Instrumental and ethical aspects of experimental research with animal models

Experimental animal models offer possibilities of physiology knowledge, pathogenesis of disease and action of drugs that are directly related to quality nursing care. This integrative review describes the current state of the instrumental and ethical aspects of experimental research with animal models, including the main recommendations of ethics committees that focus on animal welfare and raises questions about the impact of their findings in nursing care. Data show that, in Brazil, the progress in ethics for the use of animals for scientific purposes was consolidated with Law No. 11.794/2008 establishing ethical procedures, attending health, genetic and experimental parameters. The application of ethics in handling of animals for scientific and educational purposes and obtaining consistent and quality data brings unquestionable contributions to the nurse, as they offer subsidies to relate pathophysiological mechanisms and the clinical aspect on the patient.

Laboratory animal: biological reagent or living being?

The duties of humans toward non-human animals and their rights in society have been debated for a long time. However, a discussion on the terminology used for the identification of laboratory animals is usually not considered, although the employment of inadequate terminology may generate disastrous consequences for the animals before, during, and after the experiment. This study intends to defend the use of appropriate terminology, call attention to an unethical attitude of certain professionals when dealing with experimental animals, and also propose operational mechanisms, which allow for those distortions to be corrected.

Tobacco experimental model to induce urinary bladder neoplasms

OBJECTIVE: to develop an experimental model of exposure to tobacco burning (cigarette) products to assess the effects of its chronic use in relation to cancers of the bladder. METHODS: the animals were chronically exposed to the burning tobacco products in a semi-open chamber to simulate smoking. Thirty young Wistar rats were divided into two groups: one with 20 animals simulating smoking for six months, and ten not exposed control animals for the same period. After exposure by inhalation of cigarette smoke, animals were euthanized and subjected to histopathological study of the bladder wall. RESULTS: no tumor was found but mild and non significant alterations. The studies of hemo-oximetry (carboxyhemoglobin and methemoglobin) and the concentration of carbon dioxide (CO2) confirm that the animals were exposed to high concentrations of tobacco smoke and its derivatives. CONCLUSION: no bladder mucosal neoplasia was found in the pathological study of animals. The developed experimental models were highly efficient, practical and easy to use and can be used in other similar studies to determine the harmful effects caused by smoking.

Natural history of extensive diaphragmatic injury on the right side: experimental study in rats

OBJECTIVE: To evaluate the natural healing of the rat diaphragm that suffered an extensive right penetrating injury.METHODS: Animals were submitted to an extensive penetrating injury in right diaphragm. The sample consisted of 40 animals. The variables studied were initial weight, weight 21 days after surgery; healing of the diaphragm, non-healing of the diaphragm, and herniated abdominal contents into the chest.RESULTS: Ten animals were used as controls for weight and 30 animals were operated. Two animals died during the experiment, so 28 animals formed the operated group; healing of the diaphragm occurred in 15 animals (54%), 11 other animals showed diaphragmatic hernia (39%) and in two we observed only diaphragmatic injury without hernia (7%). Among the herniated organs, the liver was found in 100% of animals, followed by the omentum in 77%, small bowel in 62%, colon in 46%, stomach in 31% and spleen in 15%. The control group and the diaphragmatic healing subgroup showed increased weight since the beginning of the study and the 21 days after surgery (p <0.001). The unhealed group showed no change in weight (p = 0.228).CONCLUSION: there is a predominance of spontaneous healing in the right diaphragm; animals in which there was no healing of the diaphragm did not gain weight, and the liver was the organ present in 100% the diaphragmatic surface in all rats with healed diaphragm or not.

Analysis of natural history of the diaphragmatic injury on the right in mice

Objective: to evaluate natural evolution of right diaphragmatic injury after the surgical removal of a portion from hemi diaphragm. Methods: the animals were submitted to a surgical removal of portion from right hemi diaphragm by median laparotomy. The sample consists of 42 animals being 2 animals from pilot project and 40 operated animals. And the variables of the study were herniation, liver protection, healing, persistent diaphragm injury, evaluation of 16 channels tomography and the variables "heart rate" and "weight". Results: we analyzed 40 mice, we had two post-operative deaths; we had 17 animals in this group suffered from herniation (42.5%) and 23 animals didn't suffer from herniation (57.5%). Analyzing the tomography as image method in the evaluation of diaphragmatic hernia, we had as a method with good sensitivity (78.6%), good specificity (90.9%), and good accuracy (86.1%) when compared to necropsy. Conclusion: there was a predominance of healing of right hemi diaphragm, the size of initial injury didn't have influence on occurrence of the liver protection or hernia in mice.

The search for circadian clock components in humans: new perspectives for association studies

Individual circadian clocks entrain differently to environmental cycles (zeitgebers, e.g., light and darkness), earlier or later within the day, leading to different chronotypes. In human populations, the distribution of chronotypes forms a bell-shaped curve, with the extreme early and late types _ larks and owls, respectively _ at its ends. Human chronotype, which can be assessed by the timing of an individual's sleep-wake cycle, is partly influenced by genetic factors - known from animal experimentation. Here, we review population genetic studies which have used a questionnaire probing individual daily timing preference for associations with polymorphisms in clock genes. We discuss their inherent limitations and suggest an alternative approach combining a short questionnaire (Munich ChronoType Questionnaire, MCTQ), which assesses chronotype in a quantitative manner, with a genome-wide analysis (GWA). The advantages of these methods in comparison to assessing time-of-day preferences and single nucleotide polymorphism genotyping are discussed. In the future, global studies of chronotype using the MCTQ and GWA may also contribute to understanding the influence of seasons, latitude (e.g., different photoperiods), and climate on allele frequencies and chronotype distribution in different populations.

Experimental infection with the Toxoplasma gondii ME-49 strain in the Brazilian BR-1 mini pig is a suitable animal model for human toxoplasmosis

Toxoplasma gondii causes toxoplasmosis, a worldwide disease. Experimentation with pigs is necessary for the development of new therapeutic approaches to human diseases. BR-1 mini pigs were intramuscularly infected with T. gondii with tachyzoites (RH strain) or orally infected with cysts (ME-49 strain). Haematology and serum biochemistry were analysed and buffy coat cells were inoculated in mice to determine tachyzoite circulation. No alterations were observed in erythrocyte and platelet values; however, band neutrophils increased seven days after infection with ME-49. Serology of the mice inoculated with pig blood leucocytes revealed circulating ME-49 or RH strain tachyzoites in the pigs' peripheral blood at two and seven or nine days post-infection. The tachyzoites were also directly observed in blood smears from the infected pigs outside and inside leucocytes for longer periods. Alanine-aminotransferase was high at days 21 and 32 in the RH infected pigs. After 90 days, the pigs were euthanised and their tissue samples were processed and inoculated into mice. The mice serology revealed the presence of parasites in the hearts, ileums and mesenteric lymph nodes of the pigs. Additionally, cysts in the mice were only observed after pig heart tissue inoculation. The infected pigs presented similar human outcomes with relatively low pathogenicity and the BR-1 mini pig model infected with ME-49 is suitable to monitor experimental toxoplasmosis.

Performance assessment of the single photon emission microscope: high spatial resolution SPECT imaging of small animal organs

The single photon emission microscope (SPEM) is an instrument developed to obtain high spatial resolution single photon emission computed tomography (SPECT) images of small structures inside the mouse brain. SPEM consists of two independent imaging devices, which combine a multipinhole collimator, a high-resolution, thallium-doped cesium iodide [CsI(Tl)] columnar scintillator, a demagnifying/intensifier tube, and an electron-multiplying charge-coupling device (CCD). Collimators have 300- and 450-µm diameter pinholes on tungsten slabs, in hexagonal arrays of 19 and 7 holes. Projection data are acquired in a photon-counting strategy, where CCD frames are stored at 50 frames per second, with a radius of rotation of 35 mm and magnification factor of one. The image reconstruction software tool is based on the maximum likelihood algorithm. Our aim was to evaluate the spatial resolution and sensitivity attainable with the seven-pinhole imaging device, together with the linearity for quantification on the tomographic images, and to test the instrument in obtaining tomographic images of different mouse organs. A spatial resolution better than 500 µm and a sensitivity of 21.6 counts·s-1·MBq-1 were reached, as well as a correlation coefficient between activity and intensity better than 0.99, when imaging 99mTc sources. Images of the thyroid, heart, lungs, and bones of mice were registered using 99mTc-labeled radiopharmaceuticals in times appropriate for routine preclinical experimentation of <1 h per projection data set. Detailed experimental protocols and images of the aforementioned organs are shown. We plan to extend the instrument's field of view to fix larger animals and to combine data from both detectors to reduce the acquisition time or applied activity.

A adequação do consumo de alimentos de origem animal e sua relação com renda familiar

Foi realizado estudo sobre adequação de consumo de proteínas de alto valor biológico e seu relacionamento com preço e com renda familiar, em diferentes épocas. A diminuição de consumo de origem animal observada, foi causada, por uma modificação na distribuição orçamentária, na qual, a fração destinada à aquisição de carne, leite e ovos, em 1975, foi menor do que em 1969. O ''índice de Consumo de Proteínas de Origem Animal - ICPA", avaliado através da razão entre renda familiar e custo dos alimentos de origem animal recomendados para a família, é indicador eficiente do consumo desses alimentos e indiretamente do estado nutricional. Para sua utilização é necessária a fixação de um ICPA crítico, que permita identificar as famílias quanto à capacidade econômica, uma vez que a fração destinada a compra de alimentos de origem animal varia no decorrer do tempo.

Study on the growth promoting capacity of calf and fetal bovine serum for animal cells "in vitro" II: electrophoretic study and survey on the antiproteolytic activity of pools of calf and fetal bovine serum

Calf serum and fetal bovine serum present great variability as to its growth promoting efficiency (GPE). As supplement of culture media to cultivate cells of animal origin they stimulate the "in vitro" multiplication and maintain cell viability. When fourteen lots of calf sera of variable GPE had the total protein contents as well as the percentages of serum fractions determined, no significant differences that could possibly explain the variability of the GPE were observed. Evaluation of the antiproteolytic activity of nineteen lots of calf serum and eighteen serum lots of younger calves showed that the former exhibited lower antiproteolytic titers (1:40 to 1:80) than the latter (1:80 to 1:160). Twelve lots of fetal bovine serum studied in parallel, showed the highest concentration of antiproteolytic factors, with titers equal to 1:320. Sera of bovine origin, but not fetal sera, are usually heat-inactivated, what was demonstrated to be responsible for the decrease of the antiproteolytic activity of 75% of the lots tested. This could explain the inability of certain heat-inactivated sera in promoting multiplication of some cells "in vitro", as verified with primary monkey kidney cells. The results obtained in this study indicated the convenience of submiting each lot of serum to be introduced in cell culture to previous determination of its characteristics, such as growth promoting efficiency, antiproteolytic activity and also toxicity, absence of extraneous agents, etc., in order to minimize the possibility of using serum lots of questionable quality, thus preventing not only the loss of cell lines, but also undesirable and sometimes expensive delays.

Species and biotype distribution of thermotolerant Campylobacters in animal reservoirs in Southern Chile

The prevalence of thermotolerant Campylobacters in mammals and birds from Southern Chile was determined. Campylobacters were isolated from 46.3% of the animals studied being C. jejuni biotipe 1 the most frequent (25.7%) followed by C. coli (17.4%) and C. jejuni biotipe 2 (3.2%).

Febre purpúrica brasileira, virulência em modelo animal do Haemophilus Aegyptius (H. influenzae biogrupo aegyptius)

Febre Purpúrica Brasileira (FPB) é causada por cepas invasoras de Haemophilus aegyptius (H. influenzae biogrupo aegyptius, Hae). Estas cepas invasoras foram diferenciadas de cepas de Hae associadas apenas a conjuntivites (cepas não invasoras) através de marcadores moleculares específicos. Modelo de ratos recém nascidos depletados de complemento foi aplicado ao estudo de cepas de Hae, associadas e não associadas a FPB, com o objetivo de se caracterizar seus potenciais de virulência. Com dose infectante de 10(5) células, as cepas invasoras causaram bacteriemia em 80-100% dos ratos inoculados,.e a magnitude da bacteriemia variou de 10(2,5±0,49) a > 10(4,69) ufc/ml de sangue. Usando a mesma dose infectante as cepas controles não causaram bacteriemia frequente (0 a 50%) e a magnitude variou de 0 a 10(3,69±0,53) ufc/ml de sangue. As doses infectantes capazes de causar bacteriemia em 50% dos ratos inoculados (DB50%) para as cepas invasoras de Hae variaram de < 10³ a 10(4,2) bactérias, enquanto que para as cepas não invasoras, as DB50% variaram de 10(6,2) a > 10(7,3) bactérias. Imunização passiva com antissoros produzidos com cepas invasoras demonstrou que os ratos foram protegidos das bacteriemias causadas pelas cepas homólogas, mas não da infecção causada pela cepa heteróloga. Comparando a bacteriemia causada pelas cepas de Hae com a bacteriemia causada pelo H. influenzae b, cepa Eagan (Hib), foi demonstrado o maior potencial de invasibilidade de Hib. Este modelo animal demonstrou ser útil para esclarecer o maior potencial de virulência das cepas invasoras de Hae.

Paracoccidioides brasilienses isolates obtained from patients with acute and chronic disease exhibit morphological differences after animal passage

The basis for virulence in Paracoccidioides brasiliensis is not completely understood. There is a consensus that the sequencial in vitro subcultivation of P. brasiliensis leads to loss of its pathogenicity, which can be reverted by reisolation from animal passage. Attention to morphological and biochemical properties that are regained or demonstrated after animal passage may provide new insights into factors related to the pathogenicity and virulence of P. brasiliensis. We evaluated morphological characters: the percentage of budding cells, number of buds by cell and the diameter of 100 mother cells of yeast-like cells of 30 P. brasiliensis isolates, before and after animal passage. The isolates were obtained from patients with different clinical forms of paracoccidioidomycosis (PCM): acute form (group A, n=15) and chronic form (group C, n=15). The measurement of the yeast cell sizes was carried out with the aid of an Olympus CBB microscope coupled with a micrometer disc. We measured the major transverse and longitudinal axes of 100 viable cells of each preparation. The percentage of budding cells as also the number of buds by cell was not influenced by animal passage, regardless of the source of the strain (acute or chronic groups). The size values of P. brasiliensis isolates from groups A and C, measured before the animal passage exhibited the same behavior. After animal passage, there was a statistically significant difference between the cell sizes of P. brasiliensis isolates recovered from testicles inoculated with strains from groups A and C. The maximum diameter of mother cells from group A isolates exhibited a size of 42.1mm in contrast with 32.9mm exhibited by mother cells from group C (p<0.05). The diameter of 1500 mother cells from group A isolates exhibited a medium size of 16.0mm (SD ± 4.0), a value significantly higher than the 14.1mm (SD = ± 3.3) exhibited by 1500 mother cells from group C isolates (p<0.05). Our results reinforce the polymorphism exhibited by P. brasiliensis in biological material and the need for further investigations to elucidate the role of morphological parameters of the fungus in the natural history of the disease.