Effect of Shock Wave Therapy Without Biological and Immuno-histochemical Analysis.

ESWT procedures The participants received seven ESWT therapy sessions twice a week, the last session being about twenty minutes before the surgical procedure of bariatric surgery, the therapy was performed in an area of 150 cm² on the left side of the abdominal region, following the white line, frequently 15Hz, with 4,000,000 shots with 180mJ energy and 15mm stainless steel tip and 2,000,000 shots with 100mJ energy with 15mm plastic tip, for the tip slip a Thork ® Essential Cosmetics Industry Lotion was used LTDA- MED Anvisa nº 25351.419510 / 2017- 95.

For therapy, the THORK Shock Wave® equipment - IBRAMED- Industria Brasileira de Equipamentos Electromédicos EIRELI, approved by Anvisa nº 10360310036, was used.

The direct side of the participants' abdomen was used as a control and did not receive ESWT therapy.

Surgical procedure

After performing the anesthetic procedure at the beginning of the surgical act of bariatric surgery, the surgeon responsible removed two fragments of cutaneous tissue with an average size of 4 cm in diameter, one sample from the region previously demarcated on the left side of the abdomen and the other sample on the side contralateral where there was no demarcation considered as control.

Histological procedure After collection, the material was stored in a container with 10% formaldehyde for 48 hours. The samples were processed histotechnically, embedded in paraffin and then cut with a rotating microtome in sections of 3-5 μm thick.

For morphological evaluation of the cutaneous tissue and collagen cells, the slides were stained with Hematoxylin and Eosin (HE) and Masson's Trichrome (ab150686, Abcam, Cambridge, United Kingdom). The slides were evaluated by light microscopy with a binocular microscope (Nikon YS 100, Japan) adapted with a WSCF 10X / 18 eyepiece and Nikon 4X / 0.10, 10X / 0.25, 40X / 0.65 and 100X / 1 lenses, 25.

To differentiate the types of collagen fibers, Picrosirius Red staining (ab150681, Abcam) was used. After staining, the slides were analyzed under polarized light with a DMR microscope (Leica) and photographs were taken at 400 × magnification, the collagen fibers were differentiated by means of their staining and the type I collagen fibers were yellow-orange in color. some cases even red and type III with green coloring, to quantify the analyzes, the ImageJ® software (NIH, Bethesda, USA) was used.

All preparations were made following the protocol of antibodies and standardized by the laboratory of pathological analysis at Hospital de Clinicas da Unicamp.

Immunohistochemical procedures To perform immunohistochemical reactions, the paraffin blocks with the skin samples were cut to a thickness of 3 μm and placed on signed slides (3-aminopropyl-triethoxysilane, Sigma-Aldrich, USA). Positive and negative controls were performed in all reactions. The reactions were carried out according to previously published protocols

Briefly, the tissues were subjected to dewaxing, rehydration and endogenous peroxidase blocking processes. Antigenic recovery was performed with a solution of ethylenediamine tetraacetic acid (EDTA), pH 9.0, in an electric pressure cooker for 15 minutes.

For immunohistochemical analysis, the following antibodies, markers of fibroblast growth factors (FGFs) and their respective receptors were used: FGF1 (Antibody sc-55520, Santa Cruz Biotechnology, USA), FGF2 (Antibody sc-365106, Santa Cruz Biotechnology), FGFR1 (Antibody sc-121, Santa Cruz Biotechnology), FGFR2 (Antibody sc-122, Santa Cruz Biotechnology) and to evaluate cell proliferation, the marker Ki67 (Antibody ab92742, Abcam) was used.

The antigen-antibody reaction was developed using the chromogenic substrate (3.3 diaminobenzidine-DAB, Sigma-Aldrich). Then the slides were scanned and digitally analyzed also according to previous protocols