Influence of analgesia on inflammation and NF-κB response in the murine intestine

Abstract

Inflammatory bowel diseases, including ulcerative colitis (UC), cause chronic inflammation of the colon and/or the small intestine. The dextran sulfate sodium (DSS) induced colitis model is widely used to study UC but raises ethical concerns due to symptoms like bloody diarrhea and abdominal cramps. Following the 3R principle, animal suffering must be minimized while ensuring the accuracy of research outcomes. Providing analgesia in this model is challenging, as many analgesics can interfere with inflammation, epithelial integrity, and immune responses, potentially compromising research outcomes. The aim of this study was to identify the most suitable analgesic for use in DSS induced UC research, with limited interference with intestinal inflammation and NF-κB activity. To achieve this, a deep learning-based tool, PEDL+, was used to identify amantadine and piritramide as the most promising candidates, while tramadol and paracetamol were included as positive controls based on LAGeSo recommendations. In the healthy colon, all analgesics increased goblet cell size, suggesting a general effect on mucin production. Among them, amantadine-treated mice showed the most similar goblet cell number, goblet cell size, and NF-κB activity to untreated controls, making it the most promising candidate for minimizing interference in colitis studies. In contrast, tramadol significantly influenced epithelial cell proliferation and NF-κB activity, while paracetamol significantly affected macrophage numbers and goblet cell dynamics. This shows that commonly used analgesics can introduce significant biases in UC research, while it also validates the effectiveness of PEDL+, as the preselected drugs, amantadine and piritramide, performed better than tramadol and paracetamol. In the DSS UC model, amantadine had protective effects on epithelial integrity, reduced T-cell numbers, and decreased NF-κB activity in the colon. While these effects suggest potential anti-inflammatory properties, they also indicate interference with key research targets in UC studies. A similar trend was observed in the small intestine, where amantadine treatment led to a reduction in T-cells and NF-κB activity, despite no major structural changes in the ileum. This shows that even the most promising candidate, amantadine, is not ideal for all research applications. While it may be suitable for certain studies, it does influence NF-κB activity and immune responses. From a 3R perspective, avoiding analgesics when possible, would allow collected biopsies to be used for a broader range of analyses, reducing the need for additional animal experiments. If analgesics are used, they should be carefully chosen based on the primary research targets.