A.S. Martins

Caspase-3/-8/-9, Bax and Bcl-2 expression in the cerebellum, lymph nodes and leukocytes of dogs naturally infected with canine distemper virus.

Canine distemper is an immunosuppressive disease caused by the canine distemper virus (CDV). Pathogenesis mainly involves the central nervous system and immunosuppression. Dogs naturally infected with CDV develop apoptotic cells in lymphoid tissues and the cerebellum, but this apoptotic mechanism is not well characterized. To better understand this process, we evaluated the expression of Bax, Bcl-2, and caspase-3, -8 and -9, by evaluating mRNA levels in the peripheral blood, lymph nodes and cerebellum of CDV-infected (CDV+) and uninfected (CDV-) dogs by real-time polymerase chain reaction (PCR). Blood samples from 12 CDV+ and 8 CDV- dogs, diagnosed by reverse transcription-PCR, were subjected to hematological analysis and apoptotic gene expression was evaluated using real-time-PCR. Tissues from the cerebellum and lymph nodes of four CDV+ and three CDV-dogs were also subjected to real time-PCR. No significant differences were found between CDV+ and CDV- dogs in the hemotological results or in the expression of caspase-3, -8, -9, Bax, and Bcl-2 in the peripheral blood. However, expression of Bax, caspase-3, -8 and -9 was significantly higher in the cerebellum of CDV+ compared to CDV- dogs. Expression of caspase-3 and -8 was significantly higher in the lymph nodes of CDV+ compared to CDV- dogs. We concluded that infection with CDV induces apoptosis in the cerebellum and lymph nodes in different ways. Lymph node apoptosis apparently occurs via caspase-3 activation, through the caspase-8 pathway, and cerebellum apoptosis apparently occurs via caspase-3 activation, through the caspase-8 and mitochondrial pathways.

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

Increased expression of oxidative enzymes in adipose tissue following PPARα-activation.

OBJECTIVE Evaluate the effect of fenofibrate treatment on the expression of PPARα and oxidative enzymes in adipose tissue. MATERIALS/METHODS Wistar male rats were fed a balanced diet supplemented with 100mg.Kg-1 bw.day-1 fenofibrate (Sigma) during nine days. Plasma glucose, free fatty acids (FFA) leptin and insulin were determined. PPARα, ACO and CPT-1 mRNA expression and amount of PPARα and PPARγ protein were assessed in epididymal adipose tissue. Oral glucose tolerance test was evaluated into overnight fasted rats. Glucose uptake was measured in adipocytes isolated from epididymal fat pads in the presence or absence of insulin (25ng/mL). RESULTS Fenofibrate treatment increased PPARα and PPARγ protein abundance in adipose tissue. In addition to it well- known effect on oxidative enzymes in liver, fenofibrate treatment also induces a high expression of Acyl CoA Oxidase (ACO) and Carnitine palmitoyltransferase 1 (CPT-1) in adipose tissue. Furthermore, we have shown that the fenofibrate treatment improves the glucose tolerance and enhance the glucose uptake by adipocytes. CONCLUSION Altogether, the data suggest that fenofibrate have a direct effect in adipose tissue contributing to the low adiposity and improvement of glucose homeostasis.

Apoptose e expressão de Bcl-2 e das caspases 3 e 8 em placenta bovina, em diferentes estádios de gestação

Apoptosis and its regulating mechanisms are crucial physiological events for the maintenance of the placental homostasis; and disequilibrium of these processes may compromise placental function and the success of the pregnancy. In this study, apoptosis was investigated by histomorphometry using slides stained with HE and TUNEL reaction. Besides that, Bcl-2 and caspases 8 and 3 expression were evaluated by real time polymerase chain reaction in healthy placentas under different gestacional ages. Samples of placentones of cows at 4th, 6th, and 9th months of gestation were harvested and processed. The apoptotic index gradually increased with the advance of the gestation. Bcl-2 and caspases 3 and 8 were expressed in all the studied periods, being the expression of Bcl-2 lower than that of caspase 8, which was lower than caspase 3. These results indicate that these molecules are involved in the activated apoptotic way in the placental maturation, showing a standard expression throughout the gestation and contributing for the physiological balance of the cellularity and cellular turn over in bovine placenta.

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.

Apoptose e expressão de VP2 e GAPDH na infecção precoce pelo vírus da doença infecciosa da bursa de Fabricius em pintos SPF

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.

Expressão gênica de caspases 3 e 8 em timo e baço de ratas recém-desmamadas e imunossuprimidas por glicocorticóide

Determinou-se a expressao genica das caspases 3 e 8 mediante transcricao reversa de mRNA total e reacao em cadeia da polimerase (RT-PCR) para avaliar a apoptose em timo e baco de ratas imunossuprimidas por glicocorticoides. Utilizou-se dexametasona para inducao da apoptose e atrofia linfoide. Quarenta e cinco femeas Wistar recem-desmamadas foram separadas em tres grupos: as ratas de A (n=18) e B (n=18) foram tratadas com 250 e 500mg de glicocorticoide, via intramuscular, respectivamente, e as do C (n=9) nao foram tratadas. Apos 24, 48 e 72 horas, seis animais de cada grupo tratado e tres do controle foram anestesiados, pesados e sacrificados. O baco e o timo foram coletados e pesados. Fragmentos dos orgaos foram fixados em formol tamponado a 10% e processados segundo tecnica para inclusao em parafina. Os blocos foram seccionados em 5µm, e os cortes corados em hematoxilina e eosina. A analise histopatologica aliada ao peso dos orgaos nas diferentes doses e tempos demonstrou que a dexametasona induziu hipotrofia linfoide, que ocorreu com maior intensidade no tempo de 72 horas em animais do grupo B. Fragmentos de timo e de baco foram imediatamente congelados em nitrogenio liquido para extracao de mRNA e DNA. Para a padronizacao da tecnica 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 tecnica de RT-PCR foi sensivel o suficiente para a deteccao dos mRNAs que codificam as caspases 3 e 8, e ambas participaram do processo de apoptose induzido por dexametasona.

Genetic deletion of the Angiotensin-(1–7) receptor Mas leads to a reduced ovulatory rate

HighlightsGenetic deletion of Angiotensin (1–7) receptor Mas promotes a reduced ovulatory outcome.Knockout (Mas‐KO) mice exhibit reduced follicularpool.Mas receptor deletion promotes a lower IGF‐1 expression in the ovary. ABSTRACT Angiotensin‐(1–7) [Ang‐(1–7)] is a component of Renin‐Angiotensin System (RAS) that acts through activation of the G‐protein‐coupled receptor Mas. Recent studies highlight Ang‐(1–7) as an intermediate of gonadotropin in ovarian physiology. Genetically Mas‐deficient mice allow the investigation of Ang‐(1–7) in the ovulatory process. Therefore, the present study aimed to analyze the effects of Mas gene deletion on ovulation to confirm our hypothesis that Mas Knockout (Mas‐KO) mice exhibit impairment in the ovulatory outcome. First, we evaluated the breeding data from our animal facilities and from a breeding experiment. The ovulation was observed directly from oviducts after a superovulation protocol and in the estrus morning. We also checked the follicular pool and mRNA expression of Insulin‐like growth factor‐1 (IGF‐1) in ovaries to investigate a possible reason underlying the reduced ovulation. Mas‐KO mice showed a reduced litter size and decreased spontaneous ovulatory rate. Ovarian stimulation by gonadotropins reversed ovulation outcome in Mas‐KO mice. Mas deficiency also promoted a reduced ovarian follicular pool and lower IGF‐1 mRNA levels, suggesting that Mas receptor plays a role in the survival of ovarian follicle. The reduction of ovulatory rate highlights the relevance of Ang‐(1–7)/Mas axis in female reproduction, probably through a reduction of IGF‐1 mRNA levels.

Transcriptome of the Wistar audiogenic rat (WAR) strain following audiogenic seizures.

The Wistar Audiogenic Rat (WAR) is a model whose rats are predisposed to develop seizures following acoustic stimulation. We aimed to establish the transcriptional profile of the WAR model, searching for genes that help in understanding the molecular mechanisms involved in the predisposition and seizures expression of this strain. RNA-Seq of the corpora quadrigemina of WAR and Wistar rats subjected to acoustic stimulation revealed 64 genes differentially regulated in WAR. We validated twelve of these genes by qPCR in stimulated and naive (non-stimulated) WAR and Wistar rats. Among these, Acsm3 was upregulated in WAR in comparison with both control groups. In contrast, Gpr126 and Rtel1 were downregulated in naive and stimulated WAR rats in comparison with the Wistar controls. Qdpr was upregulated only in stimulated WAR rats that exhibited audiogenic seizures. Our data show that there are genes with differential intrinsic regulation in the WAR model and that seizures can alter gene regulation. We identified new genes that might be involved in the epileptic phenotype and comorbidities of the WAR model.

Etoposide-induced apoptosis in murine neuroblastoma (N2A) cells infected with Paramyxoviruses

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.