Hepatic fibropoiesis in dogs naturally infected with Leishmania (Leishmania) infantum treated with liposome-encapsulated meglumine antimoniate and allopurinol.

Hepatic fibropoiesis in canine visceral leishmaniasis (CVL) were evaluated by histological (morphometrical collagen deposition) and immunohistochemical assays characterizing alpha-actin (α-SMA), vimentin, calprotectin (L1 antigen), and TGF-ß in 46 naturally infected dogs with Leishmania infantum treated with liposome-encapsulated meglumine antimoniate and allopurinol separately and in combination. Six treatment groups were defined: meglumine antimoniate encapsulated in nanometric liposomes (LMA), allopurinol (ALLOP); liposome-encapsulated meglumine antomoniate combined with allopurinol (LMA+ALLOP); empty liposomes (LEMP); empty liposomes combined with allopurinol (LEMP+ALLOP) and saline. Relative liver weight was lower in LMA, LMA+ALLOP, and ALLOP groups compared to the LEMP control. Significantly lower granulomatous chronic inflammatory reaction was seen in the ALLOP group compared to a control group. Calprotectin was lowest in liver of those dogs showing lower numbers of intralobular hepatic granulomas. Collagen deposits were significantly higher in LMA compared to ALLOP, LEMP+ALLOP, and Saline groups. LMA+ALLOP group collagen deposition was higher than dogs treated only with allopurinol. Immunohistochemical analysis showed significant higher α-SMA in hepatic stellate cells (HSCs), hepatic perisinusoidal cells, in control groups than LMA+ALLOP and LEMP+ALLOP. Alpha-actin and Vimentin positive cells were diffusely distributed throughout the liver parenchyma in the hepatic lobule, mainly in HSCs. Vimentin expression was significantly higher in the saline group than in the ALLOP group. Our data suggest that allopurinol inhibits HSC and results in lower collagen deposits in liver during CVL progression, as supported by the significantly lower expression of TGF-ß in the ALLOP group compared to other groups. Results demonstrated that treatment with allopurinol inhibited chronic granulomatous inflammatory reaction and hepatic fibrosis in CVL.

Avaliação de diferentes fixadores na preservação das características histológicas de pele de orelha de cães / Evaluation of different fixatives for histological studies in canine ear skin

Os fixadores biológicos desempenham um papel importante na qualidade final da histologia. Na rotina veterinária, a biópsia de pele é um procedimento comum e a escolha do fixador é primordial para resultado final adequado. Os fixadores mais usados são à base de formalina, ainda que sejam tóxicos, cancerígenos, de baixa penetração e de fixação lenta. Mesmo assim, não existe um fixador ideal que substitua as suas qualidades. O objetivo deste trabalho foi avaliar qualitativamente a preservação das características histológicas de pele de cão utilizando diferentes fixadores de tecidos incluídos em parafina, cortados e corados pela hematoxilina-eosina. Utilizou-se uma caneta Punch de 4 milímetros para coletar amostras de pele de orelha em seis cadáveres de cães. Após coleta, os tecidos foram fixados em: (1) Bouin, durante seis horas; (2) Carnoy, durante quatro horas; (3) formaldeído tamponado 10% durante 24 horas, todos sob refrigeração (4ºC). Posteriormente, os tecidos foram processados, cortados e corados em hematoxilina e eosina. As lâminas foram avaliadas, às cegas, por quatro patologistas diferentes, que consideraram aspectos qualitativos a seguir: (1) qualidade da coloração; (2) preservação das características histológicas; e (3) preservação dos limites citoplasmáticos utilizando a escala de LIKERT de pontuação para cada lâmina. O fixador com a maior média de pontuação em todos os itens foi o formol tamponado com 3,76 pontos, seguido pelo Bouin (3,39) e pelo Carnoy (2,52). O formol pode trazer riscos à saúde do profissional que rotineiramente o manuseia, portanto se faz necessária a busca por fixadores com as mesmas qualidades, mas menos nocivos à saúde.(AU)

The biological fixatives have an important role in the final histology quality. In veterinary, routine skin biopsy is a common procedure and the choice of fixative is essential for the final result. The most common fixative is Formalin, even though it is toxic, carcinogenic, and has low and slow penetration. Still, there isn't a fixer which can replace the qualities of formalin. The aim of this study was to evaluate qualitatively the preservation of the histological features of dog skin using different tissue fixative embedded in paraffin, sectioned and stained with hematoxylin - eosin. We used a 4 mm punch pen to collect ear skin samples in six dog cadavers. After collection, the tissues were fixed in: (1) Bouin for 6 hours; (2) Carnoy for 4 hours; (3) 10% buffered formaldehyde for 24 hours, all under refrigeration (4 ° C). The tissues were then processed, sectioned and stained with hematoxylin and eosin. The slides were evaluated blindly by four different pathologists who considered the qualitative aspects below: (1) quality of coloring; (2) preservation of the histological characteristics; (3) preservation of cytoplasmic boundaries using a Likert scale score for each blade. The fixative with the highest mean score on all items was buffered formalin with 3.76 points followed by Bouin (3.39) and Carnoy (2.52). Formaldehyde can bring health a risk of professional routine handling, so it is necessary to search for a biological fixative with the same qualities being less harmful to health.(AU)

Glycol methacrylate embedding for the histochemical study of the gastrointestinal tract of dogs naturally infected with Leishmania infantum.

In canine visceral leishmaniasis a diffuse chronic inflammatory exudate and an intense parasite load throughout the gastrointestinal tract has been previously reported. However, these studies did not allow a properly description of canine cellular morphology details. The aim of our study was to better characterize these cells in carrying out a qualitative and quantitative histological study in the gastrointestinal tract of dogs naturally infected with Leishmania infantum by examining gut tissues embedded in glycol methacrylate. Twelve infected adult dogs were classified in asymptomatic and symptomatic. Five uninfected dogs were used as controls. After necropsy, three samples of each gut segment, including esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, and rectum were collected and fixed in Carnoy's solution for glycol methacrylate protocols. Sections were stained with hematoxylin-eosin, toluidine blue borate, and periodic acid-Schiff stain. Leishmania amastigotes were detected by immunohistochemistry employed in both glycol methacrylate and paraffin embedded tissues. The quantitative histological analysis showed higher numbers of plasma cells, lymphocytes and macrophages in lamina propria of all segments of GIT of infected dogs than controls. The parasite load was more intense and cecum and colon, independently of the clinical status of these dogs. Importantly, glycol methacrylate embedded tissue stained with toluidine blue borate clearly revealed mast cell morphology, even after mast cell degranulation. Infected dogs showed lower numbers of mast cells in all gut segments than did controls. Despite the glycol methacrylate (GMA) protocol requires more attention and care than the conventional paraffin processing, this embedding procedure proved to be especially suitable for the present histological study, where it allowed to preserve and observe cell morphology in fine detail.