Although increasing used in the preclinical testing of new anti-fibrotic drugs, a thorough validation of micro-computed tomography (CT) in pulmonary fibrosis models has not been performed. Moreover, no attempts have been made so far to define density thresholds to discriminate between aeration levels in lung parenchyma. In the present study, a histogram-based analysis was performed in a mouse model of bleomycin (BLM)-induced pulmonary fibrosis by micro-CT, evaluating longitudinal density changes from 7 to 21 days after BLM challenge, a period representing the progression of fibrosis. Two discriminative densitometric indices (i.e. 40th and 70th percentiles) were extracted from Hounsfield Unit density distributions and selected for lung fibrosis staging. The strong correlation with histological findings (rSpearman = 0.76, p < 0.01) confirmed that variations in 70th percentile could reflect a pathological lung condition and estimate the effect of antifibrotic treatments. This index was therefore used to define lung aeration levels in mice distinguishing in hyper-inflated, normo-, hypo- and non-aerated pulmonary compartments. A retrospective analysis performed on a large cohort of mice confirmed the correlation between the proposed preclinical density thresholds and the histological outcomes (rSpearman = 0.6, p < 0.01), strengthening their suitability for tracking disease progression and evaluating antifibrotic drug candidates.

No 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.

No abstract

Melatonin exerts well-known reproductive effects, mainly acting on hypothalamic gonadotrophin-releasing hormone release. More recent data suggest that melatonin acts directly at the ovarian level, even if, at present, these aspects have been only partly investigated. Swine follicular fluid contains melatonin and its concentration is significantly reduced during follicular growth. Therefore, the present study was undertaken to examine the effects of melatonin, used at physiological concentrations, on cultured swine granulosa cells collected from small (<3mm) and large (>5mm) follicles on the main parameters of granulosa cell function such as proliferation and steroidogenesis, namely oestradiol 17β and progesterone (P4) production. Moreover, the effects of melatonin on superoxide anion and nitric oxide (NO) generation by swine granulosa cells were also investigated. Finally, since angiogenesis is crucial for follicle growth, the effects of melatonin on new vessel growth were studied. Collected data indicate that melatonin interferes with cultured granulosa cell proliferation and steroidogenesis, specifically in terms of P4 production and NO output. In addition, the events of physiological follicular angiogenesis were stimulated by melatonin as evidenced by angiogenesis bioassay. Therefore, we suggest that physiological melatonin concentrations could potentially be involved in local modulation of swine ovarian follicle function.