Effect of Shock Wave Therapy Without Biological and Immuno-histochemical Analysis.
Condition: Obesity
MeSH terms: Obesity
Study type: Interventional

Summary: 
      Informative Summary: Extracorporeal shockwave therapy (ESWT) is a new method for treating
      aesthetic disorders within the field of aesthetic medicine, its mechanisms of action and
      physiological effects promise to combat localized fat accumulation, improve skin appearance,
      favor neocolagenesis and neovascularization. There are currently few studies on ESWT that can
      claim this benefit, but therapy has been shown to be safe and does not generate adverse
      reactions to individuals.

      Objective: To evaluate the physiological effects and mechanisms of ESWT acting on
      subcutaneous adipose tissue and adjacent structures through an experimental study.

      Methods: A randomized experimental clinical study will be performed in 30 women with grade I,
      II and III obesity who participated in the multidisciplinary preoperative preparation group
      for bariatric surgery with previously scheduled surgery. Participants will receive ESWT
      application within hours of surgery, or in the weeks prior to bariatric surgery, during the
      surgical procedure a small sample of subcutaneous adipose tissue will be taken for
      histological and immunohistochemical analysis.

      Hypotheses: Through histological and immunohistochemical analysis of the subcutaneous adipose
      tissue sample, the investigators can identify which real physiological effect and mechanism
      of action the ESWT has on subcutaneous adipose tissue, epidermis and adjacent structures,
      thus confirming the benefits of therapy.

      Statistical analysis: A descriptive analysis of the results obtained in the histological
      analysis will be performed initially, with frequency tables for categorical and descriptive
      variables (mean, standard deviation, median, minimum and maximum values) for continuous or
      numerical variables. In order to compare the main variables between groups and session
      number, the analysis of variance (ANOVA) will be used for repeated measures. For comparison
      between groups the Tukey test will be used. The significance level adopted for the
      statistical tests will be 5% or p <0.05.
    

Detailed description: 
      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
    

Intervention name(s): shock wave therapy
Intervention type(s): Device
Intervention description(s): The participants received the treatment protocol established by the manufacturer of the Thork Shock Wave® device, IBRAMED- Industria Brasileira de Equipamentos Electromédicos, Amparo, São Paulo approved by Anvisa No. 10360310036. (Figure 1). The parameters were, 4,000 thousand shots with energy of 180mJ and frequency of 15Hz with 15mm stainless steel tip and 2,000 shots with energy of 100mJ and frequency of 15Hz with 15mm plastic tip, and for the tip slip, Neutral Thork® Lotion, RMC was used. Seven ESWT sessions were held, the average time of each was seven minutes, twice a week and the seventh session was held minutes before the surgical procedure of bariatric surgery, therapy was performed in an area of 150cm² on the left side of the abdominal region following the midline. The right side of the participants' abdomen did not receive ESWT and was termed as control.
Allocation: Non-Randomized
Intervention model: Single Group Assignment
Intervention model description: experimental study
Masking: None (Open Label)
Primary purpose: Basic Science
Keywords: fat localized; sagging skin; Obesity