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Curbing our animal instinct...

Curbing our animal instinct: How 3R technology is aiming at superiority

7 Aug 2014 by qmtbqdmdjq.d qmtbqdmdjq.d

From stem cells to human-on-a-chip devices, opportunities for applying the 3Rs are intimately related to the development of new technologies but the real key to their uptake, says Dr Ian Ragan, is the acceptance of their superiority by scientists The use of animals in research continues to be a subject of great public interest. The very recently published Concordat on Openness on Animal Research in the UK¹ is a recognition that the public has a right to know more about the use of animals and the 72 organisations that signed the Concordat have pledged to explain their efforts to minimise the use and suffering of animals through applying the 3Rs – replacement, reduction and refinement. But although any progress is, or should be, welcome, it is not surprising in an area as emotionally charged and polarised as this one, that the Concordat has not yet been signed by every organisation that could have done so, and that some animal welfare bodies have been less than impressed by its scope and purported transparency. The polarisation is between those who believe that animal research is morally justified if there are no alternatives, and those who believe that it is morally unjustified under any circumstances and should stop. The former belief tends to create defensiveness (sometimes but not always merited), but can be severely undermined when standards are not of the highest quality². On the other hand, to maintain that animal research is never justified is a step too far for the great majority of people who believe that under certain circumstances it is acceptable. Somewhere in-between are those organisations which campaign for abolition on the basis that adequate alternatives to live animal experiments already exist. This resonates with the public because they can have their ethical cake and eat it, but unfortunately it is not a stance that is supported by evidence. The NC3Rs³ – the UK’s national 3Rs centre – belongs to none of these camps. It neither defends nor campaigns but operates in the belief that the ethically desirable goal of reducing dependence on animal experimentation can be met by promoting scientific research that provides tools and technologies that are superior to existing animal methods. Where animal research cannot currently be eliminated, the NC3Rs works to ensure that the procedures used maximise the information obtained through application of new technologies and minimise animal suffering through advances in animal welfare. Opportunities for applying the 3Rs are intimately related to the development and uptake of new technologies which will be driven not by legislation or campaigning, but by the acceptance of their superiority by scientists, regulators and policy makers. Before considering what the exciting new technologies are, is it possible to define in a simple fashion the ways in which animals are used in research? The diversity across public and private sectors is enormous so I have restricted this article to animal research related to human health. This is something we all take an interest in but many people are uneasy knowing that the medicines and treatments we take required the use of thousands of animals for their development. Was it all necessary? Can we do things differently in the future? Whether work is carried out in the public or private sector, all human health-related activity can be reduced to three categories in which animals are used as surrogates for (or models of) human behaviour:

Animal models of normal life processes (e.g. to understand birth, growth and development, nutrition, ageing and reproduction).
Animal models of disease and injury (e.g. for the discovery and development of medicines and new treatments).
Animal models to evaluate hazard and risk to human health from external agents (e.g. drugs, chemicals in the environment, pollution, radiation).

The use of animals as surrogates has a long history and apart from experimentation on humans themselves, for a long time it was the only option available when mechanistic understanding of human physiology, disease and response to drugs and chemicals was limited and living organisms were regarded as "black boxes". Science has moved on, though for these lingering historical reasons, some scientists, legislators and the general public still put more faith in animal experiments than the facts may support. The "just in case" argument sadly still leads to the needless use of many animals, but the further development and application of the tools and technologies below will lead in time to a greater appreciation and acceptance of non-animal methods. Retrospective data analysis The first of these is not a new technology although some applications may rely on new tools for data gathering and computational analysis in the areas of digital health and big data. In its simplest form, it involves examining the accumulated data on the use of an animal model, whether it is of a disease or a test for toxicity of chemicals, and asking the question whether the model has delivered the results expected of it. It sounds so simple but it is fraught with problems. The data may be spread among many hundreds of publications the quality of which has to be judged by someone trusted to make those decisions. The data may be regarded as confidential and their aggregation with other data may require legal agreements and processes for maintaining their anonymity. In the medical world, the Cochrane Reviews⁴ are the gold standard for evidence-based healthcare, collating all primary research data on a medical intervention. The same approach can be applied to research using animals, and it has been powerful in assessing the evidence base for the design of animal studies in toxicology. The NC3Rs has a well-developed process for dealing with the confidentiality issues of data held by pharmaceutical and chemical companies. As an example, this approach has led to recommendations about the use of non-human primates in the development of monoclonal antibodies⁵. But in addition, the analysis of large sets of human clinical data matching, for example, disease progression to changes in blood or imaging markers, can lead to the identification of biological "signatures" which can then be used to predict disease susceptibility, progression and treatment response in ways infinitely more relevant to humans than similar studies on animal models of disease⁶. Mechanistic understanding Greater mechanistic understanding helps to eliminate use of animals still based on "black box" thinking. We are beginning to build in silico models of human organs such as the heart⁷ which not only recapitulate what we know, but can be used to test hypotheses about treatments and interventions and therefore have the potential to replace animal use. Less visually dramatic but just as important, is the understanding of the mechanisms by which drugs or chemicals in the environment cause toxic effects in man. They can be used in the first place to ensure that new molecules are designed to avoid these adverse effects, and secondly to construct in vitro cell systems that recapitulate the toxic mechanism (the adverse outcome pathway or AOP) and can be used in the place of animal tests⁸. In the chemicals sector, the REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals) legislation⁹ came into force in Europe in 2007 to address the impact of chemicals on health and the environment. The potentially enormous number of animals envisaged for safety testing has been mitigated to an extent by requirements to avoid animal use through data sharing, read-across (estimation of risk by comparison with a similar chemical already tested), computational methods, preferential use of in vitro methods and a pragmatic approach to risk based on exposure. While the REACH guidelines by no means satisfy all points of view on the necessity of animal testing, other bioscience sectors would potentially benefit from application of the principles by which REACH operates. Imaging technology The advances in imaging technology in recent years are quite staggering and the increased range of techniques, coupled with greatly improved resolution and sensitivity, now mean that non-invasive methods developed initially for human use can be applied to small laboratory animals. Whatever the method, the ability to measure a time course in the same animal reduces the numbers of animals that need to be used, can replace repeat sampling methods, reduce handling, stress and pain in the animals, but importantly can also produce major improvements in speed, precision and accuracy. PET, SPECT and MRI have been in routine use for several years but recent advances in bioluminescence¹⁰, near-IR fluorescence¹¹ and photoacoustic tomography¹² are opening up new ways of following cell migration (e.g. in cancer biology and neuronal development), cell therapy (e.g. stem cell migration), infection (bacterial growth) and drug and protein distribution. The NC3Rs recently announced the award of a number of grants, co-funded by the Engineering and Physical Sciences Research Council, to advance imaging technologies, including bioluminescence, radiolabelling and implantable technology, in pre-clinical research. The grants, totalling £1.5m, will support research to extend imaging in applications not currently possible, with a view to improving animal research. Telemetry The use of telemetry has a long history in toxicological research to measure basic physiological parameters, such as blood pressure, ECG, temperature and motor activity. But telemetry also reduces handling, and allows group housing so reducing stress. In larger animals, the use of external telemetry in the form of devices attached to jackets has obvious 3Rs advantages. But even in smaller animals that require implantation, recent advances in miniaturisation, longer battery life, video recording and data analytics, permit extensive remote monitoring of behaviour and physiology even in group housed rodents^13,14. This has also been applied to in vivo electrophysiology, traditionally carried out in rodents with head implants, which were directly wired to recording devices. This obviously restricted the range of movement of the animals and had welfare considerations. Recent advances in low voltage electronics should overcome the daunting technical challenge of remote wireless recording even in mice, which can then live in more naturalistic environments, and even undergo simultaneous behavioural testing¹⁵. Microsampling Mice and rats are still used extensively for regulatory toxicology research and large numbers are required for measurements of drug concentrations in the blood. Serial sampling is possible in larger animals but this is not conventionally done in rats and especially not so in mice for which permitted blood sampling volumes are extremely small. The developments in microsampling, particularly in the use of small volume capillaries¹⁶, permit serial sampling even from mice as the sample sizes are much smaller. Combined with more sensitive analytical techniques microsampling could become the method of choice, once reassurances have been given that such sampling does not perturb toxicological findings. The advantage of microsampling is that blood sampling can be carried out in the same animals that are in the main toxicological study group thus reducing numbers appreciably and reducing variability. Stem cells The astonishing discovery that human pluripotent stem cells can be induced from skin fibroblasts and differentiated into different cell lineages has enormous implications for biomedical research and animal use. Here is a tool that can be used to create in vitro cell culture models that previously were only accessible, if at all, through use of post mortem tissue or biopsies with all the attendant ethical and supply issues. These latter difficulties were of course overcome traditionally by use of animal cells and tissues, but why would anyone do this in the future if cells from individual patients can be used to study their diseases? The hope is that iPS cells can be used as the basis of future tissue engineering (see below) but many technical problems associated with production and reproducibility of iPS cells are still to be solved before their use can be considered routine, and therefore their impact on animal use remains to be realised. Tissue engineering The final topic is tissue engineering, which in the 3Rs context means the creation of complex in vitro cell culture models that mimic the behaviour of tissues and organs in vivo and can be used for mechanistic studies as well as drug efficacy and chemical safety testing. The engineering aspect comes in when cells are seeded onto suitable scaffolds in order to recapitulate the in vivo structure and allow cells to create their own microenvironments and connectivity. Into these scaffolds devices for measuring cell function, e.g. imaging or electrical activity, can be built. A key technology in successful tissue engineering is the use of microfluidics that permits precise control over nutrient flow and geometry, as well as lending itself to automation and high throughput formats for screening purposes. The types of cells are of course critical, and while the use of immortalised cell lines, primary cultures and tissue explants has produced extraordinary devices, such as the Wyss Institute lung-on-a-chip¹⁷, it is hoped that the full potential of these advances can be realised by using patient iPS cells to study dysfunction in disease and its treatment. The ultimate aim is to use microfluidics to link organ-on-a-chip devices to create a human-on-a-chip¹⁸. Advances such as these have enormous potential to replace animal use for mechanistic studies, toxicological analysis and drug discovery. These new technologies do not stand alone. Mechanistic understanding underpins all in vitro technologies rather than aiming at replacing "black box" thinking. Complex in vitro systems based on stem cells and tissue engineering can take advantage of the new imaging and analytical technologies that are being applied to animal and human studies. The new techniques illustrate the scientific advantages that accrue when new ideas are allied to application of the 3Rs. The outcome is compelling. Scientists do not have to be convinced to apply new science to the furtherance of the 3Rs, but on the contrary, they can see that applying the 3Rs leads to scientific and technological advances.