A modified murine model of systemic sclerosis: bleomycin given by pump infusion induced skin and pulmonary inflammation and fibrosis

Abstract

Daily subcutaneous (sc) injection of bleomycin (BLM) causes dermal fibrosis but rarely causes lung changes in mice. There are also significant disadvantages to this traditional model for systemic sclerosis, including a variable distribution of lesions and a requirement for repetitive procedures. The present study was undertaken to develop a convenient method of BLM administration that yields stable dermal inflammation and fibrosis with extensive and reproducible interstitial lung disease (ILD) in mice. Osmotic minipumps containing BLM (150 mg/kg) or saline were implanted sc in C57BL/6 mice and the drug was delivered as a continuous infusion over 1B4 weeks. The time course of morphological features, collagen content, and pro-inflammatory cytokine expression in the skin and the lungs were analyzed. Pathological examination demonstrated dominant inflammatory infiltrates at week 1 and significant fibrosis at week 4. Decreased microvessel density and increased myofibroblast counts were observed in the skin of BLM-treated mice at week 4. In addition, there were obvious increases in dermal infiltration of CD45 þ leukocytes, including F4/80 þ macrophages, Gr-1 þ neutrophils, and CD3 þ T lymphocytes in BLM-treated mice. IL-1b, IL-4, and CXCL2 transcripts were continually upregulated by BLM in the skin and lung tissues. In addition, lungs from BLM-treated mice showed significant inflammatory infiltrates and confluent subpleural fibrosis at week 4. In conclusion, this modified murine model for drug-induced systemic inflammation and fibrosis uses a single procedure and provides reproducible skin and lung lesions, mimicking human systemic sclerosis (SSc) with ILD-like manifestation.

Keloids: Animal models and pathologic equivalents to study tissue fibrosis

“…In the skin, bleomycin can be administered to produce a localized area of cutaneous fibrosis, the most common route of administration being direct subcutaneous injection. Given the difficulty and variability repeated daily injections can pose, however, newer models have emerged using sustained release of bleomycin via subcutaneously implanted osmotic pumps, eliminating the need for daily injections [21,22,26]. This administration method reduces injection site variability and provides a more consistent distribution of lesions in affected skin [22,26].…”

“…Many pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-4, IL-6 and CXCL2, are found to be upregulated in the skin of mice injected with bleomycin [21,22]. Additionally, many inflammatory cells, including CD45 + leukocytes, F4/80 + macrophages, CD3 + T lymphocytes and mast cells as well as α-smooth muscle actin (αSMA) positive myofibroblasts, are found in higher numbers in the skin of bleomycin-treated animals versus control [21,22,27]. A downstream effect of this intense inflammatory process is tissue fibrosis and increased collagen deposition.…”

“…A downstream effect of this intense inflammatory process is tissue fibrosis and increased collagen deposition. Both dermal thickness and hydroxyproline content of bleomycin-injected skin are 2–3 fold higher than control [21,22,26,27]. …”

“…Many studies utilize dermal thickness as an indicator for fibrosis, and while this does reveal the effects of the drug, use of this metric is limited by sample area selection and observer bias. Dermal thickness is not uniformly increased in most specimens, with some exhibiting patches of increased dermal thickness as the bleomycin is dispersed throughout the skin [19,22]. Newer models utilizing sustained release of the drug via osmotic pump implantation have improved on this limitation, however, constant administration is still required for a long duration to achieve the desired effect [21,22].…”

Optimization of a murine and human tissue model to recapitulate dermal and pulmonary features of systemic sclerosis

“…A few reports have shown that systemic delivery of BLM via continuous diffusion from subcutaneously implanted osmotic minipumps can cause fibrosis of the skin, lungs, and other internal organs [10–12]. This approach may provide the opportunity to test the effects of potential therapeutic treatments on fibrosis in each of the affected organs simultaneously.…”