Animal studies

Animal studies:

Three different mouse models were employed: bleomycin-induced skin fibrosis, tight skin-1 mice (Tsk-1) and fibrosis induced by injections of replication-deficient type 5 adenoviruses encoding for a constitutively active TGFβ receptor I (TBR) construct (1, 2). For bleomycin-induced skin fibrosis, 6-week-old mice received repeated subcutaneous injections of bleomycin (100µl) at a concentration of 0.5 mg/ml in defined areas of 1 cm² at the upper back every other day for four weeks. Mice injected with equal volumes of 0.9 % sodium chloride served as controls. Tsk-1 mice are a genetic model of skin fibrosis with spontaneous progressive accumulation of extracellular matrix in the hypodermal layer in the skin (3, 4). Tsk-1 mice were analysed at an age of ten weeks. 

For TBR induced fibrosis, 4-week-old mice received of 6.67 x 10⁷ pfu per mouse of replication-deficient type 5 adenoviruses encoding for TBR into defined areas of 1 cm² at the upper back four times per two months. Mice injected with LacZ-expressing viruses served as controls. 

The dermal layer of the skin thickens in mice challenged with bleomycin and in mice overexpressing TBR, but does not significantly change in Tsk-1 mice. However, in Tsk-1 mice, the hypodermal layer thickens (which is not affected in the other models). We thus quantified the dermal thickness in bleomycin- and TBR-induced fibrosis and the hypodermal thickness in Tsk-1 mice. The thickness of the dermal and the hypodermal layers were analyzed using established protocols (5-13). The dermal thickness was quantified by measuring the distance from the epidermal/dermal border to the dermal/hypodermal border at four random sites per mouse; other layers of the skin were thus not included in these measurements.

To selectively inactivate FGF9 or FGFR3 in fibroblasts, mice carrying two conditional alleles of FGF9 (Fgf9^fl/fl) (14) or FGFR3 (Fgfr3^fl/fl) (15) were crossbred with Col1a2-CreER mice to generate Fgf9^fl/fl x Col1a2-CreER or Fgfr3^fl/fl x Col1a2-CreER mice, respectively. Cre-mediated recombination was induced by repeated i.p. injections of tamoxifen (TAM) at a dose of 75mg/kg per mouse for 5 days. Control groups were injected with corn oil, the vehicle of tamoxifen. For pharmacological inhibition of FGFR3 and CREB, we administered selective FGFR3 kinase inhibitor PD173074 orally (1 mg/kg every alternate day) and selective CREB inhibitor 666-15 intraperitoneally (i.p) (5 mg/kg every alternate day) respectively. All mouse experiments were approved by the governments of Mittelfranken and/or Unterfranken.

Histologic analysis

The injected skin areas of all mice were fixed in 4% formalin and embedded in paraffin. Deparaffinized histologic sections were stained with hematoxylin and eosin or Sirius red or Masson’s trichrome and visualized by Nikon Eclipse 80i microscope (Nikon, Tokyo, Japan). The hypodermal thickness for Tsk-1 model and dermal thickness for other models was determined by hematoxylin and eosin staining images captured at ×100 microscopic magnification and analyzed at four different sites in each mouse in a blinded manner (16, 17).

All images were captured in a batch mode under the same settings using an automated digital slide scanner (Nanozoomer S60, Hamamatsu, Munich, Germany). For visualization of collagen content, trichrome staining was performed and captured at ×200 microscopic magnification. 

For determining myofibroblast numbers in skin of mice, deparaffinized histologic sections were stained overnight with primary antibody against αSMA (Sigma Aldrich) followed by incubation with 3 % H₂O₂ for 10 minutes to block endogenous peroxidase activity. Species-specific, horse-radish peroxidase-conjugated antibody (Dako) and 3,3′-Diaminobenzidine (DAB) (Sigma Aldrich) substrate was used  for visualization. The number of αSMA –positive stellate-shaped myofibroblasts was determined at 200-fold magnification in atleast four different sections at ×200 microscopic magnification from each mouse by two blinded examiners (17).

Hydroxyproline content in lesional skin biopsies (3mm) were analyzed as described previously (17). After digestion of the biopsies in 6 M HCl for 3 hours at 120 °C, the pH of the samples was adjusted to 7 with 6 M NaOH and mixed with 0.06 M chloramine T for 20 min at room temperature. Next, 3.15 M perchloric acid and 20 % p-dimethylaminobenzaldehyde were added and samples were incubated for additional 20 minutes at 60 °C. The absorbance was determined with SpectraMax 190 microplate spectrophotometer (Molecular Devices) at a wavelength of 557 nm.

Reference:

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