Establishment of a liver fibrosis model in cynomolgus monkeys

Hepatic fibrosis, resulted from hepatoxicity and other diseases such as diabetes, is an important pathological characteristic of chronic liver diseases. Establishment of hepatic fibrosis animal models is of great importance and a prerequisite for human clinical studies. The common models for liver fibrosis are often established in lower small animals such as rats, but non-human primates are a much better model for human diseases because of the physiological similarity with humans. In this study, we investigated the method to induce liver fibrosis in cynomolgus monkeys using carbon tetrachloride (CCl₄) and to establish a model that more closely mimics human liver fibrosis. We successfully established the liver fibrosis model in 15 of the 20 cynomolgus monkeys (success rate 75%), by CCl₄ administration at a dose of 1.0 mL/kg (400 mL/L) twice a week. Liver biopsy showed that liver fibrosis progressed with time and gradually advanced into early-stage cirrhosis in 10 of the 15 established models at 16 weeks. Our study provides a better research platform for the prevention and treatment of chronic liver diseases.     

Development of cell therapy using autologous bone marrow cells for liver cirrhosis

The plasticity of bone marrow has been confirmed by the autopsy of a female recipient of bone marrow cell transplantation from a male donor. To establish new clinical cell therapies using autologous bone marrow cells for patients with liver failure, we developed a new in vivo model named the green fluorescent protein (GFP)/carbon tetrachloride (CCl₄) model. Using the GFP/CCl₄ model, we found that transplanted Liv8-negative cells efficiently repopulated into cirrhotic liver tissue and differentiated into albumin-producing hepatocytes under persistent liver damage induced by carbon tetrachloride. Moreover, bone marrow cell transplantation into mice with liver cirrhosis improved liver function and liver fibrosis with the strong expression of matrix metalloproteinases (MMPs), especially MMP-9 activity, resulting in an improved survival rate. Results from the GFP/CCl₄ model showed that cell therapy using autologous bone marrow cells has the potential to become an effective treatment for patients with liver failure. A summary of findings from the GFP/CCl₄ model is described.     

Hepatoprotection by malotilate against carbon tetrachloride-alcohol-induced liver fibrosis

Subchronic treatment of male rats with carbon tetrachloride (CCl₄, twice weekly 0.2 ml/kg p.o) and feeding a 5% alcohol solution instead of drinking water led to a nearly complete liver cirrhosis in all animals within 4 weeks. This was also documented by a three fold increase in hepatic total hydroxyproline content. Steatosis was quantified by enhanced liver triglyceride concentrations and acute necroses by increments of serum enzyme activities (GPT, SDH). Daily oral treatment with malotilate (100 mg/kg) totally prevented the development of liver cirrhosis, hepatic hydroxyproline accumulation and increases in serum enzyme activities induced by CCl₄-alcohol. In cianidanol-treated rats (100 mg/kg p.o.) only portoseptal fibrosis was seen, however hydroxyproline and triglyceride accumulation as well as enhanced serum enzyme activities were not suppressed. D-penicillamine (300 mg/kg p.o.) and colchicine (50 g/kg i.p.) failed to protect rats against CCl₄-alcohol induced fibrosis, necrosis and steatosis in this model.     

Growth-inhibitory activity of bone marrow cells during CCl4-induced liver cirrhosis

During CCl₄-induced liver cirrhosis, cells of the hemopoiesis-inducing microenvironment in the bone marrow of BALB/c mice produced activity inhibiting the growth of erythropoiesis and granulomonocytopoiesis precursors. Stimulation with yeast polysaccharide zymosan increased the inhibitory activity (especially in relation to granulomonocytic precursors). The highest growth-inhibitory activity was produced by the bone marrow adherent fraction (residual bone marrow macrophages). Tumor necrosis factor-α is probably responsible for the inhibition of the growth of myeloid precursors in mice with CCl₄-induced liver cirrhosis. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 6, pp. 645–647, June, 1999     

Assessing inflammatory liver injury in an acute CCl4 model using dynamic 3D metabolic imaging of hyperpolarized [1‐13C]pyruvate

To facilitate diagnosis and staging of liver disease, sensitive and non‐invasive methods for the measurement of liver metabolism are needed. This study used hyperpolarized ¹³C‐pyruvate to assess metabolic parameters in a CCl₄ model of liver damage in rats. Dynamic 3D ¹³C chemical shift imaging data from a volume covering kidney and liver were acquired from 8 control and 10 CCl₄‐treated rats. At 12 time points at 5 s temporal resolution, we quantified the signal intensities and established time courses for pyruvate, alanine, and lactate. These measurements were compared with standard liver histology and an alanine transaminase (ALT) enzyme assay using liver tissue from the same animals. All CCl₄‐treated but none of the control animals showed histological liver damage and elevated ALT enzyme levels. In agreement with these results, metabolic imaging revealed an increased alanine/pyruvate ratio in liver of CCl₄‐treated rats, which is indicative of elevated ALT activity. Similarly, lactate/pyruvate ratios were higher in CCl₄‐treated compared with control animals, demonstrating the presence of inflammation. No significant differences in metabolite ratios were observed in kidney or vasculature. Thus this work shows that metabolic imaging using ¹³C‐pyruvate can be a successful tool to non‐invasively assess liver damage in vivo. Copyright © 2015 John Wiley & Sons, Ltd.     

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 (CCl₄) 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 α‐smooth muscle actin (α‐SMA) evaluation by immunohistochemistry (IHC). In parallel with fibrogenesis, hepatic microcirculation was markedly disturbed in CCl₄ and CBDL mice with a significant decrease in sinusoidal diameter compared to control mice. In CCl₄ 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 α‐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 CCl₄ 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.     

Cytoprotective Activity in the Gastric Mucosa of Rats Exposed to Carbon Tetrachloride-Induced Liver Injury

The present study was undertaken to evaluate the cytoprotective activity in the gastric mucosa of rats subjected to CCl₄-induced liver injury. Response of gastric mucosa to absolute ethanol insult or acid (pylorus ligation) after CCl₄ challenge was analyzed. Intraperitoneal administration of CCl₄ increased plasma AST and ALT, but liver protein and glycogen levels were decreased; in addition, gastric acid secretion was significantly increased with respect to control animals (1541 ± 266 vs. 629 ± 25 eq H⁺; p < 0.001). Microscopical gastric erosions were observed in 3/10 animals after CCl₄ challenge. Pylorus-ligated as well as CCl₄-challenged rats developed increased susceptibility to gastric lesions, compared to control (lesion indices: 4.6 ± 0.20 vs 2.8 ± 0.13; p < 0.05), while showing increased resistance to absolute ethanol-induced gastric damage (30.4 ± 11.2 vs 89.7 ± 9.7 mm, p < 0.01). PGE₂ levels in the gastric mucosa were not influenced by exposure to CCl₄. Ultrastructural studies revealed the presence of continuous ethanol-resistant and apparently more adherent layer of mucus in CCl₄-challenged animals. Morphological evaluation revealed an increase in Alcian blue-stained mucus. A dual condition of enhanced sensitivity to HCl with increased tolerance to ethanol was observed in gastric mucosa of CCl₄-treated animals. These observations could be explained by the amount and/or composition of protective mucus layer in the gastric mucosa.     

N-acetylcysteine protects against liver injure induced by carbon tetrachloride via activation of the Nrf2/HO-1 pathway

Chronic liver injury is an important clinical problem which eventually leads to cirrhosis, hepatocellular carcinoma and end-stage liver failure. It is well known that cell damage induced by reactive oxygen species (ROS) is an important mechanism of hepatocyte injure. N-acetylcysteine (NAC), a precursor of glutathione (GSH), is well-known role as the antidote to acetaminophen toxicity in clinic. NAC is now being utilized more widely in the clinical setting for non-acetaminophen (APAP) related causes of liver injure. However, the mechanisms underlying its beneficial effects are poorly defined. Thus, Aim of the present study was to investigate potential hepatic protective role of NAC and to delineate its mechanism of action against carbon tetrachloride (CCl₄)-induced liver injury in models of rat. Our results showed that the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities as well as malondialdehyde (MDA) contents decreased significantly in CCl₄-induced rats with NAC treatment. GSH content and superoxide dismutase (SOD) activities remarkably increased in the NAC groups compared with those in CCl₄-induced group. Treatment with NAC had been shown to an increase in nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) mRNA levels. In conclusion, these results suggested that NAC upregulated HO-1 through the activation of Nrf2 pathway and protected rat against CCl₄-induced liver injure. The results of this study provided pharmacological evidence to support the clinical application of NAC.     

Hepatoprotective effect of Bathysa cuspidata in a murine model of severe toxic liver injury

The objective of this study was to investigate the hepatoprotective effect of a bark extract of Bathysa cuspidata extract (BCE) in a murine model of severe liver injury induced by carbon tetrachloride (CCl₄). Forty‐two Wistar rats were randomized into six groups of seven animals each: Group 1(G1): CCl₄; Group 2 (G2): dimethyl sulfoxide (DMSO) + CCl₄; Group 3 (G3): BCE 400 mg/kg alone; Group 4 (G4): BCE 200 mg/kg + CCl₄; Group 5 (G5): BCE 400 mg/kg + CCl₄; Group 6 (G6): DMSO alone. The extract was administered by gavage for 18 days beginning 6 days prior to the first application of CCl₄. After completing CCl₄ administration, the animals were euthanized. The animals in G1, G2, G4 and G5 experienced significant body weight loss and had an increased liver somatic index compared with G3 and G6 (P < 0.05). A significant reduction in serum aspartate and alanine transaminase and gamma‐glutamyl transferase (P < 0.05) and a significant increase in the activity of the anti‐oxidant enzyme superoxide dismutase were found in G5 (P < 0.05). Lower proportions of cellular necrosis and lipid droplets were found in the livers of animals in G4 and G5 compared with G1 and G2 (P < 0.05). These results confirm the marked hepatoprotective activity of the bark extract of Bathysa cuspidata in severe injuries induced by CCl₄ in rats and suggest that this effect may be associated with the inhibition of oxidative damage.     

Radon Inhalation Protects Mice from Carbon-Tetrachloride-Induced Hepatic and Renal Damage

We assessed whether radon inhalation provided protection from carbon tetrachloride (CCl₄)-induced hepatic and renal damage in mice. Mice were subjected to intraperitoneal injection of CCl₄ after inhaling approximately 18 kBq/m³ radon for 6 h. Radon inhalation significantly increased total glutathione (t-GSH) content and glutathione peroxidase (GPx) activity in the liver and kidney. Injection of CCl₄ was associated with significantly higher levels of glutamic oxaloacetic transaminase (GOT) and alkaline phosphatase (ALP) activity and creatinine level in serum, and pretreatment with radon significantly decreased the GOT and ALP activity and creatinine level associated with CCl₄ injection, suggesting that radon inhalation alleviates CCl₄-induced hepatic and renal damage. The t-GSH contents and GPx activity in the liver and kidney of animals pretreated with radon were significantly higher than those of the CCl₄-only group. These findings suggested that radon inhalation activated antioxidative functions and inhibited CCl₄-induced hepatic and renal damage in mice.     

Dose- and time-dependent effects of luteolin on carbon tetrachloride-induced hepatotoxicity in mice

Carbon tetrachloride (CCl₄) is a well-known model compound for producing chemical hepatic injury. This study investigated the protective effects of the flavonoid luteolin on the CCl₄-induced hepatotoxicity in mice. Luteolin dissolved in dimethyl sulfoxide (DMSO) was administered intraperitoneally (i.p.) at 5 or 50 mg/kg as a single dose, and once daily for 2 consecutive days. Two hours after the final treatment, the mice were treated with CCl₄ (20 mg/kg, i.p.). CCl₄-induced hepatotoxicity was reduced in a dose- and time-dependent manner, as determined by decreased serum aminotransferase activities and liver histopathology. CCl₄ intoxication resulted in an overexpression of heat shock protein gp96 in the mice liver, which was strongly attenuated by luteolin pretreatment. Luteolin has also decreased oxidative stress produced by CCl₄, as suggested by improvement in the Cu/Zn superoxide dismutase activity. The effect of luteolin on myeloperoxidase, an indicator of inflammatory cell infiltration, was also investigated. Treatment of the mice with luteolin resulted in a significant decrease in the myeloperoxidase activity. The hepatoprotective effect of luteolin against CCl₄ hepatotoxicity was higher in animals pretreated with luteolin for 2 consecutive days. This suggests that the protection might be due to induction of some adaptive mechanisms. The data indicate that luteolin could be effective in protecting mice from the hepatotoxicity produced by CCl₄.     

High-mobility group box 1 exacerbates CCl4-induced acute liver injury in mice

High-mobility group box 1 (HMGB1) is a nuclear factor that can also serve as an imflammatory mediator once released into extracellular milieu. Therefore, HMGB1 has been recognized to play a pivotal role in inflammatory diseases such as sepsis, acute lung injury, ischemia reperfusion injury and type 1 diabetes. Nevertheless, its impact on carbon tetrachloride (CCl₄)-induced hepatic injury is yet to be elucidated. In the present report, we demonstrated evidence indicating that high levels of HMGB1 were not only present in the necrotic area of liver but also in the serum after CCl₄ challenge. In line with these observations, administration of exogenous recombinant HMGB1 exacerbated CCl₄-induced hepatic injury, while HMGB1 blocking antibody provided protection for mice against CCl₄-induced acute liver injury as evidenced by the decrease of serum transaminase and reduction of hepatic tissues necrosis. Mechanistic studies revealed that blockade of HMGB1 attenuated CCl₄-induced MDA accumulation along with improved SOD and GSH activity. Treatment of mice with HMGB1 neutralizing antibody also significantly inhibited the production of proinflammatory mediators TNF-α and IL-6 along with attenuated HMGB1 expression and its extracellular release. Together, our data suggest an essential role for HMGB1 in CCl₄-induced acute liver injury, while HMGB1 neutralizing antibody could be served as an effective regimen for preventing CCl₄-induced acute liver injury.     

Functions of bone marrow hemopoiesis-inducing microenvironment during CCl4-induced liver cirrhosis

During late stages of CCl₄-induced liver cirrhosis, production of factors stimulating the growth of erythroid and granulomonocytopoietic precursors by bone marrow adherent and nonadherent fractions of BALB/c mice decreased. Stimulation with the yeast polysaccharide zymosan decreased production of these activities by various bone marrow cells, especially by the adherent fraction. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 128, No. 9, pp. 283–285, September, 1999     

Mechanism of chloramphenicol prevention of carbon tetrachloride-induced liver damage

Chloramphenicol (CAP), when given prior to CCl₄, prevented the CCl₄-induced liver necrosis observed at either 24 or 72 hr after its administration. CAP administration did not modify either body temperature of the CCl₄-treated rats or the CCl₄ levels in the liver at different times after CCl₄ administration. Prior treatment of the rats with CAP decreased the extent of irreversible binding of ¹⁴CCl₄ to liver microsomal lipids, reduced CCl₄-induced lipid peroxidation, decreased CCl₄-induced polysome breakdown, and attenuated the dilation and vesiculation of the endoplasmic reticulum induced by CCl₄. The results are analyzed in relation to the hypothesis that drug-metabolizing enzymes and activation of CCl₄ are related to its hepatotoxic effects.     

The new antioxidant 1-benzoyl-6-hydroxy-2,2,4-trimethyl-1,2-dihydroquinoline has a protective effect against carbon tetrachloride-induced hepatic injury in rats

Liver diseases with the central pathogenetic mechanism of oxidative stress are one of the main causes of mortality worldwide. Therefore, dihydroquinoline derivatives, which are precursors of hepatoprotectors and have antioxidant activity, are of interest. We have previously found that some compounds in this class have the ability to normalize redox homeostasis under experimental conditions. Here, we initially analyzed the hepatoprotective potential of the dihydroquinoline derivative 1-benzoyl-6-...     

Von Willebrand factor protects against acute CCl<Subscript>4</Subscript>-induced hepatotoxicity through phospho-p38 MAPK signaling pathway inhibition

The blood glycoprotein von Willebrand factor (vWF) is involved in coagulopathy and inflammation; however, its role in the pathogenesis of acute liver failure, as suggested by its higher expression levels in such patients, remains unknown. In this study, vWF-knockout (KO) mice showed more severe carbon tetrachloride (CCl₄)-induced liver injury than wild-type mice. Patients with acute liver injury also showed elevated vWF protein activity and expression in liver tissues, as compared to healthy individuals. Using the mouse model and cultured human umbilical vein endothelial cells (HUVECs), CCl₄ was found to directly increase vWF protein expression through interaction with the highly expressed vWF receptor, GPIbα. Microarray analysis revealed that the genes showing the most differential expression in response to CCl₄-induced liver injury and vWF deficiency were related to the MAPK signaling pathway. Subsequent inhibition of vWF protein activity in HUVECs led to activation of the MAPK signal pathway and elevated production of FGL2, and treatment with a phospho-p38 inhibitor suppressed the CCl₄-induced production of FGL2. Exposure of liver sinusoidal endothelial cells isolated from the vWF-KO acute liver injury model mice to phospho-p38 inhibitor also decreased FGL2 expression. The vWF/GPIbα axis plays a protective role against development of acute liver injury by attenuating FGL2 production through the MAPK signaling pathway. Collectively, these data provide insight into the pathogenesis of acute liver injury and a potential novel strategy for its treatment.     

Changes in mitotic activity of hepatocytes and resorption of necrotic areas during induction of cirrhosis of the liver

Cirrhosis of the liver was induced in rats weighing 120–140 g by administration of CCl₄ for 2 months. Iodinated oil was injected through the spleen of the experimental and intact animals to produce embolism of the branches of the portal vein and foci of necrosis in the liver tissue. The volume of the necrotic foci and the mitotic activity of the hepatocytes were determined. In cirrhosis of the liver the necrotic foci were resorbed more rapidly. The increase in the mitotic index on the second day after injection of the iodinated oil was greater in the control rats. The results suggest the appearance of a new cell clone in the liver which is responsible for resorption of the necrotic tissue and the reduction in mitotic activity of the hepatocytes in animals during development of cirrhosis of the liver.Department of Pathological Anatomy, I. P. Pavlov First Leningrad Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR N. A. Kraevskii.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 5, pp. 617–618, May, 1976.     

Effect of Unfiltered Coffee on Carbon Tetrachloride-Induced Liver Injury in Rats

To assess the role of unfiltered coffee upon carbon tetrachloride (CCl₄) induced hepatotoxicity in rats. All rats were randomly divided into control group, CCl₄-treated, unfiltered coffee-treated and CCl₄/unfiltered coffee-treated. Hepatic damage was induced by repeated intraperitoneal injections of CCl₄ every other day. Unfiltered coffee was given as drinking fluid for 8 days starting the day before CCl₄ administration. Liver enzymes, plasma and liver tissue malondialdehyde were analyzed. Histopathological evaluation of liver sections was performed. Serum aminotransferase level significantly increased in CCl₄/unfiltered coffee-treated group compared to CCl₄-treated group, as well as, lipid peroxidation products in the plasma and liver tissue. In addition, histopathological findings including inflammation and necrosis were significantly confirmed these findings. Unfiltered coffee potentiates acute liver injury in rats with CCl₄-induced hepatotoxicity.     

A comparison of the effects of partial surgical and partial chemical (CCl4) hepatectomy on microsomal cytochrome b5 and P450 and oxidative N-demethylation

Summary The effects of partial surgical hepatectomy and chemical (CCl₄) hepatectomy on microsomal cytochromes b₅ and P₄₅₀, and oxidative N-demethylation were measured. CCl₄-induced liver injury was associated with decreased levels of P₄₅₀ and both steps in oxidative N-demethylation of dimethylaniline (N-oxide accumulation and formaldehyde release). Cytochrome b₅ was rapidly restored to control levels. Surgical hepatectomy resulted in decreased cytochromes, decreased formaldehyde production, but increased N-oxide accumulation. These results were found in adrenalectomized animals, suggesting a direct effect of the hepatectomy.Supported in part by USPHS grants AM 08686, GM 13543 and American Cancer Society grant E480.Special fellow, USPHS 1-F3-CA-39, 395-01.