The 3Rs in research: a contemporary approach to replacement, reduction and refinement.

First promulgated in 1959, the 3Rs of Replacement, Reduction and Refinement have evolved as fundamental principles underlying the use of animals and alternatives in science throughout the modern world. This review describes a contemporary approach to delivering the 3Rs through acknowledging the contribution of new technologies and emphasising that applying the 3Rs can be beneficial to good science as well as to animal welfare. This science-led approach moves the concept of the 3Rs out of an ethical silo where they were often considered by scientists to be an inconvenient obligation. On the contrary, relevant examples demonstrate the opportunity to practise better science using 3Rs technologies which deliver faster, more reproducible and more cost-effective results. Indeed, methods harnessing Replacement approaches may permit discoveries which are simply not feasible using animals and frequently are more flexible and agile since compliance with regulatory oversight requirements is simplified. Although the necessity for rigorous oversight is well recognised, it is important that the associated bureaucracy is not allowed to become prohibitive, causing scientists to avoid pursuing justifiable and important research involving animals. Public support for research is conditional - animals should not suffer unnecessarily and sufficient potential benefit should accrue from the research. However, society also actively seeks pioneering medical and scientific advances which can only be achieved through research. Therefore, a balance must be struck between safeguarding animal welfare whilst enabling high-quality science. It is this balance which promotes and sustains public confidence that animal based research is acceptable and being appropriately managed.

Are slaughterhouse-obtained livers suitable for use in ex vivo perfusion research?

OBJECTIVES: The success of the ex vivo machine perfusion of pig livers used for preclinical research depends on organ quality and availability. In this study, we investigated whether livers obtained from slaughterhouses are suitable and equivalent to livers obtained from laboratory pigs. METHODS: Livers were obtained from slaughterhouse pigs stunned by electrocution or CO2 inhalation and from laboratory pigs. For the latter group, 45 minutes of warm ischemia was mimicked for a subgroup, ensuring a valid comparison with slaughterhouse-derived livers. RESULTS: Livers from CO2-stunned pigs showed lower indocyanine green clearance and bile production, higher blood lactate and potassium concentrations, and higher alanine aminotransferase activities than electrically stunned pigs. Furthermore, livers from electrically stunned pigs, and livers from laboratory pigs, subjected or not to warm ischemia, showed similar performance in terms of perfusion and metabolism. CONCLUSION: For an ex vivo liver model generated using slaughterhouse pigs, electrical stunning is preferable to CO2 stunning. Livers from electrically stunned slaughterhouse pigs performed similarly to laboratory pig livers. These findings support the use of livers from electrically stunned slaughterhouse pigs, which may therefore provide an alternative to livers obtained from laboratory pigs, consistent with the principle of the 3Rs.

An animal-free preclinical drug screening platform based on human precision-cut kidney slices.

OBJECTIVE: Renal fibrosis is one of the main pathophysiological processes underlying the progression of chronic kidney disease and kidney allograft failure. In the past decades, overwhelming efforts have been undertaken to find druggable targets for the treatment of renal fibrosis, mainly using cell- and animal models. However, the latter often do not adequately reflect human pathogenesis, obtained results differ per strain within a given species, and the models are associated with considerable discomfort for the animals. Therefore, the objective of this study is to implement the 3Rs in renal fibrosis research by establishing an animal-free drug screening platform for renal fibrosis based on human precision-cut kidney slices (PCKS) and by limiting the use of reagents that are associated with significant animal welfare concerns. RESULTS: Using Western blotting and gene expression arrays, we show that transforming growth factor-ß (TGF-ß) induced fibrosis in human PCKS. In addition, our results demonstrated that butaprost, SC-19220 and tamoxifen - all putative anti-fibrotic compounds - altered TGF-ß-induced pro-fibrotic gene expression in human PCKS. Moreover, we observed that all compounds modulated fairly distinct sets of genes, however they all impacted TGF-ß/SMAD signaling. In conclusion, this study revealed that it is feasible to use an animal-free approach to test drug efficacy and elucidate mechanisms of action.

[Historical and philosophical issues in animal experimentation]. / Enjeux historiques et philosophiques de l'expérimentation animale.

Initial practices involving experimentation with animals can be found in ancient Greece, but animal experimentation as understood in the modern world first emerged in the Renaissance. In the 19th century, the French scientist Claude Bernard analysed the basis for animal experimentation using the Cartesian philosophical concept of animals being equivalent to machines. Yet as Claude Bernard's work on biology developed, it showed that animals, in particular the so-called sentient animals, did have forms of sensitivity and consciousness similar to humans. This led to the present-day moral concern with animal experimentation. The moral argument is expressed in philosophical terms in the Universal Declaration of Animal Rights and the law known as the "Three Rs", while the practical measures for implementation are set out in the European Directive 2010/63/EU on the protection of animals used for scientific purposes, and then as transposed and enforced in the different European Union Member States. This has led to improvements in the treatment of animals used for experimentation, and also allows scope for further improvements to be added in the future, particularly with alternative methods.

Title: Enjeux historiques et philosophiques de l'expérimentation animale. Abstract: Les ébauches de l'expérimentation animale peuvent être trouvées dans l'Antiquité, mais sa pratique moderne s'amorce à partir de la Renaissance. C'est Claude Bernard qui en analyse au XIXe siècle les bases, fondées sur le concept philosophique cartésien de l'animal-machine. Mais le développement même de la biologie bernardienne a révélé que les animaux, notamment les animaux dits « sentients ¼, disposent de processus de sensibilité et de conscience proches de ceux des êtres humains. D'où un souci moral qui se glisse, de nos jours, dans l'expérimentation animale et qui conduit à diverses améliorations du traitement des animaux d'expérience.

Transcutaneous measurement of renal function in two rodent models of obstructive nephropathy.

OBJECTIVE: Glomerular filtration rate (GFR) is a key indicator of renal function. In both clinical practice and pre-clinical research, serum levels of endogenous filtration markers, such as creatinine, are often used to estimate GFR. However, these markers often do not reflect minor changes in renal function. In this study, we therefore set out to evaluate the applicability of transcutaneous GFR (tGFR) measurements to monitor the changes in renal function, as compared to plasma creatinine (pCreatinine), in two models of obstructive nephropathy, namely unilateral ureteral obstruction (UUO) or bilateral ureteral obstruction followed by release (BUO-R) in male Wistar rats. RESULTS: UUO animals showed a significant reduction in tGFR compared to baseline; whereas pCreatinine levels were not significantly changed. In BUO animals, tGFR drops 24 h post BUO and remains lower upon release of the obstruction until day 11. Concomitantly, pCreatinine levels were also increased 24 h after obstruction and 24 h post release, however after 4 days, pCreatinine returned to baseline levels. In conclusion, this study revealed that the tGFR method is superior at detecting minor changes in renal function as compared to pCreatinine measurements.

Invasive Research on Non-Human Primates-Time to Turn the Page.

Invasive research on primates (i.e., laboratory research that implies body manipulations causing pain or distress that is not aimed to directly improve the individuals' well-being) has a long history. Although some invasive studies have allowed answering research questions that we could not have addressed with other methods (or at least not as quickly), the use of primates in invasive research also raises ethical concerns. In this review, we will discuss (i) recent advances in the study of primates that show evidence of complex behaviour and cognition, (ii) welfare issues that might arise when using primates in invasive research, (iii) the main ethical issues that have been raised about invasive research on primates, (iv) the legal protection that primates are granted in several countries, with a special focus on the principle of the 3Rs, and (v) previous and current attempts to ban the use of primates in invasive research. Based on this analysis, we suggest that the importance of a research question cannot justify the costs of invasive research on primates, and that non-invasive methods should be considered the only possible approach in the study of primates.

Harm-Benefit Analysis May Not Be the Best Approach to Ensure Minimal Harms and Maximal Benefits of Animal Research-Alternatives Should Be Explored.

Using animals in scientific research is commonly justified on the utilitarian basis that the benefits of scientific progress to human health and society exceed by far the harm inflicted on animals. In an attempt to ensure that this is indeed the case for every research project, legislation and guidelines increasingly demand the application of harm-benefit analysis (HBA) as part of the approval process of animal research protocols. The ethical principle of HBA asserts that the costs of an action should be weighed against the expected benefits. Any action that may inflict harm can only be approved if it is associated with a greater benefit. This principle is intuitively appealing but how to use it as a practical rule for ethical decisions is a difficult question. The main difficulty is that the future benefits of most scientific research are unmeasurable, unpredictable and are not manifested at the level of the individual project. Applying HBA in such cases may impede scientific progress by inducing a bias against basic research. Moreover, it can lead to the toleration of unnecessary harm to animals in research. Given these caveats of HBA, I call policy-makers to reconsider the place of HBA in animal research. Instead, I support an alternative guideline which is based on replacing the HBA principle (that the expected benefits of the research must exceed the harms caused to the animals) with two independent but mutually necessary principles: (1) any research using an animal must carry a benefit for society and (2) the harm inflicted to an animal in an experiment must be minimal and scientifically justified. I argue that rigorous harm-analysis, which is not weighted against obscure benefits, can increase the over-all benefits of research while reducing the harms to animals.

Animal-Free Human Whole Blood Sepsis Model to Study Changes in Innate Immunity.

Studying innate immunity in humans is crucial for understanding its role in the pathophysiology of systemic inflammation, particularly in the complex setting of sepsis. Therefore, we standardized a step-by-step process from the venipuncture to the transfer in a human model system, while closely monitoring the inflammatory response for up to three hours. We designed an animal-free, human whole blood sepsis model using a commercially available, simple to use, tubing system. First, we analyzed routine clinical parameters, including cell count and blood gas analysis. Second, we demonstrated that extracellular activation markers (e.g., CD11b and CD62l) as well as intracellular metabolic (intracellular pH) and functional (generation of radical oxygen species) features remained stable after incubation in the whole blood model. Third, we mimicked systemic inflammation during early sepsis by exposure of whole blood to pathogen-associated molecular patterns. Stimulation with lipopolysaccharide revealed the capability of the model system to evoke a sepsis-like inflammatory phenotype of innate immunity. In summary, the presented model serves as a convenient, economic, and reliable platform to study innate immunity in human whole blood, which may yield clinically important insights.

Less Is Better. Avoiding Redundant Measurements in Studies on Wild Birds in Accordance to the Principles of the 3Rs.

The Principles of the 3Rs apply to animal use in research regardless where the research is conducted. In wildlife research, particularly research on wild birds, 3R implementation lags behind research using laboratory, farm, or pet animals. Raised 3R awareness and more field-adapted techniques and protocols are expected to improve the situation. Unpredictable access to animals entices the wildlife researcher to make the most of each caught animal, leading to potential over-use, and violation of the 3Rs. In this study, I statistically screened an existing set of Bean Goose biometric data for the presence of redundant measurements. The results show that it was possible to distinguish between the fabalis and rossicus subspecies (the original aim of the measurements) with fewer measurements (2 vs. 17). Avoidance of the redundant measurements was estimated to reduce both handling time and welfare impact with c. 80%. A robust scheme, supported by an R-script, is presented for continuously weeding out redundant measurements. This scheme is potentially applicable for measurement protocols in any wildlife study, and thus, contributes to the implementation of the principals of the 3Rs in wildlife research in general.

The 3Rs and Humane Experimental Technique: Implementing Change.

In 1959, the Universities Federation for Animal Welfare (UFAW) Scholars Russell & Burch published the Principles of Humane Experimental Technique in which they laid out the principles of the Three Rs. However, the Three Rs owed much to others. It was UFAW and, in particular, UFAW's Founder and Director, Major Charles Hume who identified the problem that needed to be tackled, and who developed the non-confrontational approach that was needed to both formulate the questions that needed answers and to obtain the answers from the research community. Russell & Burch's work was also guided by an expert scientific and technical committee chaired by the Nobel Prize winner Sir Peter Medawar. This essay describes the history of the Three Rs using publications by the protagonists and others as well as material from UFAW's archives. It describes the background to the employment of Russell & Burch, the methodology of Russell & Burch's approach and the impact of their work up to the present day-where the Three Rs are incorporated in legislation throughout the world.

Reducing the Number of Individuals to Monitor Shoaling Fish Systems - Application of the Shannon Entropy to Construct a Biological Warning System Model.

The present study aims at identifying the lowest number of fish (European seabass) that could be used for monitoring and/or experimental purposes in small-scale fish facilities by quantifying the effect that the number of individuals has on the Shannon entropy (SE) of the trajectory followed by the shoal's centroid. Two different experiments were performed: (i) one starting with 50 fish and decreasing to 25, 13, and 1 fish, and (ii) a second experiment starting with one fish, adding one new fish per day during 5 days, ending up with five fish in the tank. The fish were recorded for 1h daily, during which time a stochastic event (a hit in the tank) was introduced. The SE values were calculated from the images corresponding to three arbitrary basal (shoaling) periods of 3.5 min prior to the event, and to the 3.5 min period immediately after the event (schooling response). Taking both experiments together, the coefficient of variation (CV) of the SE among measurements was largest for one fish systems (CV 37.12 and 17.94% for the daily average basal and response SE, respectively) and decreased concomitantly with the number of fish (CV 8.6-10% for the basal SE of 2 to 5 fish systems and 5.86, 2.69, and 2.31% for the basal SE of 13, 25, and 50 fish, respectively). The SE of the systems kept a power relationship with the number of fish (basal: R2= 0.93 and response: R2= 0.92). Thus, 5-13 individuals should be the lowest number for a compromise between acceptable variability (<10%) in the data and reduction in the number of fish. We believe this to be the first scientific work made to estimate the minimum number of individuals to be used in subsequent experimental (including behavioral) studies using shoaling fish species that reaches a compromise between the reduction in number demanded by animal welfare guidelines and a low variability in the fish system's response.

Preclinical Evaluation of the Efficacy of Antivenoms for Snakebite Envenoming: State-of-the-Art and Challenges Ahead.

Animal-derived antivenoms constitute the mainstay in the therapy of snakebite envenoming. The efficacy of antivenoms to neutralize toxicity of medically-relevant snake venoms has to be demonstrated through meticulous preclinical testing before their introduction into the clinical setting. The gold standard in the preclinical assessment and quality control of antivenoms is the neutralization of venom-induced lethality. In addition, depending on the pathophysiological profile of snake venoms, the neutralization of other toxic activities has to be evaluated, such as hemorrhagic, myotoxic, edema-forming, dermonecrotic, in vitro coagulant, and defibrinogenating effects. There is a need to develop laboratory assays to evaluate neutralization of other relevant venom activities. The concept of the 3Rs (Replacement, Reduction, and Refinement) in Toxinology is of utmost importance, and some advances have been performed in their implementation. A significant leap forward in the study of the immunological reactivity of antivenoms against venoms has been the development of "antivenomics", which brings the analytical power of mass spectrometry to the evaluation of antivenoms. International partnerships are required to assess the preclinical efficacy of antivenoms against snake venoms in different regions of the world in order to have a detailed knowledge on the neutralizing profile of these immunotherapeutics.

Life in 3D is never flat: 3D models to optimise drug delivery.

The development of safe, effective and patient-acceptable drug products is an expensive and lengthy process and the risk of failure at different stages of the development life-cycle is high. Improved biopharmaceutical tools which are robust, easy to use and accurately predict the in vivo response are urgently required to help address these issues. In this review the advantages and challenges of in vitro 3D versus 2D cell culture models will be discussed in terms of evaluating new drug products at the pre-clinical development stage. Examples of models with a 3D architecture including scaffolds, cell-derived matrices, multicellular spheroids and biochips will be described. The ability to simulate the microenvironment of tumours and vital organs including the liver, kidney, heart and intestine which have major impact on drug absorption, distribution, metabolism and toxicity will be evaluated. Examples of the application of 3D models including a role in formulation development, pharmacokinetic profiling and toxicity testing will be critically assessed. Although utilisation of 3D cell culture models in the field of drug delivery is still in its infancy, the area is attracting high levels of interest and is likely to become a significant in vitro tool to assist in drug product development thus reducing the requirement for unnecessary animal studies.