key: cord-0697089-4jnniidz
authors: Gama, Jaciara Fernanda Gomes; Cardoso, Liana Monteiro da Fonseca; Lagrota-Candido, Jussara Machado; Alves, Luiz Anastacio
title: Animal models applied to acute-on-chronic liver failure: Are new models required to understand the human condition?
date: 2022-03-26
journal: World J Clin Cases
DOI: 10.12998/wjcc.v10.i9.2687
sha: c435893c1fe9306b446c7982a5012d71aa94075a
doc_id: 697089
cord_uid: 4jnniidz

The liver is a multifaceted organ; its location and detoxifying function expose this organ to countless injuries. Acute-on-chronic failure liver (ACLF) is a severe syndrome that affects the liver due to acute decompensation in patients with chronic liver disease. An infection environment, ascites, increased liver enzymes and prothrombin time, encephalopathy and fast-evolving multiorgan failure, leading to death, usually accompany this. The pathophysiology remains poorly understand. In this context, animal models become a very useful tool in this regard, as understanding; the disease may be helpful in developing novel therapeutic methodologies for ACLF. However, although animal models display several similarities to the human condition, they do not represent all ACLF manifestations, resulting in significant challenges. An initial liver cirrhosis framework followed by the induction of an acute decompensation by administering lipopolysaccharide and D-GaIN, potentiating liver damage supports the methodologies applied to induce experimental ACLF. The entire methodology has been described mostly for rats. Nevertheless, a quick PubMed database search indicates about 30 studies concerning ACFL models and over 1000 regarding acute liver failure models. These findings demonstrate the clear need to establish easily reproducible ACFL models to elucidate questions about this quickly established and often fatal syndrome.

The liver is a multifaceted organ that performs various functions, including protein and amino acid metabolism and plasma protein secretion, in addition to lipid oxidation and drug and xenobiotic blood detoxification [1] . Hepatic tissue is susceptible to countless injuries that may lead to liver cirrhosis. Most chronic liver lesions that culminate in cirrhosis processes are reversible, depending on their etiology, the amount of affected liver tissue and appropriate treatment, although this may lead to serious complication or be fatal in some patients [2] . Deaths caused by liver disease have increased by around 400% in the United Kingdom since the 1970s up to 2018 [3] . About 2 million people die each year due to liver disease complications, 50% of which are due to liver cirrhosis. This is mostly due to alcohol consumption and non-alcoholic fatty liver diseases in the western world and hepatitis B in China and Asian countries [4] . Acute-on-chronic liver failure (ACLF) was diagnosed in 30% of all cases among 1343 hospitalized patients presenting liver cirrhosis according to a European study carried out between February and September 2011, with a mortality rate of 32.9% at 28 d and 51.2% at 90 d. Patients with liver cirrhosis may experience acute decompensating that leads to ACLF, characterized by ascites, hepatic encephalopathy, gastrointestinal bleeding or a combination of these symptoms [5, 6] . The molecular, cellular, and immune mechanisms reported in patients with liver cirrhosis that develop ACLF, however, remain unclear. Therefore, effective therapeutic methodologies become a challenge, generating high costs, with liver transplants comprising the only effective treatment to date.

Animal models concerning hepatic disease have been very useful in preclinical research for decades, comprise an alternative in the understanding of ACLF pathophysiology, as they are reproducible, and are able to adequately mimic some ACLF events. However, many models do not faithfully reproduce human disease and require special management for understanding specific hypothesis. In this context, this review aims to provide an overview of the main animal models used in ACLF research, alongside their pros and cons. Furthermore, pathological events that do reproduce human ACLF are also discussed.

The vast majority of patients referred to specialist hepatological centers suffer from acute deterioration during chronic liver disease. Two important conditions are observed in patients with known chronic liver disease who exhibit acute decompensation, namely acutely decompensated cirrhosis and ACLF. The first is a widely accepted condition and refers to the development of ascites, encephalopathy, gastrointestinal hemorrhage, or any combination of these disorders in patients with cirrhosis [5, 7] . The second, ACLF, identifies patients with known or unknown chronic liver disease who develop rapid liver function deterioration and high short-term mortality after an acute insult. The definitions of ACLF differ, with most addressing the role of both hepatic and extra-hepatic precipitating events and including extra-hepatic organ failures [8, 9] . ACLF, a term suggested by Jalan and Williams in 2002[10] , emerged from studies indicating the development of a syndrome associated with a high risk of shortterm death (death < 28 d after hospital admission) in patients presenting acutely decompensated cirrhosis. Three major features characterize this syndrome, namely intense systemic inflammation, frequently displaying a close temporal relationship with pro-inflammatory precipitating events (e.g., infections or alcoholic hepatitis) associated with single-or multiple-organ failure. However, the first ACLF definition was only established by a consensus of the Asian Pacific Association for the study of the liver (APASL) in 2008. Unlike other definitions, this definition does not include extra-hepatic organ failures [7] . Thus, ACLF was defined as an acute hepatic insult in patients with chronic liver disease resulting in jaundice (total bilirubin ≥ 5 mg/dL) and coagulopathy (INR ≥ 1.5), complicated within four weeks by ascites and/or encephalopathy [7] . Thereafter, the North American Consortium for the Study of End-Stage Liver Disease associated ACLF with a 30-d mortality rate of 41% compared to 7% concerning acute decompensation without ACLF [11] . Meanwhile, the European Association Study Liver (EASL) defines ACLF as an acute decompensated liver insult in patients with cirrhosis or chronic liver disease that can often lead to sepsis due to bacterial infection, increasing the requirement for intensive care and resulting in a high 28-d mortality rate (≥ 15%) [12] .

The prevalence of ACLF ranges from 24% to 40% of hospitalized patients with liver cirrhosis. ACLF may develop in patients with previously compensated or decompensated cirrhosis, as well as in patients presenting the underlying chronic liver disease without cirrhosis [13] . Excessive alcohol consumption is also important in an ACLF context, as alcoholic hepatitis results in a high mortality rate and is one of the causes of acute-on-chronic liver injury [14] . Furthermore, over 50% of patients presenting alcoholic cirrhosis exhibit decompensated disease and, in the US, alcohol-associated liver diseases can lead to an indication for liver transplantation [15, 16] . In contrast, the reactivation of hepatitis B virus (HBV) is the leading cause of ACLF in the Asian region, with high prevalence where HBV-related acute-on-chronic liver failure (HBV-ACLF) accounts for over 70% of ACLF [17, 18] . However, early diagnosis and prevention measures through long-term HBV infection suppression with antiviral agents (such as lamivudine, tenofovir, entecavir or telbuvidine) or sustained eradication of hepatitis C virus (HCV) infection in patients with compensated or decompensated cirrhosis can decrease mortality and prevent the development of HBV-ACLF and HCV-related acute-on-chronic liver failure (HCV-ACLF) in this region [19, 20] . Patients with chronic hepatitis B or HBV-related cirrhosis are at risk of developing ACLF, with multi-organ failure and high short-term mortality [21] . HBV reactivation could be either a spontaneous setting of treatment cessation [22] or due to intensive chemotherapy or immunosuppressive therapy[23,24], treatment related [25] or reactivation of the occult HBV infection by rituximab (anti-CD20)-based chemotherapy [26] [27] [28] or immune restoration after highly active antiretroviral therapy for HIV [29, 30] . Similarly, HCV infection reactivation has also been reported, mainly following immune suppressive therapy [31, 32] . Drugs such as anti-tuberculosis drugs, methotrexate and antiretroviral drugs in HIV/AIDS-infected individuals have been implicated in triggering liver injury, particularly in the setting of underlying chronic liver disease due to HBV or HCV[31, [33] [34] [35] . Drugs are seen as a precipitating factor in ACLF, although databases on concerning drugs as an acute insult leading to ACLF are extremely scarce. This factor limits the study and knowledge of the effects of certain drugs and medications in ACLF development. This indicates the need for further data and assessments concerning models on hepatic injury caused by different herbal and medicinal preparations in cirrhosis patients [8] .

ACLF pathogenesis is still poorly understood, and will depend on the origin of the condition, the number of organ failures and the patient's past medical history. It can, however, be characterized as a systemic inflammatory syndrome, whose evolution and pathophysiology are directly associated with the involved immune mechanisms, such as those in response to danger associated molecular patterns and pathogen associated molecular patterns (DAMPs and PAMPs, respectively), cytokine production and the inflammatory profile [36] . Patients with hepatitis B (HB) diagnosed with ACLF exhibit an exacerbated innate and adaptive inflammatory response evidenced by increased ROS (reactive oxygen species) production by macrophages and neutrophils, increased TLR4 expression and high blood cytokine levels. The induction of cytokine storms has been associated with interleukin 33 (IL-33) upregulation, an important DAMP associated with disease severity [36, 37] . The source of this inflammatory response exacerbation, however, remains unknown. Another event consists in the formation of the NLRP3 inflammasome, as reported in a study on 70 HB patients presenting ACLF. This is composed of a NOD-like receptor family, pyrin domain containing 3 (NLRP3), an intracellular PAMP receptor responsible for activating Caspase-1 that, consequently, cleaves pro-inflammatory IL-1β and IL-18 cytokines[38]. Immune system cells involved in the inflammatory response play a key role in the ACLFcaused mechanism, as the neutrophil-leucocyte ratio is an indicator of ACLF death severity [39] . In addition, the large liver cell death that occurs in ACLF may be closely related to mitochondrial damage, mainly in organ and multi-organ failure. In three CANONICAL studies in cirrhotic patients with ACLF was observed changes in mitochondrial markers associated with ACLF follow up [40] [41] [42] . The increase of fingerprints in metabolomics assays demonstrated the fatty acylcarnitines (FA) was raised, suggesting impairment in β-mitochondrial oxidation that can cause decreasing of the FA-derived energy in peripheral organs, and then, cell death and organ failure [40] . In addition, immunometabolism was required with the increased of GDF15 and FGF21, as wells as the up-regulation of inflammatory cytokines (e.g. MIP-1 α/β, MCP-1, IL-1ra and IL-6) and ultra-structural changes with cristae rarefication in mitochondrial morphology of the peripheral blood mononuclear cells [41, 42] . These findings together indicate the impairment of mitochondrial function through the energy expenditure by systemic inflammation. Xue and coworkers demonstrated that mitofusin2 (Mtn-2) can regulated the autophagy and apoptosis levels, as well as decreasing reactive oxygen species (ROS) [43] .

Another important issue concerning the knowledge and studies of the events that lead to ACLF development in humans is directly related to the current pandemic scenario caused by the new coronavirus disease 2019 (COVID-19). The hallmark of ACLF is excessive systemic inflammation, and patients with ACLF exhibit higher levels of inflammatory markers and pro-inflammatory cytokines-IL-6, IL-1β, and IL-8. Systemic inflammation inducers can be exogenous or endogenous and viruses have been described previously as triggering inflammation [13] . A cytokine storm has been reported in patients with COVID-19, characterized by increased IL-2, IL-7, G-SCF and TNF-α [44] . Thus, it is believed that the excessive inflammatory response associated with COVID-19 can serve as a trigger for ACLF in patients with underlying chronic liver disease, which could justify the increase in liver disease patient deaths [45] . However, other mechanisms may also contribute to ACLF development in COVID-19 patients, such as hypoxic changes and iatrogenic causes such as drugs and ventilation, exacerbating underlying liver disease [46, 47] . A case study reported the development of ACLF precipitated by severe acute respiratory syndrome coronavirus-2 infection (SARS-CoV-2) in a patient with HBV-related cirrhosis without no previous anti-viral treatment. The authors suggest that the SARS-CoV-2 infection induced systemic inflammatory response syndrome, and the resultant immune dysregulation could have precipitated ACLF, in turn. Since the patient had not been on nucleoside analogs treatment for HBV prior to admission, it is possible that the ACLF was caused by HBV flare in a context of uncontrolled inflammation and dysbalance of innate and adaptive immune responses triggered by the SARS-CoV-2 infection [48] . This highlights the importance of the treatment in patients with HBV and other chronic liver disease in the current pandemic status worldwide. Nevertheless, long-term followup clinical studies are required to explore the potential relationship between ACLF development in COVID-19 patients [45] .

Despite these relevant facts concerning ACLF pathophysiology, the low number of patients assessed to date, its poor diagnosis and the use of different methodological analyses are still a challenge in understanding this condition. Focused research on strategies to prevent and treat this potentially reversible syndrome are, thus, paramount. Therefore, animal models comprise a helpful tool, as they are reproducible, easy to manage and are able of mimicking several ACLF mechanisms.

As described previously, acute and chronic liver diseases are frequent and potentially lethal conditions, displaying a high worldwide prevalence [49] . The development of new therapeutic strategies, drugs and the definition of effective potential biomarkers depends on understanding liver damage pathogenesis and progression, which can be investigated by making use of suitable animal models. Liver injury is highly complex and the absence of adequate animal models able to faithfully reproduce human liver disease characteristics limits the understanding of the mechanisms underlying this condition and treatment [50, 51] . Rodent animal models are preferred in preclinical liver research, mainly in ACLF assessments, due to easy to maintain and breed in captivity, the genetic similarity to human, similar liver morphology and simple management concerning the use of genetic manipulation tools [52] . In contrast, large animal models, like pigs, previously used mostly in acute liver failure modeling, now play an important role in the assessment of various acute and chronic liver diseases[51]. Non-human primate models like baboons (Papio hamadryas) are also widely applied due proximity of the human condition, for example, as alcohol-caused liver disease (ALD) models. They displaying significant similarity to the pathogenesis of this disease, i.e., inducing fatty liver and fibrosis, and high annexin-2 Levels, directly associated to the final stage of the disease in humans, as well as fibrinolysis and high risk of bleeding [53] . Furthermore, Rhesus monkeys also significantly reproduce the pathology, biochemistry markers and genetic expression of human ALD [54] . However, after a deep and thorough data review, we observed that no primate non-human models have been reported in the literature concerning ACLF research, and that rodent (rats and mice) models are the most assessed (Table 1) .

Animal models are useful in understanding the mechanisms surrounding immune responses and multiorgan failure in inflammatory and systemic syndromes [55] . Many studies have attempted to comprehend this inflammatory scenario [56] [57] [58] [59] [60] [61] [62] . With the aim of mimicking the human disease, techniques able to induce ACLF, resulting in a chronic liver insult and, finally, a precipitating event, have been developed. It is important to note that the main precipitating events comprise the reactivation of hepatitis viral or acute hepatitis A virus infection, acute alcoholic hepatitis or acute bacterial infection in Asian patients and alcoholism and bacterial infection that aggravate chronic cirrhosis conditions in the western world [20] . In this context, most ACLF animal models induce chronic cirrhosis and a posterior acute insult, usually through the administration of Lipopolysaccharide (LPS), LPS/D-Galactosamine hydrochloride (D-GaIN) or ethyl alcohol (EtOH), in order to reproduce events of bacterial infection (Figure 1) . However, few animal ACLF models have been established to date (described in the next section). 

A widely applied model used to induce chronic lesions consist in the use of carbon tetrachloride (CCl 4 ) as a hepatocellular damage inducer. This model leads to liver steatosis due to CCl 4 covalent binding to cell metabolites, resulting in Ca 2+ sequestration and K + /Na 2+ channel imbalances in an anaerobic environment and cytochrome P450 (CYP450) action [63] . Liver CCl 4 metabolism occurs through carbonyl chloride and free radicals formation (i.e., + CCl 3 ), the latter binding to protein and lipids in the cell cytosol, even though no acid nucleic binding occurs [64] . Thus, toxicity due to CCl 4 administrations is similar to alcohol-induced toxicity. Furthermore, fibrosis following significant liver destruction is also noted in this model, evidenced by increased hydroxiproline content and liver function failure, comprising increased alanine aminotransferase (ALT) and aspartate transaminase (AST) activities [63, 65] . Aiming to mimic ACLF events, CCl 4 in combination with the administration of D-GalN, with or without LPS, is the mostly used to induce chronic liver injury (Table 1) . LPS is a bacterial endotoxin capable of activating Kupffer cells and stimulating TNF-α and an immune response by the NF-κB pathway, while D-galactosamine is able to potentiate this response by depleting the uridine nucleotides and interfering in protein synthesis, leading to acute insults as a precipitating event [66] . The major challenge verified in this model consists in determine differences between CCl 4 and LPS/GaIN administration time and doses for ACLF induction. In one study, female Sprague-Dawley (SD) rats (180-200 g) were administered intraperitoneal (i.p.) CCl 4 dissolved in olive oil (10%) twice a week for ten weeks. After cirrhosis confirmation, the animals received 0.70 g/kg body weight (BW) D-GaIN i.p. [67] . ACLF was confirmed due to increased AST and ALT activities and higher bilirrubin and plasma ammonia levels associated with changes consistent with necrosis as revealed by histological analyses. However, modified CCl 4 doses were administered according to the liver function index and animal body weight [67] . In another study, ACLF was induced in male SD rats (150 -170 g) by the i.p. injection of 1.5 mL/kg of CCl 4 dissolved in vegetable oil (40%) twice a week for ten weeks. After cirrhosis establishment, LPS (100 µg/kg) and D-GaIN (0.5 g/kg) were i.p. administered [43] . In addition to the same biochemistry and histopathology events observed as in the first study, a macroscopic analysis also detected granules and severe liver surface adhesions, as well as increased B-cell lymphoma 2 (BCL-2) and BCL-2associated X protein apoptosis regulator genes, which could probably be handled with the control of mitochondrial damage via Mtn-2 [43, 67] . Immune responses are also a target for investigation. Thus, another study evaluated male SD rats (160-180 g) received i.p. injections comprising CCl 4 in peanut oil (1/1 w/v) once every three days for two months (1.5 mL/kg BW during the 1 st month and 2 mL/kg BW during the 2 nd month). Subsequently, 500 mg/Kg BW D-GalN and 80 μg/Kg BW LPS were i.p. administered, resulting in a Treg/Th17 ratio imbalance, necrosis and fibrotic tissue as revealed by histological assessments [59] . In addition to intense inflammatory response, the HIF-1α shows an important role in development of ACLF and the mitochondrial function was impairment. These findings was observed in a study with ACLF induced by i.p. CCl 4 in vegetable oil (1.5 mL/kg) 3 days in 4 wk, in male SD rats (approximately 200 g), and then i.p. of LPS (100 μg/kg) in combination with D-GaIN (0.5 g/kg) [68] . These models show enhanced histopathological evidences; AST and ALT levels and prothrombin time (PT) was raised, as well as the liver mitochondrial ultrastructure was damage with nuclear fragmentation [68] . Its corroborated the energy expenditure due the oxidative stress and systemic inflammation [40, 42] . Tripathi and collaborators reported that CCl 4 is capable of inducing advanced chronic liver injury, as Wistar rats (50-75 g) subjected to CCl 4 inhalation and receiving phenobarbital (0.3 g/L) in drinking water presented micronodular cirrhosis with ascites after 15 or 16 wk. Subsequently, LPS (from obtained from Escherichia coli, O111:B4 -1 mg/kg) was i.p. injected to induce an acute insult in the decompensated cirrhosis group 4 h prior to a hemodynamic study and 24 h prior in the compensated cirrhosis group [69] . LPS administration was more effective in the decompensated cirrhosis animals, resulting in portal hypertension and increased neutrophil infiltration and neutrophil extracellular trap (NET), as well as a high inflammatory response and aggravated fibrosis [69] . 73] . Briefly, 12-wk-old C57BL/6J male mice were categorized into a control group receiving an olive oil i.p. injection and an experimental group receiving a CCl 4 i.p. injection (0.2 mL/kg) twice a week during 6 wk to induce liver fibrosis. The experimental group was then submitted to surgical intragastric intubation, beginning at EtOH 16 g/kg/day, gradually increasing to 25 g/kg/day. The CCl 4 associated with EtOH was able to mimic an acute-on-chronic injury, resulting in intense inflammation, hypoxia and opportunist infection by E. coli and Candida sp., as well as chromatin and DNA modifications with epigenetic gene dysregulation. This model is, however, difficult to reproduce, due to the applied surgery and an approximate mortality rate of 30%.

It is noteworthy that CCl 4 has been widely applied to induce hepatotoxicity in experimental studies, although it is toxic. According to the National Institute of Health chemistry database (PubChem) CCl 4 is a clear, colorless, volatile and very stable chlorinated hydrocarbon and its inhalation (> 200-250 ppm CCl 4 for > 4 h) leads to accumulation mainly in fatty tissues and may result in nervous system depression, lung, kidney and liver damage and increased cancer susceptibility [74] . The intoxication level and affected tissue, however, may vary with exposure time and amount. Although no direct data concerning humans are available, animal experiments have indicated that toxic CCl 4 metabolites are produced in reactions catalyzed by CYP450 oxigenases (e.g., CYP2E1; CYP3A) [74, 75] .

Administration i.p of Thioacetamide (TAA) also has been used to induce ACLF In one assessment, male SD rats (150-200 g) received 250 mg/kg of TAA dissolved in saline twice a week for ten weeks to induce compensated cirrhosis without ascites development [69, 76] . The animals subsequently received i.p. LPS injection obtained from Escherichia coli O111:B4 -1 mg/kg) 24 h prior to a hemodynamic study. Increased liver enzymes, portal hypertension worsening and fibrosis were noted, similar to the human condition [69] . This is the only study to date applying TAA alongside LPS to induce ACLF in an animal model as an alternative methodology to CCL 4 administration. However, TAA, a crystalline solid, is highly toxic following inhalation or dermal contact and is carcinogenic to humans [77] , requiring care to avoid researcher, laboratory and environmental contamination.

In addition to chemical ACLF induction, surgery can also be used to establish ACLF. An obstructive jaundice model was developed in female Wistar rats (200-250 g) through bile duct ligation (BDL) by the dissociation of the common bile duct of the hepatoduodenal ligament[58], through a double ligature using surgical threads at the proximal end of the common bile duct, followed by a layered suturing of the abdominal wall to close the abdominal cavity [58, 78] . Reduced-size hepatic is-chemia/reperfusion injury was performed, leading to cholestasis (0 min, 15 min, 30 min and 45 min of ischemia), followed by a choledochoduodenostomy to relieve this condition. A reduction of the 70% in the hepatic area was observed. This model was able to reproduce certain ACLF pathological features, such as increased AST, ALT and total bilirubin levels after the obstruction surgery [58] . Another study demonstrated that microsurgery applied to induce cholestasis also mimics certain pathological ACLF events. For example, male Wistar rats (200-400 g) have been submitted to an extra-hepatic biliary tract dissection through hepatic lobe and lobe ligament sectioning [60, 79] . This technique is most applied in human secondary biliary hepatic cirrhosis and atresia assessments, but is also capable of inducing ACLF syndrome events, such as histological and biochemistry aspects and an international normalized ratio (INR) of up to 1.5. In addition, an increase in CD45 + leucocyte infiltration was also observed [60] . Recent studies have suggested the bile duct obstruction Wistar rat model followed by the i.p. injection of a single dose of LPS (1 mg/kg, extracted from Salmonella typhimurium) [80] or bile duct-obstructed SD rats followed by an i.p. injection of LPS (0.03 mg/kg BW). It was obtained from Klebsiella pneumoniae 28 d after cirrhosis as confirmed ACFL models presenting systemic disease. Alternatively, to induce acute decompensation, animals received a single intravenous LPS dose obtained from Escherichia coli O111:B4 -6.25 mg/kg BW 3 wk after bile duct obstruction [62] . Using a similar methodology, common bile duct ligature was performed in SD rats for 28 d, with cirrhosis displaying ascites established on the 25 th day. The animals were then injected with LPS 1 mg/kg obtained from Escherichia coli (O111:B4) [69] . An increase in plasma enzyme levels and splenomegaly were observed, mimicking pathological events and inducing the inflamassome (e.g. IL-1β; IL-18), as noted in the human syndrome [62, 80] . Although these methodologies result in similar conditions to the human disease, the need for surgical procedures and animal survival maintenance make them more challenging and costly, requiring previous surgical knowledge.

Studies have demonstrated that the albumin, the most abundant protein in the human serum, metabolized in the liver and secreted to the plasma can be an alternative to treat decompensated cirrhosis, although it can become hepatotoxic when an imbalance in albumin levels occurs. Thus, albumin may induce cirrhosis events both when interacting with LPS or when undergoing irreversible alkalization by drug metabolization [81, 82] . Therefore, human albumin serum (HSA) is a potential model for liver damage. In order to develop HSA-induced ACLF, female Wistar rats (180-220 g) were challenged with a subcutaneal HSA injection diluted in saline (8 g/L) and the same volume of incomplete Freund's adjuvant for four times (0.5 mL containing 4 mg HSA). Next, 4 mg HSA were injected into the animal tail veins twice/week for six weeks [56] . After the establishment of immune cirrhosis, the animals received 400 mg/kg of D-GaIN and 100 μg/kg LPS, both i.p., inducing acute decompensation in chronic liver disease, leading to changes in liver histology and increased serum enzymes and several inflammatory markers, such as IL-6, IL-18 and HMGB-1. It is important to note that both NF-κB and TNF-α play an important role in HMGB-1-mediated responses [56, 57, 83, 84] , similar to human ACLF. Since the HSA ACLF model results in high mortality rates, it was developed a rat model using porcine serum (PS), resulting in better induction of pathophysiological events [56, 61] . Male Wistar rats (120-150 g) received 0.5 mL i.p. PS twice/week for 11 wk and developed liver fibrosis as revealed by hepatic hydroxyproline levels. Then, following the induction of immune cirrhosis, LPS was i.p. injected at 50 μg/kg followed by 600 mg/kg i.p. of D-GaIN thirty minutes later, in order to induce acute liver insult on chronic liver damage [61] . This ACLF model presented several similarities with human ACLF, such as increased inflammatory marker (TNF-α, IL-6), plasma enzyme (AST, ALT) and ammonia levels, as well as higher prothrombin times consistent with coagulation function impairment [20, 61] . Therefore, ACLF induced by serum albumin showed to be a good model and comprises an alternative to better understand ACLF physiopathology. However, the PS model may be more adequate, it induces a closer response to the human condition, easier to manipulate and results in lower mortality rates compared to the other mentioned models.

Due to the complexity of liver injury, the understanding of underlying liver disease mechanisms and their treatment has been limited by the lack of satisfactory animal models. Currently, no model has been able to completely capture the corresponding human acute and chronic liver disorder[52, 61, 85] . The ideal ACLF model should combine bacterial infection and high short-term mortality. As described previously, several existing ACLF models have been developed by combining chronic and acute liver injury [70] . Chronic injury is most commonly induced by the injection of CCl 4 or via BDL surgery, whereas acute injury is induced by the injection of D-GaIN/LPS. The principle of these models is to reproduce the bi-factorial disease profile comprising chronic liver injury, which leads to the development of progressive liver fibrosis, and a precipitating event inducing further organ injury, resulting in ACLF and considerable mortality. This is not, however, entirely consistent with ACLF pathogenesis, and the surgery required for the BDL model is difficult. The clinical situation is often more complex, and different modulating factors may occur concurrently or sequentially [86] . Typically, 50% of patients develop bacterial infection as an ACLF complication, although the (initial) precipitating event was non-inflammatory [87] .

The significant challenge to develop an ACLF model is the ability to unite all the clinical characteristics observed in humans, as this is a multifactorial disease with multiple precipitators and complications and, therefore, varying disease phenotypes and organ failures, making it almost impossible to develop a single experimental model capable of triggering all of the most important clinical features [86] . In a recent study by Xiang and coworkers (2020), the authors developed a new ACLF model that could sequentially reproduce three important clinical ACLF disease factors. To this end, a severe liver injury model was prepared by combining chronic injury (CCl 4 injection), acute hepatic insult (injection of a CCl 4 double dose), and systemic bacterial infection (i.p. injection of bacteria Klebsiella pneumonia).

The findings indicate that this severe liver injury model developed acute-on-chronic liver injury, bacterial infection, multi-organ injury, and high mortality, some of the features of clinical ACLF. The authors highlight that the single bacterial infection step is crucial in inducing multi-organ failure in this model, as chronic-plus-acute liver injury did not drive the full course of ACLF in mice without bacterial infection [70] . In contrast, Schwarzkopf and coworkers developed a model combining chronic liver disease (CCl 4 /EtOH or CYP2D6-linked adenovirus (ADV)-induced autoimmune hepatitis) with different precipitating events [two EtOH binges or i.p. polymicrobial infection by cecal slurry (CS)]. After 7 wk of CCl 4 /EtOH, ACLF was induced with two alcohol binges (alcohol gavage with 31.5% Vol.) with an interval of 3 days between binges. Mice mortality was observed, as well as systemic inflammation and significant elevation of serum ALT levels alongside other ACLF features [73] . According to the authors, polymicrobial sepsis by CS is closer to human infection-triggered ACLF than the K. pneumonia injection employed by Xiang[70, 73] . These variabilities in current data also significantly interfere with the development of a standardized ACLF animal model. Furthermore, it is not yet possible to identify all ACLF precipitants, as over 40% of patients who develop ACLF exhibit no known precipitant, requiring further knowledge of ACLF activation events [22] .

Animal models are helpful in understanding human diseases and play a relevant role in preclinical research, as they are capable of reproducing major pathophysiological events that occur in humans. ACLF mechanisms remain unclear, displaying a poor diagnosis and high mortality rates worldwide, it becomes relevant to highlight current animal models and how they can elucidate ACLF mechanisms. These models are also crucial in testing new drugs and novel bioengineering and genetic therapies, as the only effective therapy to date is liver transplantation. In this context, few viable livers are available and the quick clinical evolution of this condition was observed. Currently, rats are the most widely applied models (Figure 1 ), as their maintenance and management is simple compared to other models (Table 1) . Furthermore, ACLF induced by PS combined with LPS/D-GaIN seems to be the most adequate methodology in the establishment of an ACLF animal model. However, further studies are required, as controversies concerning model designs, reproducibility and safety are still noted. In addition, the animal models are incapable to reproduce all manifestation of ACLF condition due to different causes of pathology. For example, the HBV infection model remains a challenge, because of use of transgenic mice that were capable to support virus replication or virus products. Thus, understanding current models in order to establish a safe and easily reproducible model to study complementary alternatives to liver transplantation is paramount. 

The liver

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Mechanism of carbon tetrachloride-induced hepatotoxicity. Hepatocellular damage by reactive carbon tetrachloride metabolites

Formation of carbonyl chloride in carbon tetrachloride metabolism by rat liver in vitro

Mechanisms of CCl4-induced liver fibrosis with combined transcriptomic and proteomic analysis

D-galactosamine/Lipopolysaccharide-induced hepatotoxicity downregulates sirtuin 1 in rat liver: role of sirtuin 1 modulation in hepatoprotection

Effects of coencapsulation of hepatocytes with adipose-derived stem cells in the treatment of rats with acute-on-chronic liver failure

HIF1a Inhibitor Rescues Acute-on-Chronic Liver Failure

Simvastatin Prevents Progression of Acute on Chronic Liver Failure in Rats With Cirrhosis and Portal Hypertension

Interleukin-22 ameliorates acute-on-chronic liver failure by reprogramming impaired regeneration pathways in mice

Development of an experimental model of liver cirrhosis in rabbits

Interleukin-22 in acute-on-chronic liver failure: A matter of ineffective levels, receptor dysregulation or defective signalling?

Carbon tetrachloride

Carbon Tetrachloride Toxicity: What Are the Toxicological Effects of Carbon Tetrachloride Exposure? | Environmental Medicine | ATSDR

Liraglutide improves liver microvascular dysfunction in cirrhosis: Evidence from translational studies

Protective effect and mechanisms of radix astragali injection on the intestinal mucosa of rats with obstructive jaundice

A model of cholestasis in the rat, using a microsurgical technique

The effect of fractal-like mechanical ventilation on vital signs in a rat model of acute-on-chronic liver failure

Human serum albumin, systemic inflammation, and cirrhosis

Irreversible alkylation of human serum albumin by zileuton metabolite 2-acetylbenzothiophene-S-oxide: a potential model for hepatotoxicity

Recombinant adenovirus containing hyper-interleukin-6 and hepatocyte growth factor ameliorates acute-on-chronic liver failure in rats

Role of hemostatic factors in hepatic injury and disease: animal models de-liver

Regeneration in acute-on-chronic liver failure -the phantom lost its camouflage

Bacterial infections in cirrhosis: a position statement based on the EASL Special Conference