The injured area was evaluated macroscopically after the burn and throughout the experiment considering skin color, presence of blisters and superficial crust formation. The histological sections stained with H&E were scanned using a Leica® DM 2500 microscope coupled to a Leica® DFC 425 camera and the Leica® Application Suite LAS v3.7 program. The images were obtained from the cross sections of four sequential fields of each slide, with the 10X and 40X objectives under a light microscope. To quantify the number of inflammatory cells (neutrophils and macrophages) and fibroblasts (young and adult), the images were analyzed using the ImageJ software, which allowed elaborating a grid and the individual marking of the cell nuclei with the aid of the manual counting tool.
The slides stained with Picrosirius Red were evaluated by digital image analysis to calculate the area occupied by the deposition of collagen types I and III, and photographed with the 10X objective, with a polarized light microscope (Leica® DM 2000) coupled to the camera (Leica® DFC 425). The Image-Pro Plus 4.5 program was used to quantify the percentage of type I and III collagens. When analyzed in association with polarized light, presence of collagen considered the following specifications for identification of the collagen types: collagen type I - yellow-reddish color; and collagen type III - green-whitish color. All histomorphometric analyses were performed blindly.
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