A prevailing animal model currently used to study severe human diseases like obstructive cholestasis, primary biliary or sclerosing cholangitis, biliary atresia, and acute liver injury is the common bile duct ligation (cBDL). Modifications of this model include ligation of the left hepatic bile duct (pBDL) or ligation of the left bile duct with the corresponding left hepatic artery (pBDL+pAL). Both modifications induce cholestasis only in the left liver lobe. After induction of total or partial cholestasis in mice, the well-being of these animals was evaluated by assessing burrowing behavior, body weight, and a distress score. To compare the pathological features of these animal models, plasma levels of liver enzymes, bile acids, bilirubin, and within the liver tissue, necrosis, fibrosis, inflammation, as well as expression of genes involved in the synthesis or transport of bile acids were assessed. The survival rate of the animals and their well-being was comparable between pBDL+pAL and pBDL. However, surgical intervention by pBDL+pAL caused confluent necrosis and collagen depositions at the edge of necrotic tissue, whereas pBDL caused focal necrosis and fibrosis in between portal areas. Interestingly, pBDL animals had a higher survival rate and their well-being was significantly improved compared to cBDL animals. On day 14 after cBDL liver aspartate, as well as alanine aminotransferase, alkaline phosphatase, glutamate dehydrogenase, bile acids, and bilirubin were significantly elevated, but only glutamate dehydrogenase activity was increased after pBDL. Thus, pBDL may be primarily used to evaluate local features such as inflammation and fibrosis or regulation of genes involved in bile acid synthesis or transport but does not allow to study all systemic features of cholestasis. The pBDL model also has the advantage that fewer mice are needed, because of its high survival rate, and that the well-being of the animals is improved compared to the cBDL animal model.

Cholestatic liver diseases, such as primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC), lead to inflammation and severe hepatic damage with limited therapeutic options. This study assessed the efficacy of the inverse RORγt agonist, GSK805, both in vitro using the hepatic stellate cell‐line LX‐2 and in vivo using male bile duct‐ligated BALB/c mice. In vitro, 0.3 μM GSK805 reduced alpha‐smooth muscle actin expression in LX‐2 cells. In vivo, GSK805 significantly decreased IL‐23R, TNF‐α, and IFN‐γ expression in cholestatic liver. Despite high concentrations of GSK805 in the liver, no significant reduction in fibrosis was noticed. GSK805 significantly increased aspartate aminotransferase and alanine aminotransferase activity in the blood, while levels of glutamate dehydrogenase, alkaline phosphatase, and bilirubin were not substantially increased. Importantly, GSK805 did neither increase an animal distress score nor substantially reduce body weight, burrowing activity, or nesting behavior. These results suggest that a high liver concentration of GSK805 is achieved by daily oral administration and that this drug modulates inflammation in cholestatic mice without impairing animal well‐being.

When pain might occur during an animal experiment, sufficient analgesia is necessary. Metamizole is the third most used postoperative pain medication in animal research. The analgesic effect of metamizole is supposed to last 6–8 h in rodents. Therefore, the supplementation of drinking water with metamizole should be the preferred method to ensure permanent pain relief without unnecessary stressors. The present exploratory study compared the voluntary intake of metamizole-supplemented drinking water (3 mg/ml) between healthy mice of three different mouse strains. After the addition of metamizole to the drinking water, a marginal reduction in body weight was observed in C57BL/6J and BALB/c mice. However, NSG mice displayed a significantly higher body weight loss and reduction of drinking behavior compared with the C57BL/6J and BALB/c strains. The acceptance of metamizole in NSG mice did not increase with a different metamizole formulation. Thus, the mice of the inbred strains C57BL/6J and BALB/c seemed to be able to adapt to the taste of metamizole, while NSG mice were not able to accustom to analgesia within 1 week. Strain-specific habituation should be considered in future animal studies when analgesia is applied via drinking water.

Preclinical studies rarely test the efficacy of therapies in both sexes. The field of oncology is no exception in this regard. In a model of syngeneic, orthotopic, metastasized pancreatic ductal adenocarcinoma we evaluated the impact of sex on pathological features of this disease as well as on the efficacy and possible adverse side effects of a novel, small molecule-based therapy inhibiting KRAS:SOS1, MEK1/2 and PI3K signaling in male and female C57BL/6J mice. Male mice had less tumor infiltration of CD8-positive cells, developed bigger tumors, had more lung metastasis and a lower probability of survival compared to female mice. These more severe pathological features in male animals were accompanied by higher distress at the end of the experiment. The evaluated inhibitors BI-3406, trametinib and BKM120 showed synergistic effects in vitro. This combinatorial therapy reduced tumor weight more efficiently in male animals, although the drug concentrations were similar in the tumors of both sexes. These results underline the importance of sex-specific preclinical research and at the same time provide a solid basis for future studies with the tested compounds.

Despite ongoing research, realistic in vitro models for acute lymphoblastic leukemia (ALL) that can mimic the complex pathology are still not available, highlighting the need for continuous animal-based investigation. As part of the 3R principles, constant refinement of animal experiments is mandatory. Therefore, reviewing the effectiveness of used analgesics is essential for animal model-specific refinement. Here we evaluate whether metamizole—previously used in our institute—or tramadol is more suitable as on-demand analgesia in mouse models of ALL. The murine orthotopic xenograft models were induced by intravenous injection of either the human precursor ALL cell lines RS4;11 or SEM into immune-deficient male and female NSG mice. Mice were weighed and checked daily for basic behavior and well-being, while detailed welfare parameters, such as burrowing behavior, nesting activity, perianal temperature, liquid intake, fecal corticosterone metabolites, mouse grimace scale and tumor cell proliferation were monitored weekly. Upon leukemic progression, when signs of pain or discomfort were observed, metamizole (3 mg/ml) or tramadol (1 mg/ml) was administered via drinking water for analgesic treatment, and detailed welfare parameters were assessed daily. Following the initiation of treatment, mice receiving either metamizole or tramadol continued to show a decline in body weight, liquid intake and other welfare parameters, suggesting that neither drug was sufficient to fully counteract the effects of late-stage ALL. Combining the data with the relative severity assessment algorithm revealed that metamizole treatment appeared less effective than tramadol in mitigating the detrimental effects of the disease. Therefore, the opioid tramadol should replace metamizole as the analgesic compound of choice for hematological xenograft models to improve animal welfare in future studies.

Antibody-mediated immune responses against platelet factor 4 (PF4) underlie a spectrum of rare but severe thromboinflammatory disorders, including heparin-induced thrombocytopenia (HIT), autoimmune HIT variants, vaccine-induced immune thrombocytopenia and thrombosis (VITT), and VITT-like syndromes. Despite differences in triggering factors and antibody-binding requirements, these conditions share a common pathogenic mechanism in which anti-PF4–IgG immune complexes engage platelet FcγRIIa, thereby triggering platelet activation, procoagulant transformation, and amplification of immunothrombosis. Experimental models have been instrumental in defining the molecular and cellular basis of anti-PF4 immunothrombosis. Platelet-centric in vitro assays and whole-blood systems established FcγRIIa-dependent platelet activation as a central effector mechanism and revealed functional heterogeneity among anti-PF4 antibodies. Flow-based vascular models further incorporate shear stress and endothelial interfaces, enabling analysis of thrombus formation under near-physiological conditions. In vivo, passive antibody transfer models in humanized mice expressing human PF4 and FcγRIIa reproduce key clinical features, including thrombocytopenia and thrombosis, and have enabled mechanistic and therapeutic studies. Despite these advances, important conceptual gaps remain. Most existing models focus on the effector phase of disease. Conversely, active immunization models can induce anti-PF4 antibodies but rarely reproduce the downstream thrombotic phenotype. As a result, the mechanisms linking antigen formation, innate immune activation, B cell recruitment, and acquisition of pathogenic antibody function remain incompletely understood. Developing integrated experimental systems that connect immune initiation with FcγRIIa-dependent effector pathways will be essential to advance mechanistic insight, improve therapeutic strategies, and establish predictive preclinical models to assess the risk of anti-PF4 antibody induction during vaccine development.