Antoine Brault

Sinusoidal endothelial cell and hepatocyte death following cold ischemia‐warm reperfusion of the rat liver

Cold ischemia‐warm reperfusion (CI‐WR) injury of the liver is characterized by marked alterations of sinusoidal endothelial cells (SECs), whereas hepatocytes appear to be relatively unscathed. However, the time course and mechanism of cell death remain controversial: early versus late phenomenon, necrosis versus apoptosis? We describe the occurrence and nature of cell death after different periods of CI with University of Wisconsin (UW) solution and after different periods of WR in the isolated perfused rat liver model. After 24‐ and 42‐hour CI (viable and nonviable livers, respectively), similar patterns of liver cell death were seen: SEC necrosis appeared early after WR (10 minutes) and remained stable for up to 120 minutes. After 30 minutes of WR, apoptosis increased progressively with WR length. Based on morphological criteria, apoptotic cells were mainly hepatocytes within liver plates or extruded in the sinusoidal lumen. In addition, only after 42‐hour CI were large clusters of necrotic hepatocytes found in areas of congested sinusoids. In these same livers, the hepatic microcirculation, evaluated by means of the multiple‐indicator dilution technique, revealed extracellular matrix disappearance with no‐flow areas. In conclusion, different time courses and mechanisms of cell death occur in rat livers after CI‐WR, with early SEC necrosis followed by delayed hepatocyte apoptosis. These processes do not appear to be of major importance in the mechanism of graft failure because they are similar under both nonlethal and lethal conditions; this is not the case for the loss of the extracellular matrix found only under lethal conditions and associated with hepatocyte necrosis. (HEPATOLOGY 2004;39:1110–1119.)

Role of Kupffer cells in cold ischemia/reperfusion injury of rat liver

BACKGROUND & AIMS Kupffer cell activation is hypothesized to play an etiopathogenic role in storage-related graft failure after liver transplantation. The aim of this study was to verify whether the elimination of Kupffer cells modifies the magnitude of cold ischemia/reperfusion injury of the liver. METHODS Rat Kupffer cells were eliminated by an intravenous injection of liposome-encapsulated dichloromethylene diphosphonate. Livers from control and treated rats were isolated and perfused before and after 24-hour cold ischemia in the University of Wisconsin solution (4 degrees C). Hepatocyte and sinusoidal endothelial cell functions were evaluated by taurocholate and hyaluronic acid elimination, respectively. Liver transplantation was also performed using control and treated donor livers stored under identical conditions. RESULTS Compared with baseline values, similar alterations were found in both groups after cold ischemia for hepatocyte function (intrahepatic resistance, bile secretion, lactate dehydrogenase release, oxygen consumption, and taurocholate intrinsic clearance) and for sinusoidal endothelial cell function (hyaluronic acid intrinsic clearance). The 10-day survival rate of animals undergoing transplantation was not different between the groups (6 of 15 vs. 4 of 15, control vs. treated donor livers, respectively). CONCLUSIONS The presence or absence of Kupffer cells does not modify the effect of 24-hour cold ischemia/reperfusion on the rat liver.

Antioxidant and Hepatoprotective Effects of Silibinin in a Rat Model of Nonalcoholic Steatohepatitis

Nonalcoholic steatohepatitis (NASH) is a progressive liver disease related to the metabolic syndrome, obesity and diabetes. The rising prevalence of NASH and the lack of efficient treatments have led to the exploration of different therapeutic approaches. Milk thistle (Silibum marianum) is a medicinal plant used for its hepatoprotective properties in chronic liver disease since the 4th century BC. We explored the therapeutic effect of silibinin, the plants most biologically active extract, in an experimental rat NASH model. A control group was fed a standard liquid diet for 12 weeks. The other groups were fed a high-fat liquid diet for 12 weeks without (NASH) or with simultaneous daily supplement with silibinin–phosphatidylcholine complex (Silibinin 200 mg kg−1) for the last 5 weeks. NASH rats developed all key hallmarks of the pathology. Treatment with silibinin improved liver steatosis and inflammation and decreased NASH-induced lipid peroxidation, plasma insulin and TNF-α. Silibinin also decreased O2 ∙− release and returned the relative liver weight as well as GSH back to normal. Our results suggest that milk thistles extract, silibinin, possesses antioxidant, hypoinsulinemic and hepatoprotective properties that act against NASH-induced liver damage. This medicinal herb thus shows promising therapeutic potential for the treatment of NASH.

Matrix metalloproteinase inhibition protects rat livers from prolonged cold ischemia–warm reperfusion injury

Matrix metalloproteinases (MMPs) have been implicated in the hepatic injury induced after cold ischemia–warm reperfusion (CI‐WR), by altering the extracellular matrix (ECM), but their precise role remains unknown. The hepatic MMP expression was evaluated after 2 conditions of CI (4°C for 24 and 42 hours: viable and nonviable livers) followed by different periods of WR, using isolated perfused rat livers. CI‐WR induced moderate changes in hepatic MMP transcript levels not influenced by CI duration, whereas gelatinase activities accumulated in liver effluents. Therefore, the protective effect of a new phosphinic MMP inhibitor, RXP409, was tested after prolonged CI. RXP409 (10 μM) was added to the University of Wisconsin solution, and livers were preserved for 42 hours (4°C), then reperfused for 1 hour in Krebs solution (37°C), containing 20% erythrocytes. Liver viability parameters were recorded, and the extent of cell necrosis was evaluated on liver biopsies, using trypan blue nuclear uptake. Treatment with RXP409 significantly improved liver function (transaminase release and bile secretion) and liver injury. In particular, the MMP inhibitor significantly modified the extent of cell death from large clusters of necrotic hepatocytes as found in control livers (2%–60% of liver biopsies; mean, 26% ± 9%) to isolated necrotic hepatocytes as found in treated livers (0.2%–12%; mean, 3% ± 2%) (P < 0.05). Conclusion: These data demonstrate that MMPs, by altering the ECM, play a major role in liver CI‐WR injury leading to extensive hepatocyte necrosis and that their inhibition might prove to be a new strategy in improving preservation solutions. (HEPATOLOGY 2007.)

Metabolism of hyaluronic acid by liver endothelial cells : effect of ischemia-reperfusion in the isolated perfused rat liver

Liver endothelial cells appear to be particularly vulnerable to cold ischemia reperfusion. However, their function has not yet been evaluated, except using electron microscopic changes and trypan blue exclusion (an index of cell death). Hyaluronic acid is a polysaccharide highly extracted by normal liver endothelial cells. We thus evaluated liver endothelial cell function by measuring hyaluronic acid elimination in a model of ischemia-reperfusion injury using isolated perfused Wistar rat livers. We compared the effects of two preservation solutions during cold ischemia (4 degrees C): normal saline with 2 mM CaCl2 (4 h and 8 h ischemia) and the University of Wisconsin solution (8 h and 24 h ischemia). Eliminations were measured during two 40-min periods before and after ischemia; during each period, hyaluronic acid (150 ng/ml) and also, to evaluate hepatocyte function, propranolol (100 ng/ml) were infused into the reservoir. We show that, whatever the preservation solution or time used, liver endothelial cell function is altered to a larger extent than hepatocyte function. University of Wisconsin solution does not appear to protect liver endothelial cells during preservation, particularly after 24 h of cold ischemia. Hyaluronic acid elimination can be a useful tool in the investigation of an ideal preservation solution to protect liver endothelial cells from ischemia-reperfusion damage.

In Vivo Anti-Diabetic Activity of the Ethanolic Crude Extract of Sorbus decora C.K.Schneid. (Rosacea): A Medicinal Plant Used by Canadian James Bay Cree Nations to Treat Symptoms Related to Diabetes

A number of potential anti-diabetic plants were identified through an ethnobotanical survey of the traditional pharmacopeia of the Cree of Eeyou Istchee (CEI—Northeastern Canada) used against symptoms of diabetes and their biological activity assessed by in vitro bioassays. Among these, Sorbus decora C.K.Schneid. (Rosacea) ranked highly and increased the transport of glucose in skeletal muscle cells in culture. The present study thus aimed at confirming the antidiabetic potential of S. decora in in vivo models of insulin resistance and diabetes, notably the streptozotocin Type 1 diabetic rat (STZ), the genetic KK-Ay Type 2 diabetic mouse and the rat rendered insulin resistant with 10% glucose water consumption for 6 weeks. Sorbus decora ethanolic crude extract (SDEE) was administered orally (200 mg kg−1) and compared to metformin (150 or 500 mg kg−1). The intragastric (i.g.) gavage of SDEE transiently decreased glycemia in STZ rats in a bi-phasic manner but the effect was cumulative over several days. In KK-Ay mice, SDEE incorporated in food (0.12%) decreased glycemia by 15% within 1 week as compared to vehicle controls. In pre-diabetic insulin-resistant rats, SDEE fed daily by i.g. gavage for 2 weeks significantly decreased the slight hyperglycemia and hyperinsulinemia, without affecting sugar water intake. Using the HOMA insulin resistance parameter, the effect of SDEE was equivalent to that of metformin. In conclusion, the ethanolic crude extract of S. decora demonstrates both anti-hyperglycemic and insulin-sensitizing activity in vivo, thereby confirming anti-diabetic potential and validating CEI traditional medicine.

COLD ISCHEMIA-REPERFUSION INJURY OF THE LIVER: Role of the Liver Donor Nutritional Status in Rats

To verify the role of donor nutritional status on the quality of liver preservation after cold storage, we assessed hepatocyte and liver endothelial cell viabilities and functions in an isolated perfused rat liver model. Livers from fed and fasted Wistar rats were isolated and perfused either immediately after liver harvesting or after a 24-hr cold (4 degrees C) preservation in University of Wisconsin solution. Hyaluronic acid (150 ng/ml) and taurocholate (11.5 micrograms/ml) were infused into the reservoir, and their eliminations were assessed to evaluate liver endothelial cell function and hepatocyte function, respectively. Liver viability was estimated by intrahepatic resistance, oxygen consumption, bile secretion, and lactate dehydrogenase release. In addition, cell viabilities were evaluated by trypan blue staining. In fed-rat livers, glycogen content did not differ before or after the cold preservation, although a reduction was observed during the subsequent perfusion period. Liver glycogen content in fed rats was markedly higher than in the fasted rats at each time point studied. In fasted and fed rats, liver viability parameters and hepatocyte function were moderately altered, whereas liver endothelial cell function was markedly impaired after cold preservation. However, feeding had no influence on either hepatocyte or liver endothelial cell functions which were similarly altered in both nutritional conditions. The present data show that the nutritional status of liver donors does not play an important role in the preservation of liver endothelial cells after cold ischemia-reperfusion and, thus, should not affect the overall resistance of livers to hypothermic-ischemic injury.

Populus balsamifera L. (Salicaceae) mitigates the development of obesity and improves insulin sensitivity in a diet-induced obese mouse model.

ETHNOBOTANICAL RELEVANCE : In previous in vitro bioassay studies, Populus balsamifera L. (Salicaceae), a medicinal plant ethnobotanically identified from the traditional pharmacopoeia of the Cree of Eeyou Istchee (Eastern James Bay area of Canada), exhibited a strong anti-obesity potential by potently inhibiting adipogenesis in 3T3-L1 adipocytes. The aim of the study is to evaluate the effectiveness of this plant extract in mitigating the development of obesity and the metabolic syndrome in diet-induced obese (DIO) C57BL/6 mice. MATERIALS AND METHODS Mice were subjected for eight weeks to a standard diet (CHOW), a high fat diet (HFD; DIO group), or HFD to which Populus balsamifera was incorporated at 125 and 250 mg/kg. RESULTS The results showed that Populus balsamifera decreased in a dose-dependent manner the weight gain of whole body, retroperitoneal fat pad and liver as compared to DIO controls and reduced the severity of hepatic macrovesicular steatosis and triglyceride accumulation. This plant extract also decreased glycemia in the second half of the feeding period and improved insulin sensitivity by diminishing insulin levels and the leptin/adiponectin ratio, as well as augmenting adiponectin levels. These effects were associated with slightly but significantly reduced food intake with 250 mg/kg Populus balsamifera as well as with an increase in energy expenditure (increase in skin temperature and increased expression of uncoupling protein-1; UCP-1). Data also suggest other mechanisms, such as inhibition of adipocyte differentiation, decrease of hepatic inflammatory state and potential increase in hepatic fatty acid oxidation. CONCLUSION Taken together, these results confirm the potential of Populus balsamifera as a culturally adapted therapeutic approach for the care and treatment of obesity and diabetes among the Cree.

Effects of acute physical exercise on hepatocyte volume and function in rat

The goal of the present experiment was to measure the volume of the different compartments in liver of exercised rats and to get some insights into the appropriate working of the hepatic function following exercise. Hence, livers from male rats were isolated and perfused after treadmill exercise or rest. This procedure was performed on rats that were overnight semifasted (50% food restriction) or well fed. To evaluate the hepatocyte cell volume, the multiple-indicator dilution curve technique was used after 40 min of perfusion. Radioactive tracers for red blood cells, sucrose, and water were used to measure liver vascular space, liver interstitial space, and water cellular space, respectively. The hepatocyte function was assessed by taurocholate and propanolol clearance. Oxygen consumption, intrahepatic resistance, bile secretion, and lactate dehydrogenase release estimated liver viability. Liver viability and hepatocyte function were not changed following exercise either in the fed or in the semifasted animals. As expected, liver glycogen levels were significantly (P < 0.01) reduced in the food-restricted rats. Consequently, liver glycogen levels following exercise were decreased significantly (P < 0.01) only in the fed rats. Despite this, exercise decreased the hepatocyte water space in both food-restricted and fed groups ( approximately 15%; P < 0.01) without altering the sinusoidal and interstitial space. The present data show that acute exercise decreased the hepatocyte volume and that this volume change is not entirely linked to a decrease in hepatic glycogen level.

Larix laricina, an Antidiabetic Alternative Treatment from the Cree of Northern Quebec Pharmacopoeia, Decreases Glycemia and Improves Insulin Sensitivity In Vivo

Larix laricina K. Koch is a medicinal plant belonging to traditional pharmacopoeia of the Cree of Eeyou Istchee (Eastern James Bay area of Canada). In vitro screening studies revealed that, like metformin and rosiglitazone, it increases glucose uptake and adipogenesis, activates AMPK, and uncouples mitochondrial function. The objective of this study was to evaluate the antidiabetic and antiobesity potential of L. laricina in diet-induced obese (DIO) C57BL/6 mice. Mice were subjected for eight or sixteen weeks to a high fat diet (HFD) or HFD to which L. laricina was incorporated at 125 and 250 mg/kg either at onset (prevention study) or in the last 8 of the 16 weeks of administration of the HFD (treatment study). L. laricina effectively decreased glycemia levels, improved insulin resistance, and slightly decreased abdominal fat pad and body weights. This occurred in conjunction with increased energy expenditure as demonstrated by elevated skin temperature in the prevention study and improved mitochondrial function and ATP synthesis in the treatment protocol. L. laricina is thus a promising alternative and complementary therapeutic approach for the treatment and care of obesity and diabetes among the Cree.