2.8. Histopathological Evaluation

The end of the experiment occurred on the fourteenth day after the surgical specimen was obtained. The burned area of each animal was measured by the previously mentioned method. During the burn healing process, a skin fragment was carefully removed from each animal using a no. 15 knife blade to cut the skin and perform the necropsy excision. The margins were demarcated using the square ceramic pattern with a 220 mm extension on each side.

The specimens were placed in flasks containing 10% buffered formalin (Biotec™, Pinhais, Brazil) for 48 hours. Afterward, the fragments were transferred to properly identified cassettes and sent to the Laboratory of Experimental Pathology. The tissue repair process was evaluated through histological sections stained with hematoxylin & eosin (HE), Masson's trichrome (EasyPath, São Paulo, Brazil), Picrosirius-hematoxylin (EasyPath, São Paulo, Brazil), and LYVE-1 polyclonal antibody (Bioss Antibodies Inc., Woburn, USA). The slides were scanned in the Axio Scan.Z1 equipment (Zeiss, Oberkochen, Germany) with a 20x objective, and the skin slices were analyzed in the region adjacent to the wound using the ZEN Lite software (Zeiss, Oberkochen, Germany).

By H&E staining, we evaluated the presence and distribution of inflammatory infiltrate, blood vessels, and lymphatic vessels in the lesion area, by morphological criteria. The total inflammatory cells were quantified in the central burn area in three areas: area one, which represents the borders between the epidermis and the first portion of the dermis, area two, which refers to the central region of the dermis, and area three, which constitutes the hypodermis. We use as a resource a ZEN Lite software selection tool, with a scale bar of 20 μm, to aid in the counting of the inflammatory infiltrate. The total cell number of each animal was used as a comparison parameter between the study groups. The blood vessels were quantified in ten consecutive fields in the region of the superficial dermis, middle and lower, in the central area of the burn. Lymphatic vessels and small caliber veins have similarities. However, in order to differentiate them, we used as reference the endothelial cell shape and the distance between the nuclei [11]. We use as a resource an area selection tool in ZEN Lite software, with a scale bar of 50 μm, to aid in vessel counting. The mean blood and lymphatic vessels of each animal were used as a comparison parameter between the study groups.

Using Masson's trichrome technique, it was possible to verify the collagen production and distribution in the region of the burned skin. Histological sections were also scanned in the Axio Scan.Z1 equipment with a 20x objective and analyzed in the region adjacent to the wound using the ZEN Lite software, and collagen was quantified with the Image Pro Plus 5.1 program (Media Cybernetics, Rockville, USA).

The staining with Picrosirius-hematoxylin determined the presence of type I and III collagen in the burned tissue matrix. For this purpose, the skin sections were evaluated in an optical microscope under polarized light (Axio Scope.A1, Zeiss, Oberkochen, Germany). Type I collagen fibers showed a red-orange coloration, while type III collagen showed a greenish coloration. Collagen fibers were quantified in the Image Pro Plus 5.1 program (Media Cybernetics, Rockville, USA) to determine the proportion of collagen types I and III in the burn region.

Lymphatic vessels quantified by H&E were subsequently qualitative identified by immunofluorescence with LYVE-1 polyclonal antibody (Bioss Antibodies Inc., Woburn, USA) conjugated to the ALEXA fluorescent molecule FLUOR™ 350.