepatic encephalopathy (HE) due to chronic liver failure; however its pathogenetic mechanisms are poorly described. Hyperammonemia is considered the central component in the pathogenesis of HE, but ammonia levels are not correlated with severity of HE. Other factors such as oxidative stress and impaired lactate metabolism are believed to be involved. Temporal resolution of these factors involved in the progression of the disease and brain edema might elucidate the relationship between these pathogenetic factors. The present study investigates temporal resolution of ammonia, oxidative stress and lactate in relation to the development of brain edema in bile-duct ligated rats (BDL), a widely used rat model of chronic liver failure and HE. Methods: Ammonia (commercial kit), reactive oxygen species (ROS) (DCFDA fluorescence), lactate (Amplex Red fluorescence) and brain edema (specific gravimetric technique) were measured in BDL rats vs sham operated controls at 2, 4 and 6 weeks after surgical intervention in plasma and cerebrospinal fluid or brain tissue. Results: See table.Conclusions: Following 2 weeks of BDL, an increase in plasma oxidative stress is observed with no appearance of hyperammonemia, hyperlactataemia, increased brain lactate and ammonia or brain edema. Following 4 weeks of BDL, in addition to an increase in systemic oxidative stress, an increase in plasma and brain ammonia and brain (not plasma) lactate is demonstrated but still no evidence of brain edema. Brain edema appears at 6 weeks, along with an increase in all 3 pathogenetic factors. The further increase in brain lactate and ammonia compared to 4 weeks, superimposed on systemic oxidative stress, may be responsible the development of brain edema in BDL rats. Our results support the multifactorial pathogenesis of brain edema in HE and suggests systemic oxidative stress might be an important “first hit”, followed by increases in ammonia and lactate, in the pathogenesis of brain edema in chronic liver failure.

Aims: Chronic liver failure leads to hyperammonemia, a central component in the pathogenesis of hepatic encephalopathy (HE). Neurotoxic effects of ammonia induce numerous metabolic alterations such as increased brain lactate (Lac) and glutamine (Gln) levels. Brain edema is present in patients with chronic liver failure however the pathophysiological mechanisms are not clearly understood. The present study investigates the role of brain ammonia, Lac and Gln in pathogenesis of brain edema in 2 different rat models of chronic liver failure/HE; 1) portacaval anastomosis (PCA), and 2) bile-duct ligation (BDL). Methods: Ammonia (commercial kit), brain edema (specific gravimetric technique) Lac (Amplex Red fluorescence) and Gln (1H nuclear magnetic spectroscopy) were measured in brain tissue of PCA and BDL rats vs sham operated controls. De novo synthesis of Lac and Gln was assessed in brain tissue by 13C nuclear magnetic resonance spectroscopy, after injecting PCA and BDL rats along with their respective sham operated controls with [U-13C] glucose (500 mg/kg, i.p) 30 minutes before sacrifice. Lac, Gln and brain edema were also investigated in BDL rats at 2, 4 and 6 weeks after surgery and after reducing ammonia levels with AST-120 (spherical carbon adsorbent), at 1g/kg/day for 6 weeks. Results: Hyperammonemia developed in both PCA (177.3±8.9μM vs sham: 52.1±11.3μM; p<0.0001) and BDL (155.0±8.9μM vs sham: 41.0±11.2μM; p<0.0001); with no significant difference between the groups. An increase in brain water content was observed in rats with BDL (81.88±0.12% vs sham: 81.13±0.15%, p<0.05), as well as total Lac levels; whereas no significant change in brain water and total Lac was found in PCA. Gln levels significantly increased in PCA rats and BDL rats vs respective sham-operated controls. A significant increase in de novo synthesis of 13C Lac and 13C Gln from 13C glucose was found in BDL rats, whereas in PCA rats only de novo synthesis of 13C Gln increased. In addition, total Lac levels were increased in BDL rats before the apparition of brain edema. Following AST-120 treatment, Lac levels and brain edema significantly decreased, while Gln levels remained unchanged. Conclusions: BDL and PCA rats both presented with similar levels of hyperammonemia and Gln, as well as increased de novo synthesis of Gln from glucose. However only BDL rats demonstrated an increase in brain water and Lac as well as increased de novo synthesis of Lac from glucose. Our findings suggest increased brain Lac, not increased brain Gln, is associated with brain edema. We conclude that impaired Lac metabolism may contribute to the pathogenesis of brain edema in HE.

Introduction: L’insuffisance hépatique mène à l’hyperammonémie, la composante centrale dans la pathogénèse de l’encéphalopathie hépatique (EH). Les niveaux neurotoxiques d’ammoniaque induisent des nombreuses altérations métaboliques telles que l’augmentation du lactate et glutamine au niveau cérébral. Des nouvelles techniques d’imagerie ont démontré que l’œdème cérébral est présent chez les patients avec insuffisance hépatique chronique, mais les mécanismes pathophysiologiques ne sont pas bien connus. Dans cette étude, nous avons investigué l’implication de la synthèse de novo du lactate et glutamine dans la pathogénèse de l’œdème cérébral en utilisant 2 modèles différents d’insuffisance hépatique chronique/EH chez le rat: 1) la dérivation portosystémique (PCA); et 2) la ligature de voie biliaire (BDL). Méthodes: Des rats PCA et BDL, ainsi que contrôles SHAM-opérés ont été injectés avec [U-13C] glucose (500 mg/kg, i.p) et sacrifiés après 30 minutes. Les niveaux totaux, ainsi que la synthèse de novo de glutamine et du lactate ont été mesurés dans le tissu cérébral par spectroscopie de résonance magnétique nucléaire 1H et 13C. L’ammoniaque a été mesurée dans le plasma artériel et le liquide céphalorachidien par un kit commercial. Le contenu en eau du cerveau a été mesuré dans le cortex frontal par une technique spécifique gravimétrique. Résultats: L’hyperammonémie s’est développée dans les deux groupes; PCA: 177.3 ± 8.9 μM vs SHAM: 52.1 ± 11.3 μM; p<0.001 et BDL: 155.0 ± 8.9 μM vs SHAM: 41.0 ± 11.2 μM; p<0.001, sans différence significative entre les 2 groupes. Des résultats similaires ont été observés dans le liquide céphalorachidien; PCA: 179.5 ± 10.3 μM vs SHAM: 34.9 ± 6.3 μM; p<0.001 et BDL: 142.6 ± 28.3 μM vs SHAM: 27.9 ± 4.9 μM; p<0.001. Le contenu en eau du cerveau a augmenté dans les rats BDL (BDL: 81.88 ± 0.12% vs SHAM: 81.13 ± 0.15%, p<0.05), par contre dans les rats PCA il n’y avait pas une différence significative comparativement au rats SHAM-opérés. Les niveaux de glutamine étaient augmentés 3 fois chez les rats PCA vs SHAM et 2 fois chez les rats BDL vs SHAM. Une augmentation significative de la synthèse de novo du 13C lactate et 13C glutamine provenant du 13C glucose administré a été observée chez les BDL (1.6 fois, respectivement 2 fois vs SHAM), tandis que chez les rats PCA seulement une augmentation de la synthèse de novo de 13C glutamine a été observée. Conclusions: Les rats PCA et BDL présentent des taux similaires d’ammoniaque plasmatique et cérébral et une augmentation de la synthèse de novo de glutamine cérébrale provenant du glucose, mais seulement les rats BDL présentent l’œdème cérébral ainsi qu’une augmentation de la synthèse de novo du lactate cérébral provenant du glucose. Nos résultats suggèrent que le lactate est un important facteur dans la pathogénèse de l’œdème cérébral dans l’insuffisance hépatique chronique. Projet subventionné par: Instituts de recherche en santé du Canada

Background: Chronic liver failure leads to hyperammonemia, a central component in the pathogenesis of hepatic encephalopathy. Neurotoxic effects of ammonia have been shown to induce numerous metabolic alterations such as increased brain lactate and glutamine levels. Newly developed imaging techniques have demonstrated brain edema is present in patients with chronic liver failure however the pathophysiological mechanisms are not clearly understood. The present study investigates the role of de novo synthesis of brain lactate and glutamine in pathogenesis of brain edema in 2 different rat models of chronic liver failure/hepatic encephalopathy; 1) portacaval anastomosis (PCA), and 2) bile-duct ligation (BDL). Methods: PCA and BDL rats along with their respective sham-operated controls were injected with [1-13C] glucose (500 mg/kg, i.p) and sacrificed 30 minutes later. Glutamine and lactate were assessed in brain tissue by 1H and 13C nuclear magnetic resonance spectroscopy. Ammonia levels were measured in arterial plasma and cerebrospinal fluid using a commercially available kit. Brain water content was measured in frontal cortex using a specific gravimetric technique. Results: Hyperammonemia developed in both PCA (177.3 ± 8.9μM vs sham: 52.1 ± 11.3μM; p< 0.0001) and BDL (155.0 ± 8.9μM vs sham: 41.0 ± 11.2μM; p< 0.0001); with no significant difference between the groups (p> 0.05). Similar results were found in cerebrospinal fluid (PCA: 179.5 ± 10.3μM vs sham: 34.9 ± 6.3μM; p< 0.0001 and BDL: 142.6 ± 28.3μM vs sham: 27.9 ± 4.9μM; p< 0.0001); with no significant difference between the groups (p> 0.05). An increase in brain water content was observed in rats with BDL (81.88 ± 0.12% vs sham: 81.13 ± 0.15%, p< 0.05) whereas no significant change in brain water content was found in PCA vs their respective sham-operated controls. Glutamine levels significantly increased 3 fold in PCA rats and 2 fold in BDL rats vs respective sham-operated controls. A significant increase in de novo synthesis of 13C lactate and 13C glutamine from 13C glucose was found in BDL rats (1.6 fold and 2 fold respectively vs sham), whereas in PCA rats only de novo synthesis of 13C glutamine increased. Conclusion: BDL and PCA rats both presented with similar levels of hyperammonemia and increased brain ammonia, increased de novo synthesis of glutamine from glucose however only BDL rats demonstrated an increase in brain water as well as an increase in de novo synthesis of lactate from glucose. Our findings suggest lactate is an important pathogenic factor implicated in the pathogenesis of brain edema in chronic liver failure.