Cirrhotic Model Research Articles

Alleviation of CCl4-induced cirrhosis in rats by tetramethylpyrazine is associated with downregulation of leptin and TGF-β1 pathway

This study was conducted to investigate the effect of tetramethylpyrazine (TMP) on CCl4-induced fibrosis in rats and the possible roles of leptin, TGF-β1, Smad3, and Smad7 in this process. Liver fibrosis in rats was induced by the subcutaneous injection of 60% CCl4 (0.3 mL /100 g body weight, biweekly ) for 12 weeks. Rats in TMP prevention and treatment groups were given TMP (10 mg /100 g body weight, daily) by gavage from days 1 and 31 after the start of CCl4 injection, respectively. The mRNA expression of leptin, OB-Rb, TGF-β1, and TGF-βRII in the liver were detected by RT-PCR, whereas Smad3 and Smad7 protein were determined by Western blot. The results showed that hepatic cirrhosis was obviously alleviated in both TMP prevention and treatment groups. The mRNA expression of leptin, OB-Rb, TGF-β1 and -βRII, and Smad3 protein were higher in the cirrhotic models. In TMP prevention and treatment groups, these markers of expression were higher, compared with that of the normal control, but were lower when compared with that of the cirrhotic model group. Smad7 protein expression was lower in the cirrhotic model group than in the normal control. Smad7 expression in TMP prevention and treatment groups was higher, compared with that in the cirrhotic model group. Liver collagen in the TMP prevention group was the lowest among all CCl4 injection groups. In conclusion, TMP can prevent and alleviate the development of liver fibrosis in rats. The possible mechanism could involve the downregulation of leptin, Ob-Rb, TGF-β1, TGF-βRII, and Samd3, and upregulation of Smad7.

Physiopathology of splanchnic vasodilation in portal hypertension

In liver cirrhosis, the circulatory hemodynamic alterations of portal hypertension significantly contribute to many of the clinical manifestations of the disease. In the physiopathology of this vascular alteration, mesenteric splanchnic vasodilation plays an essential role by initiating the hemodynamic process. Numerous studies performed in cirrhotic patients and animal models have shown that this splanchnic vasodilation is the result of an important increase in local and systemic vasodilators and the presence of a splanchnic vascular hyporesponsiveness to vasoconstrictors. Among the molecules and factors known to be potentially involved in this arterial vasodilation, nitric oxide seems to have a crucial role in the physiopathology of this vascular alteration. However, none of the wide variety of mediators can be described as solely responsible, since this phenomenon is multifactorial in origin. Moreover, angiogenesis and vascular remodeling processes also seem to play a role. Finally, the sympathetic nervous system is thought to be involved in the pathogenesis of the hyperdynamic circulation associated with portal hypertension, although the nature and extent of its role is not completely understood. In this review, we discuss the different mechanisms known to contribute to this complex phenomenon.

An intravital microscopic study of the hepatic microcirculation in cirrhotic mice models: relationship between fibrosis and angiogenesis

This intravital fluorescence microscopy (IVFM) study validates cirrhotic mice models and describes the different intrahepatic alterations and the role of angiogenesis in the liver during genesis of cirrhosis. Cirrhosis was induced by subcutaneous injection of carbon tetrachloride (CCl(4)) and by common bile duct ligation (CBDL) in mice. Diameters of sinusoids, portal venules (PV), central venules (CV) and shunts were measured at different time points by IVFM. Thereafter, liver samples were taken for sirius red, CD31, Ki67, vascular endothelial growth factor (VEGF) and alpha-smooth muscle actin (alpha-SMA) evaluation by immunohistochemistry (IHC). In parallel with fibrogenesis, hepatic microcirculation was markedly disturbed in CCl(4) and CBDL mice with a significant decrease in sinusoidal diameter compared to control mice. In CCl(4) mice, CV were enlarged, with marked sinusoidal-free spaces around CV. In contrast, PV were enlarged in CBDL mice and bile lakes were observed. In both mice models, intrahepatic shunts developed gradually after induction. During genesis of cirrhosis using CD31 IHC we observed a progressive increase in the number of blood vessels within the fibrotic septa area and a progressively increase in staining by Ki67, VEGF and alpha-SMA of endothelial cells, hepatocytes and hepatic stellate cells respectively. In vivo study of the hepatic microcirculation demonstrated a totally disturbed intrahepatic architecture, with narrowing of sinusoids in both cirrhotic mice models. The diameters of CV and PV increased and large shunts, bypassing the sinusoids, were seen after both CCl(4) and CBDL induction. Thus present study shows that there is angiogenesis in the liver during cirrhogenesis, and this is probably due partially to an increased production of VEGF.

Nitric oxide and vascular remodeling modulate hepatic arterial vascular resistance in the isolated perfused cirrhotic rat liver

Hepatic arterial resistance is modulated by the hepatic arterioles but the role of NO and vascular remodeling in hepatic arterial resistance in cirrhosis is unknown. Cirrhosis was induced by CCl(4) or BDL. Using a bivascular liver perfusion dose-responses curves to methoxamine were obtained from the hepatic artery in absence and presence of L-NMMA. Lumen-diameter, wall thickness and number of smooth muscle nuclei were quantitated in the arteries using image analysis. Hepatic arterial resistance and the response to methoxamine were lower in cirrhosis compared to controls (p< or = 0.04) and lower in BDL compared to CCl(4) (p< or = 0.01). L-NMMA increased the response to methoxamine in CCl(4) (p=0.002) and BDL (p=0.05) but corrected the response only in CCl(4) (p=n.s. vs. control). Wall thickness and the number of smooth muscle nuclei were significantly smaller in cirrhosis compared to controls (p<0.05) and the number of nuclei was also lower in BDL compared to CCl(4) (p=0.005). NO is the main modulator of hepatic arterial resistance in CCl(4) but not in BDL. Intrahepatic arterial remodeling is present in both cirrhotic models but is greater in BDL. This indicates a larger role of structural changes in the control of hepatic arterial resistance in BDL.

Reduction of fibrosis in a rat model of non‐alcoholic steatohepatitis cirrhosis by human HGF gene transfection using electroporation

To study the histological changes caused by transfection of the hepatocyte growth factor (HGF) gene using electroporation (EP) in a non-alcoholic steatohepatitis (NASH) cirrhotic liver model. NASH cirrhotic livers were prepared by administering a choline-deficient diet to 5-week-old male Wister rats for 12 weeks. Three groups of rats were used: rats in the G(+) group were transfected with the GFP gene using EP, rats in the H(+) group were transfected with the HGF gene using EP, and rats in the H(-) group were only injected with the HGF gene. Rats were sacrificed 2 days after gene transfection, and the Azan positive rate (APR) and Sudan positive rate (SPR) were calculated to evaluate fibrosis and fatty changes. The APR of the NASH cirrhotic livers was significantly higher than that in the normal livers. The APR did not decrease in the G(+) group and the H(-) group, but decreased significantly in the nonelectroporated as well as electroporated areas of the H(+) group. For SPR, there were no significant differences between the G(+), H(-), and H(+) groups. The improvement of fibrosis was not significant when a direct injection of the HGF gene was used alone, but it was enhanced by the concomitant use of EP. However, no efficacy was observed in fat components. These findings suggest that transfection of the HGF gene by EP may lead to an improvement of irreversible cirrhotic livers to reversible fatty livers.

Comparison of three research models of portal hypertension in mice: macroscopic, histological and portal pressure evaluation

The characterization of mice models of portal hypertension (PHT) is lacking in the literature. Therefore, the aim of the present study was to make a histological approach during development of PHT in two models of cirrhosis with PHT compared with one model of isolated PHT. The model of isolated PHT was developed by partial portal vein ligation (PPVL). Two portal hypertensive cirrhotic mice models were developed either by common bile duct ligation (CBDL) or administration of carbon tetrachloride (CCl(4)) subcutaneously (twice weekly, 1 ml/kg). These models represent, respectively, a secondary biliary cirrhosis and alcoholic cirrhosis. Mice were killed at several time points to evaluate liver changes by histological and ultrastructural methods. A correlation was made with portal pressure measurements. Histology revealed the absence of fibrosis or cirrhosis in PPVL mice. They developed an isolated portal hypertension. After CBDL induction, the mice developed the characteristics of cirrhosis after 6 weeks, with simultaneous increase in portal pressures. Fifty percent of the mice had ascites at that time point. Sixteen weeks after administration of CCl(4), a micronodular cirrhotic aspect of the liver was seen associated with signs of portal hypertension. This is the first descriptive study of three widely used animal models in mice, allowing the study of pathophysiological changes in cirrhosis and portal hypertension. The PPVL in mice leads to a model of isolated portal hypertension. Secondary biliary cirrhosis developed after 6 weeks of common bile duct ligation in 50% of the mice that developed ascites. Subcutaneous injection of CCl(4) for 16 weeks induces cirrhosis and portal hypertension, without ascites. Moreover, the present study is the first description of a cirrhotic model in mice developed by subcutaneous injections of CCl(4). Well-described mice models will facilitate use of knock-out or transgenic mice and lead to a better understanding of the underlying molecular pathways in the field of portal hypertension and cirrhosis.

Brain cholinergic impairment in liver failure

The cholinergic system is involved in specific behavioural responses and cognitive processes. Here, we examined potential alterations in the brain levels of key cholinergic enzymes in cirrhotic patients and animal models with liver failure. An increase (∼30%) in the activity of the acetylcholine-hydrolyzing enzyme, acetylcholinesterase (AChE) is observed in the brain cortex from patients deceased from hepatic coma, while the activity of the acetylcholine-synthesizing enzyme, choline acetyltransferase, remains unaffected. In agreement with the human data, AChE activity in brain cortical extracts of bile duct ligated (BDL) rats was increased (∼20%) compared to controls. A hyperammonemic diet did not result in any further increase of AChE levels in the BDL model, and no change was observed in hyperammonemic diet rats without liver disease. Portacaval shunted rats which display increased levels of cerebral ammonia did not show any brain cholinergic abnormalities, confirming that high ammonia levels do not play a role in brain AChE changes. A selective increase of tetrameric AChE, the major AChE species involved in hydrolysis of acetylcholine in the brain, was detected in both cirrhotic humans and BDL rats. Histological examination of BDL and non-ligated rat brains shows that the subcellular localization of both AChE and choline acetyltransferase, and thus the accessibility to their substrates, appears unaltered by the pathological condition. The BDL-induced increase in AChE activity was not parallelled by an increase in mRNA levels. Increased AChE in BDL cirrhotic rats leads to a pronounced decrease (∼50–60%) in the levels of acetylcholine. Finally, we demonstrate that the AChE inhibitor rivastigmine is able to improve memory deficits in BDL rats. One week treatment with rivastigmine (0.6 mg/kg; once a day, orally, for a week) resulted in a 25% of inhibition in the enzymatic activity of AChE with no change in protein composition, as assessed by sucrose density gradient fractionation and western blotting analysis. In conclusion, this study is the first direct evidence of a cholinergic imbalance in the brain as a consequence of liver failure and points to the possible role of the cholinergic system in the pathogenesis of hepatic encephalopathy.

Effects of Fuzheng Huayu Decoction on plasma proteome in cirrhosis: preliminary experimental study with rats

The aim of this paper is to study the effects of Fuzheng Huayu Decoction on the plasma proteome in cirrhotic rats. Twenty-six male Sprague-Dawley (SD) rats were randomly divided into three groups: cirrhotic model group (n = 10), treated with CCl4 (CCl4/olive oil: v/v = 1:1); Fuzheng Huayu Decoction intervention group (n = 10), treated with CCl4 + Fuzheng Huayu Decoction; and normal control group (n = 6), treated with olive oil only. After 8 weeks, blood samples were collected from the inferior vena cava to undergo bi-dimensional electrophoresis (2DE) and analysis by PDQuest 7.3 software. Differential protein spots were cut, enzyme hydrolysis was conducted, and peptide fragments extracted from the mixture underwent mass spectrometry (MS) with MALDI-TOF-TOF-MS. The liver fibrogenesis was assessed using a digital image analysis instrument of Masson’s trichrome stained sections. The fibrosis area of the Fuzheng Huayu Decoction was (8.9 ± 3.7)%, significantly smaller than that of the cirrhotic model group [(12.4 ± 4.7)%, P<0.05]. Ten markedly changed protein spots were identified by MALDI-TOF-TOF-MS. Eight of the 10 proteins, including plasma glutathione peroxidase, plasma glutathione peroxidase precursor, prealbumin, haptoglobin, apolipoprotein A-IV precursor, complement C4, inter-alpha-inhibitor H4 heavy chain, and serine/threonine-protein kinase microtubule-affinity regulating kinase 1 (MARK1) were expressed very lowly in the cirrhotic model group while they were expressed highly in the Fuzheng Huayu Decoction group. The expression of liver regeneration-related protein LRRG03 and vimentin increased in the cirrhotic model group, and reduced in the Fuzheng Huayu Decoction group. Some proteins related to oxidative stress, cell proliferation and transformation have changed in the plasma of cirrhosis induced by CCl4. Fuzheng Huayu Decoction promotes protein synthesis and plays an anti-fibrotic role by anti-oxidation and accommodation of cell proliferation and transformation.

FR-167653, a selective p38 MAPK inhibitor, exerts salutary effect on liver cirrhosis through downregulation of Runx2

Liver cirrhosis remains a difficult-to-treat disease with a substantial morbidity and mortality rate. There is an emerging body of data purporting a pivotal role of the activated p38 mitogen-activated protein kinase (MAPK) in the process of cirrhosis. Several anticirrhotic agents have been developed over the past few years, and most of them exert their effects by indirectly inhibiting the p38 pathway. Effect of a selective p38 inhibitor is yet to be reported. In this study, we evaluated the salutary effect of FR-167653 (FR), a selective p38 inhibitor, in a carbon tetrachloride (CCl4)-induced rat cirrhotic model. Twenty rats were assigned into four groups: Sham, olive oil only; Control, CCl4 in olive oil; FR50, FR 50 mg/kg/day and CCl4; and FR100, FR 100 mg/kg/day and CCl4. FR dose-dependently inhibited activation of p38 and had an ameliorating effect on cirrhosis formation. Significant dose-dependent reduction in α-smooth muscle actin immunostaining and hydroxyproline content of the liver was noticed in the FR-treated rats. Also densitometric analysis showed a significant reduction in azan-stained area in the FR-treated rats. These fibrotic changes were observed in the myofibroblasts including the hepatic stellate cells and portal fibroblasts. mRNA expression of runt-related protein 2 (Runx2), a profibrogenic transcription factor, was significantly low in FR-treated livers, indicating that Runx2 might be a key downstream regulator of the p38 pathway. A similar reduction in expression of Smad4 and tissue inhibitor of metalloproteinase-1 was noticed in the FR-treated rats. In conclusion, FR treatment exerted a significant beneficial effect in a CCl4-induced rat cirrhotic model. The ameliorating effect of FR could be partially attributable to an inhibition of the Smad4/p38/Runx2 axis in the cirrhotic liver.

Effects of Fuzheng Huayu Decoction on plasma proteome in cirrhosis: preliminary experimental study with rats

To study the effects of Fuzheng Huayu Decoction on plasma proteome in cirrhosis. Twenty-six male S-D rats were randomly divided into three groups, cirrhotic model group (n = 10), treated with CCl4 (CCl4/olive oil: v/v = 1:1), Fuzheng Huayu Decoction intervention group (n = 10), treated with CCl4 + Fuzheng Huayu Decoction, and normal control group (n = 6), treated with olive oil only. After 8 weeks, blood sample was collected from the vena cava inferior to undergo bi-dimensional electrophoresis (2DE) and analysis by PDQuest 7.3 software. Differential protein spots were cut, enzyme hydrolysis was conducted, and peptide fragments extracted from the mixture underwent mass spectrometry with MALDI-TOF-TOF-MS. The liver fibrogenesis was assessed by digital image analysis instrument of Masson's trichrome stained sections. The fibrosis area of the Fuzheng Huayu Decoction was 9% +/- 4%, significantly smaller than that of the cirrhotic model group (12% +/- 5%, P < 0.05). Ten markedly changed protein spots were identified by MALDI-TOF-TOF-MS. Eight of the 10 proteins, including plasma glutathione peroxidase, plasma glutathione peroxidase precursor, prealbumin, haptoglobin, apolipoprotein A-IV precursor, complement C4, inter-alpha-inhibitor H4 heavy chain, and serine/threonine-protein kinase MARK1 (microtubule- affinity regulating kinase 1) were expressed very lowly in the cirrhotic model group while were expressed highly in the Fuzheng Huayu Decoction group. The expression of liver regeneration-related protein LRRG03 and vimentin increased in the cirrhotic model group, and reduced in the Fuzheng Huayu Decoction group. Some proteins related to oxidative stress, cell proliferation and transformation have changed in the plasma of cirrhosis induced by CCl4. Fuzheng Huayu Decoction promotes protein synthesis and plays an anti-fibrotic role by antioxidation and accommodation of cell proliferation and transformation.

Retracted: Rapamycin Attenuates Liver Graft Injury in Cirrhotic Recipient—The Significance of Down-Regulation of Rho-ROCK-VEGF Pathway

To investigate whether rapamycin could attenuate hepatic I/R injury in a cirrhotic rat liver transplantation model, we applied a rat orthotopic liver transplantation model using 100% or 50% of liver grafts and cirrhotic recipients. Rapamycin was given (0.2 mg/kg, i.v.) at 30 min before graft harvesting in the donor and 24 h before operation, 30 min before total hepatectomy and immediately after reperfusion in the recipient. Rapamycin significantly improved small-for-size graft survival from 8.3% (1/12) to 66.7% (8/12) (p = 0.027). It also increased 7-day survival rates of whole grafts (58.3%[7/12] vs. 83.3%[10/12], p = 0.371). Activation of hepatic stellate cells was mainly found in small-for-size grafts during the first 7 days after liver transplantation. Rapamycin suppressed expression of smooth muscle actin, which is a marker of hepatic stellate cell activation, especially in small-for-size grafts. Intragraft protein expression and mRNA levels of vascular endothelial growth factor (VEGF) were down-regulated by rapamycin at 48 h both in whole and small-for-size grafts. Consistently, mRNA levels and protein expression of Rho and ROCK I were decreased by rapamycin during the 48 h after liver transplantation. In conclusion, rapamycin attenuated graft injury in a cirrhotic rat liver transplantation model by suppression of hepatic stellate cell activation, related to down-regulation of Rho-ROCK-VEGF pathway.

A stable model of cirrhotic portal hypertension in the rat: thioacetamide revisited

Cirrhotic animal models are vital to investigate complications of chronic liver disease. We chronologically characterized the effect of thioacetamide, administrated orally and adapted weekly to weight changes, focusing on the optimal moment to obtain all typical features of portal hypertension and cirrhosis. Male Wistar rats, 200-250 g, were intoxicated for 6, 12 or 18 weeks (n = 8 per group), respectively, and compared with age-matched controls (n = 4 per group). An in-situ perfusion model was used to evaluate intrahepatic resistance and endothelial function. Splanchnic blood flow and portosystemic shunting were assessed by a perivascular flow probe. Rats intoxicated for 6 or 12 weeks had no mortality and histologically showed hepatitis and advanced fibrosis, respectively. At 18 weeks, mortality was 16% (on a total of 56 animals) and only at that moment all animals showed homogenous macronodular cirrhosis with signs of high-grade hepatocellular dysplasia. Portal hypertension was present at 12 weeks (11 +/- 0.4 vs. 5.9 +/- 0.4 mmHg, P < 0.001), but was not associated with the hyperdynamic state until 18 weeks (12.1 +/- 0.8 vs. 5.6 +/- 0.5 mmHg, P < 0.001). At this latter time-point, we also observed increased intrahepatic resistance associated with endothelial dysfunction, hyperresponsiveness to vasoconstrictors, splanchnic hyperaemia and portosystemic shunting. These alterations were associated with increased systemic levels of nitrate/nitrite and thromboxane A(2). Thioacetamide, adapted to weekly weight changes, leads to a homogenous, reproducible model of cirrhosis in the rat in 18 weeks, which is associated with all the typical characteristics of portal hypertension, including endothelial dysfunction.

Impact of cirrhosis on the development of experimental hepatic metastases by B16F1 melanoma cells in C57BL/6 mice.

Metastases rarely occur in human livers with cirrhosis in clinical studies. We postulated that this phenomenon would also occur in experimental cirrhosis. Cirrhosis was established in C57BL/6 mice by carbon tetrachloride (CCl(4)) gastrogavage. B16F1 melanoma cells were injected into the mesenteric vein to induce hepatic metastases. Contrary to our postulate, there was greater than 4-fold increase in metastasis in animals with cirrhosis compared to controls. Intravital videomicroscopy showed that the hepatic sinusoids were narrower and more tumor cells were retained in the terminal portal vein (TPV) in cirrhotic livers. Immunohistochemistry demonstrated that the expression of vascular adhesion molecules was significantly increased in cirrhosis. Using confocal microscopy and the fluorescent nitric oxide (NO) probe 4,5-diaminofluorescein diacetate, a significantly lower level of NO release was detected in livers with cirrhosis both in basal conditions and after tumor cell arrest. Eight hours after mesenteric vein tumor cell injection, the percentage of apoptotic tumor cells in the sinusoids was 17% +/- 2% in livers with cirrhosis and 30% +/- 5% in normal livers. More mitotic and Ki-67 labeled tumor cells were seen in livers with cirrhosis. In conclusion, the changes in architecture and adhesion molecule expression in livers with cirrhosis may cause more tumor cells to arrest in the TPV. Lower levels of NO production may reduce apoptosis of B16F1 cells in livers with cirrhosis. As a result, these changes may promote the growth of metastasis in this cirrhotic model.

Thalidomide salvages lethal hepatic necroinflammation and accelerates recovery from cirrhosis in rats

The authors investigated the feasibility of thalidomide employed to treat liver fibrosis. A cirrhotic model was established using Sprague-Dawley rats fed thioacetamide. Thalidomide-treated group was given thalidomide (10mg/kg/day) intraperitoneally for 10 consecutive days. Mortality, histopathological changes, TNFalpha, TGFbeta1, TIMP-1 and TIMP-2 were determined. Expression of TNFalpha and TGFbeta1 mRNA of Kupffer's cells derived from the experimental rats were determined. The mortality rates of thalidomide-treated group and vehicle-treated group were 8 and 32%, respectively. The total Knodell score of thalidomide-treated rats was lower than those of vehicle-treated rats. Micro-nodular cirrhosis resolved grossly in thalidomide-treated rats on day 28; while vehicle-treated rats continued to display uneven liver surface on day 28. Expression of TNFalpha, TGFbeta1, TIMP-1, and TIMP-2 was decreased in thalidomide-treated rats compared to those treated with vehicles. Finally, the expression of TNFalpha and TGFbeta1 mRNA of Kupffer's cells derived from rats treated with thalidomide were much lower than those treated with vehicle. Thalidomide salvages lethal hepatic necroinflammation, accelerates recovery from cirrhosis in rats, and works by suppressing of TNFalpha and TGFbeta1 production of Kupffer's cells.

Branched-chain amino acids as a protein- and energy-source in liver cirrhosis

Protein-energy malnutrition (PEM) is a common manifestation in cirrhotic patients with reported incidences as high as 65–90%. PEM affects largely the patients’ quality of life and survival. Thus, diagnosis of and intervention for PEM is important in the clinical management of liver cirrhosis. Supplementation with branched-chain amino acids (BCAA) is indicated to improve protein malnutrition. As an intervention for energy malnutrition, frequent meal or late evening snack has been recently recommended. Plasma amino acid analysis characterizes the patients with liver cirrhosis to have decreased BCAA. Such reduction of BCAA is explained by enhanced consumption of BCAA for ammonia detoxication and for energy generation. Supplementation with BCAA raises in vitro the synthesis and secretion of albumin by cultured rat hepatocytes without affecting albumin mRNA expression. BCAA recover the impaired turnover kinetics of albumin both in rat cirrhotic model and in cirrhotic patients. Longer-term supplementation with BCAA raises plasma albumin, benefits quality of life issues, and finally improves survival in liver cirrhosis. Recent interests focused on the timing of administration of BCAA, since daytime BCAA are usually consumed by energy generation for physical exercise of skeletal muscles. Nocturnal BCAA seem to be more favorable as a source of protein synthesis by giving higher nitrogen balance. This minireview focuses on the basic and clinical aspects of BCAA as a pharmaco-nutritional source to control PEM in liver cirrhosis.

High expression of the CD14 gene and interleukin-1β gene in the liver and lungs of cirrhotic rats after partial hepatectomy

Background. A hepatic resection is invasive for cirrhotic patients because postoperative complications, such as hepatic disturbance sometimes resulting in hepatic failure and pulmonary disturbances, are frequent and serious. We investigated here the alteration of the CD14 and inflammatory cytokine genes expressed in the liver and lungs after partial hepatectomy (PH) of a cirrhotic rat model to help elucidate the pathophysiological change occurring during the postoperative course of hepatectomized cirrhotic patients. Materials and methods. Wistar rats were orally administrated carbon tetrachloride once a week for 14 weeks to induce liver cirrhosis. In comparison with cirrhotic and normal rats, we analyzed the expression of the CD14, tumor necrosis factor-α, and interleukin (IL)-1β genes in remnant liver and whole lung tissue during 48 h after 30% partial hepatectomy with Northern blottings and measured asparatate aminotransferase (AST) in serum for evaluation of postoperative hepatic injury. Gadolinium chloride (GdCl3; 7 mg/kg body weight) was intravenously injected 24 h before partial hepatectomy to suppress Kupffer cells (KC) activation. Results. The expression of the CD14 and IL-1β genes moderately increased at 6 h and peaked at 12 h in parallel with the time course of AST values after PH only in cirrhotic rats. GdCl 3 significantly inhibited the elevation of AST similar to the inhibition of the expression of the CD14 and IL-1β genes after PH. In addition, the expression of these genes showed marked enhancement in the lungs of the cirrhotic hepatectomy model. Conclusions. KC activation was responsible for hepatic injury after PH, and the CD14 system appeared to be an early trigger for KC activation followed by induction of inflammatory cytokine IL-1β synthesis leading to hepatic injury. Furthermore, the CD14 system was suggested to participate in respiratory disturbances after hepatectomy in cirrhosis.

Hepatic Cirrhosis Occurring in a Young Woodchuck (Marmota monax) Due to Vertical Transmission of Woodchuck Hepatitis Virus (WHV)

Histologic and clinicopathologic findings of a woodchuck (Marmota monax) vertically infected with woodchuck hepatitis virus (WHV) are presented. The liver exhibits marked cirrhotic changes, which is characteristic of the pre-transformation phase of WHV. At necropsy, the woodchuck exhibited ascites and the liver had a grossly nodular appearance. Microscopically, focal hepatocyte necrosis and inflammatory cells were observed in midzonal and periportal areas in the liver. In Macchiavellos stained sections, cytoplasmic inclusion bodies appeared reddish granular materials. We believe that this may represent a new suitable and cost-effective cirrhotic model for the disease processes associated with hepadnaviruses in a number of other species, most notably Hepatitis B virus infection in man.

Gastric emptying of solids in cirrhotic and peritoneal dialysis patients: influence of peritoneal volume load.

A delay in gastric emptying rate has been reported in peritoneal dialysis patients, often normalizing after evacuation of the dialysate. To evaluate the effect of the intraperitoneal volume, we compared this finding with a cirrhotic model in which gastric emptying was studied before and after a large-volume paracentesis. We used the 13C-octanoic acid breath test to measure gastric half-emptying time (T1/2) for solids in patients with alcoholic cirrhosis, non-diabetic peritoneal dialysis patients, and a control population (asymptomatic volunteers). Cirrhotic patients underwent the test on two consecutive mornings before and after an evacuating paracentesis. Peritoneal dialysis patients were studied twice on consecutive days: once with the dialysate present intra-abdominally ("full"), and once with an emptied abdomen ("empty"). Biochemical analysis was carried out on blood samples before the first test. All cirrhotics underwent a 13C-aminopyrine breath test to assess residual liver function. Gastric emptying in cirrhotics showed no difference before or after paracentesis (median T1/2 108.0 min v. 117.9 min), but it was delayed significantly versus normal in both tests. There was no correlation with biochemical parameters, Child-Pugh score, or 13C-aminopyrine breath test results. Gastric half-emptying times of "full" peritoneal dialysis patients (median T1/2 103.1 min) were significantly higher than those of "empty" peritoneal dialysis patients (median T1/2 68.9 min) and asymptomatic volunteers (median T1/2 60.1 min). "Empty" peritoneal dialysis patients showed no gastroparesis. In alcoholic cirrhotic patients with ascites, gastric emptying of solids is delayed, independently of the volume of ascites. In peritoneal dialysis patients, gastric emptying was delayed when "full" and normalized after drainage of the dialysate.

Expression patterns of cell cycle-related proteins in a rat cirrhotic model induced by CCl4 or thioacetamide.

To analyze the aberrant expression of cell cycle-related proteins and their biological significance in relation to cirrhosis, we compared the cirrhotic patterns induced by two different types of cirrhotic agents, CCl4 and thioacetamide (TAA) in rats. CCl4 or TAA treatment was given to rats for 8 or 30 weeks, respectively, and the livers were removed at 9, 20, and 30 weeks after the experiment began. The TAA-induced fibrotic pattern was different from the CCl4-induced one, in terms of the formation of fibrous connective tissue and the proliferation of bile ductule cells. Cholangiofibrosis and clear cell foci were also observed in TAA-treated rats at 30 weeks. Histological examination revealed severe cirrhotic changes at 9 weeks in CCl4-treated rats and at 30 weeks in TAA-treated rats. Immunoblotting for cyclin D1, E, A, B, and proliferating cell nuclear antigen (PCNA) and their counterpart protein kinases (CDK2, 4, and CDC2) showed significant overexpression in rats with severely cirrhotic livers. The p53 tumor suppressor protein increased dramatically in the CCl4-treated group, while it was not detected in the livers of TAA-treated rats. Upregulation of p21WAF1, a CDK inhibitory protein, was detected in TAA-treated rats, but not in CCl4-treated rats. Immunohistochemical data for cyclin D1, E, and PCNA were well correlated with immunoblotting data; these proteins were increased in hepatocytes surrounding the cirrhotic lesions, suggesting that hepatocyte regeneration is correlated with cell cycle-related protein expression in cirrhotic liver. In the TAA-treated rats, the expression of these proteins was increased both in hepatocytes and in ductule cells. Our data suggest that liver cirrhosis induced by CCl4 or TAA is associated with alterations in cell cycle-related proteins, and that the expression of these proteins is responsible for hepatocyte regeneration in the damaged liver and may be involved in liver carcinogenesis.

Effect of liver blood flow and function on hepatic indocyanine green clearance measured directly in a cirrhotic animal model.

Peripheral blood clearance of indocyanine green (ICG) has been used as a test of liver function but gives little information on biliary excretion. Hepatic ICG uptake and clearance can be measured directly by near-infrared spectroscopy (NIRS). Direct ICG measurement has not previously been correlated with liver blood flow and function in cirrhosis. Two groups of New Zealand white rabbits (n = 12) underwent laparotomy for liver exposure. Cirrhosis was induced by feeding animals (n = 6) with a high-cholesterol (2 per cent) diet for 16 weeks. Hepatic blood flow and microcirculation were measured. Hepatic ICG concentration was measured directly using NIRS probes on the liver surface. From the ICG concentration-time curve, hepatic ICG uptake and excretion rates were calculated by a non-linear least square curve fitting method. There was a significant reduction in ICG uptake rate (mean(s.d.) 0.300(0.130) versus 2.040(0.420) min-1; P = 0.0001) and ICG excretion rate (0.007(0.009) versus 0.227(0.096) min-1; P = 0. 002) in cirrhotic animals. The hepatic ICG uptake rate correlated with hepatic blood flow and flow in the microcirculation (r = 0.81, P = 0.002; r = 0.92, P < 0.001, respectively). The hepatic ICG excretion rate was significantly associated with indicators of impaired liver function including bilirubin (r = - 0.86, P = 0.0004), aspartate aminotransferase (r = - 0.81, P = 0.001) and lactate dehydrogenase (r = - 0.83, P = 0.0008). ICG uptake measured directly by NIRS reflects the reduced liver blood flow and perfusion in cirrhosis and its excretion correlates with the degree of liver parenchymal dysfunction. This technique may allow a more accurate method of liver function assessment than peripheral blood ICG clearance.