Establishment of a protocol for obtention of neuronal stem cells lineages from the dog olfactory epithelium

A morphological and cell culture study from nasal mucosa of dogs was performed in order to establish a protocol to obtain a cell population committed to neuronal lineage, as a proposal for the treatment of traumatic and degenerative lesions in these animals, so that in the future these results could be applied to the human species. Twelve mongrel dogs of 60-day aged pregnancy were collected from urban pound dogs in São Paulo. Tissue from cribriform ethmoidal lamina of the fetuses was collected at necropsy under sterile conditions around 1h to 2h postmortem by uterine sections and sections from the fetal regions described above. Isolated cells of this tissue were added in DMEM/F-12 medium under standard conditions of incubation (5% CO², >37ºC). Cell culture based on isolated cells from biopsies of the olfactory epithelium showed rapid growth when cultured for 24 hours, showing phase-bright sphere cells found floating around the fragments, attached on culture flasks. After 20 days, a specific type of cells, predominantly ellipsoids or fusiform cells was characterized in vitro. The indirect immunofluorescence examination showed cells expressing markers of neuronal precursors (GFAP, neurofilament, oligodendrocyte, and III â-tubulin). The cell proliferation index showed Ki67 immunostaining with a trend to label cell groups throughout the apical region, while PCNA immunostaining label predominantly cell groups lying above the basal lamina. The transmission electron microscopy from the olfactory epithelium of dogs revealed cells with electron-dense cytoplasm and preserving the same distribution as those of positive cell staining for PCNA. Metabolic activity was confirmed by presence of euchromatin in the greatest part of cells. All these aspects give subsidies to support the hypothesis about resident progenitor cells among the basal cells of the olfactory epithelium, committed to renewal of these cell populations, especially neurons.

Vaccination protocol and bacterial strain affect the serological response of beef calves against blackleg

The serological response of beef calves was evaluated with different vaccination regimens against blackleg, using an official strain (MT) and a field-collected strain of Clostridium chauvoei as antigens. Sixty calves were randomly allocated to four different groups and were submitted to distinct vaccination protocols with a commercial polyvalent vaccine. Group G1 was first vaccinated at four months of age and a booster shot was given after weaning, at eight months. Group G2 was given the first dose at eight months and a booster shot 30 days later. Group G3 was vaccinated only once at eight months and the control group was not vaccinated. These alternative vaccination regimens were proposed in an effort to adequately protect cattle under open-field farming conditions. Serological evaluations were made by Elisa at 4, 8, 9 and 10 months of age. Both groups receiving booster shots had a significantly increased serological response 30 days later. However, the serum IgG levels against C. chauvoei were significantly higher in the calves that were first vaccinated at four months. At 10 months, the two booster shot groups (G1 and G2) had similar serological responses, while the calves that were treated with a single dose of vaccine at weaning (G3) had a response that was similar to that of the control group. The serological response of the calves was significantly inferior at several of the evaluation times when the field strain of the bacteria was used as a challenge antigen instead of the official MT strain. The serological response of calves that are vaccinated twice was found to be satisfactory, independent of the first injection being made at four or eight months of age. It was also concluded that it would be useful to include local bacterial strains in commercial vaccine production.

Protocol for collection and separation of bone marrow mononuclear cells in Chlorocebus aethiops

Abstract: Chlorocebus aethiops is a species of non-human primate frequently used in biomedical research. Some research involves this species as an experimental model for various diseases and possible treatment with stem cells. The bone marrow is one of the main sources of these cells and provides easy access. The aim of this study was to standardize the protocol of collection and separation of bone marrow in C. aethiops. Ten animals were submitted to puncture of bone marrow with access to the iliac crest and cell separation by density gradient. The bone marrow of C. aethiops had an average of 97% viability. From the results achieved, we can conclude that C. aethiops is an excellent model to obtain and isolate mononuclear cells from bone marrow, fostering several studies in the field of cell therapy.

The modified 2VO ischemia protocol causes cognitive impairment similar to that induced by the standard method, but with a better survival rate

Permanent bilateral occlusion of the common carotid arteries (2VO) in the rat has been established as a valid experimental model to investigate the effects of chronic cerebral hypoperfusion on cognitive function and neurodegenerative processes. Our aim was to compare the cognitive and morphological outcomes following the standard 2VO procedure, in which there is concomitant artery ligation, with those of a modified protocol, with a 1-week interval between artery occlusions to avoid an abrupt reduction of cerebral blood flow, as assessed by animal performance in the water maze and damage extension to the hippocampus and striatum. Male Wistar rats (N = 47) aged 3 months were subjected to chronic hypoperfusion by permanent bilateral ligation of the common carotid arteries using either the standard or the modified protocol, with the right carotid being the first to be occluded. Three months after the surgical procedure, rat performance in the water maze was assessed to investigate long-term effects on spatial learning and memory and their brains were processed in order to estimate hippocampal volume and striatal area. Both groups of hypoperfused rats showed deficits in reference (F(8,172) = 7.0951, P < 0.00001) and working spatial memory [2nd (F(2,44) = 7.6884, P < 0.001), 3rd (F(2,44) = 21.481, P < 0.00001) and 4th trials (F(2,44) = 28.620, P < 0.0001)]; however, no evidence of tissue atrophy was found in the brain structures studied. Despite similar behavioral and morphological outcomes, the rats submitted to the modified protocol showed a significant increase in survival rate, during the 3 months of the experiment (P < 0.02).

Influence of population and exercise protocol characteristics on hemodynamic determinants of post-aerobic exercise hypotension

Due to differences in study populations and protocols, the hemodynamic determinants of post-aerobic exercise hypotension (PAEH) are controversial. This review analyzed the factors that might influence PAEH hemodynamic determinants, through a search on PubMed using the following key words: “postexercise” or “post-exercise” combined with “hypotension”, “blood pressure”, “cardiac output”, and “peripheral vascular resistance”, and “aerobic exercise” combined only with “blood pressure”. Forty-seven studies were selected, and the following characteristics were analyzed: age, gender, training status, body mass index status, blood pressure status, exercise intensity, duration and mode (continuous or interval), time of day, and recovery position. Data analysis showed that 1) most postexercise hypotension cases are due to a reduction in systemic vascular resistance; 2) age, body mass index, and blood pressure status influence postexercise hemodynamics, favoring cardiac output decrease in elderly, overweight, and hypertensive subjects; 3) gender and training status do not have an isolated influence; 4) exercise duration, intensity, and mode also do not affect postexercise hemodynamics; 5) time of day might have an influence, but more data are needed; and 6) recovery in the supine position facilitates systemic vascular resistance decrease. In conclusion, many factors may influence postexercise hypotension hemodynamics, and future studies should directly address these specific influences because different combinations may explain the observed variability in postexercise hemodynamic studies.

Optimization of random amplified polymorphic DNA protocol for molecular identification of Lophius gastrophysus

Lophius gastrophysus has important commercial value in Brazil particularly for foreign trade. In this study, we described the optimization of Random Amplified Polymorphic DNA (RAPD) protocol for identification of L. gastrophysus. Different conditions (annealing temperatures, MgCl concentrations, DNA quantity) were tested to find reproducible and adequate profiles. Amplifications performed with primers A01, ² A02 and A03 generate the best RAPD profiles when the conditions were annealing temperature of 36ºC, 25 ng of DNA quantity and 2.5 mM MgCl2. Exact identification of the species and origin of marine products is necessary and RAPD could be used as an accurate, rapid tool to expose commercial fraud.

Identification of main contamination points by hygiene indicator microorganisms in beef processing plants

The microbiological quality of beef and meat products is strongly influenced by the conditions of hygiene prevailing during their production and handling. Without proper hygienic control, the environment in slaughterhouses and butcher shops can act as an important source of microbiological contamination. To identify the main points of microbiological contamination in the beef processing chain, 443 samples of equipment, installations and products were collected from 11 establishments (1 slaughterhouse and 10 butcher shops) located in the state of Paraná, Brazil. The microbiological quality of all the samples was evaluated using Petri dishes to obtain counts of mesophilic aerobes (AC), total coliforms, Escherichia coli (EC), yeasts and molds (YM). The main contamination points identified in butcher shops, in decreasing order, were stainless steel boxes, beef tenderizers, grinders, knives, mixers, sausage stuffers, plastic boxes, floors and drains. In the slaughterhouse, these points were sausage stuffers, platforms, floors and drains. The most severely contaminated products were fresh sausages and ground beef. This information about the main points of microbiological contamination in the beef processing chain is expected to aid professionals responsible for hygiene in similar establishments to set up proper hygienic procedures to prevent or reduce microbiological contamination of beef and meat products.

Microbiological quality of hamburgers sold in the streets of Cuiabá - MT, Brazil and vendor hygiene-awareness

This study evaluated the microbiological quality of hamburgers and the microbe community on the hands of vendors in Cuiabá, Mato Grosso, Brazil, in relation to vendors´ awareness as to what constitute acceptable food-handling practices as part of a broad-spectrum research programme on street foods in Brazil . Sale of the hamburger known as the 'baguncinha' is common and widespread in urban Cuiabá, Mato Grosso, Brazil. Food inspectors encounter various difficulties in carrying out inspections. One hundred and five hamburgers samples were evaluated using conventional methods including tests for facultative aerobic and/or anaerobic mesophytic bacteria, coliform counts at 45 °C, the coagulase test for Staphylococcus, Gram-staining for the presence of Bacillus cereus, Clostridium sulphite reductase and Salmonella spp. The hamburgers were categorized as unsuitable for human consumption in 31.4% of samples, with those testing positive for coliforms and Staphylococcus at unacceptably high levels by Brazilian standards. High levels of microbiological contamination were detected on the hands of the food handlers and mesophytic bacterial counts reached 1.8 × 10(4) CFU/hand. Interviews were carried out by means of questionnaires to evaluate levels of awareness as to acceptable food handling practices and it was found that 80,1% of vendors had never participated in any kind of training.