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

Abstract

The murine bleomycin (BLM)-induced fibrosis model is the most widely used in systemic sclerosis (SSc) studies. It has been 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, by employing the pump model in C57BL/6J mice, we show a dose-dependent increase in lung fibrosis by day 28 and a transient increase in dermal thickness. Dermal thickness and the level of collagen in skin treated with high-dose BLM was significantly higher than in skin treated with low dose BLM or vehicle. A reduction in the thickness of the adipose layer was noted in both high and low dose groups at earlier time points suggesting that the loss of the fat layer precedes the onset of fibrosis. High-dose BLM also induced dermal fibrosis and increased expression of fibrosis-associated genes ex vivo in human skin, thus confirming and extending the in vivo findings, and demonstrating that a human organ culture model can be used to assess the effect of BLM on skin. In summary, our findings suggest that the BLM pump model is an attractive model to analyze the underlying mechanisms of fibrosis and test the efficacy of potential therapies. However, the choice of mouse strain, duration of BLM administration and dose must be carefully considered when using this model.

Fig 1. Dose-dependent changes in mouse lung and skin using the pump model.

Mice treated with the indicated doses of BLM were sacrificed on day 28. (A) The amount of collagen in the lungs was quantified using hydroxyproline assay (PBS, N = 12; 1.0 U/kg, N = 9; 10 U/kg, N = 5; 60 U/kg, N = 5; 110 U/kg, N = 5). (B) Representative images of hematoxylin and eosin (H&E) (upper) and Masson trichrome (MT) (lower) stained sections of control and BLM–treated lung tissues (original magnification ×40). (C) Dermal thickness was measured in dorsal mouse skin (PBS, N = 16; 1.0 U/kg, N = 5; 10 U/kg, N = 4; 60 U/kg, N = 5; 110 U/kg, N = 5). (D) Representative images of H&E stained sections of BLM–treated skin tissues (original magnification ×40).

Fig 2. The effect of low dose BLM on dermal and pulmonary fibrosis.

Mice treated with BLM (1 U/kg) were sacrificed on days 7 (N = 7), 10 (N = 4), 14 (N = 4), 21 (N = 4), and 28 (N = 4). (A) Hydroxyproline levels were measured in lungs using hydroxyproline assay. (B) Dermal thickness was measured in dorsal skin. (C) Subcutaneous adipose layer thickness was measured in dorsal skin.

Fig 3. The effects of high dose BLM on dermal fibrosis.

Mice treated with BLM (60 U/kg) were sacrificed on days 7 (N = 7), 10 (N = 12), 14 (N = 7), 21 (N = 12), and 28 (N = 6). (A) Dermal thickness was measured in the dorsal skin. (B) Hydroxyproline content was measured in skin tissues shown in (A). (C) Representative H&E (left) and MT (right) images of mouse skin treated with BLM (original magnification ×40). (D) Subcutaneous adipose layer thickness was measured in the dorsal skin.

Fig 4. Expression levels of fibrosis-related genes.

Mice treated with BLM (60 U/kg) were sacrificed on days 7, 10, 14, 21, and 28. Expression levels of fibrosis-related genes were measured in skin tissues on the indicated days. (A) Coll1α1mRNA levels. (B) Fibronectin mRNA levels. (C) Ctgf mRNA levels. (D) Tgfb1 mRNA levels.

Fig 5. BLM-induced fibrosis in human skin ex vivo.

Human skin from three different donors was injected with, or immersed in, media containing BLM (1 or 10 mU/ml) and maintained in culture for one week. (A) The amount of collagen in skin injected with BLM was quantified using hydroxyproline assay. (B) Representative images of H&E (upper)–and MT (lower)–stained sections of BLM–injected human skin (original magnification ×25). (C) The amount of collagen in skin immersed in BLM was quantified using hydroxyproline assay. (D) Representative images of H&E (upper)–and MT (lower)–stained sections of BLM–immersed human skin (original magnification ×25). (E) Expression levels of fibrosis-related genes were measured in human skin treated with 10 mU/ml BLM for 48 hours.