Background/Purpose: The murine bleomycin (BLM)-induced fibrosis model is the most widely used in systemic sclerosis (SSc) studies. Traditionally, daily subcutaneous injections of BLM for 4-6 weeks are commonly used to induce local dermal inflammation and fibrosis. However, this model has disadvantages such as localized cutaneous fibrosis, rare involvement of the lung, and a requirement for repeated local injections. It was recently reported that systemic delivery of BLM via continuous diffusion from subcutaneously implanted osmotic minipumps can cause fibrosis of the skin, lungs, and other internal organs. However, the mouse strain, dosage of BLM, administration period, and additional important features differ from one report to the next. In this study, we have investigated the dosage of BLM, extent of fibrosis, and time-dependent changes in dermal and pulmonary fibrosis in C57BL/6J mice using the pump model.

Methods: BLM was administered to 8 week-old, male C57BL/6J mice using osmotic minipumps implanted subcutaneously and containing either 100μl saline as vehicle or BLM for 7 days. BLM was administered at 1.0 U/kg, 10 U/kg, 60 U/kg, or 110 U/kg. Pumps were implanted for 7 days. Lung and skin tissues were harvested on days 10, 14, 21, and 28 post-implantation. The extent of pulmonary fibrosis was measured using hydroxyproline assay. The extent of dermal fibrosis was quantified using both hydroxyproline assay and measurement of dermal thickness. Furthermore, the mRNA levels of fibrosis-related genes were measured using real-time PCR.

Results: Mice treated with different concentrations of BLM showed a dose-dependent increase in lung fibrosis by day 28. The levels of collagen in lungs from mice treated with high dose BLM were significantly greater than levels in lungs from mice treated with vehicle or lower doses of BLM. On the other hand, dermal fibrosis was not increased in a dose- or time-dependent manner. Interestingly, marked dermal thickness was induced by low dose BLM while no significant changes were noted with high-dose BLM by day 28. However, dermal thickness was significantly increased 10 days post BLM. Hydroxyproline assay also revealed that the amount of collagen in skin on day 10 was significantly higher than in skin treated with vehicle. However, by day 21 there were no significant differences in dermal thickness or collagen content between the treatment groups.

Conclusion: Our findings show that dermal fibrosis in C57BL/6J mice using the pump model differs with mouse strains, dose of BLM, and duration of the model. The BLM pump model is a powerful tool for the functional analysis of systemic fibrosis and the testing of potential therapies. However, the choice of mouse strains, duration of BLM administration and dose must be carefully considered when using this model.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/optimization-of-a-murine-model-to-recapitulate-dermal-and-pulmonary-features-of-ssc/