Modeling pulmonary fibrosis through bleomycin delivered by osmotic minipump: a new histomorphometric method of evaluation

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.

Pharmacological validation of SSc-ILD mouse model bleomycin-induced by osmotic minipump

“…Even though Hyp is commonly considered an important readout, in our drug screening experiments concerning BLM-induced lung brosis models, we always observed high variability and poor inter-experiments reproducibility. As previously reported, the brotic lesions, mainly localized in the subpleural area, could be underestimated if evaluated over the whole parenchyma 29 . Similarly, the quanti cation of Hyp could be affected by the size of the sampling site.…”

“…Their tissue inhibitor, TIMP-1, is expressed by the interstitial cells in the brotic areas during wound healing 35 . TIMP-1 levels remain high in the BALF up to day 21 29 , corresponding to the peak of activated macrophages in the alveolar spaces, but decrease after 28 days, probably caused by the progressive depletion of cellular reserves of the inhibitor. For this reason, it is acceptable that NINT may not have any effect upon it.…”

“…In a recently published paper, an experimental model of human SSc-ILD in C57BL/6 mice has been described. This model involved a continuous and controlled release of BLM by subcutaneously implanted osmotic mini-pumps 15 . Before using a new animal model in drug discovery, its reproducibility, robustness and ability to respond to pharmacological treatments should be validated.…”

“…A model inducing subacute -chronic brosis through the release of BLM by osmotic mini-pumps allows evaluation about the effects of anti brotic compounds, either in the skin or in the lungs, since the target brotic lesions have been identi ed in both organs [15][16][17] .…”

Extracellular CIRP Induces an Inflammatory Phenotype in Pulmonary Fibroblasts via TLR4

“…The tissues were collected on 7, 14, 21, 28, and 42 days after the first injection (51). While the exact timing of peak inflammation and fibrosis in the lung, in this model, is not clear, based on previous studies, inflammation occurs between days 7 and 14, and peak fibrosis likely happens between days 21 and 28 (52)(53)(54). Day 14, which we call the inflammatory-to-fibrotic transition phase of exposure to bleomycin, is a time when both processes are active.…”