2015 AASLD presentations

Accepted abstract

Two students from the lab, Marc-André and Cristina, will present an oral and a poster to AASLD meeting. Two others posters from collaboration's project will also be presented.

We are glad to have been choosen for an oral presentation of our results at the Liver Meeting 2015 who is currently held at San Francisco, USA, from November 13th to 17th. Marc-André Clément will present his master's project at parallel session 3, about liver transplantation. Cristina will present her poster on encephalopathy's session. Don't miss them!

Related Publications

Long term oral treatment of ornithine phenylacetate increases lean mass and attenuates brain edema in bile-duct ligated rats.

Cristina R. Bosoi, Mariana Oliveira, Marc-André Clément, Mélanie Tremblay, Gabrie Ten Have, Nicolaas E. P. Deutz, Christopher F. Rose.

Background: Chronic liver disease (cirrhosis; CLD) is characterized by numerous metabolic disturbances which lead to complications that impact the clinical outcome. Among these, loss of muscle, characterized by a deterioration of muscle quantity and quality, leads to a decrease in functional capacity, adversely affecting survival, quality of life and outcome following liver transplantation. Hyperammonemia is central in the development of hepatic encephalopathy, a major complication of cirrhosis. However, it is speculated the toxic effect of ammonia extends beyond the brain, possibly affecting muscle. Therefore, we hypothesized that lowering blood ammonia will attenuate muscle mass loss in cirrhotic rats. Ornithine phenylacetate (OP; OCR-002) was used to lower blood ammonia. Methods: We induced CLD in rats following 6-week bile-duct ligation (BDL). Four experimental groups were tested; 1) Sham; 2) BDL; 3) Sham + OP; 4) BDL + OP. One week following BDL, rats were orally administered (gavage) OP (1g/kg) daily for 5 weeks. Body weight, fat and lean mass (EchoMRI), blood ammonia, cerebral edema (specific gravity method), fractional synthesis of protein (FSR) in muscle (with D2O) and locomotor activity (day/night) were measured. Results: At the end of the 6-weeks experiment, BDL rats demonstrated a 4-fold increase in blood ammonia vs Sham-operated controls. This increase was reduced by 40% in OP-treated BDL rats. BDL rats gained less body weight compared to sham-operated controls (body weight of 360.2g  13.6 vs 476.8g  10.38; p<0.001) which was accompanied with a lower gain of lean mass and a lower muscle FSR. OP-treated BDL rats showed a significant increase in body weight (429.6g  117.9; p<0.001 vs BDL) with a significant higher lean mass (303.1g  10.7 in BDL+OP vs 264.4g  10.5 in BDL, p<0.01). Fat mass remained unchanged between the treated and untreated BDL groups. OP treatment normalized brain water content in BDL rats. In contrast, OP-treatment reduced muscle FSR in SHAM animals, but not in BDL rats. Locomotor activity in BDL rats was reduced compared with sham-operated controls but no significant change was found between BDL+OP and SHAM+OP. Conclusion: This is the first study demonstrating the efficient ammonia-lowering effect of an oral formulation of OP. Long-term treatment with OP is a safe, non-antibiotic alternative demonstrating a significant ammonia-lowering effect, as well as a protective effect on the development of brain edema and muscle mass loss in rats with CLD. Whether the effect of OP on muscle mass loss attenuation is a result of lowering blood ammonia or directly improves muscle metabolism remains to be established.

Oral

Minimal hepatic encephalopathy leads to hypotension-induced neuronal cell loss in BDL rats.

Marc-André Clément, Cristina Bosoi, Mélanie Tremblay, Chantal Bémeur, Christopher F. Rose.

Background: Hepatic encephalopathy (HE) is a major neuropsychiatric complication caused by liver disease characterized by cognitive and motor dysfunction. The only curative treatment to date remains liver transplantation (LT). Historically, HE has always been considered to be a reversible metabolic disorder and has therefore been expected to completely resolve following LT. However, persisting neurological complications remain a common problem affecting as many as 47% of LT recipients. LT is a major surgical procedure accompanied by intraoperative stress and confounding factors, including blood loss and hypotension. We hypothesize, in the setting of minimal HE (MHE), the compromised brain becomes susceptible to hypotensive insults, resulting in cell injury and death. Methods: Six-week bile-duct ligated (BDL) rats with MHE and respective controls (SHAM) were used. Blood is withdrawn from the femoral artery (inducing hypovolemia) until an mean arterial pressure of 30 and 60 mmHg (hypotension) and maintained for 120 minutes. Cerebral blood flow (BCF) was assessed by injecting fluorescent microspheres (1x106 microspheres/ml) through the brachial artery. Upon sacrifice, brains were extracted for apoptotic analysis (western blot) and neuronal cell count (immunohistochemistry). In a separate group, BDL rats were treated for MHE with ornithine phenylacetate (OP; OCR-002) (1g/kg) for 3 weeks. Results: Both BDL rats and SHAM-operated controls without hypotension did not display any cell injury or neuronal loss. However, BDL rats following hypotension (30 and 60mmHg) demonstrated a significant decrease in neuronal cell count in the frontal cortex (using NeuN+DAPI and Cresyl Violet) compared to hypotensive SHAM-operated controls. In addition, neuronal loss was associated with an increased in cellular stress protein, hsp32, hsp70 and caspase-3, suggesting apoptotic cell death. CBF decreased in BDL rats compared to SHAM and correlated with degree of hypotension insult. BDL rats treated with OP did not lead to neuronal cell death following hypotension. Discussion: These findings strongly suggest that cirrhotic patients with MHE are more susceptible to hypotension-induced neuronal cell loss. Moreover, these results suggest a patient with HE (even MHE), with a “frail brain”, will fare worse during liver transplantation and consequently result in poor neurological outcome. Combination of MHE and hypotension may account for the persisting neurological complications observed in a number of cirrhotic patients following LT. Therefore, MHE, i) should not to be ignored and ii) deserves to be treated in order to reduce the risk of neurological complications occurring post-LT.

Enoxaparin treatment does not ameliorate liver fibrosis or portal hypertension in experimental models of cirrhosis in rats.

Jose Ignacio Fortea, Alexander Zipprich, Carolina-Mena Fernandez, Christopher F. Rose, Juan Bañares, Marta Puerto, Cristina R. Bosoi, Jorge Almagro, Marcus Hollenbach, Marc-André Clément, Javier Vaquero, Rafael Bañares, Cristina Ripoll.

Recent clinical and experimental studies suggest that the administration of low molecular weight heparin may reduce the risk of decompensation of liver disease and liver fibrosis. Our aim was to evaluate the effects of enoxaparin on liver fibrosis and hemodynamics in several experimental models of cirrhosis. Methods: Cirrhosis was induced in male SD rats using 3 protocols: 1) Oral gavage with carbon tetrachloride (CCl4) twice a week for 12 weeks, 2) Inhalation of CCl4 twice a week for 12 weeks, and 3) Bile duct ligation (BDL) surgery. Rats (n= 8-15/group) underwent daily sc treatment with saline or diverse protocols of enoxaparin: 40 U/kg bw from 8th week of CCl4, 180 U/kg bw from 1st week or 8th week of CCl4, and 180 U/kg from 2nd week of BDL. Control groups of rats gavaged with water or sham-operated rats followed the same protocols. Portal pressure was measured in the ileocolic vein, liver fibrosis was assessed in Sirius Red and Masson’s Trichrome stained liver sections. Ascitic fluid was evaluated for bacterial growth in thioglycolate medium. Ex-vivo liver perfusion experiments were performed for assessing endothelium-dependent and -independent reactivity. Results: Compared with controls, rats with oral CCl4 gavage tended to show decreased survival and body weight gain, both of which were further worsened by enoxaparin 180 U/kg bw (p< 0.01). Rats with CCL4-induced cirrhosis showed altered laboratory parameters (increased INR, AST, ALT, bilirubin / decreased albumin, total proteins, glucose and platelets) regardless of enoxaparin treatment. In all experimental models, cirrhotic rats receiving saline and those receiving enoxaparin showed similar increases in the area of liver fibrosis compared with controls (p< 0.001). Rats with cirrhosis induced by oral CCl4 gavage and by BDL surgery developed increases of portal pressure and spleen-to-bw ratios compared with control rats (p< 0.001), regardless of enoxaparin treatment. Among rats with ascites, a similar proportion presented positive bacterial cultures (CCl4+saline 2 of 7 vs. CCl4+enoxaparin 4 of 8, NS; BDL+saline 1 of 6 vs. BDL+enoxaparin 2 of 7, NS). Potential effects of enoxaparin on hepatic vascular reactivity were only observed in rats receiving enoxaparin 180 U/kg bw from the beginning of CCl4 administration, and consisted of increased portal venous resistance after addition of acetylcholine or S-nitroso acetyl-penicillamine (SNAP, both p< 0.05) and increased sinusoidal resistance after addition of SNAP (p< 0.05). Conclusion: Our experimental data do not support a role of long-term treatment with enoxaparin for improving liver fibrosis, portal hypertension or endothelialdysfunction in cirrhosis.

Sarcopenia and Sarcopenic-Obesity are Associated with Hyperammonemia and Increased Risk of Hepatic Encephalopathy in Cirrhotics.

Aldo J. Montano-Loza, Andres Duarte-Rojo, Rahima A. Bhanji, Christopher F. Rose.

Background/Aims: Muscle mass functions as an alternative site of ammonia detoxification in patients with cirrhosis. In this study we aimed to investigate if sarcopenia, myosteatosis, and obesity are associated with hyperammonemia and hepatic encephalopathy (HE) in patients with cirrhosis. Methods: A total of 204 cirrhotic patients were studied. Muscularity assessment was analyzed using CT scans at the level of the 3rd lumbar vertebral body. Sarcopenia was defined using the skeletal muscle index and myosteatosis according to the muscle attenuation index. Overweight-obesity was defined as a body mass index ≥25 kg/m2. Sarcopenic-obesity was defined as concomitant sarcopenia and overweight-obesity. HE was evaluated clinically (West-Heaven criteria) and defined as absent in patients not using specific treatment (i.e. lactulose, rifaximin) and with no prior episodes of HE in the preceding year. Ammonia blood levels were also performed (nl. 0-35 mmol/L) at the time of the muscularity assessment. Results: Mean age was 56±1 years and 141 were males (69%). Cirrhosis was caused by HCV in 78 patients (38%), alcohol in 56 (28%), NASH in 34 (16%), and other etiology in 36 patients (18%). Sarcopenia was noted in 96 patients (47%), 137 had myosteatosis (67%), 136 were overweight-obese (67%), and 53 (28%) had sarcopenic-obesity. Patients with sarcopenia (87±6 vs. 61±4 mmol/L, P<0.001), and sarcopenic-obesity (95±9 vs. 40±3 mmol/L, P<0.001) had higher levels of ammonia. Levels of ammonia were not different among patients with myosteatosis (77±5 vs. 67±5 mmol/L, P=0.2), and overweight-obesity (76±5 vs. 67±5 mmol/L, P=0.2). Patients with sarcopenia (84 vs. 63%, P=0.001) and sarcopenic-obesity (93 vs. 65%, P<0.001) had higher frequency of hyperammonemia. Patients with myosteatosis had a trend (77 vs. 64%, P=0.06), and overweight-obesity was not associated with hyperammonemia (77 vs. 66%, P=0.1). Lastly, patients with sarcopenia (42 vs. 26%, P<0.001), and sarcopenic-obesity (47 vs. 30%, P<0.001) had higher frequency of HE. Sarcopenia and sarcopenic-obesity increased the risk of hyperammonemia (OR 3.2, 95% CI 1.66.2, P=0.001, and OR 7.0, 95% CI 2.4-21, P<0.001, respectively). Also, sarcopenia and sarcopenic-obesity increased the risk of HE (OR 2.0, 95% CI 1.5-2.8, P<0.001, and OR 2.1, 95% CI 1.4-3.0, P<0.001, respectively). Conclusions: Cirrhotic patients with sarcopenia and sarcopenic-obesity have higher ammonia levels and risk for HE. Muscle mass plays a protective role for hyperammonemia and therapeutic strategies to avoid muscle depletion might decrease the risk of HE in cirrhosis.