Abstract
The systemic delivery of bleomycin (BLM) to mice through subcutaneously implanted osmotic minipumps may be used to experimentally mimic the typical features of systemic sclerosis and related interstitial lung diseases. The published studies on this model principally have focused on induced dermal modifications, probably because lung lesions are typically mild, subpleurally localized, and difficult to analyze. The use of high BLM doses to increase their severity has been proposed but is ethically questionable because of the compromising of animal welfare. We propose a tailored histomorphometric method suitable to detect and quantify this type of mild lung lesions. Using a two-step automated image analysis, a peripheral region of interest with a depth of 250 µm from the pleural edge was defined on whole slide images, and the fibrotic foci were histomorphometrically characterized. The effects of different BLM doses on lung alterations were evaluated in C57BL/6 mice and 60 U/kg resulted in a fair compromise between fibrotic lesions and animal welfare. This dose was also tested in time course experiments. The analysis revealed a peak of histological fibrotic-like alterations, cytokine expression, metalloprotease, and macrophagic activation between the 21st and 28th day after pump implant. The induced dermal fibrosis was characterized by the progressive loss of the white dermal adipose layer, an increase in dermal thickness, dermal hyperplasia, and more compacted collagen fibers. Despite the trend toward spontaneous resolution, our model allowed a double organ readout of the BLM effect and the identification of a therapeutic window for testing pharmacological compounds without using life-threatening doses.
Highlights
Systemic sclerosis or scleroderma (SSc) is a systemic inflammatory disorder of uncertain origin characterized by vascular damage, autoimmunity, and fibrosis of the skin, joints, and internal organs
The present study describes a murine model usually used for SSc and related ILD, in which BLM is administrated through
Very few studies had already proposed a similar approach [6, 12, 13, 28] but assessed lung fibrosis using conventional histological methods, which are not able to discriminate slight pulmonary structural changes caused by different BLM concentrations or which arise during time
Summary
Systemic sclerosis or scleroderma (SSc) is a systemic inflammatory disorder of uncertain origin characterized by vascular damage, autoimmunity, and fibrosis of the skin, joints, and internal organs. One of the most widely reported complications arising in scleroderma patients, frequently leading to death, is related interstitial lung disease (SSc-ILD) [26]. ILD consists of a group of chronic and progressive disorders in which the pulmonary parenchyma can show varying degrees of fibrosis and inflammation with consequent gas exchange impairment. A predominance of fibrosis indicates an advanced stage of the disease, which becomes refractory to treatments, rendering patients with poor prognoses [3, 7]. It is imperative to find highly relevant animal models that are able to reproduce the chronic and progressive aspect of the disease. BLM is used in cancer chemotherapy, but it causes adverse side effects, including fibrosis in organs like lungs, in which there is a scarce presence of BLM hydrolase, the deactivating enzyme [11, 13]
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