L'ischémie dans l'encéphalopathie hépatique.
B.Sc., Sciences biomédicales, Université de Montréal
Direction:
- Dr Christopher Rose
Été 2011
Publications connexes
Jimmy Huynh, Cristina R. Bosoi, Christian Parent-Robitaille, Mélanie Tremblay, Christopher F. Rose.
Background: Brain edema is a serious complication associated with hepatic encephalopathy (HE) due to chronic liver disease (CLD). An increase in blood brain barrier (BBB) ion permeability can occur across an intact BBB through alterations in transport mechanisms. NH4+ has very similar ionic properties to K+ and can be transported through K+ channels and cotransporters, implying that hyperammonemia could result in BBB hyperpermeability. An increase in BBB permeability via transport Na+-K+-2Cl- (NKCC1) has shown to promote brain edema and astrocyte swelling under pathophysiological conditions such as ischemia. Aim: To study the BBB integrity (vasogenic vs cytotoxic) and the role of NKCC1 in the pathogenesis of brain edema in cirrhotic rats. Methods: Two distinct animal models of HE are used in the present study; 1) biliary cirrhosis model (6 weeks bile duct ligation (BDL)). 2) portacaval shunt model (4 weeks portacaval anastomosis (PCA)). Both models develop hyperammonemia however brain edema is only observed in BDL. BBB breakdown was assessed by measuring brain extravasation of Evans blue and sodium fluorescein (injected i.v). Expression of BBB tight junction proteins (occludin, claudin-5, ZO-1 and ZO-2) were assessed by Western blot. Bumetanide was administered (i.p) for 10 days in BDL and SHAM animals. Brain water content was measured in the frontal cortex using the specific gravimetric method. Levels of brain NKCC1 mRNA were evaluated by RT-PCR in cerebral microvessels. Results: Extravasation of Evans blue and sodium fluorescein was not detected and no significant change in all tight junction protein was observed in both BDL and PCA models. Brain water content was reduced in bumetanide-treated BDL rats compared to control (77.66±0.15% vs 78.12±0.21%). In brain microvessels, NKCC mRNA increased in BDL rats compared to BDL SHAM (0.78±0.09 vs. 1.92±0.42) whereas no change was found in PCA compared to PCA SHAM (1.72±0.52 vs. 1.53±0.23). Conclusions: BDL rats did not demonstrate a change in BBB integrity or expression of tight junction proteins concluding brain edema in BDL is not of vasogenic origin. Furthermore, since brain edema was only observed in BDL rats (vs PCA), this implies additional factors aside ammonia, are involved in the pathogenesis of brain edema. Moreover, an increase of NKCC1 mRNA and an attenuation of brain edema following bumetanide treatment were demonstrated in BDL rats suggesting NKCC1 plays a role in the development of brain edema in CLD. These results demonstrate the potential therapeutic use of bumetanide for the treatment of HE.
Jimmy Huynh, Cristina R. Bosoi, Christian Parent-Robitaille, Mélanie Tremblay, Christopher F. Rose.
Background: Brain edema is a serious complication associated with hepatic encephalopathy (HE) due to chronic liver disease. An increase in BBB ion permeability (increase uptake of ions and accompanied by water fluxes towards the brain) can occur across an intact BBB through alterations in transport mechanisms. Endothelial cells that comprise the BBB secrete up to 40% of brain interstitial fluid as they transport Na+, K+ and 2 Cl- across the BBB. NH4+ has very similar ionic properties to K+ (similar ionic radius and diffusion coefficient) and can be transported through K+ channels and cotransporters, implying that hyperammonemia could result in BBB hyperpermeability. An increase in BBB permeability via transport Na+-K+-2Cl- (NKCC1) has shown to promote brain edema and astrocyte swelling under pathophysiological conditions such as ischemia. Aim: To study the BBB integrity (vasogenic vs cytotoxic) and the role of NKCC1 in the pathogenesis of brain edema in cirrhotic rats. Methods: Two distinct animal models of chronic liver failure and HE are used in the present study; 1) biliary cirrhosis model (6 weeks bile duct ligation (BDL)). 2) portacaval shunt model (4 weeks portacaval anastomosis (PCA)). Both models develop hyperammonemia however brain edema is only observed in BDL. BBB breakdown was assessed by measuring brain extravasation of Evans blue and sodium fluorescein (injected i.v). Expression of BBB tight junction proteins (occludin, claudin-5, ZO-1 and ZO-2) were assessed by Western blot. Bumetanide was administered (i.p) for 10 days in BDL and BDL SHAM. Brain water content was measured in the frontal cortex using the specific gravimetric method. Levels of brain NKCC1 mRNA were evaluated by RT-PCR in cerebral microvessels. Results: Extravasation of Evans blue and sodium fluorescein was not detected and there was no significant change in all tight junction protein levels measured in both BDL and PCA models. Brain water content was reduced in bumetanide-treated BDL rats compared to control (77.66±0.15% vs 78.12±0.21%). In brain microvessels, NKCC mRNA increased in BDL rats compared to BDL SHAM (0.78±0.09 vs. 1.92±0.42) whereas no change was found in PCA compared to PCA SHAM (1.72±0.52 vs. 1.53±0.23). Conclusions: BDL rats did not demonstrate a change in BBB integrity or in expression of BBB tight junction proteins. This suggests brain edema in BDL is not of vasogenic origin. Furthermore, since brain edema was only observed in BDL rats (vs PCA), this implies additional factors aside ammonia, are involved in the pathogenesis of brain edema. Moreover, an increase of NKCC1 mRNA and an attenuation of brain edema following bumetanide treatment were demonstrated in BDL rats suggesting NKCC1 plays a role in the development of brain edema in chronic liver disease. Furthermore, these results demonstrate the potential therapeutic use of bumetanide for the treatment of HE.
Cristina R. Bosoi, Claudia Zwingmann, Helen Marin, Christian Parent-Robitaille, Jimmy Huynh, Mélanie Tremblay, Christopher F. Rose.
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.
Systemic oxidative stress induction leads to brain edema in hyperammonemic portacaval-shunted rats.
Cristina Bosoi, Christian Parent-Robitaille, Mélanie Tremblay, Christopher F. Rose.
Aims: Although ammonia is the central component in the pathogenesis of hepatic encephalopathy, it is believed oxidative stress (OS) plays a role in the pathogenesis of brain edema during liver disease. We previously demonstrated that portacaval shunted hyperammonemic rats do not develop OS or brain edema (Bosoi et al., FRBM, 2012). To define whether a synergistic effect exists between hyperammonemia and OS in the development of brain edema, the present study investigates the role of OS in the pathogenesis of brain edema in PCA rats following glutathione depletion by diethyl maleate (DEM). Methods: We evaluated the effect of DEM (1 mg/kg/day intraperitoneally for 10 days starting day 18 after surgery) in PCA and SHAM-operated rats. Rats were sacrificed at day 28 and OS was evaluated by measuring arterial and cerebral malon-dialdehyde (MDA, commercial kit), reactive oxygen species (ROS, DCFDA fluorescence), and 4-hydroxy-2-nonenal (HNE, Western blot). Ammonia levels (commercial kit) were also assessed. Frontal cortex brain water was measured using a specific gravimetric technique. Results: DEM induced a significant increase in plasma MDA, ROS and HNE levels in PCA rats. No increase was detected in DEM-treated SHAM-operated controls. No changes in cerebral OS markers were observed in any group. Ammonia levels increased in non-treated and DEM-treated PCA vs SHAM-operated rats (p<0.001) and remained unchanged between non-treated and DEM-treated PCA groups. Brain water content increased in DEM-treated PCA rats vs non-treated PCA rats (PCA+DEM: 78.45±0.13% vs PCA: 77.38±0.11%, p< 0.001). Conclusions: DEM treatment in PCA rats induced systemic, not central OS. This, superimposed on hyperammonemia, led to the development of brain edema. Similar DEM doses did not result in increased OS and brain edema in SHAM-operated rats, suggesting PCA rats are more susceptible to OS. Our findings support a synergistic effect between hyperammonemia and systemic OS in the pathogenesis of brain edema in hepatic encephalopathy.
Cristina R. Bosoi, Christian Parent-Robitaille, Mélanie Tremblay, Christopher F. Rose.
Introduction: L’insuffisance hépatique mène à l’hyperammonémie, le facteur principal de l’encéphalopathie hépatique (EH). Toutefois, le stress oxydatif peut avoir un rôle important dans l’exacerbation des effets neuropsychologiques de l’hyperammonémie chez les patients atteints de maladies hépatiques, dont l’œdème cérébral. Les rats cirrhotiques développent l’hyperammonémie et l’œdème cérébral, mais aussi une augmentation des espèces réactives d’oxygène (ROS) plasmatiques suite à une augmentation de l’activité de la xanthine oxydase. Afin de mieux comprendre la relation entre l’ammoniaque et le stress oxydatif dans la pathogenèse de l’œdème cérébral, dans cette étude nous avons investigué l’effet de l’allopurinol, un inhibiteur de la xanthine oxydase, chez des rats cirrhotiques. Méthodes: La cirrhose a été induite suite à une ligature de voie biliaire (BDL) pendant 6 semaines. Des rats BDL et contrôles SHAM-opérés ont reçu l’allopurinol (100 mg/kg/jour pour les derniers 10 jours). Des groupes contrôles BDL et SHAM-opérés ont reçu de la saline. L’ammoniaque et les ROS ont été mesurés dans le plasma (artériel) par, respectivement, un kit commercial et une technique de fluorescence. Le contenu en eau du cerveau a été mesuré dans le cortex frontal par une technique spécifique gravimétrique. Résultats: Chez les rats BDL traités avec allopurinol, nous avons observé une diminution significative des ROS et de l’ammoniaque systémique vs les rats BDL non-traités, aux valeurs similaires de celles des rats SHAM. Le contenu en eau du cerveau a été normalisé suite au traitement avec allopurinol. Conclusion: Le traitement avec allopurinol diminue les ROS et l’ammoniaque plasmatique et atténue l’œdème cérébral. Cela a démontré que le stress oxydatif joue un rôle dans le métabolisme de l’ammoniaque et dans la pathogenèse de l’œdème cérébral. Nos résultats suggèrent qu’un traitement antioxydant dirigé vers l’inhibition de la production des ROS pourrait être bénéfique dans le traitement de l’EH.
Christian Parent-Robitaille, Cristina R. Bosoi, Mélanie Tremblay, Christopher F. Rose.
Introduction: L'encéphalopathie hépatique (EH), une complication grave de la maladie du foie, se manifeste par un syndrome métabolique neuropsychiatrique complexe mais ayant toujours été considérée comme réversible. La transplantation hépatique (TH) est le seul traitement efficace pour traiter les patients de maladie hépatique, par contre les complications neurologiques survenant suite à une TH restent un problème persistant. Avec l’EH pré-TH, l'hypotension et une hypoxie cérébrale pendant la période péri-opératoire peuvent affecter les fonctions neurologiques. Ces observations nous ont conduits à l'hypothèse suivante: un patient cirrhotique atteint d’EH est plus à risque de subir des dommages cérébraux suite à une insulte d’hypotension/hypoxie. Méthodes: Pour vérifier cette hypothèse, nous évaluons l’effet d’une hypotension/hypoxie cérébrale chez un rat avec ligature de la voie biliaire (BDL) de 6 semaines (modèle établi de la maladie chronique du foie et de l’EH). Les animaux ont été sacrifiés et leurs cerveaux ont été évalués pour une perte/altération cellulaire par marquage neuronal (au violet de crésyl et par immunohistochimie avec NeuN) ainsi que pour détecter des protéines pro- et anti-apoptotiques (par western blot). Résultats: Jusqu’à maintenant, nous avons démontré qu’une hypotension/hypoxie cérébrale de 90 minutes ne cause pas de perte neuronale ni une augmentation de marqueurs apoptotiques dans le cerveau de rats encéphalopatiques comparativement aux cerveaux de rats normaux. Conclusions: Nous sommes présentement en train d’évaluer les cerveaux de rats ayant subi une hypotension/hypoxie de 30 minutes. À cause de la fragilité d’un cerveau encéphalopatique, nous croyons qu’une hypotension/hypoxie moindre pourrait donner lieu à une plus grande perte/altération cellulaire que dans un cerveau normal. Nous allons par la suite utiliser d’autres techniques telles que le marquage avec fluorojade, un marqueur d’apoptose et TUNEL une méthode pour détecter fragmentation de l'ADN
Glutathione depletion leads to brain edema in portacaval-shunted rats.
Cristina R. Bosoi, Christian Parent-Robitaille, Mélanie Tremblay, Christopher F. Rose.
Aims: The pathogenesis of hepatic encephalopathy (HE) is multifactorial. Even though ammonia is the central component in the pathogenesis of HE, oxidative stress is believed to play a role in exacerbating the neuropsychological effects of ammonia in patients with liver disease. With new, highly sensitive imaging techniques, brain edema is observed in HE patients. We previously demonstrated that portacaval shunted hyperammonemic rats do not develop oxidative stress or brain edema. In order to define a synergistic effect between hyperammonemia and systemic oxidative stress, the present study investigates the role of oxidative stress in the pathogenesis of brain edema in PCA rats following glutathione depletion by diethyl maleate (DEM). Methods: In the first set of experiments, we evaluated the effect of DEM in PCA and SHAM-operated control rats by injecting DEM at a dose of 0.4 and 1 mg/kg/day intraperitoneally for 10 days starting at day 18 after surgery. Rats were sacrificed at day 28 and oxidative stress was evaluated by arterial malon-dialdehyde (MDA, commercial kit). In the second set of experiments, 1 mg/kg/day DEM was used to induce oxidative stress. Ammonia (commercial kit) as well as other different oxidative stress markers: reactive oxygen species (DCFDA fluorescence technique), and 4-hydroxy-2-nonenal (HNE, Western blot) were assessed in arterial plasma and frontal cortex tissue. Brain water content was measured in the frontal cortex using a specific gravimetric technique. Results: DEM at 1 mg/kg/day (not 0.4 mg/kg/day) induced a significant increase in MDA levels in PCA rats. No increase in MDA was detected following either dose of DEM in SHAM-operated controls. Ammonia levels in both DEM-treated and non-treated PCA rats were significantly increased vs respective sham-operated controls (p<0.001) and remained unchanged between non-treated and DEM-treated PCA groups (p>0.05). An increase in brain water content was observed in DEM-treated PCA rats vs non-treated PCA rats (PCA+DEM: 78.45 ± 0.13% vs PCA: 77.38 ± 0.11, p< 0.001). Although no significant changes in reactive oxygen species were observed, there was an increase in plasma levels of HNE in DEM-treated PCA rats compared to non-treated PCA rats. No significant changes in any oxidative stress markers were observed in the frontal cortex. Conclusions: DEM treatment in PCA rats induced systemic oxidative stress but not central oxidative stress. This, imposed on hyperammonemia, was accompanied by the onset of brain edema in rats with PCA. Oxidative stress and brain edema were not detected in SHAM-operated rats, which were not hyperammonemic. Our findings suggest a synergistic effect between hyperammonemia and systemic oxidative stress is implicated in the pathogenesis of brain edema in hepatic encephalopathy.
Xanthine oxidase inhibition attenuates brain edema in in rats with liver failure.
Cristina R. Bosoi, Christian Parent-Robitaille, Mélanie Tremblay, Christopher F. Rose.
Aims: Liver disease leads to hyperammonemia, a central component in the pathogenesis of hepatic encephalopathy (HE). There is increasing evidence that systemic oxidative stress may exacerbate the neuropsychological effects of hyperammonemia in patients with liver disease. With new, highly sensitive imaging techniques, brain edema is commonly being observed in patients with cirrhosis and HE. Bile duct ligated rats, six-week after intervention, present with 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 xanthine oxidase inhibition in rats with cirrhosis. Methods: 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 (xanthine oxidase inhibitor) during the last 10 days before sacrifice. As control groups, separate BDL and SHAM-operated rats received equivalent volume of 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.
Cristina R. Bosoi, Xiaoling Yang, Jimmy Huynh, Christian Parent-Robitaille, Wenlei Jiang, Mélanie Tremblay, Christopher F. Rose.
Chronic liver failure leads to hyperammonemia, a central component in the pathogenesis of hepatic encephalopathy (HE); however, a correlation between blood ammonia levels and HE severity remains controversial. It is believed oxidative stress plays a role in modulating the effects of hyperammonemia. This study aimed to determine the relationship between chronic hyperammonemia, oxidative stress, and brain edema (BE) in two rat models of HE: portacaval anastomosis (PCA) and bile-duct ligation (BDL). Ammonia and reactive oxygen species (ROS) levels, BE, oxidant and antioxidant enzyme activities, as well as lipid peroxidation were assessed both systemically and centrally in these two different animal models. Then, the effects of allopurinol (xanthine oxidase inhibitor, 100mg/kg for 10days) on ROS and BE and the temporal resolution of ammonia, ROS, and BE were evaluated only in BDL rats. Similar arterial and cerebrospinal fluid ammonia levels were found in PCA and BDL rats, both significantly higher compared to their respective sham-operated controls (p<0.05). BE was detected in BDL rats (p<0.05) but not in PCA rats. Evidence of oxidative stress was found systemically but not centrally in BDL rats: increased levels of ROS, increased activity of xanthine oxidase (oxidant enzyme), enhanced oxidative modifications on lipids, as well as decreased antioxidant defense. In PCA rats, a preserved oxidant/antioxidant balance was demonstrated. Treatment with allopurinol in BDL rats attenuated both ROS and BE, suggesting systemic oxidative stress is implicated in the pathogenesis of BE. Analysis of ROS and ammonia temporal resolution in the plasma of BDL rats suggests systemic oxidative stress might be an important "first hit", which, followed by increases in ammonia, leads to BE in chronic liver failure. In conclusion, chronic hyperammonemia and oxidative stress in combination lead to the onset of BE in rats with chronic liver failure.
Cristina R. Bosoi, Christian Parent-Robitaille, Helen Marin, Mélanie Tremblay, Christopher F. Rose.
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
Allopurinol attenuates systemic oxidative stress and hyperammonemia in bile-duct ligated rats.
Cristina R. Bosoi, Christian Parent-Robitaille, Helen Marin, Mélanie Tremblay, Christopher F. Rose.
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.
Jimmy Huynh, Cristina R. Bosoi, Christian Parent-Robitaille, Mélanie Tremblay, Christopher F. Rose.
Background: Brain edema is a serious complication associated with hepatic encephalopathy (HE) due to chronic liver failure (CLF) and its pathogenesis remains undefined. NKCC1, a Na-K-Cl cotransporter, located on the blood-brain barrier (BBB) has been demonstrated to be implicated in the pathogenesis of brain edema in experimental models of ischemia. Therefore, our aim was to 1) investigate the relationship of hyperammonemia, 2) study the integrity of the BBB and 3) determine the role of NKCC1, in association with brain edema in rats with CLF. Methods: Two distinct animal models of CLF and HE were used; i) biliary cirrhosis model (6 weeks bile duct ligation (BDL)) ii) portacaval shunt model (4 weeks portacaval anastomosis (PCA)). Brain water content was measured using the specific gravimetric method. BBB breakdown was assessed by measuring brain extravasation of injected BBB permeability tracers (Evans blue and sodium fluorescein). Expression of BBB tight junction proteins (occludin, claudin-5, ZO-1 and ZO-2) were assessed by immunoblot. Levels of brain NKCC1 mRNA were evaluated by RT-PCR in isolated cerebral microvessels. Rats were treated with bumetanide (an NKCC inhibitor; administered (i.p) for 10 days). Results: Similar degree of hyperammonemia was measured in both BDL and PCA rats however brain edema was only found in BDL rats. In brains of both BDL and PCA rats, extravasation of Evans blue and sodium fluorescein was not detected and no significant change in the levels of all tight junction proteins was found. Brain water content was reduced in bumetanide-treated BDL vs BDL-non treated (77.35±0.18% vs 78.89±0.25%). A 2.4 fold increase in NKCC1 mRNA was detected in BDL vs BDL-sham rats whereas no change was found in PCA vs PCA-sham rats. Conclusions: Chronic hyperammonemia independently does not lead to an increase in brain water. Brain edema, present in BDL rats, is not associated with a change in either BBB integrity or expression of BBB tight junction proteins and is therefore not of vasogenic origin. Furthermore, an increase in NKCC1 mRNA and attenuation of brain edema following bumetanide treatment, suggests NKCC1, independent of hyperammonemia, plays a role in the development of brain edema in CLF.
Claudia Zwingmann, Jimmy Huynh, Cristina R. Bosoi, Christian Parent-Robitaille, Helen Marin, Mélanie Tremblay, Christopher F. Rose.
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.
Christian Parent-Robitaille, Cristina R. Bosoi, Claudia Zwingmann, Jimmy Huynh, Helen Marin, Mélanie Tremblay, Christopher F. Rose.
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.
Jimmy Huynh, Cristina R. Bosoi, Christian Parent-Robitaille, Mélanie Tremblay, Christopher F. Rose.
Aims: Brain edema is a serious complication associated with hepatic encephalopathy (HE) due to chronic liver failure. It is unclear whether brain edema is of vasogenic (blood brain barrier (BBB) breakdown) or of cytotoxic (disturbance of neurocellular metabolism) origin. It has been demonstrated that the Na-K-Cl cotransporter (NKCC) located on the luminal side of the BBB is implicated in the pathogenesis of brain edema in different animal models of ischemia. Furthermore, following the administration of bumetanide, an inhibitor of NKCC, brain edema is attenuated. Therefore, our aim was to study the BBB integrity and the role of NKCC in the pathogenesis of brain edema in cirrhotic rats. Methods: Two distinct animal models of chronic liver failure and HE are used in the present study; 1) biliary cirrhosis model (6 weeks bile duct ligation (BDL)). 2) portacaval shunt model (4 weeks portacaval anastomosis (PCA)). Both models develop hyperammonemia however brain edema is only observed in BDL. BBB breakdown was assessed by measuring brain extravasation of BBB permeability tracers. Evans blue and sodium fluorescein were injected (i.v), brains were perfused and extravasation was determined by spectophotometry. Expression of BBB tight junction proteins (occludin, claudin-5, ZO-1 and ZO-2) were assessed by immunoblot. Bumetanide was administered (i.p) for 10 days in BDL and BDL SHAM. Brain water content was measured in the frontal cortex using the specific gravimetric method. Levels of brain NKCC mRNA were evaluated by RT-PCR in microvessels isolated using centrifugation methods. Results: Extravasation of Evans blue and sodium fluorescein was not detected and there was no significant change in all tight junction protein levels measured in both BDL and PCA (negative control) models. Brain water content was reduced in bumetanide-treated BDL rats compared to control (77.66±0.15% vs 78.12±0.21%). In brain microvessels, NKCC mRNA increased in BDL rats compared to BDL SHAM (0.78±0.09 vs. 1.92±0.42) whereas no change was found in PCA compared to PCA SHAM (1.72±0.52 vs. 1.53±0.23). Conclusions: BDL rats did not demonstrate a change in BBB integrity or a change in expression of BBB tight junction proteins. This suggests brain edema in BDL is not of vasogenic origin. Moreover, an increase of NKCC mRNA and an attenuation of brain edema following bumetanide treatment were demonstrated in BDL rats suggesting NKCC plays a role in the development of brain edema in chronic liver failure.
Cristina R. Bosoi, Christian Parent-Robitaille, Keith Anderson, Mélanie Tremblay, Christopher F. Rose.
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 m(2) /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;).
Jimmy Huynh, Cristina Bosoi, Christian Parent-Robitaille, Mélanie Tremblay, Christopher F. Rose.
Objectif : L’œdème cérébral est un symptôme sévère de l’encéphalopathie hépatique (EH) lors d’une insuffisance hépatique chronique. La cause de l’œdème cérébral est ambigüe parce qu’elle peut être de type vasogénique, la rupture des jonctions serrées de la barrière hémato-encéphalique (BHE), ou de type cytotoxique, la perturbation du métabolisme neurocellulaire. Plusieurs recherches démontrent que le cotransporteur Na-K-Cl 1 (NKCC1) serait impliqué dans la pathogénèse de l’œdème cérébral dans différents modèles animaux d’ischémie cérébrale et qu’un traitement avec de la bumetanide, un inhibiteur de NKCC1, atténue l’augmentation d’eau dans le cerveau. Ainsi, notre objectif était d’étudier l’intégrité de la BHE et le rôle de NKCC1 dans la pathogénèse de l’œdème cérébral chez des rats cirrhotiques. Méthodes : Deux modèles animaux d’insuffisance hépatique chronique sont utilisés dans cette étude. Le premier modèle (cirrhose biliaire de 6 semaines (BDL)), est une ligature de la voie biliaire. Alors, la bile synthétisée par les hépatocytes s’accumule dans le foie et provoque la cirrhose du foie. Ce modèle présente une hyperammonémie et du stress oxydatif plasmatique ainsi que l’œdème cérébral. Le deuxième modèle (dérivation portocave de 4 semaines (PCA)), est une anastomose de la veine porte à la veine cave inférieure. Ainsi, le sang provenant du système digestif n’est pas filtré par le foie, ce qui provoque une hyperammonémie mais elle n'est pas accompagnée par une induction de stress oxydatif et d’œdème cérébral alors ce sera un contrôle négatif. Pour déterminer la perméabilité générale de la barrière hémato-encéphalique, l’extravasation de l’Evans blue, un colorant qui se lie aux protéines (dont l’albumine sérique), et du fluorophore, fluorescéine de sodium, a été évaluée par spectrophotométrie et l’expression des protéines des jonctions serrées (occludin, claudin-5, ZO-1, ZO-2) a été mesurée par immunobuvardage dans les deux modèles. La bumetanide a été administrée i.p pendant 10 jours chez les BDL et BDL SHAM. La quantité d’eau dans le cortex cérébral a été mesurée par gravimétrie. L’expression d’ARNm de NKCC1 a été mesurée dans les microvaisseaux sanguins isolés à partir de cerveaux des deux modèles à l’aide d’un protocole de centrifugation. Résultats : L’extravasation des colorants n’a pas été détectée et l’expression des protéines de jonctions serrées est demeurée inchangée dans les deux modèles. L’œdème cérébral a été atténué lorsque la bumetanide a été administrée chez les BDL comparé aux contrôles (77.66±0.15% vs 78.12±0.21%). Dans les microvaisseaux sanguins cérébraux, le niveau d’ARNm de NKCC1 était augmenté chez les BDL comparé aux BDL SHAM (1.92±0.42 vs. 0.78±0.09) tandis qu’il restait inchangé chez les PCA comparé aux PCA SHAM (1.72±0.52 vs. 1.53±0.23). Conclusion : L’extravasation est absente et l’expression des protéines de jonctions serrées est inchangée donc l’œdème cérébral n’est pas de type vasogénique. L’augmentation d’ARNm de NKCC1 et l’atténuation de l’œdème cérébral par la bumetanide suggèrent que l’œdème cérébral est de type cytotoxique chez les rats avec une insuffisance hépatique chronique.
Cristina R. Bosoi, Christian Parent-Robitaille, Mélanie Tremblay, Christopher F. Rose.
Background: Ammonia is central in the pathogenesis of hepatic encephalopathy (HE) and clinical sequelae are exacerbated by systemic inflammation. Sequestration of ammonia in the gut represents a primary treatment target for HE. AST-120 (spherical carbon adsorbent), an oral adsorbent of engineered activated carbon microspheres with surface areas exceeding 1600m2/g works as a sink for neuro- and hepato–toxins present in the gut. In this study, we evaluated the capacity of AST-120 to lower arterial ammonia, brain edema and systemic inflammatory markers in cirrhotic rats. Methods: Cirrhosis was induced in rats by bile duct ligation (BDL). BDL and SHAM-operated rats received AST-120 dispersed in methylcellulose by gavage at a dose of 0.1, 1 or 4g/kg/day for 6 weeks. Ammonia and the pro-inflammatory cytokine TNF-α were measured in arterial plasma using a commercially available kit and ELISA technique respectively. Brain water content was measured in the frontal cortex, cerebellum and brain stem using the specific gravimetric technique. Results: Arterial ammonia increased in non-treated BDL vs SHAM-operated controls (166.9±21.1uM vs 70.5±14.4uM, p<0.01). All doses of AST-120 decreased ammonia in BDL to similar levels found in respective SHAM-operated controls; 0.1g/kg/day (117.2±12.6uM vs 100.4±7.3uM, p>0.05); 1g/kg/day (78.9±22.3uM vs 78.1±8.9uM, p>0.05) and 4g/kg/day (48.8±19.6uM vs 49.9±14.7uM, p>0.05). Ammonia levels significantly correlated with doses of AST-120 (r=-0.66, p<0.0001). Brain water content increased in frontal cortex, cerebellum and brain stem in BDL vs SHAM-operated and was normalized in all AST-120 treatment groups compared to respective controls. Circulating levels of TNF-α were significantly increased in BDL vs SHAM-operated rats (p<0.05). AST-120 treatment did not attenuate TNF-α levels compared to respective SHAM-operated controls. Conclusion: AST-120 treatment dose-dependently decreased arterial ammonia levels, which resulted in normalization of multi-foci brain edema. This response was not correlated with differences in levels of circulating TNF-α. AST-120 treatment is a safe, non-antibiotic alternative demonstrating a significant ammonia-lowering effects, as well as a protective effect on the development of brain edema in rats with chronic liver disease. AST-120 represents a useful tool to elucidate further the relationship between circulating ammonia and gut-derived or systemic inflammatory components of liver disease.
Cristina R. Bosoi, Christian Parent-Robitaille, Mélanie Tremblay, Christopher F. Rose.
AIMS: The pathogenesis of hepatic encephalopathy (HE) is multifactorial involving gut-derived toxins such as ammonia. Liver disease leads to hyperammonemia and neurotoxic levels of ammonia which have been demonstrated to induce oxidative stress. Therefore a primary treatment target for HE is to decrease ammonia production in the gastrointestinal tract. AST-120 (spherical carbon adsorbent), an oral adsorbent of engineered activated carbon microspheres with surface areas exceeding 1600m2/g acts as a sink for neuro- and hepato-toxins present in the gut. In this study, we evaluated the protective effect of AST-120 in lowering arterial ammonia and oxidative stress and in attenuating brain edema 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 AST120 (dispersed in methylcellulose (MC)) by gavage at a dose of 0.1, 1 or 4g/kg/day for 6 weeks. Control BDL and SHAM-operated groups received MC only. AST and ALT were measured to assess liver function. Ammonia and reactive oxygen species (ROS) were measured in arterial plasma using a commercially available kit and DCFDA-fluorescent technique respectively. Brain water content was measured in the frontal cortex using the specific gravimetric technique. RESULTS: AST-120 was well-tolerated with no deaths in either of the treatment groups. Brain water content increased in BDL vs SHAM-operated rats (79.2±0.2% vs 78.7±0.1%, p<0.05). All AST-120 treatments, 0.1, 1 and 4g/kg/day, normalised brain water content in BDL rats. Arterial ammonia levels increased in the BDL control group vs SHAM-operated controls (169.9±21.1uM vs 70.5±14.4uM, p<0.05) and decreased significantly to SHAM levels in 0.1g/kg/day (117.2±12.6uM, p<0.05), 1g/kg/day (78.9±22.3uM, p<0.05) and 4g/kg/day (48.7±19.5uM, p<0.05) AST-120 treated BDL rats. Circulating levels of ROS were significantly increased in BDL vs SHAM-operated rats (11 fold, p<0.001). AST-120 treatments did not attenuate arterial levels of ROS compared to respective SHAM-operated controls. Liver functions enzymes, AST and ALT, were increased in all BDL rats compared to their respective SHAM-operated controls (p<0.001) but were not attenuated following AST120 treatments. CONCLUSIONS: AST-120 treatment normalized brain water content and decreased arterial ammonia levels but did not demonstrate an effect on systemic ROS. Long-term treatment with AST-120 is a safe, non-antibiotic alternative with significant ammonia-lowering effect as well as a protective effect on the development of brain edema in rats with chronic liver failure. Additional studies are warranted to evaluate the effects of AST-120 on HE in patients with advanced liver disease.
Cristina R. Bosoi, Christian Parent-Robitaille, Mélanie Tremblay, Christopher F. Rose.
Introduction: La pathogénèse de l'encéphalopathie hépatique (EH) est multifactorielle et implique des toxines provenant des intestins, telle que l'ammoniaque. L'insuffisance hépatique mène à l'hyperammonémie et aux niveaux neurotoxiques d’ammoniaque qui peuvent induire le stress oxydatif. Ainsi, la première cible du traitement de l'EH est la diminution de la production intestinale d'ammoniaque. AST-120 (adsorbant sphérique de charbon), un adsorbant oral de microsphères activées par ingénierie ayant une surface de plus de 1600m2/g, adsorbe les neuro- et hépato-toxines présentes dans les intestins. Dans cette étude, nous avons évalué l'effet protecteur d'AST-120 sur l'œdème cérébral et sa capacité à diminuer l'ammoniaque artérielle et le stress oxydatif chez les rats cirrhotiques. Méthodes: La cirrhose a été induite par la ligature de la voie biliaire (BDL) pour 6 semaines. Les rats BDL et SHAM ont reçu durant 6 semaines une dose d'AST-120 de 0.1 et 1mg/kg/jour dispersé dans la méthylcellulose (MC) par gavage. Des rats contrôles BDL et SHAM ont reçu seulement le véhicule. L'AST et ALT ont été mesurés à la fin du traitement pour évaluer la fonction du foie. L'ammoniaque et les espèces réactives d'oxygène (ROS) ont été mesurées dans le plasma artériel par un kit commercial et une technique de fluorescence. Le contenu en eau du cerveau a été mesuré dans le cortex frontal par une technique spécifique gravimétrique. Résultats: Le traitement avec AST-120 a été bien toléré. Les marqueurs de la fonction hépatique, AST et ALT, étaient augmentés dans tous les groupes de rats BDL vs SHAM (p<0.001). Le contenu en eau du cerveau a augmenté dans les rats BDL (BDL: 81.8±0.1% vs SHAM: 81.3±0.2%, p<0.05). Les deux dosages d'AST-120 de 0.1 et 1mg/kg/jour ont normalisé le contenu en eau du cerveau dans les rats BDL. L'ammoniaque artérielle était augmentée chez les rats BDL (175.5± 48.1uM vs SHAM: 105.3±27.3uM, p<0.05) et diminuée significativement dans les BDL 0.1mg/kg/jour (118.0±6.4uM, p<0.05) et BDL 1mg/kg/jour (75.4±31.4uM, p<0.05). Les niveaux de ROS étaient augmentés chez les rats BDL vs SHAM (augmentation de 11 fois vs SHAM). AST-120 n'a pas diminué les niveaux plasmatiques de ROS comparativement aux SHAM. Conclusions: Le traitement avec AST-120 normalise le contenu en eau du cerveau et diminue les niveaux artériels d'ammoniaque, mais n'a pas d'effet sur les ROS systémiques. AST-120 administré à long terme a été prouvé une alternative sûre de traitement non-antibiotique pour l'EH, en démontrant un effet significatif sur la diminution de l'ammoniaque, en même temps qu'un effet protecteur sur l'œdème cérébral chez les rats avec ligature de voie biliaire. Projet subventionné par: Instituts de recherche en santé du Canada et Ocera Therapeutics Inc. (CA, USA)
Christopher F. *Rose, Cristina Bosoi, Christian Parent-Robitaille, Mélanie Tremblay, Christopher F. Rose.
Background: The pathogenesis of hepatic encephalopathy (HE) is multifactorial involving gut-derived toxins such as ammonia. Liver disease leads to hyperammonemia and neurotoxic levels of ammonia which have been demonstrated to induce oxidative stress. Therefore a primary treatment target for HE is to decrease ammonia production in the gastrointestinal tract. AST-120 (spherical carbon adsorbent), an oral adsorbent of engineered activated carbon microspheres with surface areas exceeding 1600m2/g works as a sink for neuro- and hepato–toxins present in the gut. In this study, we evaluated the protective effect of AST-120 in attenuating brain edema and the capacity of AST-120 to lower arterial ammonia and oxidative stress 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 AST-120 by gavage in methylcellulose (MC) at a dose of 0.1 or 1g/kg/day for 6 weeks. Control BDL and SHAM-operated groups received MC only. AST and ALT were measured at the end of the treatment period to assess liver function. Ammonia and reactive oxygen species (ROS) were measured in arterial plasma using a commercially available kit and DCFDA-fluorescent technique respectively. Brain water content was measured in the frontal cortex using the specific gravimetric technique. Results: AST-120 was well-tolerated with no deaths in either of the treatment groups. Brain water content increased in BDL vs SHAM-operated rats (81.8 ± 0.1% vs 81.3 ± 0.2%, p<0.05). Both 0.1 and 1g/kg/day AST-120 treatments normalised brain water content in BDL rats. Arterial ammonia increased in the BDL control group vs SHAM-operated controls (175.5 ± 48.1uM vs 105.3 ± 27.3uM, p<0.05) and decreased significantly in BDL 0.1g/kg/day (118.0 ± 6.4uM, p<0.05) and BDL 1g/kg/day (75.4 ± 31.4uM, p<0.05). Circulating levels of ROS were significantly increased in BDL vs SHAM-operated rats (11 fold, p<0.001). AST-120 treatment did not attenuate arterial levels of ROS compared to respective SHAM-operated controls. Liver functions enzymes, AST and ALT, were increased in all BDL rats compared to their respective SHAM-operated controls (p<0.001). Conclusions: AST-120 treatment normalized brain water content and decreased arterial ammonia levels but did not demonstrate an effect on systemic ROS. Long-term treatment with AST-120 is a safe, non-antibiotic alternative demonstrating a significant ammonia-lowering effect, as well as a protective effect on the development of brain edema in rats with chronic liver failure. Additional studies are warranted to evaluate the effects of AST-120 on HE in patients with advanced liver disease.
Cristina Bosoi, Christian Parent-Robitaille, Mélanie Tremblay, Christopher F. *Rose.
Background: The pathogenesis of hepatic encephalopathy (HE) is multifactorial involving gut-derived toxins such as ammonia. Liver disease leads to hyperammonemia and neurotoxic levels of ammonia which have been demonstrated to induce oxidative stress. Therefore a primary treatment target for HE is to decrease ammonia production in the gastrointestinal tract. AST-120 (spherical carbon adsorbent), an oral adsorbent of engineered activated carbon microspheres with surface areas exceeding 1600m2/g works as a sink for neuro- and hepato-toxins present in the gut. In this study, we evaluated the protective effect of AST-120 in attenuating brain edema and the capacity of AST-120 to lower arterial ammonia and oxidative stress 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 AST-120 by gavage in methylcellulose (MC) at a dose of 0.1 or 1g/kg/day for 6 weeks. Control BDL and SHAM-operated groups received MC only. AST and ALT were measured at the end of the treatment period to assess liver function. Ammonia and reactive oxygen species (ROS) were measured in arterial plasma using a commercially available kit and DCFDA-fluorescent technique respectively. Brain water content was measured in the frontal cortex using the specific gravimetric technique. Results: AST-120 was well-tolerated with no deaths in either of the treatment groups. Brain water content increased in BDL vs SHAM-operated rats (81.8 ± 0.1% vs 81.3 ± 0.2%, p<0.05). Both 0.1 and 1g/kg/day AST-120 treatments normalised brain water content in BDL rats. Arterial ammonia increased in the BDL control group vs SHAM-operated controls (175.5 ± 48.1uM vs 105.3 ± 27.3uM, p<0.05) and decreased significantly in BDL 0.1g/kg/day (118.0 ± 6.4uM, p<0.05) and BDL 1g/kg/day (75.4 ± 31.4uM, p<0.05). Circulating levels of ROS were significantly increased in BDL vs SHAM-operated rats (11 fold, p<0.001). AST-120 treatment did not attenuate arterial levels of ROS compared to respective SHAM-operated controls. Liver functions enzymes, AST and ALT, were increased in all BDL rats compared to their respective SHAM-operated controls (p<0.001). Conclusions: AST-120 treatment normalized brain water content and decreased arterial ammonia levels but did not demonstrate an effect on systemic ROS. Long-term treatment with AST-120 is a safe, non-antibiotic alternative demonstrating a significant ammonia-lowering effect, as well as a protective effect on the development of brain edema in rats with chronic liver failure. Additional studies are warranted to evaluate the effects of AST-120 on HE in patients with advanced liver disease.
Cristina R. Bosoi, Xiaoling Yang, Wenlei Jiang, Christian Parent-Robitaille, Mélanie Tremblay, Christopher F. Rose.
Background: Liver failure/disease leads to hyperammonemia, a central component in the pathogenesis of hepatic encephalopathy. However, it is believed systemic oxidative/nitrosative stress may exacerbate the neuropsychological effects of hyperammonemia observed in patients with liver disease. The present study aims to investigate the role of oxidative/nitrosative stress in 2 rat models of liver disease/hepatic encephalopathy; 1) portacaval anastomosis (PCA), and 2) bile-duct ligation (BDL). Methods: Rats were sacrificed 4 weeks following PCA and 6 weeks following BDL. Arterial plasma and cerebrospinal fluid (CSF) were collected to measure ammonia, reactive oxygen species (ROS), hydrogen peroxide (H2O2) and nitric oxide (NO). S-nitrosylation of cystein thiols was measured in the frontal cortex and plasma proteins by immunoblotting. Brain water content was measured in the frontal cortex using a specific gravimetric technique. Results: Hyperammonemia developed in both PCA (PCA: 181.0 ± 8.5uM vs sham: 60.5 ± 7.0uM; p< 0.0001) and BDL (BDL: 101.1 ± 11.0uM vs sham: 41.7 ± 9.2uM; p< 0.05) rats with PCA rats being significantly higher compared to BDL rats (p< 0.001). Consequently, ammonia increased in CSF in both PCA (PCA: 159.5 ± 15.8uM vs sham: 32.5 ± 3.7uM; p< 0.0001) and BDL (BDL: 123.5 ± 21.7uM vs sham: 23.3 ± 6.1uM; p< 0.05), but no significant change was observed between PCA and BDL. Only rats with BDL demonstrated an increase in plasma levels of ROS (5.8-fold increase vs sham; p< 0.001), H2O2 (3.2-fold increase vs sham; p< 0.05) and NO (2.9-fold increase vs sham; p< 0.05). This was accompanied by an increase in S-nitrosylation of plasma proteins. An increase in brain water content was observed in rats with BDL (BDL: 81.91 ± 0.19% vs sham: 81.23 ± 0.20, p< 0.05) whereas no significant change in brain water content was found in PCA vs sham-operated control rats. Conclusion: Hyperammonemia together with systemic oxidative stress are associated with brain edema in rats with BDL which is not demonstrated in PCA rats where only hyperammonemia is observed. Our findings suggest systemic oxidative stress is implicated in the pathogenesis of brain edema and its synergistic effect with ammonia may lead to progression of hepatic encephalopathy.
Cristina R. Bosoi, Wenlei Jiang, Christian Parent-Robitaille, Mélanie Tremblay, Christopher F. Rose.
Introduction: L'insuffisance/maladie hépatique mène à l'hyperammonémie, le facteur central dans la pathogénèse de l'encéphalopathie hépatique. Toutefois, le stress oxydatif/nitrosatif peut avoir un rôle important dans l'exacerbation des effets neuropsychologiques de l'hyperammonémie chez les patients atteints de maladies hépatiques. Cette étude a comme objectif l'investigation du rôle du stress oxydatif/nitrosatif dans deux modèles de maladie du foie/encéphalopathie hépatique chez le rat; 1) dérivation portosystémique (PCA), et 2) ligature de la voie biliaire (BDL). Méthodes: Les rats ont été sacrifiés 4 semaines après la PCA ou 6 semaines après la BDL. L'ammoniaque, les espèces réactives d'oxygène (ROS), le H2O2 et l'oxyde nitrique (NO) ont été mesurés dans le plasma (artériel) et l'ammoniaque, les ROS et le NO dans le liquide céphalorachidien (CSF), respectivement par un kit commercial, une technique de fluorescence et la réaction Griess. 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 (PCA: 173.7 ± 13.6 uM vs sham: 77.8 ± 11.2 uM; p<0.001) et BDL (BDL: 114.1 ± 19.2 uM vs sham: 33.2 ± 4.7 uM; p<0.05) et les valeurs pour les rats PCA sont significativement plus élevées que celles des BDL (p<0.05). En conséquence, l'ammoniaque est élevée dans le CSF chez les rats PCA (PCA: 146.6 ± 26.0 uM vs sham: 31.5 ± 6.9 uM; p<0.001) et BDL (BDL: 128.4 ± 36.7 uM vs sham: 23.3 ± 6.1 uM; p<0.05). On n'observe pas de différences significatives entre les PCA et BDL. Seulement les rats BDL montrent une augmentation des niveaux plasmatiques de ROS (augmentation de 5.8 fois vs sham; p<0.001) et H2O2 (augmentation de 3.2 fois vs sham; p<0.05). On n'observe pas de différences significatives entre les valeurs des ROS dans le CSF chez les PCA et BDL, comparativement aux groupes contrôles. De plus, on observe une augmentation du contenu en eau du cerveau seulement dans les rats BDL (BDL: 81.52 ± 0.15% vs sham: 80.43 ± 0.21%, p<0.05). Aucun changement significatif dans les niveaux plasmatiques des ROS, H2O2 ou du contenu en eau cérébrale dans les PCA vs les contrôles sham-opérés n'a été trouvé. Conclusion: Le stress oxydatif superposé à l'hyperammonémie entraîne une augmentation du contenu cérébral en eau dans les rats BDL qui n'est pas observée chez les rats PCA, où seulement l'hyperammonémie est présente. Nos résultatss suggèrent que le stress oxydatif systémique est impliqué dans la pathogénèse de l’œdème cérébrale et que son effet synergique avec l'ammoniaque peut mener à la progression de l'encéphalopathie hépatique.
Cristina R. Bosoi, Xiaoling Yang, Wenlei Jiang, Christian Parent-Robitaille, Mélanie Tremblay, Christopher F. Rose.
Background: Liver failure/disease leads to hyperammonemia which is central in the pathogenesis of hepatic encephalopathy. However, it is believed systemic oxidative/nitrosative stress may play an important role in exacerbating the neuropsychological effects of hyperammonemia observed in patients with liver disease. The present study aims to investigate the role of oxidative/nitrosative stress in 2 rat models of liver disease/hepatic encephalopathy; 1) portacaval anastomosis (PCA), and 2) bile-duct ligation (BDL). Methods: Rats were sacrificed 4 weeks following PCA and 6 weeks following BDL. Arterial plasma and cerebrospinal fluid (CSF) were collected to measure ammonia, reactive oxygen species (ROS), hydrogen peroxide (H2O2) (only measured in plasma) and nitric oxide (NO) using a commercially available kit, the DCFDA and Amplex Red fluorescent techniques and the Griess reaction respectively. S-nitrosylation of cystein thiols was measured in the frontal cortex and plasma proteins by immunoblotting. Brain water content was measured in the frontal cortex using a specific gravimetric technique. Results: Hyperammonemia developed in both PCA (PCA: 181.0 ± 8.5uM vs sham: 60.5 ± 7.0uM; p<0.0001) and BDL (BDL: 101.1 ± 11.0uM vs sham: 41.7 ± 9.2uM; p<0.05) rats with PCA rats being significantly higher compared to BDL rats (p<0.001). Consequently, ammonia increased in CSF in both PCA (PCA: 159.5 ± 15.8uM vs sham: 32.5 ± 3.7uM; p<0.0001) and BDL (BDL: 123.5 ± 21.7uM vs sham: 23.3 ± 6.1uM; p<0.05), but no significant change was observed between PCA and BDL. Only rats with BDL demonstrated an increase in plasma levels of ROS (5.8-fold increase vs sham; p<0.001), H2O2 (3.2-fold increase vs sham; p<0.05) and NO (2.9-fold increase vs sham; p<0.05). This was accompanied by an increase in S-nitrosylation of plasma proteins. No significant changes in CSF ROS and NO, nor in S-nitrosylation of frontal cortex proteins were found in BDL or PCA compared to their respective control groups. In addition, an increase in brain water content was observed in rats with BDL (BDL: 81.91 ± 0.19% vs sham: 81.23 ± 0.20, p<0.05). No significant change in brain water content was found in PCA rats vs sham-operated controls. Conclusion: Systemic oxidative stress together with hyperammonemia results in an increase in brain water in rats with BDL which is not demonstrated in PCA rats where only hyperammonemia is observed. Our findings suggest systemic oxidative stress is implicated in the pathogenesis of brain edema and its synergistic effect with ammonia may lead to progression of hepatic encephalopathy.
Cristina R. Bosoi, Christian Parent-Robitaille, Mélanie Tremblay, Christopher F. Rose.
Background: Liver failure/disease leads to hyperammonemia which is central in the pathogenesis of hepatic encephalopathy. However, it is believed systemic oxidative/nitrosative stress may play an important role in exacerbating the neuropsychological effects of hyperammonemia observed in patients with liver disease. The present study aims to investigate the role of oxidative/nitrosative stress in 2 rat models of liver disease/hepatic encephalopathy; 1) portacaval anastomosis (PCA), and 2) bile-duct ligation (BDL). Methods: Rats were sacrificed 4 weeks following PCA or 6 weeks following BDL. Ammonia, reactive oxygen species (ROS), H2O2 and nitric oxide (NO) were measured in plasma (arterial) and ammonia, ROS and NO were measured in cerebrospinal fluid (CSF) using respectively a commercially available kit, a fluorescent technique and the Griess reaction. Brain water was measured in the frontal cortex using a specific gravimetric technique. Results: Hyperammonemia developed in both PCA (PCA: 173.7 ± 13.6 uM vs sham: 77.8 ± 11.2 uM; p<0.001) and BDL (BDL: 114.1 ± 19.2 uM vs sham: 33.2 ± 4.7 uM; p<0.05) rats with PCA rats being significantly higher compared to BDL rats (p<0.05). Consequently, ammonia increased in CSF in both PCA (PCA: 146.6 ± 26.0 uM vs sham: 31.5 ± 6.9 uM; p<0.001) and BDL (BDL: 128.4 ± 36.7 uM vs sham: 23.3 ± 6.1 uM; p<0.05). No significant change was observed between PCA and BDL. Only rats with BDL demonstrated an increase in plasma levels of ROS (5.8-fold increase vs sham; p<0.001) and H2O2 (3.2-fold increase vs sham; p<0.05). No significant change in CSF ROS was found in BDL or PCA compared to their respective control groups. In addition, an increase in brain water content was only observed in rats with BDL (BDL: 81.52 ± 0.15% vs sham: 80.43 ± 0.21%, p<0.05). No significant change in plasma ROS, H2O2 or brain water was found in PCA rats vs sham-operated controls. Conclusion: Oxidative stress in addition to hyperammonemia results in an increase in brain water in rats with BDL which is not in PCA rats where only hyperammonemia is observed. Our findings suggest systemic oxidative stress is implicated in the pathogenesis of brain edema and its synergistic effect with ammonia may lead to progression of hepatic encephalopathy.
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