Histochemical and immunohistochemical evaluation of angiogenesis in rabbits (Oryctolagus cuniculus) submitted to skin grafts associated with platelet-rich plasma

Abstract

Pazzini J.M., Serafim E.L., Gärtner F., Amorim I., Faria F., Rêma A., Moraes P.C. & De Nardi A.B. 2017. Histochemical and immunohistochemical evaluation of angiogenesis in rabbits (Oryctolagus cuniculus) submitted to skin grafts associated with platelet-rich plasma. Pesquisa Veterinária Brasileira 37(12):1519-1525. Histochemical staining consists of a set of specific chemical reactions of structures or tissueendogenous substances. Immunohistochemistry allows verification of proteins in tissues related to biological and pathological factors. The standardization of methods to assess angiogenesis resulting from formation of new blood vessels in procedures with sti mulants is important to facilitate the implementation of research as well as to assist inter pretation of data. In rabbits some markers of angiogenesis antibodies in the skin are not standardized because of crossreactions that may occur because the antibodies are made from such animals.The aim of this study was to analyze the immunohistochemical metho ds through dyes and immunohistochemical markers angiogenesis in rabbits (Oryctolagus cuniculus) having undergone reconstructive surgery with skin grafts associated with plas ma angiogenesis stimulator rich in platelets, in order to evaluate which method would be better to visualize the vessels, as well as to evaluate which antibody would promote better immunostaining, and find the differences between the methods and to standardize the me thodology to be applied in experiments using rabbits. Sixteen rabbits were used, split into two groups of eight animals: Gprp (plasma rich in platelets) and Gc (control, saline solution, 9%). The same technique of reconstructive surgery using graft mesh was performed on each rabbit. The groups differed only in the application of plateletrich plasma before the surgical wound synthesis. Samples for evaluation of angiogenesis were collected 15 days after the surgical procedure. The dyes Hematoxylin & Eosin and Masson's Trichrome were used in the histochemical study to evaluate vascular proliferation. Markers CD31, CD34 and Caveolin1 was used for the immunohistochemical study. The evaluation between the groups (Gprp and Gc) in regard to the categorical variable (vascular proliferation intensity) used the KruskalWallis test with p values equal to or less than 0.05 being considered sig nificant. The immunohistochemistry was subjected to analysis of variance for a completely randomized design, with two groups and five repetitions (medium) and 5% significance 1

Reparação do retalho axial dorsal em ratos tratados com óleo ozonizado

“…A quantificação de vasos foi determinada pela técnica de contagem de microvasos, sendo considerado um vaso unitário qualquer grupo de células endoteliais, assim como vasos contendo hemácias em seu lúmen. A contagem foi realizada em cinco campos, previamente selecionados pela maior densidade vascular na profundidade da lesão com microscópio ajustado para uma ampliação de 400x e expresso em um valor médio de vasos para cada caso estudado semelhante ao estudo realizado por Pazzini et al (2017).…”

“…A contagem de microvasos para a determinação do índice de angiogênese foi determinada pelo método quantitativo, seguindo padronização proposta por Pazzini et al (2017). Observou-se que o grupo GO apresentou porcentagem média de vasos maior que o grupo controle o que corrobora com o trabalho Kirkil et al (2016), comprovando efeitos benéficos do óleo ozonizado para a neovascularização e consequentemente melhor reparo de feridas dérmicas em ratos.…”

“…A maior reepitelização observada no GO, permitiu considerar o óleo de girassol ozonizado como fator positivo para a reepitelização tecidual, sendo esse achado condizente com Pazzini et al (2016), que demonstrou que a aplicação de plasma rico em plaquetas promoveu maior angiogênese e consequente maior reepitelização em enxertos cutâneos.…”

Skin graft associated with platelet-rich plasma in correcting extensive injuries resulting from the resection of skin cancer chemically induced in rats

“…The presence of mononucleates, polymorphonucleated cells, proliferation of fibroblasts, haemorrhage and necrosis was evaluated 26 . In addition, differentiation of the epithelium was evaluated by using an immunohistochemical method 27 , and CD31 proliferation 28 was evaluated by using the monoclonal antibody Ae1/Ae3.…”

“…The angiogenic index for CD31 was determined by the microvascular counting technique, as recommended by the literature 28 , 29 . The vessels were counted in five fields, which were previously selected, with high-vascular density at x400 magnification using an optical light microscope.…”