Astrócitos imunorreativos à proteína glial fibrilar ácida (GFAP) em SNC de bovinos normais e de bovinos com raiva. I. Hipocampo e giro dentato

Glial fibrillary acidic protein (GFAP) belongs to the group of intermediate filaments of the cell cytoskeleton. It is present in astrocytes and in some ependymal cells. Detection of GFAP is useful for the identification of astrocytes in a highly specific manner. In the present study, peroxidase antiperoxidase (PAP) immunohistochemical staining, with a specific antibody against GFAP was employed to determine the distribution and morphology of astrocytes in the hippocampus and dentate gyrus of normal cattle and cattle naturally infected with rabies. Paraffin sections of buffered formalin-fixed tissue were used. Positively brown coloured astrocytes in each region were classified into four levels of intensity. The number of astrocytes per mm2 was determined using a straight scale ocular micrometer. Nuclear area, larger and smaller diameter of astrocytes were also determined with an image analyser system. It was observed that the morphology of astrocytes varied in the différents microregions of hippocampus and dentate gyrus. Cattle with rabies showed astrogliosis represented by strong immunoreaction, enlargement of cell body, cytoplasm and processes. The nuclei were also enlarged and light in colour. Astrocitosis was also present. Neurons showed no alterations. These results represent a contribution to the knowledge of the normal distribution and morphology of astrocytes which permits a better understanding of pathological conditions.A proteína glial fibrilar ácida (GFAP) pertence ao grupo dos filamentos intermediários do citoesqueleto celular. Está presente nos astrócitos e em algumas células do epêndima. A identificação da GFAP é empregada para identificar astrócitos com excelente especificidade. Neste trabalho, o método imunohistoquímico da peroxidase-antiperoxidase (PAP), utilizando anticorpo primário anti-GFAP, foi empregado para estudar a distribuição e a morfologia de astrócitos no hipocampo e giro dentato de bovinos normais e também de bovinos com raiva, naturalmente adquirida. Foram utilizados cortes em parafina de material fixado em formalina tamponada. Astrócitos corados positivamente em marrom foram classificados em quatro níveis de intensidade. O número de astrócitos por mm2 foi determinado com uso de ocular integradora. Área, maior e menor diâmetro do núcleo dos astrócitos foram determinados com sistema analisador de imagens. Observou-se que bovinos com raiva mostraram astrogliose, apresentando forte imunorreatividade, aumento de tamanho do corpo celular e dos processos citoplasmáticos. Mostraram também astrocitose, sendo que, em todas as camadas do hipocampo e do giro dentato, o número de astrócitos foi maior que nos animais normais. O núcleo também se apresentava aumentado e pouco corado.Células nervosas da região estudada não apresentavam alterações importantes. Os resultados colaboram para o conhecimento da distribuição e da morfologia de astrócitos imunorreativos à GFAP, em bovinos normais e em bovinos com raiva

Revisão: filamentos intermediários

Os filamentos intermediários (FIs) têm se tornado objeto de interesse considerável para os biologistas celulares e moleculares. Muitos dados indicam que as proteínas dos FIs fazem parte de uma família multigênica, extremamente, heterogênea. A complexidade dos FIs pode estar relacionada com a sua diversidade de funções dentro das células. O artigo relaciona os principais filamentos intermediários de interesse para estudos sobre embriogênese, fisiologia e patologia dos tecidos de origem animal.Intermediate filaments (IF) has become subject of considerable interest to cell and mollecular biologists. A lot of date indicates that IF proteins constitute an extremely heterogeneous multigene family. This complexity means that IF are functionally diverse component of cells. This paper describes the majors intermediate filaments and their application in some studies linked to embriogenesis, physiology and pathology of animals tissues

Astrócitos imunorreativos à proteína glial fibrilar ácida (GFAP) em SNC de bovinos normais e de bovinos com raiva. I. Hipocampo e giro dentato

A proteína glial fibrilar ácida (GFAP) pertence ao grupo dos filamentos intermediários do citoesqueleto celular. Está presente nos astrócitos e em algumas células do epêndima. A identificação da GFAP é empregada para identificar astrócitos com excelente especificidade. Neste trabalho, o método imunohistoquímico da peroxidase-antiperoxidase (PAP), utilizando anticorpo primário anti-GFAP, foi empregado para estudar a distribuição e a morfologia de astrócitos no hipocampo e giro dentato de bovinos normais e também de bovinos com raiva, naturalmente adquirida. Foram utilizados cortes em parafina de material fixado em formalina tamponada. Astrócitos corados positivamente em marrom foram classificados em quatro níveis de intensidade. O número de astrócitos por mm2 foi determinado com uso de ocular integradora. Área, maior e menor diâmetro do núcleo dos astrócitos foram determinados com sistema analisador de imagens. Observou-se que bovinos com raiva mostraram astrogliose, apresentando forte imunorreatividade, aumento de tamanho do corpo celular e dos processos citoplasmáticos. Mostraram também astrocitose, sendo que, em todas as camadas do hipocampo e do giro dentato, o número de astrócitos foi maior que nos animais normais. O núcleo também se apresentava aumentado e pouco corado. Células nervosas da região estudada não apresentavam alterações importantes. Os resultados colaboram para o conhecimento da distribuição e da morfologia de astrócitos imunorreativos à GFAP, em bovinos normais e em bovinos com raiva

Immunohistochemical detection of GFAP and TGF ?1 in C57Bl6 mice during acute vesicular stomatitis virus encephalitis

Activation of astrocytes or astrogliosis is a prominent component of the inflammatory response and an indicator of injury in the brain. These astrocytes produce a large array of inflammatory mediators, growth and neuroprotective factors. This study was an investigation from astrocyte (GFAP) response and TGF- ?1 involvement during VSV acute encephalitis using immunohistochemistry to verify relation between astrocytes and TGF-?1. Animals developed symptoms around 6th day after VSV inoculation. Viral proteins were mainly detected at olfactory bulb, ventricular cell layer and disseminated to hippocampus, mesencephalon and diecephalon areas and brain stem. Also at 6th day post inoculation GFAP and TGF-?1 staining was observed in good association with virus-detected areas of brain. However, in mice with severe symptoms we observed reduction in the intensity of GFAP labeling at the same areas where TGF-?1 upregulation was observed. These areas show correlation with areas of necrosis and where are astrocytes with degenerative aspect. We observed TGF-?1 staining in damaged astrocytes, suggesting an effort of those cells in controlling inflammation in acute phase of VSV encephalitis

Ki-67 labeling in canine perianal glands neoplasms: a novel approach for immunohistological diagnostic and prognostic

Background: The antibody Ki-67 is a reliable and easy tool to accurately assess the growth fraction of neoplasms in humans and animals, and it has been used to predict the clinical outcome. Therefore, the aim of the present study was to investigate the immunohistochemical expression pattern of Ki-67 in normal and neoplastic perianal glands of dogs to evaluate the possible use of this proliferation marker as an ancillary method of perianal tumor diagnosis. We studied 42 cases of perianal gland neoplasms including adenomas (n = 15), epitheliomas (n = 15), and carcinomas (n = 12). As controls, 13 tissue samples from normal perianal glands were used. A Ki-67 index was established by a computer-assisted image analysis and compared with manual counting. Results: Out of the 42 cases of perianal gland neoplasms, 34 were from males and eight from females. Recurrence was reported in 14 cases, being higher (8/12) in carcinomas. Immunostaining for Ki-67 revealed that the carcinomas showed a higher proliferation rate (9.87%) compared to groups of epitheliomas (2.66%) and adenomas (0.36%). For adenomas and epitheliomas of the perianal glands the computer-assisted counting and the manual counting gave similar results; however, only the computer-assisted image analysis was efficient to predict the perianal gland carcinoma recurrence.Conclusion: Since there were significant differences in the number of Ki-67-positive nuclei, this marker proved to be effective in helping the classification of perianal gland neoplasms and to refine the diagnosis criteria, especially in those samples with high variation in morphology/area. Also, higher Ki-67 index is related to recurrence in cases of perianal gland carcinomas. Further, the computer-assisted image analysis proved to be a fast and reliable method to assess the Ki-67 index in perianal gland neoplasms. © 2013 Pereira et al.; licensee BioMed Central Ltd

Lymphocytic hypophysitis in dogs infected with Leishmania spp.

BackgroundMorphological involvement of endocrine glands, such as the pituitary gland, remain uninvestigated in dogs with canine visceral leishmaniasis. Therefore, this study investigated the presence of amastigotes of Leishmania spp. and characterized inflammatory changes, highlighting the involvement of TCD3+ lymphocytes in different regions of the pituitary gland of dogs.MethodsSamples were collected from 21 naturally infected dogs and 5 control, uninfected dogs. The different pituitary regions were analyzed in histological sections stained with hematoxylin and eosin (HE) under light microscopy. Inflammation was classified by intensity in a score from 0 to 3, absent (0), mild (1), moderate (2), and marked (3). The immunohistochemical (IHC) evaluation was performed in five high-power fields (hot spot) in a 40x objective of each region with manual counting (Image J1.52ª) of the TCD3+ lymphocytes and for amastigotes analyzed in 40x and 100x objectives. The Shapiro–Wilk test was used to assess the normality of the data. Differences between groups were determined by the Mann Whitney test. The correlation between variables was assessed by Sperman’s correlation test. p < 0.05 were considered statistically significant.ResultsAmastigotes from the pituitary glands of two infected dogs were identified using IHC. The histopathological evaluation stained with hematoxylin and eosin showed greater intensity of inflammation in the pars distalis and pars intermedia regions of infected dogs. IHC for TCD3+ lymphocytes showed a higher median number of immunolabeled cells in pars nervosa in the infected group than in the control group (p < 0.05); and expecting a variation in the distribution and number of these cells in naturally infected dogs, the median of the control group was considered a cut-off point, an increase in T lymphocytes (p < 0.05) was also observed in the pars intermedia and pars distalis of an infected subgroup (n = 10). A moderate significant correlation between the intensity of inflammation and the number of immunolabeled TCD3+ lymphocytes was established in the analyzed pituitary regions, characterizing the occurrence of hypophysitis.ConclusionThese findings presuppose that inflammation and/or the parasite in the pituitary region can result in gland dysfunction, worsening the clinical condition of the patient and compromising the efficiency of treatment and prognosis

Estudo da imunopatogenia das lesões no sistema nervoso central de cães naturalmente acometidos por leishmaniose visceral

Visceral leishmaniasis in dogs is described as a chronic disease whose main symptoms are progressive weigth loss, cachexy and dermatologic lesions. Recently, the disease has been associated to neurologic disorders. A total of 40 dogs with visceral leishmaniasis were divided into two groups. The first composed of dogs without neurological signs (n=30) and the second by dogs with neurological disorders (n=10). Brain samples were collected, stored in 10% buffered formalin and subjected to immunohistochemical examination for amastigotes forms of Leishmania (Leishmania) infantum chagasi, CD3+, CD4+ and CD8+ T lymphocytes and macrophages. Imunnohistochemistry evaluation revealed no amastigote forms of the parasite. CD3+ T lymphocytes were present in 24/30 (80%) dogs without neurological signs and in all dogs from the second group (p=0.0011). CD4+ and CD8+ were rarely observed, with CD4+ immunostaining in 10/40 (25%) dogs, from which half of them had neurological disease (p=0.0090). The presence of CD8+ was detected only in 4/10 (40%) dogs from neurological group (p=0.0021). Macrophages were detected in 38/40 (95%) dogs, without significant differences between groups (p=0.7664).A Leishmaniose visceral em cães é descrita como uma doença de caráter crônico na qual os principais sintomas são perda progressiva de peso, caquexia e lesões dermatológicas. Recentemente, a doença tem sido relacionada com alterações neurológicas. Um total de 40 cães portadores de leishmaniose visceral foi dividido em dois grupos. O primeiro composto por cães sem sintomas neurológicos (n=30) e o segundo grupo composto por cães com sintomas neurológicos (n=10). Amostras de encéfalo foram coletadas e armazenadas em formalina tamponada, para realização de imunoistoquímica para a pesquisa de formas amastigotas de Leishmania (Leishmania) infantum chagasi, linfócitos T CD3+, CD4+ e CD8+ e macrófagos. A reação de imunoistoquímica não revelou formas amastigotas do parasita. Linfócitos T estavam presentes em 24/30 (80%) dos cães sem sintomas neurológicos e em todos os cães do segundo grupo (p=0,0011). Linfócitos CD4+ e CD8+ raramente foram observados, apresentando imunomarcação para CD4+ em 10/40 (25%) dos cães e em metade dos animais do grupo neurológico (p=0,0090). A presença de CD8+ foi detectada em 4/10 (40%) cães com doenças neurológicas (p=0,0021). Macrófagos foram observados em 38/40 (95%) cães, sem diferença estatística significante entre os dois grupos (p= 0,7664)