Inovação biomédica e o animal não humano em pesquisa: controvérsias científicas e reflexões éticas para contribuição na implementação de políticas públicas de técnicas substitutivas ao uso de animais em pesquisa, ensino e indústria

A experimentação animal é uma prática extremamente incorporada à ciência biomédica moderna, com raízes desde a Antiguidade, mas que foi cada vez mais se desenvolvendo, adquirindo características bem definidas. O animal não humano, no contexto da experimentação, é um ser vulnerado, enclausurado dentro de uma estrutura muito bem definida, que mantém um discurso focado na excelência do modelo do animal na pesquisa e testes, com o objetivo de garantir a manutenção dessas práticas. No entanto, ao analisarmos os dados científicos obtidos com a experimentação animal, que visa primariamente um possível benefício humano, assim como as argumentações éticas relativas ao respeito à vida e ao sofrimento do animal envoltas nesta situação, a argumentação ética prepondera sobre uma base científica cuja eficácia é também extremamente questionável. Partindo de uma análise crítica da aplicação do modelo animal como padrão da pesquisa científica e da concepção ética do reconhecimento da vulnerabilidade do animal não humano na experimentação, propomos que esta reflexão colabore com políticas públicas que incentivem e implementem as técnicas substitutivas ao uso de animais não humanos em pesquisa, testes da indústria e ensino na área biomédica no Brasil.

Progress in animal experimentation ethics. A case study from a brazilian medical school and from the international medical literature

PURPOSE: This study describes in Brazil and in the global biomedical community the time course of the development of animal research welfare guidelines. METHODS: The database of the Ethics Committee of the Faculty of Medicine of Ribeirao Preto (EC/FMRP-USP), Brazil, was surveyed since its inception in 2002 as the regulations became more stringent to provide better protection of animal research welfare at this institution. Medline database was evaluated to identify the number of publications in the period between 1968 and 2008 that used research animals and were in compliance with established ethics guidelines. RESULTS: The EC/FMRP-USP evaluated 979 projects up until 2009. Most of the applications came from Department of Physiology and the most frequently requested species was the rat. In 2004, national research funding agencies started to request prior approval from institutional review ethics committees prior to application review and this requirement became federal law in Brazil in 2008. The analysis of international publications revealed a relative reduction in studies involving research animals (18% in 1968 to 7.5% in 2008). CONCLUSIONS: The present work showed that in the last four decades major changes occurred in the guidelines dictating use of research animals occurred and they are being adopted by developing countries. Moreover, animal welfare concern in the scientific community preceded the introduction of journal guidelines for this purpose. Furthermore, in Brazil it was anticipated that laws were needed to protect animal research welfare from being not upheld.

Tracking the impact of translational research in psychiatry: state of the art and perspectives

Personalized treatments have become a primary goal in translational psychiatric research. They include the identification of neural circuits associated with psychiatric disorders and definition of treatment according to individual characteristics. Many new tools and technologies have been developed but further efforts are required to provide clues on how these scientific advances in psychiatry may be translated into more effective therapeutic approaches. Obstacles to the progress of translational psychiatry also involve numerous scientific, financial, ethical, logistics and regulatory aspects. Also, the goal of DSM-5 to expand “signs and symptoms” classification to incorporate biological measures may help the development of new multifactorial and dimensional models able to better understand the pathophysiology of psychiatric disorders and develop improved treatments. Finally, a better understanding on the significant response variability, cognitive functioning, role of comorbidities and treatment-resistant cases are critical for the development of prevention and intervention strategies that are more effective.

Progress in animal experimentation ethics: a case study from a Brazilian medical school and from the international medical literature

PURPOSE: This study describes in Brazil and in the global biomedical community the time course of the development of animal research welfare guidelines. METHODS: The database of the Ethics Committee of the Faculty of Medicine of Ribeirao Preto (EC/FMRP-USP), Brazil, was surveyed since its inception in 2002 as the regulations became more stringent to provide better protection of animal research welfare at this institution. Medline database was evaluated to identify the number of publications in the period between 1968 and 2008 that used research animals and were in compliance with established ethics guidelines. RESULTS: The EC/FMRP-USP evaluated 979 projects up until 2009. Most of the applications came from Department of Physiology and the most frequently requested species was the rat. In 2004, national research funding agencies started to request prior approval from institutional review ethics committees prior to application review and this requirement became federal law in Brazil in 2008. The analysis of international publications revealed a relative reduction in studies involving research animals (18% in 1968 to 7.5% in 2008). CONCLUSIONS: The present work showed that in the last four decades major changes occurred in the guidelines dictating use of research animals occurred and they are being adopted by developing countries. Moreover, animal welfare concern in the scientific community preceded the introduction of journal guidelines for this purpose. Furthermore, in Brazil it was anticipated that laws were needed to protect animal research welfare from being not upheld.

Establishment and characterisation of an open mini-thoracotomy surgical approach to an ovine thoracic spine fusion model

Background A large animal model is required for assessment of minimally invasive, tissue engineering based approaches to thoracic spine fusion, with relevance to deformity correction surgery for human adolescent idiopathic scoliosis. Here we develop a novel open mini–thoracotomy approach in an ovine model of thoracic interbody fusion which allows assessment of various fusion constructs, with a focus on novel, tissue engineering based interventions. Methods The open mini-thoracotomy surgical approach was developed through a series of mock surgeries, and then applied in a live sheep study. Customized scaffolds were manufactured to conform with intervertebral disc space clearances required of the study. Twelve male Merino sheep aged 4 to 6 years and weighing 35 – 45 kg underwent the abovementioned procedure and were divided into two groups of six sheep at survival timelines of 6 and 12 months. Each sheep underwent a 3-level discectomy (T6/7, T8/9 and T10/11) with randomly allocated implantation of a different graft substitute at each of the three levels; (i) polycaprolactone (PCL) based scaffold plus 0.54μg rhBMP-2, (ii) PCL-based scaffold alone or (iii) autograft. The sheep were closely monitored post- operatively for signs of pain (i.e. gait abnormalities/ teeth gnawing/ social isolation). Fusion assessments were conducted post-sacrifice using Computed Tomography and hard-tissue histology. All scientific work was undertaken in accordance with the study protocol has been approved by the Institute's committee on animal research. Results. All twelve sheep were successfully operated on and reached the allotted survival timelines, thereby demonstrating the feasibility of the surgical procedure and post-operative care. There were no significant complications and during the post-operative period the animals did not exhibit marked signs of distress according to the described assessment criteria. Computed Tomographic scanning demonstrated higher fusion grades in the rhBMP-2 plus PCL-based scaffold group in comparison to either PCL-based scaffold alone or autograft. These results were supported by histological evaluation of the respective groups. Conclusion. This novel open mini-thoracotomy surgical approach to the ovine thoracic spine represents a safe surgical method which can reproducibly form the platform for research into various spine tissue engineered constructs (TEC) and their fusion promoting properties.

Adjustable stiffness, external fixator for the rat femur osteotomy and segmental bone defect models

The mechanical environment around the healing of broken bone is very important as it determines the way the fracture will heal. Over the past decade there has been great clinical interest in improving bone healing by altering the mechanical environment through the fixation stability around the lesion. One constraint of preclinical animal research in this area is the lack of experimental control over the local mechanical environment within a large segmental defect as well as osteotomies as they heal. In this paper we report on the design and use of an external fixator to study the healing of large segmental bone defects or osteotomies. This device not only allows for controlled axial stiffness on the bone lesion as it heals, but it also enables the change of stiffness during the healing process in vivo. The conducted experiments have shown that the fixators were able to maintain a 5 mm femoral defect gap in rats in vivo during unrestricted cage activity for at least 8 weeks. Likewise, we observed no distortion or infections, including pin infections during the entire healing period. These results demonstrate that our newly developed external fixator was able to achieve reproducible and standardized stabilization, and the alteration of the mechanical environment of in vivo rat large bone defects and various size osteotomies. This confirms that the external fixation device is well suited for preclinical research investigations using a rat model in the field of bone regeneration and repair.

CowLog

We have developed CowLog, which is open-source software for recording behaviors from digital video and is easy to use and modify. CowLog tracks the time code from digital video files. The program is suitable for coding any digital video, but the authors have used it in animal research. The program has two main windows: a coding window, which is a graphical user interface used for choosing video files and defining output files that also has buttons for scoring behaviors, and a video window, which displays the video used for coding. The windows can be used in separate displays. The user types the key codes for the predefined behavioral categories, and CowLog transcribes their timing from the video time code to a data file. CowLog comes with an additional feature, an R package called Animal, for elementary analyses of the data files. With the analysis package, the user can calculate the frequencies, bout durations, and total durations of the coded behaviors and produce summary plots from the data.

CowLog

We have developed CowLog, which is open-source software for recording behaviors from digital video and is easy to use and modify. CowLog tracks the time code from digital video files. The program is suitable for coding any digital video, but the authors have used it in animal research. The program has two main windows: a coding window, which is a graphical user interface used for choosing video files and defining output files that also has buttons for scoring behaviors, and a video window, which displays the video used for coding. The windows can be used in separate displays. The user types the key codes for the predefined behavioral categories, and CowLog transcribes their timing from the video time code to a data file. CowLog comes with an additional feature, an R package called Animal, for elementary analyses of the data files. With the analysis package, the user can calculate the frequencies, bout durations, and total durations of the coded behaviors and produce summary plots from the data.

Ética en la investigación psicológica : una mirada a los códigos de ética de Argentina, Brasil y Colombia

Resumen: En esta propuesta se discuten las consideraciones éticas sobre la investigación, hechas en tres códigos, a saber, la ley 1090 de 2006, que rige el ejercicio profesional del psicólogo en Colombia, el Código de Ética Profissional do Psicólogo do Conselho Federal de Psicología en Brasil y el Código Nacional de Ética de la Federación de Psicólogos de la República Argentina. El análisis de los preceptos sobre trato con personas, consentimiento informado y manejo de información, así como las investigaciones con animales, permiten apreciar carencias y fortalezas en cada país, además de las particularidades propias de una concepción del rol del psicólogo como investigador en la sociedad actual.

Ischemic preconditioning before lower limb ischemia-reperfusion protects against acute lung injury

Objective: Prolonged limb ischemia followed by reperfusion (I/R) is associated with a systemic inflammatory response syndrome and remote acute lung injury. Ischemic preconditioning (IPC), achieved with repeated brief periods of I/R before the prolonged ischemic period, has been shown to protect skeletal muscle against ischemic injury. The aim of this study was to ascertain whether IPC of the limb before I/R injury also attenuates systemic inflammation and acute lung injury in a fully resuscitated porcine model of hind limb I/R. Methods: This prospective, randomized, controlled, experimental animal study was performed in a university-based animal research facility with 18 male Landrace pigs that weighed from 30 to 35 kg. Anesthetized ventilated swine were randomized (n = 6 per group) to three groups: sham-operated control group, I/R group (2 hours of bilateral hind limb ischemia and 2.5 hours of reperfusion), and IPC group (three cycles of 5 minutes of ischemia/5 minutes of reperfusion immediately preceding I/R). Plasma was separated and stored at -70° C for later determination of plasma tumor necrosis factor-a and interleukin-6 with bioassay as markers of systemic inflammation. Circulating phagocytic cell priming was assessed with a whole blood chemiluminescence assay. Lung tissue wet-to-dry weight ratio and myeloperoxidase concentration were markers of edema and neutrophil sequestration, respectively. The alveolar-arterial oxygen gradient and pulmonary artery pressure were indices of lung function. Results: In a porcine model, bilateral hind limb (I/R) injury significantly increased plasma interleukin-6 concentrations, circulating phagocytic cell priming, and pulmonary leukosequestration, edema, and impaired gas exchange. Conversely, pigs treated with IPC before the onset of the ischemic period had significantly reduced interleukin-6 levels, circulating phagocytic cell priming, and experienced significantly less pulmonary edema, leukosequestration, and respiratory failure. Conclusion: Lower limb IPC protects against systemic inflammation and acute lung injury in lower limb I/R injury.

Caractérisation et objectivation de l’acouphène subjectif chronique idiopathique

Objectif: Cette thèse avait pour objectif principal la mise en oeuvre et la validation de la faisabilité, chez l'humain, du paradigme de modulation du réflexe acoustique de sursaut par un court silence (GPIAS) afin de l'utiliser comme mesure objective de l'acouphène. Pour ce faire, trois expériences ont été réalisées. L'expérience 1 avait pour objectif de valider l'inhibition du réflexe de sursaut par un court silence chez des participants humains normo-entendants (sans acouphène) lors de la présentation d'un bruit de fond centré en hautes et en basses fréquences afin de déterminer les paramètres optimaux du paradigme. L'expérience 2 avait pour objectif de valider la précision et la fidélité d'une méthode de caractérisation psychoacoustique de l'acouphène (appariement en intensité et en fréquence). Finalement, l'expérience 3 avait pour objectif d'appliquer le paradigme d'objectivation de l'acouphène par le réflexe de sursaut à des participants atteints d'acouphènes chroniques en utilisant les techniques développées lors des expériences 1 et 2. Méthodologie : L'expérience 1 incluait 157 participants testés dans l'une des conditions de durée du court silence (5, 25, 50, 100, 200 ms) et dans l'un des deux paradigmes (court silence à l'intérieur du bruit de fond ou suivant celui-ci) à l'aide de bruits de fond en hautes et en basses fréquences. L'expérience 2 incluait deux groupes de participants avec acouphène, l'un musicien (n=16) et l'autre sans expérience musicale (n=16) ainsi qu'un groupe de simulateur sans acouphène (n=18). Ils tous ont été évalués sur leur capacité d'appariement en fréquence et en intensité de leur acouphène. Les mesures ont été reprises chez un sous-groupe de participants plusieurs semaines plus tard. L'expérience 3 incluait 15 participants avec acouphène et 17 contrôles évalués à l'aide du paradigme d'inhibition du réflexe de sursaut à l'aide d'un court silence (GPIAS). Les paramètres psychoacoustiques de l'acouphène ont également été mesurés. Toutes les mesures ont été reprises plusieurs mois plus tard chez un sous-groupe de participants. Résultats : Expérience 1 : le paradigme d'inhibition du réflexe acoustique de sursaut par un court silence est applicable chez l'humain normo-entendant. Expérience 2 : les mesures psychoacoustiques informatisées de l'acouphène incluant l'appariement en fréquence et en intensité sont des mesures précises et fidèles du percept de l'acouphène. Expérience 3 : un déficit d'inhibition au paradigme du GPIAS a été retrouvé chez le groupe de participants avec acouphène pour les bruits de fond en hautes et en basses fréquences au test et au retest. Les mesures d'appariement en fréquence ont révélé un acouphène dont la fréquence prédominante était d'environ 16 000 Hz chez la plupart des participants. Discussion : Il est possible d'appliquer le paradigme d'inhibition du réflexe acoustique de sursaut par un court silence à des participants humains atteints d'acouphène, tel qu'il est utilisé en recherche animale pour « objectiver » la présence d'acouphène. Toutefois, le déficit d'inhibition mesuré n'est pas spécifique à la fréquence de l'acouphène lorsque validé à partir des données d'appariement psychoacoustique. Nos résultats soulèvent des questions quant à l'interprétation originale du paradigme pour détecter la présence d'un acouphène chez les animaux.

Dietary supplements of whole linseed and vitamin E to increase levels of alpha-linolenic acid and vitamin E in bovine milk

The potential to increase the concentrations of n-3 polyunsaturated fatty acids (PUFAs) in milk fat was investigated by studying the effects of feeding a xylose-treated, whole cracked linseed supplement ( rich in alpha-linolenic acid) to dairy cows. Also the effect of increasing the dietary intake of vitamin E on the vitamin E status of milk was investigated. The effect of pasteurisation on milk fatty acid composition was also examined. Using a 3 x 2 factorial design, a total of 60 Holstein dairy cows were fed a total mixed ration based on grass silage supplemented with one of three levels of whole cracked linseed (78, 142 or 209 g . kg(-1) diet dry matter (DM); designated LL, ML or HL, respectively) in combination with one of two levels of additional dietary vitamin E intake ( 6 or 12 g vitamin E . animal(-1) . day(-1); designated LE or HE, respectively). Increasing lipid supplementation reduced (P < 0.01) diet DM intake and milk yield, and increased (P < 0.001) the overall content of oleic, vaccenic, alpha-linolenic and conjugated linoleic acids, and total PUFAs and monounsaturated fatty acids (MUFA). Myristic and palmitic acids in milk fat were reduced ( P < 0.001) through increased lipid supplementation. While α-linolenic acid concentrations were substantially increased this acid only accounted for 0.02 of total fatty acids in milk at the highest level of supplementation (630 g α-linolenic acid &BULL; animal(-1) &BULL; day(-1) for HL). Conjugated linoleic acid concentrations in milk fat were almost doubled by increasing the level of lipid supplementation (8.9, 10.4 and 16.1 g &BULL; kg(-1) fatty acids for LL, ML and HL, respectively). Although milk vitamin E contents were generally increased there was no benefit (P > 0.05) of increasing vitamin E intake from 6 to 12 g . animal(-1) . day(-1). The fatty acid composition of milk was generally not affected by pasteurisation.

Enhancement of oleic acid and vitamin E concentrations of bovine milk using dietary supplements of whole rapeseed and vitamin E

With the aim of reducing the degree of saturation and increasing the C18:1 cis fatty acid content of milk fat, the effects of feeding high levels of whole cracked rapeseed to dairy cows was investigated together with the effect of increasing dietary intake of vitamin E on the vitamin E content of milk. Using a 3 x 3 factorial design, 90 Holstein dairy cows were fed one of three levels of whole cracked rapeseed (0 (ZR), 134 (MR) and 270 g . kg(-1) diet dry matter (DM) (HR)) in combination with one of three intakes of supplementary vitamin E (0 (ZE), 2 (ME) and 4 g . cow(-1) . d(-1) (HE)). Supplementing with up to almost 2 kg . d(-1) of rapeseed oil (diet HR) significantly (P < 0.001) increased C18: 1cis in milk fat, from 181 (ZR) to over 400 g &BULL; kg(-1) (HR) of total milk fatty acids. Concentrations of C18: 0, C18: 2 and C18: 3 fatty acids were also increased ( P < 0.001) but by a much lesser degree, and the saturated fatty acids C4: 0 to C16: 0 decreased substantially. Vitamin E supplementation increased ( P < 0.01) milk vitamin E concentrations from 1.29 (ZE) to 1.68 mg &BULL; kg(-1) whole milk (HE). Thus substantial changes in milk fat composition with potentially beneficial effects on human health were achieved and without any adverse effects on milk taste. However, these improvements must be offset against the substantial reductions ( P < 0.001) observed in voluntary feed DM consumption (ZR, 20.6; HR, 15.2 kg DM . d(-1)), milk yield (ZR, 22.9; HR, 13.2 kg . d(-1)) and milk fat concentration (ZR, 42.1; HR, 33.4 g . kg(-1)) which would not be commercially sustainable unless a considerable premium was paid for this modified milk. It seems likely that the optimum dose of dietary rapeseed is lower than used in this study.

Exposure to postnatal depression predicts elevated cortisol in adolescent offspring

Background. Animal research shows that early adverse experience results in altered glucocorticoid levels in adulthood, either raised basal levels or accentuated responses to stress. If a similar phenomenon operates in humans, this suggests a biological mechanism whereby early adversity might transmit risk for major depression, glucocorticoid elevations being associated with the development of this disorder. Methods. We measured salivary cortisol at 8:00 Am and 8:00 Pm over 10 days in 13-year-old adolescents who had (n = 48) or bad not (n = 39) been exposed to postnatal maternal depression. Results: Maternal postnatal depression was associated with higher, more variable morning cortisol in offspring, a pattern previously found to predict major depression. Conclusions. Early adverse experiences might alter later steroid levels in humans. Because maternal depression confers added risk for depression to children, these alterations might provide a link between early events and later psychopathology.

Disturbances in early parenting of depressed mothers and cortisol secretion in offspring: A preliminary study

Background: Disturbances in cortisol secretion are associated with risk for psychiatric disorder, including depression. Animal research indicates that early care experiences influence hypothalamic-pituitary-adrenal (HPA) axis functioning in offspring. Similar effects are suggested in human development, but evidence of longitudinal associations between observed early parenting and offspring cortisol secretion is extremely limited. We studied associations between parenting disturbances occurring in the context of maternal postnatal depression (PND), and elevations in morning cortisol secretion in the adolescent offspring of PND mothers. Methods: We observed maternal parenting behaviour on four occasions through the first year and at five-year follow up in postnatally depressed (n = 29) and well (n = 20) mothers. Observations were coded for maternal sensitivity and withdrawal. Basal offspring salivary cortisol secretion was measured at 13-years, using collections over 10-days. Results: Postnatal, but not five-year, maternal withdrawal predicted elevated mean and maximum morning cortisol secretion in 13-year-old offspring. There were no significant associations between maternal sensitivity and offspring cortisol secretion. Limitations: The sample size was relatively small, and effects tended to be reduced to trend level when covariates were considered. The correlational nature of the study (albeit longitudinal) limits conclusions regarding causality. Conclusions: Individual differences in early maternal parenting behaviour may influence offspring cortisol secretion, and thereby risk for depression. Parenting interventions that facilitate active maternal engagement with the infant may be indicated for high risk populations.