F.G.A. Santos

Determination of the minimal representative number of microscopic fields to quantify apoptosis in canine lymph nodes

Determinou-se o numero minimo de campos por lâmina para se ter uma amostragem representativa para o estudo de apoptose em linfonodos de caes com cinomose. Um linfonodo traqueobronquico de cao foi colhido e processado segundo tecnica para inclusao em parafina. Seccoes de 5µm foram coradas pelo tricromico de Shorr. Utilizou-se um analisador de imagens para registrar o numero de celulas em apoptose e os indices apoptoticos de 300 campos, na mesma lâmina. Obtiveram-se, entao, valores medios para 10 amostras de 5, 10, 15, 20, 25, 30, 35, 40, 45 e 50 campos ao acaso. Realizou-se o estudo dos desvios-padrao das medias (DP) em relacao ao tamanho da amostra que os originou. Quando as oscilacoes de DP entre amostras consecutivas foram menores que 5%, o ganho em representatividade produzido pelo acrescimo no numero de campos nao se justificou, dispensando amostragens maiores. Desse modo, a analise de 40 campos por lâmina, na pesquisa de indices apoptoticos em linfonodos caninos, e suficientemente representativa, uma vez que apresenta instabilidade aceitavel.

Apoptosis in the transplanted canine transmissible venereal tumor during growth and regression phases

ABSTRACT Twelve male, mongrel, adult dogs were subcutaneously transplanted with cells originated from two canine transmissible venereal tumors (TVT). The aim was to demonstrate and to quantify the occurrence of apoptosis in the TVT regression. After six months of transplantation, a tumor sample was obtained from each dog, being six dogs with TVT in the growing phase and six in the regression phase as verified by daily measurements. Samples were processed for histological and ultrastructural purposes as well as for DNA extraction. Sections of 4µm were stained by HE, Shorr, methyl green pyronine, Van Gieson, TUNEL reaction and immunostained for P53. The Shorr stained sections went through morphometry that demonstrated an increase of the apoptotic cells per field in the regressive tumors. It was also confirmed by transmission electron microscopy, which showed cells with typical morphology of apoptosis and by the TUNEL reaction that detected in situ the 3’OH nick end labeling mainly in the regressive tumors. The regressive TVTs also showed an intensified immunostaining for P53 besides a more intense genomic DNA fragmentation detected by the agarose gel electrophoresis. In conclusion, apoptosis has an important role in the regression of the experimental TVT in a way that is P53-dependent. Keywords: dog, venereal tumor, apoptosis, cell death

Porcentagem de parênquima e índice apoptótico da bolsa cloacal em frangos de corte em ambiente de conforto e estresse térmico

Ninety six broiler chickens were kept in climatic chambers and submitted to three different environmental temperatures 24h/day: 29oC (hot), 24oC (neutral) and 15oC (cold). Eight chickens of each temperature group were sacrificed at 1, 7, 14, 21, 28, 35 and 42 days, and the cloacal bursae (CB) were collected, processed and embedded in paraffin. Sections were stained with HE and histometric evaluation was carried out. From each section, parenchyma ratio (PR) and apoptotic index (AI) were measured. The CB of five week-old chickens kept in lower temperature showed PR lower than CB of chickens kept in neutral temperature. The CB of five week-old chickens kept in lower temperature showed an AI greater than CB of chickens submitted to neutral temperature. The present study shows that environmental temperature stress induces lymphocytes apoptosis, cloacal bursae atrophy and affects CB maturation.

Morphometric analysis of the thymus of puppies infected with the Snyder Hill Strain of canine distemper virus

The thymic morphometry analysis was used for determining apoptosis and atrophy of the thymus of eight puppies inoculated with canine distemper virus (CDV). Three healthy dogs were used as negative controls. Sections, 5µm thick, were stained by HE and Shorr, and the latter were evaluated by morphometry. CDV nucleoprotein was detected by immunohistochemistry. Morphometric results confirmed lymphoid hypotrophy in CDV inoculated dog thymuses, more stroma, less parenchyma and higher apoptotic index/field than negative control (not inoculated) puppies. Apoptosis plays a role in the mechanism of thymus atrophy that develops in canine distemper.

Cell proliferation markers in the transplanted canine transmissible venereal tumor

Caes machos, adultos, mesticos, foram transplantados com celulas do tumor venereo transmissivel canino (TVTC), na regiao hipogastrica. Foram coletados doze especimes do TVTC, sendo metade durante a fase proliferativa e metade durante a fase regressiva. Fragmentos do tumor foram fixados em formol a 10%, tamponado e processado rotineiramente para microscopia de luz. Seccoes de 4µm foram coradas pelo Shorr, ou pela AgNOR, ou ainda, imunocorado para MIB 1 (Ki67). As coloracoes pelo Shorr, AgNOR, ou MIB 1 mostraram um aumento do indice mitotico, coloracao da proteina argirofilica nuclear e celulas tumorais ciclando em tumores em crescimento, respectivamente. Todos os tres marcadores de proliferacao celular foram capazes de distinguir o TVTC em ambas as fases de evolucao. O anticorpo monoclonal MIB 1 foi o melhor na avaliacao morfologica, de crescimento e regressao do TVTC. Isto resultou em um valor maior que a contagem de AgNOR e do indice mitotico. A imunomarcacao com MIB1 foi o parâmetro mais efetivo da atividade proliferativa. No entanto, so foi observada uma correlacao positiva entre a contagem de mitose e a AgNOR.

Ocorrência de apoptose em leucócitos no esfregaço de sangue periférico e em sincícios na infecção in vivo pelo vírus da cinomose canina

The aim of this paper is to report the occurrence of apoptosis in peripheral blood leukocytes and in syncytia induced by experimental infection of dogs with canine distemper virus. Blood was collected from nine puppies that were inoculated with the Snyder Hill strain of CDV, and stained with May Grunwaldt-Giemsa. Dogs were necropsied and retropharyngeal lymph nodes were collected and processed for paraffin embedding. Five micrometers thick sections were stained with hematoxylin-eosin and methyl green pyronin. Other sections were submitted to TUNEL reaction for in situ detection of apoptosis, and immunohistochemistry to detect CDV antigen. Immunohistochemistry confirmed the presence of viral antigen. Syncytia were present in all retropharyngeal lymph nodes. Several syncytia contained apoptotic nuclei as detected by hematoxylin-eosin and methyl green pyronin stains. Apoptotic nuclei were labeled inside syncytia by the TUNEL reaction. Blood smear analyses indicated higher proportion of apoptotic leukocytes in the peripheral blood of infected dogs.