Experimental Models Of Liver Disease Research Articles

The 3Rs in Experimental Liver Disease.

Patients with cirrhosis present multiple physiological and immunological alterations that play a very important role in the development of clinically relevant secondary complications to the disease. Experimentation in animal models is essential to understand the pathogenesis of human diseases and, considering the high prevalence of liver disease worldwide, to understand the pathophysiology of disease progression and the molecular pathways involved, due to the complexity of the liver as an organ and its relationship with the rest of the organism. However, today there is a growing awareness about the sensitivity and suffering of animals, causing opposition to animal research among a minority in society and some scientists, but also about the attention to the welfare of laboratory animals since this has been built into regulations in most nations that conduct animal research. In 1959, Russell and Burch published the book "The Principles of Humane Experimental Technique", proposing that in those experiments where animals were necessary, everything possible should be done to try to replace them with non-sentient alternatives, to reduce to a minimum their number, and to refine experiments that are essential so that they caused the least amount of pain and distress. In this review, a comprehensive summary of the most widely used techniques to replace, reduce, and refine in experimental liver research is offered, to assess the advantages and weaknesses of available experimental liver disease models for researchers who are planning to perform animal studies in the near future.

Model of Acute Liver Failure in an Isolated Perfused Porcine Liver—Challenges and Lessons Learned

Acute liver failure (ALF) is a rare but devastating disease associated with substantial morbidity and a mortality rate of almost 45%. Medical treatments, apart from supportive care, are limited and liver transplantation may be the only rescue option. Large animal models, which most closely represent human disease, can be logistically and technically cumbersome, expensive and pose ethical challenges. The development of isolated organ perfusion technologies, originally intended for preservation before transplantation, offers a new platform for experimental models of liver disease, such as ALF. In this study, female domestic swine underwent hepatectomy, followed by perfusion of the isolated liver on a normothermic machine perfusion device. Five control livers were perfused for 24 h at 37 °C, while receiving supplemental oxygen and nutrition. Six livers received toxic doses of acetaminophen given over 12 h, titrated to methemoglobin levels. Perfusate was sampled every 4 h for measurement of biochemical markers of injury (e.g., aspartate aminotransferase [AST], alanine aminotransferase [ALT]). Liver biopsies were taken at the beginning, middle, and end of perfusion for histological assessment. Acetaminophen-treated livers received a median dose of 8.93 g (8.21–9.75 g) of acetaminophen, achieving a peak acetaminophen level of 3780 µmol/L (3189–3913 µmol/L). Peak values of ALT (76 vs. 105 U/L; p = 0.429) and AST (3576 vs. 4712 U/L; p = 0.429) were not significantly different between groups. However, by the end of perfusion, histology scores were significantly worse in the acetaminophen treated group (p = 0.016). All acetaminophen treated livers developed significant methemoglobinemia, with a peak methemoglobin level of 19.3%, compared to 2.0% for control livers (p = 0.004). The development of a model of ALF in the ex vivo setting was confounded by the development of toxic methemoglobinemia. Further attempts using alternative agents or dosing strategies may be warranted to explore this setting as a model of liver disease.

Role of Glycogen Synthase Kinase-3 in Interferon-γ-Mediated Immune Hepatitis.

Glycogen synthase kinase-3 (GSK-3), a serine/threonine kinase, is a vital glycogen synthase regulator controlling glycogen synthesis, glucose metabolism, and insulin signaling. GSK-3 is widely expressed in different types of cells, and its abundant roles in cellular bioregulation have been speculated. Abnormal GSK-3 activation and inactivation may affect its original bioactivity. Moreover, active and inactive GSK-3 can regulate several cytosolic factors and modulate their diverse cellular functional roles. Studies in experimental liver disease models have illustrated the possible pathological role of GSK-3 in facilitating acute hepatic injury. Pharmacologically targeting GSK-3 is therefore suggested as a therapeutic strategy for liver protection. Furthermore, while the signaling transduction of GSK-3 facilitates proinflammatory interferon (IFN)-γ in vitro and in vivo, the blockade of GSK-3 can be protective, as shown by an IFN-γ-induced immune hepatitis model. In this study, we explored the possible regulation of GSK-3 and the potential relevance of GSK-3 blockade in IFN-γ-mediated immune hepatitis.

Cerium Oxide Nanoparticles: A New Therapeutic Tool in Liver Diseases.

Oxidative stress induced by the overproduction of free radicals or reactive oxygen species (ROS) has been considered as a key pathogenic mechanism contributing to the initiation and progression of injury in liver diseases. Consequently, during the last few years antioxidant substances, such as superoxide dismutase (SOD), resveratrol, colchicine, eugenol, and vitamins E and C have received increasing interest as potential therapeutic agents in chronic liver diseases. These substances have demonstrated their efficacy in equilibrating hepatic ROS metabolism and thereby improving liver functionality. However, many of these agents have not successfully passed the scrutiny of clinical trials for the prevention and treatment of various diseases, mainly due to their unspecificity and consequent uncontrolled side effects, since a minimal level of ROS is needed for normal functioning. Recently, cerium oxide nanoparticles (CeO2NPs) have emerged as a new powerful antioxidant agent with therapeutic properties in experimental liver disease. CeO2NPs have been reported to act as a ROS and reactive nitrogen species (RNS) scavenger and to have multi-enzyme mimetic activity, including SOD activity (deprotionation of superoxide anion into oxygen and hydrogen peroxide), catalase activity (conversion of hydrogen peroxide into oxygen and water), and peroxidase activity (reducing hydrogen peroxide into hydroxyl radicals). Consequently, the beneficial effects of CeO2NPs treatment have been reported in many different medical fields other than hepatology, including neurology, ophthalmology, cardiology, and oncology. Unlike other antioxidants, CeO2NPs are only active at pathogenic levels of ROS, being inert and innocuous in healthy cells. In the current article, we review the potential of CeO2NPs in several experimental models of liver disease and their safety as a therapeutic agent in humans as well.

Non-invasive assessment of liver disease in rats using multiparametric magnetic resonance imaging: a feasibility study.

ABSTRACTNon-invasive quantitation of liver disease using multiparametric magnetic resonance imaging (MRI) could refine clinical care pathways, trial design and preclinical drug development. The aim of this study was to evaluate the use of multiparametric MRI in experimental models of liver disease. Liver injury was induced in rats using 4 or 12 weeks of carbon tetrachloride (CCl4) intoxication and 4 or 8 weeks on a methionine and choline deficient (MCD) diet. Liver MRI was performed using a 7.0 Tesla small animal scanner at baseline and specified timepoints after liver injury. Multiparametric liver MRI parameters [T1 mapping, T2* mapping and proton density fat fraction (PDFF)] were correlated with gold standard histopathological measures. Mean hepatic T1 increased significantly in rats treated with CCl4 for 12 weeks compared to controls [1122±78 ms versus 959±114 ms; d=162.7, 95% CI (11.92, 313.4), P=0.038] and correlated strongly with histological collagen content (rs=0.717, P=0.037). In MCD diet-treated rats, hepatic PDFF correlated strongly with histological fat content (rs=0.819, P<0.0001), steatosis grade (rs=0.850, P<0.0001) and steatohepatitis score (rs=0.818, P<0.0001). Although there was minimal histological iron, progressive fat accumulation in MCD diet-treated livers significantly shortened T2*. In preclinical models, quantitative MRI markers correlated with histopathological assessments, especially for fatty liver disease. Validation in longitudinal studies is required.This article has an associated First Person interview with the first author of the paper.

Deregulation of small intestine permeability by miRNA-21 in liver disease

The gut-liver axis influences a wide variety of liver diseases. High fat, choline-deficient (HFCD) diet and common bile duct ligation (BDL) are experimental models of liver disease associated with early increased small intestinal permeability (SIP), disturbed bile acid homeostasis and gut microbiota (GM) dysbiosis. Here we investigate the impact of whole body genetic deletion or therapeutic modulation of miR-21 in SIP as well as in the GM, which may influence miR-21 effects in the liver. We have previously demonstrated that miR -21 contributes to BDL-induced liver injury and fibrosis. Here we reveal that occludin and claudin-1 mRNA levels were decreased in small intestine of WT mice after BDL, suggesting increased SIP. Moreover, zonula occludens (ZO-1) and occludin mRNA levels were also decreased in HFCD-fed mice. Strikingly, both genetic deletion of miR-21 and Anti-miR-21 treatment reverted mRNA levels of tight junction proteins to control. Moreover, the GM of WT mice became dysbiotic, with decreased Bacteroidetes and increased Proteobacteria and Firmicutes. In miR-21 KO mice, the GM was not affected by BDL surgery. Our results suggest miR-21 involvement in liver injury by impacting on liver and intestinal targets.

The Receptor Interacting Protein Kinases in the Liver.

The receptor interacting serine/threonine kinase1 and 3 (RIPK1, RIPK3) are regulators of cell death and survival. RIPK1 kinase activity is required for necroptosis and apoptosis, while its scaffolding function is necessary for survival. Although both proteins can mediate apoptosis, RIPK1 and RIPK3 are most well-known for their role in the execution of necroptosis via the mixed lineage domain like pseudokinase. Necroptosis is a caspase-independent regulated cell death program which was first described in cultured cells with unknown physiologic relevance in the liver. Many recent reports have suggested that RIPK1 and/or RIPK3 participate in liver disease pathogenesis and cell death. Notably, both proteins have been shown to mediate inflammation independent of cell death. Whether necroptosis occurs in hepatocytes, and how it is executed in the presence of an intact caspase machinery is controversial. In spite of this controversy, it is evident that RIPK1 and RIPK3 participate in many experimental liver disease models. Therefore, in addition to cell death signaling, their necroptosis-independent role warrants further examination.

No significant impact of Foxf1 siRNA treatment in acute and chronic CCl4 liver injury.

Chronic liver injury of any etiology is the main trigger of fibrogenic responses and thought to be mediated by hepatic stellate cells. Herein, activating transcription factors like forkhead box f1 are described to stimulate pro-fibrogenic genes in hepatic stellate cells. By using a liver-specific siRNA delivery system (DBTC), we evaluated whether forkhead box f1 siRNA treatment exhibit beneficial effects in murine models of acute and chronic CCl4-induced liver injury. Systemic administration of DBTC-forkhead box f1 siRNA in mice was only sufficient to silence forkhead box f1 in acute CCl4 model, but was not able to attenuate liver injury as measured by liver enzymes and necrotic liver cell area. Therapeutic treatment of mice with DBTC-forkhead box f1 siRNA upon chronic CCl4 exposition failed to inhibit forkhead box f1 expression and hence lacked to diminish hepatic stellate cells activation or fibrosis development. As a conclusion, DBTC-forkhead box f1 siRNA reduced forkhead box f1 expression in a model of acute but not chronic toxic liver injury and showed no positive effects in either of these mice models. Impact statement As liver fibrosis is a worldwide health problem, antifibrotic therapeutic strategies are urgently needed. Therefore, further developments of new technologies including validation in different experimental models of liver disease are essential. Since activation of hepatic stellate cells is a key event upon liver injury, the activating transcription factor forkhead box f1 (Foxf1) represents a potential target gene. Previously, we evaluated Foxf1 silencing by a liver-specific siRNA delivery system (DBTC), exerting beneficial effects in cholestasis. The present study was designed to confirm the therapeutic potential of Foxf1 siRNA in models of acute and chronic CCl4-induced liver injury. DBTC-Foxf1 siRNA was only sufficient to silence Foxf1 in acute CCl4 model and did not ameliorate liver injury or fibrogenesis. This underlines the significance of the experimental model used. Each model displays specific characteristics in the pathogenic nature, time course and severity of fibrosis and the optimal time point for starting a therapy.

Brain magnetic resonance in experimental acute‐on‐chronic liver failure

Acute-on-chronic liver failure is the term that refers to sustained liver injury with acute decompensation, usually induced by a precipitating factor. A common link between ensuing failures of various organs is impairment of the vascular supply, which may also induce vasogenic oedema in the brain. The aim of this study was to perform magnetic resonance (MR) study of the brain in a rat model combining bile duct ligation (BDL) and lipopolysaccharide (LPS) administration to investigate brain oedema in liver failure. Bile duct-ligated rats underwent in vivo brain MR imaging at 4, 5 and 6 weeks, and after superimposed administration of LPS. The MR techniques applied enabled assessment of brain metabolites, and intra- or extracellular water distribution. Brain water content was assessed by gravimetry. MR spectroscopy showed an increase in brain glutamine and a decrease in myo-inositol and choline in relation to progression of liver disease. BDL rats showed a slight, progressive increase in the amount of cortical brain water that was significant after LPS injection. These changes did not modify the apparent diffusion coefficient, supporting a mixed origin of brain oedema (vasogenic and cytotoxic). The mechanisms leading to the development of brain oedema in an experimental liver disease model were related to the time course of liver failure and to pro-inflammatory stimuli. MR findings support the presence of cytotoxic and vasogenic mechanisms in induced brain oedema in BDL rats exposed to LPS.

Methionine metabolic pathway in alcoholic liver injury

To outline recent advances in the understanding of the consequences of the alterations in the methionine metabolic pathway and to present new treatment options for alcoholic liver disease (ALD). ALD is a major healthcare problem worldwide. Findings in many laboratories, including ours, have demonstrated that ethanol consumption impairs several of the multiple steps in methionine metabolism that ultimately impairs the activity of many methyltransferases critical for normal functioning of the liver. Recent studies buttress the important role genetics may play in the development and progression of alcoholic liver injury. Treatment modalities using two important metabolites of the pathway, S-adenosylmethionine and betaine, have been shown to attenuate ethanol-induced liver injury in a variety of experimental models of liver disease. S-adenosylmethionine has been used in several clinical studies; however, the outcomes have been unclear and its efficacy in liver diseases continues to be debated. To date, no clinical trials have been conducted for treatment of ALD with betaine. Future treatment modalities for ALD should consider loss-of-function polymorphisms in the enzymes of the methionine metabolic and related pathways. Further new treatment modalities for ALD should consider supplementation with betaine that may prove to be a promising therapeutic agent.

Peptide-Functionalized Gold Nanorods Increase Liver Injury in Hepatitis

Targeted nanomedicine holds enormous potential for advanced diagnostics and therapy. Although it is known that nanoparticles accumulate in liver in vivo, the impact of cell-targeting particles on the liver, especially in disease conditions, is largely obscure. We had previously demonstrated that peptide-conjugated nanoparticles differentially impact macrophage activation in vitro. We thus comprehensively studied the distribution of gold nanorods (AuNR) in mice in vivo and assessed their hepatotoxicity and impact on systemic and hepatic immune cells in healthy animals and experimental liver disease models. Gold nanorods were stabilized with either cetyltrimethylammonium bromide or poly(ethylene glycol) and additional bioactive tripeptides RGD or GLF. Gold nanorods mostly accumulated in liver upon systemic injection in mice, as evidenced by inductively coupled plasma mass spectrometry from different organs and by non-invasive microcomputerized tomography whole-body imaging. In liver, AuNR were only found in macrophages by seedless deposition and electron microscopy. In healthy animals, AuNR did not cause significant hepatotoxicity as evidenced by biochemical and histological analyses, even at high AuNR doses. However, flow cytometry and gene expression studies revealed that AuNR polarized hepatic macrophages, even at low doses, dependent on the respective peptide sequence, toward M1 or M2 activation. While peptide-modified AuNR did not influence liver scarring, termed fibrosis, in chronic hepatic injury models, AuNR-induced preactivation of hepatic macrophages significantly exacerbated liver damage and disease activity in experimental immune-mediated hepatitis in mice. Bioactively targeted gold nanoparticles are thus potentially harmful in clinically relevant settings of liver injury, as they can aggravate hepatitis severity.

Measuring Altered Disposition of Xenobiotics in Experimental Models of Liver Disease

Understanding the metabolic pathway and excretion mechanisms governing the disposition of a compound is essential to the safe use of pharmaceutical agents. Because the liver is the primary organ responsible for the metabolism and elimination of xenobiotics, chronic liver disease can have a significant effect on the disposition of many xenobiotics due to changes in the expression or function of drug metabolizing enzymes and transporters. Liver disease can result in increased retention of a xenobiotic within the body, causing greater exposure of the individual to a potentially harmful compound, which may lead to toxicity. On the other hand, liver disease may also up-regulate the elimination processes of a xenobiotic, accelerating its removal from the body. With regard to a pharmaceutical agent, enhanced elimination may result in a decreased pharmacologic effect. Such alterations may necessitate dosage adjustments to achieve the desired therapeutic outcome.

Unique pathway for anandamide synthesis and liver regeneration

The endocannabinoid system (ECS) includes cannabinoid (CB1 and CB2) receptors and their endogenous ligands (i.e., the endocannabinoids anandamide and 2-arachydonylglycerol) as well as proteins involved in endocannabinoids biosynthesis and degradation (1). The ECS is present in the liver and undergoes adaptive changes in response to noxious stimuli. Endocannabinoids as well as CB1 and CB2 receptors (which are, respectively, either faintly or not expressed in normal livers) are up-regulated in experimental liver injury and liver cirrhosis of various etiologies. In vivo, CB1 receptor activation promotes fat accumulation, triggers inflammation in nonalcoholic and alcoholic fatty liver diseases, contributes to the progression of chronic hepatitis to cirrhosis by stimulating fibrogenesis, and is also implicated in hemodynamic and neurological consequences associated with liver cirrhosis, including portal hypertension, encephalopathy, and cardiomyopathy (2–4). Conversely, activation of CB2 receptors exerts antifibrogenic and antiinflammatory effects in experimental models of liver disease (5). These pharmacological effects make CB2 agonists and CB1 antagonists promising candidates for the treatment of fibrosis in chronic liver pathologies. By disclosing a prominent CB1-mediated role of anandamide in the early phase of liver regeneration, Mukhopadhyay et al. (6) provide additional and significant support to the prominent role of the ECS in liver biology in an article in PNAS.

Therapeutic potential of stem cell in liver regeneration

Liver transplantation is the only life-saving procedure for patients with end-stage liver disease. However, its potential benefits are hampered by many disadvantages, such as the relative shortage of donors, operative risks, and high costs. These issues have prompted the search for new alternative therapies for irreversible liver disease. Stem cell therapy, with the ability for self-renewal and potential for multilineage differentiation, is a promising alternative approach. Several studies have demonstrated that transplantation of hepatic stem/progenitor cells or hepatocyte-like cells derived from multipotent stem cells leads to donor cell-mediated repopulation of the liver and improved survival rates in experimental models of liver disease. However, a registered clinical application based on stem cell technology will take at least an additional 5 to 10 years because of some limitations; e.g. the lack of suitable cell sources and risk of teratoma formation. This review summarizes the general understanding of the therapeutic potentials of stem cells in liver disease, including the sources, mechanisms, and delivery methods of hepatic stem cells in liver regeneration, and discusses some challenges for their therapeutic application.

Functional role of chemokines in liver disease models

Chemokines are a class of small cytokine-like molecules that orchestrate immune cell infiltration into the liver in response to acute and chronic injuries. Apart from their chemotactic effect, however, chemokines seem to mediate many other aspects of liver diseases, including a direct activation of stellate cells, the modulation of hepatocyte proliferation and angiogenesis. The identification of specific biological functions for chemokines in liver diseases has been hampered by the finding that resident and infiltrating cells in the liver are often a source, as well as a target, of chemokines. Furthermore, chemokines might cause differing effects depending on the etiology of liver damage, their local concentrations and their ability to form multimers and heterodimers. Nevertheless, the functions of a number of important chemokines and their associated receptors have been identified in both in vivo and in vitro studies. Indeed, harmful (proinflammatory, profibrogenic) and beneficial (antifibrogenic, antiangiogenic) effects of chemokines have been discovered in experimental liver disease models. In this Review, the current knowledge of chemokines in experimental liver disease models is summarized. Advances that might lead to preclinical applications are discussed, as are the roles of chemokine receptors as promising pharmacologically targetable molecules.

Alcoholic Liver Disease and Methionine Metabolism

Alcoholic liver disease is a major health care problem worldwide. Findings have demonstrated that ethanol feeding impairs several of the multiple steps in methionine metabolism that leads to progressive liver injury. Ethanol consumption has been reported to predominantly inhibit the activity of a vital cellular enzyme, methionine synthase, involved in remethylating homocysteine. By way of compensation in some species, ethanol can also increase the activity of the enzyme, betaine homocysteine methyltransferase. This enzyme catalyzes an alternate pathway in methionine metabolism and utilizes hepatic betaine to remethylate homocysteine to form methionine and maintain levels of S-adenosylmethionine, the key methylating agent. Under extended periods of ethanol feeding, however, this alternate pathway cannot be maintained. This results in a decrease in the hepatocyte level of S-adenosylmethionine and increases in two toxic metabolites, S-adenosylhomocysteine and homocysteine. These changes in the various metabolites of methionine metabolism, in turn, result in serious functional consequences. These include decreases in essential methylation reactions by inhibiting various methyltransferases critical to normal functioning of the liver and upregulation of the activation of endoplasmic reticulum-dependent apoptosis and lipid synthetic pathways. The ultimate outcome of these consequences is increased fat deposition, increased apoptosis, accumulation of damaged proteins, and alterations in various signaling pathways, all of which can ultimately result in progressive liver damage. Of all the therapeutic modalities that are presently being used to attenuate ethanol-induced liver injury, betaine has been shown to be the most effective in a variety of experimental models of liver disease. Betaine, by virtue of aiding in the remethylation of homocysteine, removes both toxic metabolites (homocysteine and S-adenosylhomocysteine), restores S-adenosylmethionine level, reverses steatosis, prevents apoptosis and reduces both damaged protein accumulation and oxidative stress. Thus, betaine is a promising therapeutic agent in relieving the methylation and other defects associated with alcoholic abuse.

Hepatic stem/progenitor cells and stem-cell transplantation for the treatment of liver disease

Allogeneic liver transplantation is still the only effective treatment available to patients with liver failure. However, because there is a serious shortage of liver donors, an alternative therapeutic approach is needed. Transplantation of mature hepatocytes has been evaluated in clinical trials, but the long-term efficacy remains unclear and the paucity of donor cells limits this strategy. Stem-cell transplantation is a more promising alternative approach. Several studies have provided information about the mechanism underlying the proliferation and differentiation of hepatic stem/progenitor cells. Moreover, in experimental models of liver disease, transplantation of hepatic stem/progenitor cells or hepatocyte-like cells derived from multipotent stem cells led to donor cell-mediated repopulation of the liver and improved survival rates. However, before stem-cell transplantation can be applied in the clinic to treat liver failure in humans, it will be necessary to overcome several difficulties associated with the technique.