Envenomation by Loxosceles bites is characterized by dermonecrotic and/or systemic features that lead to several clinical signs and symptoms called loxoscelism. Dermonecrotic lesions are preceded by thrombosis of the dermal plexus. Recent studies show that atheromatous plaque is prone to thrombosis due to endothelial cell apoptosis. To the best of our knowledge, there are no reports of microscopic dermal lesion and endothelial cell apoptosis induced by Loxosceles similis venom in the literature. Thus, the aim of the present study is to describe histological lesions induced by L. similis venom in rabbit skin and to elucidate whether apoptosis of endothelial cells is involved in the pathogenesis of loxoscelism. Forty male rabbits were split into two groups: the control group (intradermally injected with 50 µL of PBS) and the experimental group (intradermally injected with 0.5 µg of L. similis crude venom diluted in 50 µL of PBS). After 2, 4, 6 and 8 hours of injection, skin fragments were collected and processed for paraffin or methacrylate embedding. Sections of 5 µm thick were stained by HE, PAS or submitted to TUNEL reaction. Microscopically, severe edema, diffuse heterophilic inflammatory infiltrate, perivascular heterophilic infiltrate, thrombosis, fibrinoid necrosis of arteriolar wall and cutaneous muscle necrosis were observed. Two hours after venom injection, endothelial cells with apoptosis morphology were evidenced in the dermal plexus. Apoptosis was confirmed by TUNEL reaction. It seems that endothelial cell apoptosis and its consequent desquamation is an important factor that induces thrombosis and culminates in dermonecrosis, which is characteristic of cutaneous loxoscelism.

Cães machos, adultos, mestiços, foram transplantados com células do tumor venéreo transmissível canino (TVTC), na região hipogástrica. Foram coletados doze espécimes 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. Secções de 4µm foram coradas pelo Shorr, ou pela AgNOR, ou ainda, imunocorado para MIB 1 (Ki67). As colorações pelo Shorr, AgNOR, ou MIB 1 mostraram um aumento do índice mitótico, coloração da proteína argirofílica nuclear e células tumorais ciclando em tumores em crescimento, respectivamente. Todos os três marcadores de proliferação celular foram capazes de distinguir o TVTC em ambas as fases de evolução. O anticorpo monoclonal MIB 1 foi o melhor na avaliação morfológica, de crescimento e regressão do TVTC. Isto resultou em um valor maior que a contagem de AgNOR e do índice mitótico. A imunomarcação com MIB1 foi o parâmetro mais efetivo da atividade proliferativa. No entanto, só foi observada uma correlação positiva entre a contagem de mitose e a AgNOR.

Adult male mongrel dogs were subcutaneously transplanted with the canine transmissible venereal tumor (TVT) on the hypogastric region. Twelve specimens of tumors were collected, half during the proliferative phase and the other half during the regressive phase. Fragments of the tumor were fixed in 10% buffered formalin and routinely processed for light microscopy. Sections of 4µm were stained by Schorr or AgNOR or either immunostained for MIB1 (Ki67). Schorr stain, AgNOR and MIB1 showed an increased proliferative activity through mitotic index, nuclear argyrophilic protein stain and cycling tumoral cells in the growing tumors, respectively. All of the three cell proliferation markers were able to distinguish the TVT in both evolution phases. MIB1 monoclonal antibody was the best in the morphologic evaluation of growth and regression of TVT. This resulted in higher values than AgNORs counting and mitotic index. MIB1 immunostaining was the most effective parameter of the proliferative activity of TVT. However, a significant correlation has been detected only between mitosis counting and AgNORs.

Avaliaram-se o mielograma, o hemograma e a ocorrência de apoptose no sangue periférico e na medula óssea de cães com cinomose de ocorrência natural. Foram utilizados 15 cães distribuídos em dois grupos: (a) controle - seis animais clinicamente saudáveis com RT-PCR negativa para o vírus da cinomose canina (CC); (b) infectado - nove animais com manifestações clínicas de CC e RT-PCR positiva. Dos cães com CC, oito (88,9%) apresentaram anemia discreta a moderada (hematócrito: 30,6%), normocítica (VCM: 67,9fL) e normocrômica (CHCM: 34,1g/dL). Todos os animais apresentaram contagens médias normais de leucócitos totais (11600 células/µL) e neutrófilos segmentados (8802 células/µL). Linfopenia foi observada em cinco animais (55,6%) e desvio nuclear dos neutrófilos para a esquerda em oito (88,9%). As contagens médias de linfócitos e neutrófilos bastonetes foram, respectivamente, 1054 e 1508células/µL. No mielograma, todos os animais apresentaram celularidade e relação M:E dentro dos limites de referência. O hemograma e a medula óssea dos cães-controle não apresentaram alteração e não havia células em apoptose no esfregaço sanguíneo desses animais. Nos cães com CC, a média do índice apoptótico foi 0,73% no esfregaço sanguíneo e 1,87% na medula óssea. A apoptose, portanto, pode estar envolvida na patogênese das alterações hematológicas observadas na CC.

The myelogram, the hemogram, and the occurrence of apoptosis in peripheral blood and bone marrow in dogs with canine distemper (CD) of natural occurrence were studied. Fifteen dogs were distributed into two groups: (a) control - six clinically healthy animals with RT-PCR negative for canine distemper virus (CDV); and (b) infected - nine animals showing clinical CD manifestations and RT-PCR positive. The majority of dogs with CD (88.9%) presented discrete to moderate (hematocrit: 30.6%), normocytic (MCH: 67.9fL) and normochromic (MCHC: 34.1g/dL) anemia. All animals showed total leukocytes counting (11,600 cells/µL) and segmented neutrophils (8,802 cells/µL) within the limits of reference. Lymphopenia and left shift neutrophils were observed in 55.6% and 88.9% of the dogs, respectively. Additionally, the average counts of lymphocytes and neutrophils were 1,054 and 1,508cells/µL, respectively. The myelogram of all animals presented cellularity and M:E relation within the limits of reference. Haemogram and bone marrow of the control dogs had no alteration. Moreover, no apoptotic cells were detected in the smear of the peripheral blood of control animals. On the other side, dogs with CD presented a higher apoptotic index (AI), both in the peripheral blood (AI: 0.73%) and in the bone marrow (AI: 1.87%). Therefore, apoptosis may contribute to hematological changes observed in CD.

Doze cães, adultos, machos e sem raça definida foram transplantados subcutaneamente, na região hipogástrica, com células originadas de dois tumores venéreos transmissíveis caninos (TVT). O objetivo do estudo foi demonstrar e quantificar a ocorrência de apoptose na regressão do TVT. Após seis meses, foi obtido um tumor de cada animal, totalizando seis em crescimento e seis em regressão. Fragmentos dos tumores foram processados para avaliação histológica, ultra-estrutural e também para extração de DNA. Cortes de 4µm foram corados em HE, Shorr, verde de metila pironina e Van Gieson e alguns foram submetidos à reação do TUNEL e à imunoistoquímica para P53. Secções coradas pelo Shorr, submetidas à morfometria, demonstram maior índice apoptótico nos tumores em regressão. Esse achado foi confirmado pela microscopia eletrônica de transmissão que evidenciou células com morfologia típica de apoptose e pela reação de TUNEL que marcou mais células nos tumores em regressão que naqueles em crescimento. A imunomarcação para P53 foi mais intensa nos tumores em regressão, assim como a fragmentação internucleossômica do genoma mostrada pela eletroforese em gel de agarose. Concluiu-se que a apoptose tem importante papel na regressão do TVT transplantado experimentalmente, sendo, nesse caso, dependente de P53 para a sua execução.

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.

Vinte e nove pintos SPF de um dia foram inoculados com o vírus da doença infecciosa da bursa de Fabricius (VDIB) para avaliar a ocorrência precoce de apoptose e a expressão da proteína viral 2 (VP2) e da enzima gliceraldeído fosfato dehidrogenase (GAPDH). Os animais foram distribuídos em cinco grupos: 1-controle; e 2 a 5- com 24, 48, 72 e 96 horas pós-inoculação, respectivamente. Fragmentos da bursa de Fabricius foram colhidos para processamento histológico e extração de RNA. Lâminas coradas em HE e TUNEL (marcação in situ da fragmentação do genoma com transferase terminal de deoxinucleotídeo) foram utilizadas na morfometria do índice apoptótico. Amostras de mRNA foram testadas para a expressão dos genes VP2 e GAPDH utilizando-se transcrição reversa e RT-PCR. Utilizou-se um kit SYBR GREEN PCR, e a reação foi desenvolvida em ABI Prism 7000 SDS. Os índices apoptóticos cresceram progressivamente indicando uma relação na atrofia bursal causada pelo VDIB. Paralelamente, os resultados da PCR em tempo real demonstraram queda da carga viral nas células linfóides da bursa nos diferentes intervalos de tempo do experimento. Esses resultados sugerem um papel protetor da apoptose na diminuição da replicação viral.

Twenty-nine SPF 1-day-old chicks were inoculated with infectious bursal disease virus (IBDV) to evaluate early apoptosis and the expression of viral protein 2 (VP2) and glyceraldehyde-3-phosphate dehydrogenease (GAPDH). Five groups were formed: G1-control -and G2 to G5, - 24, 48, 72 and 96 hours post inoculation, respectively. Half of each BF was fixed and processed by routine techniques. To quantify apoptosis, 5µm-thick sections were stained with HE and submitted to TUNEL (terminal transferase UDP nick end labeling) technique. mRNA was extracted from pooled samples of 3 animals/group and used for the expression of VP2 and GADPH genes using the reverse transcription and real-time polymerase chain reaction (RT-PCR). A SYBR GREEN PCR kit was used and the reaction was carried out in an ABI Prism 7000 SDS. Apoptotic indexes progressively increased indicating a role of IBDV in inducing hypotrophy of the BF. Also, it was showed that as long as apoptosis increased, viral protein expression decreased, which suggests that apoptosis plays a role as a defense mechanism against viral replication.

Determinaram-se a apoptose e a atrofia no timo de oito cães novos, inoculados experimentalmente com o vírus de cinomose. Três cães saudáveis foram usados como controle negativo. Secções coradas pelo Shorr foram avaliadas por morfometria. A nucleoproteína viral foi detectada por imunoistoquímica. Os resultados morfométricos confirmaram a hipotrofia e mostraram que o timo dos cães inoculados tinha mais estroma, menos parênquima e maior índice apoptótico/campo que o dos animais-controle. Pode-se concluir que a apoptose desempenha importante papel no mecanismo de hipotrofia tímica que se desenvolve na cinomose.

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.

Determinou-se a expressão gênica das caspases 3 e 8 mediante transcrição reversa de mRNA total e reação em cadeia da polimerase (RT-PCR) para avaliar a apoptose em timo e baço de ratas imunossuprimidas por glicocorticóides. Utilizou-se dexametasona para indução da apoptose e atrofia linfóide. Quarenta e cinco fêmeas Wistar recém-desmamadas foram separadas em três grupos: as ratas de A (n=18) e B (n=18) foram tratadas com 250 e 500mg de glicocorticóide, via intramuscular, respectivamente, e as do C (n=9) não foram tratadas. Após 24, 48 e 72 horas, seis animais de cada grupo tratado e três do controle foram anestesiados, pesados e sacrificados. O baço e o timo foram coletados e pesados. Fragmentos dos órgãos foram fixados em formol tamponado a 10% e processados segundo técnica para inclusão em parafina. Os blocos foram seccionados em 5µm, e os cortes corados em hematoxilina e eosina. A análise histopatológica aliada ao peso dos órgãos nas diferentes doses e tempos demonstrou que a dexametasona induziu hipotrofia linfóide, que ocorreu com maior intensidade no tempo de 72 horas em animais do grupo B. Fragmentos de timo e de baço foram imediatamente congelados em nitrogênio líquido para extração de mRNA e DNA. Para a padronização da técnica de RT-PCR, utilizaram-se pool de amostras de mRNA dos animais-controle e pool de mRNA de animais tratados em cada tempo de experimento. A técnica de RT-PCR foi sensível o suficiente para a detecção dos mRNAs que codificam as caspases 3 e 8, e ambas participaram do processo de apoptose induzido por dexametasona.

Expression of caspases 3 and 8 in spleen and thymus of immunosuppressed rats was analyzed by the reverse transcriptase-polymerase chain reaction (RT-PCR). Forty-five weaned female Wistar rats were divided into three groups: A (n=18) and B (n=18) rats were injected with 250µg and 500µg per rat, respectively; and C (n=9) rats were non-treated control animals. After 24, 48 and 72 hours, six animals from A and B groups and three controls were anaesthetized with chloroform, weighed and euthanized. Thymus and spleens were collected, weighed and a sample of each organ was fixed by immersion in 10% buffered formaline and embedded in paraffin. Thin sections (5µm) were stained with HE. Thymus and spleen samples were snap frozen in liquid nitrogen and stored at -80ºC for total RNA and DNA extraction. Apoptotic indices were calculated using sections stained with HE. Apoptotic indices and organs weights showed that hypotrophy happened mainly at 72h post treatment with 500µg/rat. RT-PCR was standardized using a pool of control animals and another from treated animals with A and B doses at each time period, respectively. Specific oligonucleotides for caspases 3 and 8 were drawn to obtain fragments of 271bp and 368bp, respectively. The results demonstrated that technique RT-PCR is sensitive enough to detect caspases 3 and 8 mRNAs and that caspase 3 and 8 participate in the apoptotic process induced by dexamethasone in weaned rats.

Introduction: it is during Medical Residency Training (MIR) that knowledge, abilities and habits are acquired, which will shape professional activity in the future. It is therefore very likely that residents who do not acquire the necessary habits and knowledge for research activities will eventually not carry out these activities in the future. The aim of this study was to analyze the level of satisfaction of residents with his or her scientific and research training, and to determine any deficiencies with respect to this training. Materials and methods: the aim of the questionnaire used was to determine the level of satisfaction of residents regarding their scientific and research training during their residency period. Questionnaires were usually distributed via internal mail to all residents (MIR physicians) registered at a third level teaching hospital, with a completion rate of 78% (n = 178). Results: as far as the evaluation of scientific training is concerned, 68% of residents were dissatisfied or very dissatisfied. With respect to scientific studies carried out, 49% of residents had not taken part in any, but the number of studies carried out increases as the residency progresses. On the other hand, 22% of residents reported not having started their doctoral thesis, 50% having attended doctorate courses, 24% having a title for their thesis, and only 4% having written a thesis. Doctorate courses, thesis topics, and written theses increase with the year of residency, and a greater activity may be seen in this respect in surgical departments. If we analyze help available to residents for their carrying out scientific activities, 55% reported that only selected assistant doctors would offer help, and 21% reported that no doctors would offer help. Dissatisfaction with research training increases with the year of residency. With regard to main specialist fields, it can be seen that residents in surgical fields carry out more theses, whereas central fields report less facilities. Finally, if we evaluate the influence that these variables may have on the general satisfaction of residents with his or her residency, these variables are seen to be significant factors of dissatisfaction. Conclusions: most residents are dissatisfied with their scientific training and have relatively few facilities for developing such skills, which in turn results in a scarce number of scientific studies and doctoral theses.

Introducción: durante la residencia MIR se van a adquirir los conocimientos, habilidades y hábitos que van a marcar la actividad profesional en el futuro. Por ello, es muy probable que los residentes que no adquieran el hábito y los conocimientos necesarios para realizar actividades investigadoras no realicen dichas actividades en el futuro. El objetivo es analizar la satisfacción de los MIR con su formación científica e investigadora, y determinar los déficits al respecto en su formación. Material y método: se aplicó un cuestionario destinado a constatar la satisfacción de los residentes respecto a su formación científica e investigadora durante el transcurso de su residencia. Los cuestionarios se distribuyeron nominalmente por correo interno a todos los MIR registrados en un hospital de tercer nivel acreditado para docencia, siendo el grado de cumplimentación del 78% (n=178). Resultados: en cuanto a la valoración de la formación científica, el 68% están insatisfechos o muy insatisfechos. Respecto a la realización de trabajos científicos, el 49% de los residentes no han escrito ninguno, aunque aumenta el número de trabajos realizados conforme avanza el tiempo de residencia. Por otro lado, el 22% refiere no tener iniciada la tesis doctoral, el 50% haber realizado los cursos de doctorado, el 24% tiene tema de tesis, y sólo el 4% la ha escrito. Los cursos, el tema de tesis y el haberla escrito van aumentando con el año de residencia, y se observa una mayor actividad en este sentido en los servicios quirúrgicos. Si analizamos las facilidades que tienen los residentes para realizar actividades científicas, el 55% refieren que sólo con determinados adjuntos, y el 21% que ninguna. La insatisfacción con la formación investigadora aumenta conforme aumenta el año de residencia. En cuando al área troncal de la especialidad, se observa que realizan más tesis los residentes de las áreas quirúrgicas, y que los de áreas centrales refieren menos facilidades. Por último, si se valora la influencia que estas variables pueden tener en la satisfacción general del residente con su residencia, se observa que son factores significativos de insatisfacción. Conclusiones: la mayoría de los residentes están insatisfechos con su formación científica y tienen relativamente pocas facilidades para poder desarrollarla, lo cual se plasma en un escaso número de trabajos científicos y tesis doctorales.

Determinou-se o número mínimo de campos por lâmina para se ter uma amostragem representativa para o estudo de apoptose em linfonodos de cães com cinomose. Um linfonodo traqueobrônquico de cão foi colhido e processado segundo técnica para inclusão em parafina. Secções de 5µm foram coradas pelo tricrômico de Shorr. Utilizou-se um analisador de imagens para registrar o número de células em apoptose e os índices apoptóticos de 300 campos, na mesma lâmina. Obtiveram-se, então, valores médios para 10 amostras de 5, 10, 15, 20, 25, 30, 35, 40, 45 e 50 campos ao acaso. Realizou-se o estudo dos desvios-padrão das médias (DP) em relação ao tamanho da amostra que os originou. Quando as oscilações de DP entre amostras consecutivas foram menores que 5%, o ganho em representatividade produzido pelo acréscimo no número de campos não se justificou, dispensando amostragens maiores. Desse modo, a análise de 40 campos por lâmina, na pesquisa de índices apoptóticos em linfonodos caninos, é suficientemente representativa, uma vez que apresenta instabilidade aceitável.

Noventa e seis pintos de corte foram mantidos em diferentes temperaturas ambientais (calor, 29ºC; neutro, 24ºC; frio, 15ºC) e oito aves de cada grupo de temperatura (tratamento) foram sacrificadas nos dias 1, 7, 14, 21, 28, 35 e 42, e suas bolsas cloacais (BC) coletadas e processadas segundo técnica de rotina para inclusão em parafina. Cortes de 4m m, corados em HE, foram submetidos à análise histométrica utilizando-se o programa Kontron KS300, V 2.0. De cada corte foram mensurados a porcentagem de parênquima e o índice apoptótico nas BC. As médias da porcentagem de parênquima nas BC das aves com cinco semanas foram significativamente menores quando mantidas em ambiente frio (53,0%), quando comparadas com as do ambiente neutro (78,5%). As médias do índice apoptótico foram significativamente maiores nas BC de aves mantidas no ambiente frio (0,26), em relação às do neutro (0,12). Conclui-se que o estresse térmico induz apoptose de linfócitos e, conseqüentemente, hipotrofia da bolsa cloacal, alterando sua maturação.

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.

O presente trabalho foi realizado tendo como objetivo determinar se o vírus de sarampo induz apoptose em células de neuroblastoma murino e avaliar o comportamento de células de neuroblastoma murino agudamente infectadas com vírus do sarampo ou persistentemente infectadas com o vírus da cinomose canina quando tratadas com etoposídeo. A infecção pelo vírus de sarampo induziu principalmente necrose em células de neuroblastoma murino. A infecção persistente pelo vírus de cinomose canina não alterou o comportamento de células de neuroblastoma murino tratadas com etoposídeo.

The present study aimed to determine whether measles virus can induce apoptosis in murine neuroblastoma cells and the behavior of these cells under acute infection with measles virus or persistent infection with canine distemper virus upon treatment with etoposide. Measles virus induced necrosis in murine neuroblastoma cells. Canine distemper virus-persistent infection did not alter murine neuroblastoma cells behavior when treated with etoposide.

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.

Fragmentos de tumor venéreo transmissível canino (TVTC) de ocorrência natural, com localização genital, foram obtidos de cinco animais, machos, adultos, sem raça definida. "Imprints" da superfície de corte em lâmina de microscopia foram fixadas em metanol, coradas pelo Giemsa e submetidas à avaliação citológica. Os fragmentos foram fixados e processados rotineiramente para inclusão em parafina e coloração com HE e Shorr, para confirmação histológica do tumor e identificação da apoptose. Outros fragmentos foram envolvidos com papel alumínio e acondicionados dentro de frascos de vidro em gelo seco, para serem processados no mesmo dia, visando à extração de DNA e eletroforese em gel de agarose. Análises cito e histológica do TVTC mostraram a distribuição e o padrão celular e tecidual característicos dessa neoplasia, sobressaindo-se a presença de vacúolos claros, bem definidos no citoplasma à análise citológica. Pela coloração com Shorr pôde-se identificar células retraídas, com aumento da acidofilia citoplasmática e condensação da cromatina nuclear, às vezes com fragmentação do núcleo e das células, caracterizando apoptose. A coloração pelo Shorr mostrou ser mais eficiente na distinção de células apoptóticas do que a coloração por HE. A eletroforese de DNA em gel de agarose demonstrou a fragmentação internucleossômica do genoma, que pôde ser reconhecida pelo clássico "padrão em escada".

Fragments of canine transmissible venereal tumors, from natural cases and genital localization, were obtained from five adult male mongrel dogs. Imprints of the tumors were fixed, stained by Giemsa and submitted to cytological analysis to confirm the diagnosis. Representative samples of the tumoral tissue were fixed, embedded in paraffin and processed routinely for microscopic examination. Sections were stained with hematoxylin - eosin and Shorr. Another set of fragments was packed and maintained in dry ice, until DNA could be extracted for agarose gel electrophoresis. Cytological and histological results confirmed the diagnosis of the neoplasia and showed characteristic cellular and tissular patterns, with well-defined clear vacuoles in the cytoplasm. Shorr stained sections showed several shrunken cells, with cytoplasmic acidophilia, chromatin condensation, besides nuclear and cellular fragmentation, typical of apoptosis. Shorr was better than hematoxylin - eosin to distinguish apoptotic cells. Agarose gel electrophoresis of DNA showed the internucleosomal fragmentation of the genome, which was recognized as the classic "ladder pattern". Apoptosis does occur in the natural evolution of canine transmissible venereal tumor.