An optimized peritonitis-induced ACLF model that reproduces the full spectrum of extrahepatic organ failures in mice

Abstract

Background: Acute-on-chronic liver failure (ACLF), which develops in patients with acutely decompensated cirrhosis, is characterized by multiple extrahepatic organ failures leading to high short-term mortality. Although major advances in the understanding of ACLF have been accomplished in the last years, the understanding of driving mechanisms underlying ACLF is hindered by the lack of proper animal models that faithfully reproduce both the systemic hyperinflammatory response and the full spectra of extrahepatic organ failures present in this condition.

Methods: ACLF was induced by acute induction of polymicrobial peritonitis secondary to the ligation and puncture of the cecum (CLP) in mice with chronic carbon tetrachloride (CCl4)-induced cirrhosis. The study included three groups: CCl4+CLP (n=10) mice with cirrhosis which underwent CLP surgery; CCl4+sham mice (n=10) and control mice (n=10).

Results: As compared to CCl4+sham, CCl4+CLP mice had higher short-term mortality and exhibited more severe hypoalbuminemia and hyperbilirubinemia, significantly higher AST and GGT levels and higher liver inflammatory burden. CCl4+CLP mice also showed increased serum creatinine and BUN levels and up-regulated expression of Kim-1, Il-6 and Tnf in the kidney, lower oxygen saturation (SpO2), higher serum renin concentration, higher international normalized ratio (INR) and worse neurological behavior test scores than CCl4+sham and control mice. In addition, CCl4+CLP mice showed widespread bacterial tissue colonization and exhibited increased serum cytokine levels, which correlated with the intensity of organ impairments.

Conclusion: The CCl4+CLP model reproduces the full spectra of extrahepatic organ impairments present in patients with ACLF and represents an optimized murine model to experimentally explore the pathophysiology of this disease as well as new therapeutic approaches.

Keywords: decompensated cirrhosis; experimental model; liver; multi-organ failure.

Graphical abstract

FIGURE 1

Changes in body and adipose tissue weight and serum biochemistry in mice with CCl₄-induced cirrhosis undergoing CLP. (A) Body weight progression during the 12 weeks of treatment in control mice (n=10) and CCl₄-induced cirrhotic mice (n=20). (B) End-point body weight, (C) epididymal white adipose tissue (eWAT), and (D) interscapular brown adipose tissue (iBAT) in control mice (n=10) and CCl₄-induced cirrhotic mice (n=20). (E–I) Serum glucose, total cholesterol, lactate dehydrogenase (LDH), inorganic phosphorus, and calcium levels. Results from body weight progression are expressed as mean±SEM. Results are represented in box plots; the boxes show the interquartile range, the median values (horizontal lines), and the bars denote the highest and lowest values of the distribution. Abbreviations: CCl₄, chronic carbon tetrachloride; CLP, cecal ligation and puncture; eWAT, epididymal white adipose tissue; iBAT, interscapular brown adipose tissue; LDH, lactate dehydrogenase.

FIGURE 2

Liver changes after CLP in mice with CCl₄-induced cirrhosis. (A) Liver and spleen weight, expressed as tissue-to-body weight ratio and serum albumin. (B) Albumin, bilirubin, ALT, AST, GGT, and ALP levels in mice from control (n=10), CCl₄+sham (n=10), and CCl₄+CLP (n=10) groups. (C) Representative photomicrographs (40× magnification) of liver sections stained with H&E. Necroinflammation score was calculated by a registered pathologist by analyzing the H&E-stained sections. (D) Hepatic Il6 and Tnf gene expression determined by real-time PCR. (E) Representative photomicrographs (40× magnification) of liver sections stained with Sirius red. Scale bar=500 μm (left panel) and representative photomicrographs (15,000× magnification) of liver sections obtained in the TEM. Scale bar=2 μm (right panel). Results are represented in box plots; the boxes show the interquartile range, the median values (horizontal lines), and the bars denote the highest and lowest values of the distribution. Abbreviations: CCl₄, chronic carbon tetrachloride; CLP, cecal ligation and puncture; H&E, hematoxylin and eosin; TEM, transmission electron microscopy.

FIGURE 3

Renal changes after CLP in mice with CCl₄-induced cirrhosis. (A) Kidney weight expressed as tissue-to-body weight ratio. (B) Serum creatinine and BUN levels. (C) Il6, Il1b, and Tnf expression in renal tissue as determined by real-time PCR. (D) Representative photomicrographs (40× and 100× magnifications) of kidney sections stained with H&E. (E) Kim1 expression in the kidney as determined by real-time PCR. Results are represented in box plots; the boxes show the interquartile range, the median values (horizontal lines), and the bars denote the highest and lowest values of the distribution. Abbreviations: BUN, blood urea nitrogen; CCl₄, chronic carbon tetrachloride; CLP, cecal ligation and puncture; H&E, hematoxylin and eosin.

FIGURE 4

Changes in lung and brain function and in circulatory and coagulation systems after CLP in mice with CCl₄-induced cirrhosis. (A) Pulmonary parameters, namely lung weight expressed as tissue-to-body weight ratio and oxygen saturation expressed as SpO₂, were measured in the animals prior to sacrifice. (B) Representative photomicrographs (40× magnification) of lung sections stained with H&E. (C) Expression of Il6 in the lung as determined by real-time PCR. (D) INR was measured in the mice prior to sacrifice. (E) Serum renin-1 concentration. (F) Brain parameters, namely brain weight expressed as tissue-to-body weight ratio, brain water content expressed as percentage of water in the tissue (see the Methods section), and neurological behavior test were performed and scored as the increase of failure in all the mice of the study (see the Methods section). Results are represented in box plots; the boxes show the interquartile range, the median values (horizontal lines), and the bars denote the highest and lowest values of the distribution. Abbreviations: CCl₄, chronic carbon tetrachloride; CLP, cecal ligation and puncture; H&E, hematoxylin and eosin; INR, international normalized ratio.

FIGURE 5

Exacerbated systemic inflammation in mice with CCl₄-induced cirrhosis undergoing CLP. (A) Serum G-CSF, IL-1β, IL-6, IL-8 (KC), IL-17, IP-10, MCP-1, MIP-1α, and TNF-α protein levels were determined by Milliplex technology. (B) Keratinocyte-derived chemokine (PCA) displays data from cytokines in all the animals from the study. Results are expressed as box plots, the boxes show the interquartile range, the median values (horizontal lines), and the bars denote the highest and lowest values of the distribution. Abbreviations: CCl₄, chronic carbon tetrachloride; CLP, cecal ligation and puncture; G-CSF, granulocyte colony-stimulating factor; IP-10, interferon (IFN)-γ-induced protein 10; KC, keratinocyte-derived chemokine; MCP-1α, monocyte chemoattractant protein-1; MIP-1, macrophage inflammatory protein-1; TNF-α, tumor necrosis factor-α.

FIGURE 6

Correlation of inflammatory markers with organ impairments. (A) Correlation plots showing the Spearman correlation between the serum cytokines and the parameters used to define the organ failures in control (n=10), CCl₄+sham (n=10), and CCl₄+CLP (n=10). (B) Heatmap showing the strength of Spearman correlation between the serum cytokines and the parameters used to define the organ failures in all the mice from the study. (C) Heatmap showing the statistical significance of Spearman correlation between the serum cytokines and the parameters used to define the organ failures in all the mice from the study. (D) PCA displaying data from cytokines and parameters used to define the organ failures [liver: bilirubin, kidney: creatinine, circulation: serum renin concentration, coagulation: INR, brain: neurological behavior test, and respiration: oxygen saturation (SpO₂)] in all the animals from the study. Abbreviations: CCl₄, chronic carbon tetrachloride; CLP, cecal ligation and puncture; INR, international normalized ratio; NBT, neurological behavior test; PCA, principal component analysis.

FIGURE 7

Prevalence of organ impairment, bacterial colonization, and survival rates in mice with CCl₄-induced cirrhosis undergoing CLP. (A) Adapted definition of organ impairments according to the criteria described in the Methods section. (B) Heatmap of the intensity of organ impairments in control, CCl₄+sham, and CCl₄+CLP mice, calculated from the mean values of the scores using the criteria defined in panel A. (C) Heatmap of the prevalence of organ impairment in each animal, using the criteria described above. (D) A stacked column graph showing the prevalence of the number of organ impairments in control, cirrhotic, and cirrhotic mice undergoing CLP. Results are expressed as a percentage of the total. (E) Venn diagram displaying the microorganisms detected in peritoneal fluid and hepatic, renal, and pulmonary tissues from CCl₄+CLP mice. (F) Survival of mice induced to cirrhosis by CCl₄ administration (n=20) and control mice (n=10), and 24-hour survival of sham-operated cirrhotic mice (n=10) and cirrhotic mice undergoing CLP surgery (n=10). Abbreviations: BWC, brain water content; CCl₄, chronic carbon tetrachloride; CLP, cecal ligation and puncture; INR, international normalized ratio.