2.6. Induction and Treatment of Experimental Scleroderma

Skin fibrosis was induced in female C57BL/6 mice (6–8 weeks-old) under slight anesthesia (induced with 4% isoflurane) by repetitive intradermal (i.d.) injections of bleomycin (3.2 U per kg mouse, in 100 µL of saline, three times per week, for four weeks) in a localized place in the right side of saved back of mice as previously described [19]. Moreover, saline (in 100 µL volume) was repetitively injected i.d. in a localized point in the left side of the mouse back, contralateral to bleomycin-induced lesion (used as basal control of reference). Treatment consisted in subcutaneous (s.c.) injections of 100 µL of culture supernatants of cells transfected with either pLAP (without cortistatin) or pLatent-CST (containing 0.3 pmol of cortistatin-29 in latent form), every seven days, for four weeks, starting five days after the first bleomycin injection, around the bleomycin-induced skin lesion. We used as reference controls of treatment, animals that were s.c. injected with 100 µL of PBS (untreated, vehicle) or cortistatin-29 (1 nmol), three times per week, for four weeks, starting five days after the first bleomycin injection, around the bleomycin-induced skin lesion. Four weeks after the first bleomycin injection, animals were sacrificed and skin (0.7 cm² of the bleomycin-induced lesion and of the saline-injected contralateral site) and lungs were dissected, fixed in 10% buffered formalin, embedded in paraffin and sectioned. Skin and lung cross-sections (5 µm) were stained with Masson’s trichrome using standard technique and images were acquired in an Axio Scope A1 microscope using 5X objective and 10X ocular and analyzed with Zen 2011 Light Edition software in a blinded manner by two independent researchers/pathologists as previously described [19]. Skin fibrosis was determined in at least three sections per biopsy by measuring dermal thickness (distance in µm from the base membrane of epidermal layer to the hypodermal junction with subcutaneous fat, determined as the mean of three random measurements in each section) using the Fiji-ImageJ software (http://imagej.net/Fiji, accessed on 30 May 2017). Lung fibrosis was scored in whole lung sections according to a semi-quantitative scale (0 to 4) that evaluates alveolar thickness, damage of lung structure, and fibrosis extension as previously described [19]: 0, normal lung or minimal fibrous thickening of alveolar or bronchial walls; 1, moderate thickening of the wall, with less than 25% of fibrotic area, but without obvious damage to lung architecture; 2, formation of fibrous bands, fibrous masses in 25–50% of lung area, and definitive damage of lung structure; 3, severe distortion of the structure and large fibrous areas (>50% of the cross-section involved); 4, total fibrous obliteration of the field. Results show the mean value of at least three no overlapping randomly selected areas per lung section (with 5X objective) and three representative sections per mouse (discarding at least 200 μm between sections).