Evaluation of cell proliferation and apoptosis markers as predictive factors for electrochemotherapy in cutaneous squamous cell carcinoma of cats

ABSTRACT: Determining cell proliferation rates and tumor apoptosis through immunohistochemistry allows the evaluation of the biological behavior of the tumor, optimizing the patient’s clinical course. This study aimed to analyze the immunohistochemical expression of Ki-67, COX-2 and caspase-3 and correlate them with the type of response to ECT in feline cutaneous squamous cell carcinoma (SCC), thus determining the predictive potential of these variables. For this, 13 samples of feline cutaneous SCC were evaluated before ECT, and statistical analyses of the correlation intensity between the variables were performed using the Spearman correlation coefficient, with a significance level of 95%. The results indicate a significant negative correlation between histopathological grade and response to ECT (ρ=-0.6; p=0.03); there was no significant correlation between Ki-67, COX-2 and caspase-3 immunoexpression with the response to ECT (ρ=-0.18; p=0.54/ρ=-0.23; p=0.44/ρ=-0.12; p=0.69, respectively). Therefore, the study shows that the histopathological grade, tumor size and staging, degree of cellular pleomorphism and degree of inflammatory infiltrate can be considered negative prognostic factors for cutaneous SCC and negative predictors for response to ECT. However, the markers Ki-67, COX-2 and caspase-3 are not considered predictive factors for the type of response to ECT. In addition, no relationship between these immunoexpressions and greater tumor aggressiveness was observed. The SCCs evaluated in this study showed significant COX-2 labeling, indicating a potential therapeutic target. ECT has been shown to be safe and effective for local control of feline cutaneous SCC but with reduced effectiveness in larger and invasive lesions

Retalho de padrão axial ilíaco circunflexo empregado após ressecção de hemangiopericitoma em cão - relato de caso

Os hemangiopericitomas fazem parte dos sarcomas de tecido mole, e correspondem a 14% das neoplasias mesenquimais em cães e gatos. Apresenta etiologia desconhecida, crescimento lento, aspecto infiltrativo e não metastático. Acometem principalmente animais de raças grandes, de meia idade a idosos e não apresenta predileção por raça e sexo. Os tratamentos mais indicados são a amputação, ressecção local associada à radioterapia e cirurgias reconstrutivas. O uso de técnicas cirúrgicas reconstrutivas, permite que o animal retorne sua rotina normal com maior rapidez e com resultados estéticos satisfatórios. O presente trabalho tem como objetivo relatar o emprego de retalho de padrão axial da artéria ilíaca circunflexa profunda após a ressecção de hemangiopericitoma em região de membro pélvico esquerdo de um cão. Esta modalidade de tratamento empregada no presente relato, juntamente com quimioterapia mostraram resultados satisfatórios, mantendo a funcionalidade do membro e qualidade de vida da paciente. Palavras-chave: cirurgia reconstrutiva, neoplasia, quimioterapia, canino

Method to obtain platelet-rich plasma from rabbits (Oryctolagus cuniculus )

Abstract: Platelet-rich plasma (PRP) is a product easy and inxpesnsive, and stands out to for its growth factors in tissue repair. To obtain PRP, centrifugation of whole blood is made with specific time and gravitational forces. Thus, the present work aimed to study a method of double centrifugation to obtain PRP in order to evaluate the effective increase of platelet concentration in the final product, the preparation of PRP gel, and to optimize preparation time of the final sample. Fifteen female White New Zealand rabbits underwent blood sampling for the preparation of PRP. Samples were separated in two sterile tubes containing sodium citrate. Tubes were submitted to the double centrifugation protocol, with lid closed and 1600 revolutions per minute (rpm) for 10 minutes, resulting in the separation of red blood cells, plasma with platelets and leucocytes. After were opened and plasma was pipetted and transferred into another sterile tube. Plasma was centrifuged again at 2000rpm for 10 minutes; as a result it was split into two parts: on the top, consisting of platelet-poor plasma (PPP) and at the bottom of the platelet button. Part of the PPP was discarded so that only 1ml remained in the tube along with the platelet button. This material was gently agitated to promote platelets resuspension and activated when added 0.3ml of calcium gluconate, resulting in PRP gel. Double centrifugation protocol was able to make platelet concentration 3 times higher in relation to the initial blood sample. The volume of calcium gluconate used for platelet activation was 0.3ml, and was sufficient to coagulate the sample. Coagulation time ranged from 8 to 20 minutes, with an average of 17.6 minutes. Therefore, time of blood centrifugation until to obtain PRP gel took only 40 minutes. It was concluded that PRP was successfully obtained by double centrifugation protocol, which is able to increase the platelet concentration in the sample compared with whole blood, allowing its use in surgical procedures. Furthermore, the preparation time is appropriate to obtain PRP in just 40 minutes, and calcium gluconate is able to promote the activation of platelets

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

ABSTRACT: Histochemical staining consists of a set of specific chemical reactions of structures or tissue-endogenous 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 stimulants is important to facilitate the implementation of research as well as to assist interpretation of data. In rabbits some markers of angiogenesis antibodies in the skin are not standardized because of cross-reactions that may occur because the antibodies are made from such animals.The aim of this study was to analyze the immunohistochemical methods through dyes and immunohistochemical markers angiogenesis in rabbits (Oryctolagus cuniculus) having undergone reconstructive surgery with skin grafts associated with plasma 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 methodology 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 platelet-rich 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 Caveolin-1 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 Kruskal-Wallis test with p values equal to or less than 0.05 being considered significant. The immunohistochemistry was subjected to analysis of variance for a completely randomized design, with two groups and five repetitions (medium) and 5% significance level. Multiple comparison of groups resulted in the Tukey test (p=0.05) used. The amount of vascular proliferation assessed by histochemical method HE and Masson’s Trichrome was found to be a significant variable in Gprp when compared with group Gc. When evaluating the methods used, there was no significant difference. There was no difference in the three markers which were used for correlating microvessels; however, there was more intense staining of vessels when Caveolin-1 Antibody was used. This caused intense marking of the capillaries and small vessels, as well as of larger vessels. When using CD31 and CD34, the same was observed, but it was not as intense as with Caveolin-1; though some cases showed sincere and discreet marking. The results of this study demonstrated that the histochemical methods performed are effective for semi-quantitative assessment of angiogenesis. The immunohistochemical comparison of Caveolin-1, CD31, and CD34 as markers of angiogenesis in rabbits showed that both antibodies could immunostain the newly formed vessels; but the Caveolin-1 showed better immunostaining in small and medium-sized vessels, as well as a minor presence in the background. Although not specific markers for angiogenesis, they can be used as immunohistochemical markers of vascular endothelium in rabbits

Method to obtain platelet-rich plasma from rabbits (Oryctolagus cuniculus )

Abstract: Platelet-rich plasma (PRP) is a product easy and inxpesnsive, and stands out to for its growth factors in tissue repair. To obtain PRP, centrifugation of whole blood is made with specific time and gravitational forces. Thus, the present work aimed to study a method of double centrifugation to obtain PRP in order to evaluate the effective increase of platelet concentration in the final product, the preparation of PRP gel, and to optimize preparation time of the final sample. Fifteen female White New Zealand rabbits underwent blood sampling for the preparation of PRP. Samples were separated in two sterile tubes containing sodium citrate. Tubes were submitted to the double centrifugation protocol, with lid closed and 1600 revolutions per minute (rpm) for 10 minutes, resulting in the separation of red blood cells, plasma with platelets and leucocytes. After were opened and plasma was pipetted and transferred into another sterile tube. Plasma was centrifuged again at 2000rpm for 10 minutes; as a result it was split into two parts: on the top, consisting of platelet-poor plasma (PPP) and at the bottom of the platelet button. Part of the PPP was discarded so that only 1ml remained in the tube along with the platelet button. This material was gently agitated to promote platelets resuspension and activated when added 0.3ml of calcium gluconate, resulting in PRP gel. Double centrifugation protocol was able to make platelet concentration 3 times higher in relation to the initial blood sample. The volume of calcium gluconate used for platelet activation was 0.3ml, and was sufficient to coagulate the sample. Coagulation time ranged from 8 to 20 minutes, with an average of 17.6 minutes. Therefore, time of blood centrifugation until to obtain PRP gel took only 40 minutes. It was concluded that PRP was successfully obtained by double centrifugation protocol, which is able to increase the platelet concentration in the sample compared with whole blood, allowing its use in surgical procedures. Furthermore, the preparation time is appropriate to obtain PRP in just 40 minutes, and calcium gluconate is able to promote the activation of platelets

Utilização de plasma rico em plaquetas para estimulação da angiogênese em flape de padrão axial toracodorsal em coelhos (Oryctolagus cuniculus)

Resumo: Feridas de grandes extensões, com perda da viabilidade tecidual e retardo na cicatrização por segunda intenção são casos que se faz necessário o emprego de técnicas cirúrgicas reconstrutivas. O plasma rico em plaquetas (PRP) é um produto com maior concentração plaquetária, adjuvante no processo cicatricial de cirurgias reconstrutivas, auxiliando nos processos de hemostasia e estimulação da angiogênese. Dessa forma, delineou-se um estudo a fim de avaliar a eficácia do uso do gel produzido a partir do plasma rico em plaquetas (PRP) em flapes de avanço de padrão axial toracodorsal em coelhos, para avaliar a possibilidade de favorecer a integração do retalho no leito receptor. Utilizaram-se 30 coelhos da raça Nova Zelândia branco, separados em dois grupos de 15 animais, compreendendo os grupos plasma rico em plaquetas (GPRP), na qual empregou-se o gel antes da síntese da ferida cirúrgica, e controle (GC), na qual utilizou-se apenas solução fisiológica. Para obtenção do PRP, coletou-se sangue dos animais, e determinou-se a contagem plaquetária antes da preparação do gel. No início e término do experimento os animais foram pesados para posterior análise de ganho peso médio. Após o procedimento cirúrgico iniciou-se as avaliações macroscópicas no 3º, 7º e 14º dia, e avaliou-se presença ou ausência de exsudato, integridade da pele, edema, rubor e necrose. Após esta etapa, coletou-se o material da ferida cirúrgica para confecção das lâminas histológicas e posterior avaliação microscópica. Avaliou-se a proliferação vascular, presença de células mononucleares e polimorfonucleares, proliferação fibroblástica, colagenização, reepitelização e hemorragia. Os dados obtidos foram submetidos à análise estatística (Teste t Student, t emparalhado, e Kruskall Walis, sendo p<0,05). O ganho de peso médio não foi significativo entre os grupos; a concentração plaquetária da amostra final do PRP foi significativamente maior quando comparada com a inicial; exsudato e necrose foram significativamente maior no grupo controle quando comparado ao grupo PRP; proliferação vascular e reepitelização foram significativamente maior no grupo PRP, enquanto que a presença de mononucleares, polimorfonucleares, proliferação de fibroblastos, colagenização e hemorragia não foram significativas entre os grupos. Obteve-se no terceiro dia, diferença significativamente maior quanto à variável exsudato no grupo controle quando comparado ao grupo PRP, no sétimo dia exsudato e rubor foram significativamente maior no grupo controle, e ao décimo quarto dia exsudato e integridade da pele foram significativamente maior no grupo controle. Os resultados obtidos neste estudo evidenciaram que a utilização do plasma rico em plaquetas na forma de gel em cirurgia reconstrutiva foi capaz de estimular a angiogênese na ferida favorecendo o processo de cicatrização

Effects of canine adipose-derived mesenchymal stem cells on the epithelialization of rabbits’ skin autograft (Oryctolagus cuniculus)

ABSTRACT: The present study aimed to evaluate the effects of mesenchymal stem cells derived from canine adipose tissue in the healing process of full-thickness mesh skin grafts in rabbits. The stem cells were collected from young dogs; and, after characterization, remained in cryopreservation, in independent doses containing 2 x 106 cells. The mesh distal limb graft technique was performed in 60 rabbits, divided into three groups, CG (Control Group), GT1 (Intralesional Stem Cell Treated Group), and GT2 (Intravenous Stem Cell Treated Group), containing 20 animals each. After grafting, each group was randomly divided into four subgroups according to euthanasia time 3, 7, 14, and 30 days, containing five animals in each group. Animals of GT1_14, GT1_30, and GT2_14, GT2_30 subgroups received a second dose of xenogeneic cells on the seventh day. Meanwhile, animals from GT1_30 and GT2_30 received the third dose of xenogeneic cells on day 14. The groups treated with xenogeneic stem cells positively affected type III collagen re-epithelialization and deposition, and possibly GT1 had a controlled inflammatory response. However, no effect on angiogenesis. Thus, it was possible to demonstrate tolerance and therapeutic action of mesenchymal stem cells from canine adipose tissue in skin grafts in rabbits