Foreword

The mouse liver injury model is a crucial tool for studying the pathogenesis of liver diseases, drug screening, and therapeutic strategies. Mouse liver injury models can be classified into several types, including chemical, drug-induced, immune, and alcohol-related injuries, each with specific establishment methods and evaluation criteria. The inducers of chemical injuries include alcohol, carbon tetrachloride (CCl4), acetaminophen (APAP), α-naphthylisothiocyanate (ANIT), and 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC). Among these, the CCl4-induced model is widely used due to its simplicity and clear response.

CCl4 can be metabolized by cytochrome P450 enzymes in the liver producing free radicals, which can trigger lipid peroxidation reactions and lead to liver cell damage. By intraperitoneal CCl4 injection, significant liver damage can be induced within a relatively short period. This model exhibits good reproducibility and has morphological and biochemical characteristics similar to human liver diseases, making it an ideal model for studying liver necrosis and cirrhosis, as well as for drug screening and efficacy evaluation.

Materials

1. Experimental Animals: C57BL/6J, male, 6–8 weeks old.

2. Modeling Reagents: Dilute CCl4 in olive oil to prepare a 0.3% CCl4

3. solution, and filter it using a 0.2 μm filter.

Precautions: CCl4 is listed as a Group 2B carcinogen by the International Agency for Research on Cancer (IARC). Safety precautions should be taken when handling.

Modeling Method

For the acute liver injury group, mice are received intraperitoneal injection of 0.3% CCl4 at a volume of 10 ml/kg. For the control group, administer a 10 mL/kg intraperitoneal injection of olive oil.

Endpoint Timing: After administering CCl4, collect serum and liver tissue from the mice at the following time points: 2, 6, 12, 24, and 48 hours. Ensure mice are anesthetized before sample collection at each time point.

Model Evaluation Methods

Survival Rate:

Monitor Mouse Survival Rate: The survival rate of mice decreases as the duration of model induction increases. The survival rate can drop to 40% by 24 hours after modeling.

Serum Biochemical Tests

Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST)

In the model group, levels of ALT and AST significantly increase, reaching their peak approximately 24 hours after modeling.

Cytokine Measurement: TNF-α and IL-6

In the model group, serum levels of the inflammatory cytokines TNF-α and IL-6 are significantly elevated and continue to rise with the extension of the modeling period.

Histological Examination of Liver Injury

After euthanasia, the mouse liver is harvested. At 2 hours post-modeling, the liver shows mild damage with some cellular necrosis around the blood vessels. At 24 hours, there is evident severe liver damage, with extensive cellular necrosis, loss of liver architecture, and infiltration of inflammatory cells around the blood vessels [1].

Application of RWD Sectioning Machine in Liver Tissue Sectioning

Cryosectioning

1. Temperature settings: Chamber at -20℃, sample head at -15 to -18℃.

2. Mouse liver sections: Prepared using the RWD FS800 Cryostat.

Paraffin Sectioning

Fixation: Choose the appropriate fixation time and temperature to ensure tissue structure integrity.

Dehydration and clarification: Carefully manage the processing times for alcohol and clarifying agents to prevent tissue damage from excessive dehydration.

Embedding and sectioning: Select the right type of paraffin and melting point to maintain the continuity and integrity of the sections.

Staining: The choice of staining agents and the control of staining time significantly impact the results, so optimization should be based on specific conditions.

Deparaffinization and mounting: Ensure thorough deparaffinization and mount the sections flat and without bubbles.

References:【1】Zhao L, Jin Y, Donahue K, et al. Tissue Repair in the Mouse Liver Following Acute Carbon Tetrachloride Depends on Injury-Induced Wnt/β-Catenin Signaling. Hepatology. 2019;69(6):2623-2635. doi:10.1002/hep.30563