Animal Models Of Liver Disease Research Articles

Impact of new-generation parenteral lipid emulsions in pediatric nutrition.

Advancements in the care of premature infants and infants with severe bowel disease have occurred in which long-term use of i.v. nutrition is a cornerstone of successful therapy. Concern about the role of i.v. lipid emulsions in causing severe liver damage to high-risk infants receiving long-term i.v. nutrition has led to a variety of intervention strategies. These have had relatively limited success until the recent introduction of omega-3 (n-3) fatty acid-containing forms of lipid emulsions in place of the current omega-6 fatty acid-predominant lipid emulsions currently exclusively used in the United States. Preliminary data based on nonrandomized trials performed using compassionate-use protocols in the United States suggest very high rates of resolution of cholestasis with the use of an omega-3 fatty acid-predominant lipid emulsion. This result is supported by animal models of liver disease that demonstrate decreased liver damage when animals are provided omega-3 fatty acid-containing lipid emulsions compared with those primarily omega-6 fatty acid based. However, human trials are limited at this time and further research is needed to establish the best approach to preventing liver damage in infants receiving i.v. nutrition and the optimal dose and timing of intervention with novel lipid emulsions.

Deriving Hepatocyte-like Cells from Placental Cells for Transplantation

Human hepatocyte transplantation is being trialled in lieu of orthotopic liver transplants for patients with acute and chronic liver diseases. Stem cells that can be differentiated into hepatocyte-like cells may replace human hepatocytes that are difficult to source, culture and in critically short supply. Hepatocyte-like cells have been derived from embryonic and adult tissue stem cells using a combination of growth factors and chemical inducers. Stem cells derived from the human placenta have gained interest due to the unlimited supply of placental tissue, minimal issues associated with stem cell retrieval from placental tissue and the large yields of stem cells that can be obtained. Placental stem cells have been characterised and differentiated into hepatocyte-like cells. This review summarises the literature relating to the differentiation of human placental stem cells into hepatocyte-like cells, the characterisation of the differentiated cells, testing the functionality of the hepatocyte-like cells in pre-clinical animal models of liver disease and biomaterials used for culturing and transplantation of these cells into extra-hepatic sites.

Model systems and clinical applications of hepatic stem cells for liver regeneration.

Hepatic stem cells are capable to self-renew and to differentiate into all types of cells in the liver, including hepatic progenitors, mature hepatocytes and cholangiocytes. This review summarizes the recent advances in animal studies and the results of clinical trials over the last few decades. Studies in numerous animal models of liver diseases have demonstrated the efficacy of hepatic stem cells. Clinical studies of hepatic stem cells are also promising for a variety of hepatic diseases. With the accumulation of innovative and creative works, hepatic stem cells therapy will be a new approach for liver diseases.

Dimethylnitrosamine-Induced Liver Fibrosis and Recovery in NOD/SCID Mice

There is a need for a new liver fibrosis model of immunodeficient mice to study the effects of cell therapy on liver disease because there are not many animal models available to study the effects of cell therapy. In this study, we induced liver fibrosis using dimethylnitrosamine (DMN) in NOD/SCID mice to create an animal model for liver disease. DMN (5 mg/kg, i.p.) was injected intraperitoneally for three consecutive days per week for 6 or 8 weeks, and the mice were sacrificed at weeks 0, 4 and 8 after the last DMN injection. The 6-week DMN-treated group gradually recovered from serum biochemical changes, histopathological toxic effects and lesions in the liver at weeks 4 and 8 after the last DMN injection. However, the progression of liver fibrosis and toxic levels were maintained in the 8-week DMN-treated group at week 4 after the last DMN injection. The increases in iron and extracellular matrix (collagen) in the DMN-treated group were confirmed by Prussian blue (PB) and Masson's trichrome (MT) staining, respectively. Additionally, activation of hepatic stellate cells was observed by alpha smooth muscle actin (α-SMA) immunostaining and western blot. In conclusion, treatment of NOD/SCID mice with 5 mg/kg of DMN for 8 weeks can be used to induce an appropriate animal model of disease for liver fibrosis. This model may be useful for evaluation of the efficacy and safety of cell therapies such as human mesenchymal stem cell therapy.

Current Status of Stem Cell Therapy for Liver Diseases

Liver failure is one of the main causes of death worldwide and is a growing health problem. Since the discovery of stem cell populations capable of differentiating into specialized cell types, including hepatocytes, the possibility of their utilization in the regeneration of the damaged liver has been a focus of intense investigation. A variety of cell types were tested both in vitro and in vivo, but the definition of a more suitable cell preparation for therapeutic use in each type of liver lesions is yet to be determined. Here we review the protocols described for differentiation of stem cells into hepatocytes, the results of cell therapy in animal models of liver diseases, as well as the available data of the clinical trials in patients with advanced chronic liver disease.

Study of copper metabolism and liver damage in TX Mice-an animal model for liver disease

To provide right time points in selection of right aged animals and the normal physiological data of TX mice. 7-12 months old TX and DL mice were studied, each group contained 3 female and 3 male mice of TX or DL mice. The concentration of copper in the serum, dry tissues (liver, brain and kidney), together with copper biochemistry indexes were measured. The liver histopathology was observed under light microscopy and electron microscope. Transaminase increased significantly only in 10 and 11-month- old (AST(TX10) = 218.3 U/L, AST(TX11) = 197.5 U/L, AST(DL10) = 171.5 U/L, AST(DL11) = 165.0 U/L, P(10) less than 0.001, P(11) = 0.022), but the copper concentration of liver, brain and kidney was significantly increased during 7-12 month old (the average concentration of copper, Liver(TX) = (750.0 +/- 85.5) mg/kg, Brain(TX) = (39.7 +/- 2.2)mg/kg, Kidney(TX) = (29.8 +/- 5.0) mg/kg, Liver(DL) = (11.6 +/- 1.5) mg/kg, Brain(DL) = (16.8 +/- 0.9) mg/kg, Kidney(DL) = (14.2 +/- 1.0) mg/kg, t = 21.16, 23.60, 7.47, for all these organs P less than 0.05). TX mice is a suitable model of liver disease with natural recovery, so selecting animal model of suitable time point is very important.

Coordinate regulation of metabolic enzymes and transporters by nuclear transcription factors in human liver disease

It has been hypothesised, mainly from studies with animal models of liver disease, that the transport of substrates for metabolic enzymes and their subsequent metabolism and elimination in hepatic bile or blood is co-ordinated, but there is little information on this process in diseased human liver. In this study we have measured by reverse transcription polymerase chain reaction (RT-PCR) major genes involved in drug metabolism from UDP-glucuronosyltransferases (UGT1A1, UGT1A6, UGT1A9, and UGT2B4) and cytochrome P450 (CYP) families (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4), transport (OATP-C, MRP2, MRP3, and MDR1) and major transcription factors (PXR, CAR, HNF1alpha, HNF4alpha, RXR, and AHR) involved in their regulation. Liver biopsy tissue from patients with viral hepatitis was scored for inflammation and fibrosis by the METAVIR system, and separated into groups with mild (A0-1; F0-1, n = 20) or severe (A2-3; F3-4, n = 19) liver disease. Correlation analysis (Spearman rank-test, P < 0.05) was used to identify metabolic enzymes and transporters which shared significant correlation with transcription factors. Our results show an extensive correlation between transcription factors, transporters, and metabolic enzymes. An unexpected finding was that this was substantially greater in the severely diseased liver. Cross-talk between transcription factors was markedly increased in tissue from patients with severe liver disease, particularly between CAR, HNF4alpha, and PXR. Our results support the hypothesis of co-ordinate regulation of metabolic enzymes and transporters in diseased human liver, as part of a widespread co-ordinated process under the control of nuclear receptor transcription factors.

Stem cells in liver regeneration, fibrosis and cancer: the good, the bad and the ugly

The worldwide shortage of donor livers to transplant end stage liver disease patients has prompted the search for alternative cell therapies for intractable liver diseases, such as acute liver failure, cirrhosis and hepatocellular carcinoma (HCC). Under normal circumstances the liver undergoes a low rate of hepatocyte 'wear and tear' renewal, but can mount a brisk regenerative response to the acute loss of two-thirds or more of the parenchymal mass. A body of evidence favours placement of a stem cell niche in the periportal regions, although the identity of such stem cells in rodents and man is far from clear. In animal models of liver disease, adopting strategies to provide a selective advantage for transplanted hepatocytes has proved highly effective in repopulating recipient livers, but the poor success of today's hepatocyte transplants can be attributed to the lack of a clinically applicable procedure to force a similar repopulation of the human liver. The activation of bipotential hepatic progenitor cells (HPCs) is clearly vital for survival in many cases of acute liver failure, and the signals that promote such reactions are being elucidated. Bone marrow cells (BMCs) make, at best, a trivial contribution to hepatocyte replacement after damage, but other BMCs contribute to the hepatic collagen-producing cell population, resulting in fibrotic disease; paradoxically, BMC transplantation may help alleviate established fibrotic disease. HCC may have its origins in either hepatocytes or HPCs, and HCCs, like other solid tumours appear to be sustained by a minority population of cancer stem cells.

Methionine Metabolism and Liver Disease

In the early 1930s, Banting and Best, the discoverers of insulin, found that choline could prevent the development of fatty liver disease (steatosis) in pancreatectomized dogs treated with insulin. Later work indicated that in rats and mice, diets deficient in labile methyl groups (choline, methionine, betaine, folate) produced fatty liver and that long-term administration of diets deficient in choline and methionine also caused hepatocellular carcinoma. These experiments not only linked steatosis and diabetes but also provided evidence, for the first time, of the importance of labile methyl group balance to maintain normal liver function. This conclusion is now amply supported by the observation of mice devoid of key enzymes of methionine and folate metabolism and in patients with severe deficiencies in these enzymes. Moreover, treatments with various methionine metabolites in experimental animal models of liver disease show hepatoprotective properties.

IFATS Collection: In Vivo Therapeutic Potential of Human Adipose Tissue Mesenchymal Stem Cells After Transplantation into Mice with Liver Injury

Mesenchymal stem cells (MSCs), largely present in the adult human body, represent an attractive tool for the establishment of a stem cell-based therapy for liver diseases. Recently, the therapeutic potential and immunomodulatory activity of MSCs have been revealed. Adipose tissue-derived mesenchymal stem cells (AT-MSCs), so-called adipose-derived stem cells or adipose stromal cells, because of their high accessibility with minimal invasiveness, are especially attractive in the context of future clinical applications. The goal of the present study was to evaluate the therapeutic potential of AT-MSCs by their transplantation into nude mice with CCl(4)-caused liver injury. We observed that after transplantation, AT-MSCs can improve liver functions, which we verified by changes in the levels of biochemical parameters. Ammonia, uric acid, glutamic-pyruvic transaminase, and glutamic-oxaloacetic transaminase concentrations returned to a nearly normal level after AT-MSC transplantation. These results raised the question of how AT-MSCs can achieve this. To discover the possible mechanisms involved in this therapeutic ability of AT-MSCs, in vitro production of cytokines and growth factors was analyzed and compared with MSCs from bone marrow (BM-MSCs) and normal human dermal fibroblasts (NHDFs). As a result we observed that AT-MSCs secrete interleukin 1 receptor alpha (IL-1Ralpha), IL-6, IL-8, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemotactic protein 1, nerve growth factor, and hepatocyte growth factor in a volume higher than both BM-MSCs and NHDFs. Thus, our findings suggest that AT-MSCs may account for their broad therapeutic efficacy in animal models of liver diseases and in the clinical settings for liver disease treatment. Disclosure of potential conflicts of interest is found at the end of this article.

Application of liver stem cells for cell therapy

The worldwide shortage of donor livers to transplant end stage liver disease patients has prompted the search for alternative cell therapies for intractable liver disease. Embryonic stem cells can be readily differentiated into hepatocytes, and their transplantation into animals has improved liver function in the absence of teratoma formation: their use in bioartificial liver support is an obvious application. In animal models of liver disease, adopting strategies to provide a selective advantage for transplanted foetal or adult hepatocytes have proved highly effective in repopulating recipient livers, but the poor success of today's hepatocyte transplants can be attributed to the lack of a clinically applicable procedure to force a similar repopulation of the human liver. The activation of bipotential hepatic progenitor cells is clearly vital for survival in many cases of acute liver failure, but surprisingly little progress has been made with these cells in terms of transplantation. Finally there is the controversial subject of autologous bone marrow, and while the contribution of these indigenous cells to liver turnover seems at best, trivial, results from a small number of phase 1 studies of transplantation of bone marrow to cirrhotic patients have been moderately encouraging.

In vivo confocal laser laparoscopy allows real time subsurface microscopy in animal models of liver disease

Histopathology is essential in the diagnostic workup of most liver diseases. However, biopsy sampling might carry risks, is subject to sampling error, and does not provide dynamic tissue imaging. Therefore a newly developed miniaturised confocal probe was evaluated for in vivo microscopic imaging in rodent models of human liver diseases. The handheld laparoscopy probe used a 488nm single line laser for fluorophore excitation. Optical slice thickness was 7 microm, lateral resolution 0.7 microm. Imaging depth was 0-250 microm below the tissue surface. Imaging using different fluorescent staining protocols was performed in healthy mice, IFN-gamma- and IL-12-induced hepatitis, after bile duct ligation, and in ob/ob mice. Confocal imaging permitted microscopic imaging of the liver in vivo at high resolution. Landmarks of liver diseases such as inflammatory infiltration, steatosis, bile duct proliferation, pericellular fibrosis and perfusion anomalies could be characterised, localised and differentiated in vivo. In vivo confocal microscopy with this newly developed probe allows real-time subsurface imaging and characterisation of normal and pathologic liver tissue at high resolution. Since this technology is suited for laparoscopy in humans, it has the potential to impact on the diagnostic workup of liver diseases and to permit dynamic monitoring of (patho-)physiologic events in vivo.

Activation of soluble guanylate cyclase by nitric oxide in lymphocytes correlates with minimal hepatic encephalopathy in cirrhotic patients

Patients with liver cirrhosis with normal neurological and mental status examination may present minimal forms of hepatic encephalopathy, showing intellectual function impairment that cannot be detected through general clinical examination but can be unveiled using specific neuropsychological or neurophysiological examination. Evaluation of minimal hepatic encephalopathy (MHE) in cirrhotic patients would have prognostic value. The psychometric hepatic encephalopathy score (PHES) has been recommended as the "gold standard" in the diagnosis of MHE. Altered modulation of cyclic GMP (cGMP) levels in the brain seems to be responsible for the impairment of some types of cognitive function in liver disease. In animal models of liver disease, some of the alterations in modulation of cGMP levels in the brain are reproduced in lymphocytes. The aim of the present work was to assess whether there is a correlation between the alterations in different parameters involved in modulation of cGMP levels and the presence of MHE in patients with liver disease. We studied in 46 patients with liver cirrhosis and 26 controls the performance in the PHES battery of psychometric tests and the critical flicker frequency (CFF), the concentration of cGMP in plasma and lymphocytes, activation of guanylate cyclase by nitric oxide (NO) in lymphocytes, and several parameters likely involved in altered cGMP homeostasis in liver disease such as ammonia, NO metabolites, and atrial natriuretic peptide (ANP). Activation of guanylate cyclase by NO in lymphocytes and cGMP in plasma were higher and CFF lower in patients with MHE than in patients without MHE. Ammonia, ANP, and metabolites of NO were higher in patients than in controls but were no different in patients with or without MHE. Alteration in activation of guanylate cyclase by NO in lymphocytes correlates with PHES performance, CFF, and ammonia levels. This suggests that altered modulation of guanylate cyclase by NO in lymphocytes would reflect a parallel alteration in the brain occurring in patients with MHE that would be involved in their cognitive impairment.

Isolation of multipotent progenitor cells from human fetal liver capable of differentiating into liver and mesenchymal lineages

Little is known about the differentiation capabilities of nonhematopoietic cells of the human fetal liver. We report the isolation and characterization of a human fetal liver multipotent progenitor cell (hFLMPC) population capable of differentiating into liver and mesenchymal cell lineages. Human fetal livers (74-108 days of gestation) were dissociated and maintained in culture. We treated the colonies with geneticin and mechanically isolated hFLMPCs, which were kept in an undifferentiated state by culturing on feeder layers. We derived daughter colonies by serial dilution, verifying monoclonality using the Humara assay. hFLMPCs, which have been maintained in culture for up to 100 population doublings, have a high self-renewal capability with a doubling time of 46 h. The immunophenotype is: CD34+, CD90+, c-kit+, EPCAM+, c-met+, SSEA-4+, CK18+, CK19+, albumin-, alpha-fetoprotein-, CD44h+, and vimentin+. Passage 1 (P1) and P10 cells have identical morphology, immunophenotype, telomere length, and differentiation capacity. Placed in appropriate media, hFLMPCs differentiate into hepatocytes and bile duct cells, as well as into fat, bone, cartilage, and endothelial cells. Our results suggest that hFLMPCs are mesenchymal-epithelial transitional cells, probably derived from mesendoderm. hFLMPCs survive and differentiate into functional hepatocytes in vivo when transplanted into animal models of liver disease. hFLMPCs are a valuable tool for the study of human liver development, liver injury, and hepatic repopulation.

The Effects of S-Adenosylmethionine on Clinical Pathology and Redox Potential in the Red Blood Cell, Liver, and Bile of Clinically Normal Cats

S-adenosylmethionine (SAMe), an important hepatic metabolite and glutathione (GSH) donor, has been studied mechanistically in vitro, in humans with clinical liver disease, and in experimental animal models of liver disease. Collective findings encourage its therapeutic use in necroinflammatory and cholestatic liver disorders. A chronic longitudinal study (pre- and posttreatment parameters compared) was undertaken with 15 clinically healthy cats given a stable 1,4-butanedisulfonate (SS isomer) SAMe salt (enteric coated tablets providing 180 mg SAMe), dosage 48 mg/kg PO q24h, on an empty stomach for 113 days. Routine physical and clinicopathologic assessments, red blood cell (RBC) osmotic fragility, liver function and histology, hepatic concentrations of reduced GSH (RGSH) and its oxidized disulfide form (GSSG), protein, glycogen, and deoxyribonucleic acid, GSH concentrations in RBCs, total bile acids in serum and bile, oxidative membrane products (TBARS) in RBCs and liver, and plasma SAMe concentrations were evaluated. SAMe administered PO significantly increased plasma SAMe concentrations, and peak concentrations usually occurred 2–4 hours after dosing. Chronic SAMe administration did not change peak or cumulative plasma SAMe concentrations and did cause overt signs of toxicity. A positive influence on RBC and hepatic redox status (RBC TBARS reduced 21.1% [P <.002], liver GSH increased 35% [P <.002], liver RGSH:GSSG ratio increased 69% [P <.03]) and improved RBC resilience to osmotic challenge (P <.03) were observed. Results prove that this SAMe PO product is enterically available and suggest that it imparts biologic effects that might be useful for attenuating systemic or hepatic oxidant challenge

The Effects of S-Adenosylmethionine on Clinical Pathology and Redox Potential in the Red Blood Cell, Liver, and Bile of Clinically Normal Cats

S-adenosylmethionine (SAMe), an important hepatic metabolite and glutathione (GSH) donor, has been studied mechanistically in vitro, in humans with clinical liver disease, and in experimental animal models of liver disease. Collective findings encourage its therapeutic use in necroinflammatory and cholestatic liver disorders. A chronic longitudinal study (pre- and posttreatment parameters compared) was undertaken with 15 clinically healthy cats given a stable 1,4-butanedisulfonate (S'S isomer) SAMe salt (enteric coated tablets providing 180 mg SAMe), dosage 48 mg/kg PO q24h, on an empty stomach for 113 days. Routine physical and clinicopathologic assessments, red blood cell (RBC) osmotic fragility, liver function and histology, hepatic concentrations of reduced GSH (RGSH) and its oxidized disulfide form (GSSG), protein, glycogen, and deoxyribonucleic acid, GSH concentrations in RBCs, total bile acids in serum and bile, oxidative membrane products (TBARS) in RBCs and liver, and plasma SAMe concentrations were evaluated. SAMe administered PO significantly increased plasma SAMe concentrations, and peak concentrations usually occurred 2-4 hours after dosing. Chronic SAMe administration did not change peak or cumulative plasma SAMe concentrations and did not [corrected] cause overt signs of toxicity. A positive influence on RBC and hepatic redox status (RBC TBARS reduced 21.1% [P < .002], liver GSH increased 35% [P < .002], liver RGSH: GSSG ratio increased 69% [P < .03]) and improved RBC resilience to osmotic challenge (P < .03) were observed. Results prove that this SAMe PO product is enterically available and suggest that it imparts biologic effects that might be useful for attenuating systemic or hepatic oxidant challenge.

Cardiac Changes in An Animal Model of Liver Disease Using Bile Duct Ligation

Cardiac Changes in An Animal Model of Liver Disease Using Bile Duct Ligation

HEPATOCYTE TRANSPLANTATION: Advancing Biology and Treating Children

Key advances over the past three decades have allowed the evolution of hepatocyte transplantation from its use as an experimental tool to study liver cell biology to the initial application as a potential treatment modality for patients with liver disease. Although little is known about the cellular and molecular mechanisms regulating the fate of transplanted cells, studies in animal models of liver disease clearly suggest that transplanted hepatocytes have the potential to repopulate diseased livers and correct metabolic defects. Based on these experiments, human hepatocytes have been used in the treatment of children and adults with metabolic disease and liver failure. In initial trials, the improved clinical course following hepatocyte transplantation points to a potential role of the technique as an adjunct to liver transplantation.

Evidence of a systemic phenomenon for oxidative stress in cholestatic liver disease

BACKGROUNDThere is considerable evidence indicating that the severity of hepatic damage in individuals with cholestatic liver disease is causally associated with the extent of intrahepatic oxidative stress. Increased levels or...

Effect of pentoxifylline in rat and swine models of hepatic fibrosis: role of fibroproliferation in its mechanism

Fibroproliferation was studied in two animal models of liver disease. Oral feeding of yellow phosphorus to pigs reproducibly results in fibrosis after 8 weeks of feeding, extensive fibrosis after 12 weeks and cirrhosis after 16 weeks of yellow phosphorus. Bile duct ligation was used to induce cirrhosis in the rat. Fibroproliferation was assessed as uptake of tritiated thymidine into fibroblasts which had been incubated with monocyte-conditioned medium obtained from monocytes of pigs treated with yellow phosphorus or bile duct-ligated rats and compared to the corresponding controls. Fibrosis was assessed by collagen content of liver sections obtained from the two animal models. The collagen content was determined by quantitation of Sirius red/Fast green-stained liver sections. In both animal models collagen content was significantly elevated at the conclusion of the treatment. Collagen content of liver sections of yellow phosphorus-treated animals were elevated (40 ± 2.7, n = 15) compared to mineral oil-treated controls (23 ± 1.2, n = 12) and collagen levels in the bile duct-ligated rat model liver sections were elevated (31.2 ± 1.6, n = 6) compared to sham-operated controls (21.6 ± 0.7, n = 6). The results of the fibroproliferation assay indicate that monocytes obtained from pigs treated with yellow phosphorus produce fibroproliferative factors during the development of fibrosis. This is in contrast to the bile duct-ligated rat model where no differences were observed in the production of fibroproliferative factors in the bile duct-ligated rats compared to sham operated controls suggesting that this may not be a key event in this model of fibrosis. Pentoxifylline treatment of the yellow phosphorus induced swine model of hepatic fibrosis has been associated with a marked improvement in fibrosis. In this study treatment of fibrotic pigs with pentoxifylline was associated with an improvement in liver function tests, a reduction of collagen content of liver sections, and reduction in fibroproliferation in pigs receiving yellow phosphorus treatment. Fibroproliferative factors were produced during the development of fibrosis in the swine model of fibrosis and their effect was blocked by pentoxifylline administered in vivo. This is in contrast to the bile duct-ligated rat model where pentoxifylline treatment was not associated with improvement in liver function tests or reduction of collagen content of liver sections and did not alter the fibroproliferative activity of monocyte-conditioned media. Taken together these results suggest that fibroproliferation and increased synthesis of collagen are key events in the yellow phosphorus-induced pig model of hepatic fibrosis and that the action of pentoxifylline in this animal model is likely to be related to its effects on fibroproliferation with a subsequent effect on collagen production. This is in contrast to the bile duct-ligated rat model where the results from this study suggest that fibroproliferation per se does not appear to be a key event and where pentoxifylline treatment does not alter fibroproliferative activity nor alter the course of fibrosis in this animal model.