Replacement, refinement and reduction
The MRC plays an active role in developing and disseminating the principles of the 3Rs (replacement, refinement and reduction):
Replacement refers to methods that avoid or replace the use of animals defined as 'protected' under the Animals (Scientific Procedures) Act 1986 (ASPA) in an area where they would otherwise have been used. 'Protected' animals are all living vertebrates -except man and cephalopods, such as the octopus
Replacement methods can be absolute replacements - techniques which do not involve animals at any point, such as computer modelling, in vitro methodologies (e.g. tissue engineering), or human volunteers - or relative replacements, which avoid or replace the use of 'protected' animals with organisms not protected under ASPA, such as Drosophila (flies) or worms.
Growing 'mini-brains' from stem-cells
An example of Replacement in practice
Dr Madeline Lancaster and her team at MRC Laboratory of Molecular Biology employ cerebral organoids, also known as ‘mini brains’, grown from pluripotent stem cells to model human brain development in vitro. Using in vitro models has the potential to capture the intricacies of the human brain more accurately than animals, which do not have the anatomical and functional complexity of human brains. The challenge with using in vitro models has been to model the whole brain and its complex functions, rather than single cell types or processes individually. Using brain organoids is a way of overcoming this.
The process for growing these 3D tissues was first described in a paper by Dr Lancaster and colleagues published in Nature in September 2013. Her team has previously focussed on modelling neurodevelopmental disorders, such as microcephaly, a disorder characterized by a significantly reduced brain size (some cases may be associated with Zika virus infection). Her work is currently concentrated on other neurodevelopmental disorders such as autism and intellectual disability, by introducing mutations seen in these disorders into the cells used to make the organoids. This will give us a further understanding of the role these mutations play in the development of these conditions. The cerebral organoids are a major step towards reducing reliance on animals in studying neurological diseases and the development of new treatments, and have already been up taken by 16 other research laboratories. The paper authored by Dr Lancaster, who has recently given a TEDx talk about her research, won the 2015 NC3R’s 3Rs Prize for outstanding published research with 3Rs impact.
Refinement refers to improvements to scientific procedures and husbandry which minimise actual or potential pain, suffering, distress or lasting harm and/or improve animal welfare in situations where the use of animals is unavoidable. It applies to the lifetime experience of the animal. There is evidence that refinement not only benefits animals, but can also improve the quality of research findings.
An example of Refinement in practice
Dr Simon Milling’s lab at the University of Glasgow aim to better understand the adaptive immune responses against infections in the intestine, to help manage inflammatory bowel diseases and food allergies. It is thought that specialised dendritic cells, which travel from the intestine to the lymph nodes, are important in mediating these responses, but they can often only be obtained via surgical procedures. In addition to replacing the use of larger animals to surgically collect these cells, Dr Milling’s team have worked hard to refine the surgical cannulation technique in mice. Improvements in the surgical management of the mice have enabled larger volumes of lymph to be collected after surgery, and improved post-operative procedures and the use of a thoracic harness have enabled cannulated animals the full range of normal movement, within their normal cages. This also prevents the need for restraint in the post-surgical period. The three published papers using these refined techniques have been cited over 100 times in the last three years, and staff from laboratories in the UK, Sweden, and the USA have been trained to use the refined procedures.
Project reference G0900270
The Mouse House
An example of Refinement in practice
All mice like to create nests for sleeping in, giving birth and as somewhere to feel safe and secure from predators. However, animal technicians and scientists also need to check and observe the animals with the least amount of disturbance. Following work carried out by the National Institute for Medical Research (NIMR), the Medical Research Council (MRC) developed a red tinted plastic shelter as a means of providing an environmental enrichment strategy for mice.
Mice lack the ability to distinguish red from black, behaving the same in red light as they do in darkness (Spalding, J. (1969). Influence of the Visible Colour Spectrum on Activity in Mice. Laboratory Animal Care. Vol 19, No1, 50-54). After testing a range of red photographic filters, the final mouse house, launched in 2001, was made from red tinted plastic that has a wavelength of 593nm. This allows the mice to see some light through it and perceive shadows and movement. The suggestion made was that mice benefit from having a retreat that offers some ability to detect the presence of an external predator, a natural behaviour expressed even in the laboratory environment. The mouse house is used in many different countries improving the welfare of many thousands of laboratory mice.
Reduction refers to methods which minimise animal use and enable researchers to obtain comparable levels of information from fewer animals or to obtain more information from the same number of animals, thereby reducing the future use of animals.
Single cell modifications
An example of Reduction in practice
Dr Gavin Kelsey and his team at the Babraham Institute use genetically altered strains of mice to investigate how environmental factors, such as diet and early life experiences, alter epigenetic programming and contribute to disease development later in life. A novel method, developed and published by Dr Kelsey’s team in 2014, allowed profiling of these epigenetic modifications in single cells (Smallwood et al. Nat. Methods 2014). Therefore, sufficient cells can be obtained from a small number of mice, meaning that larger groups of animals are no longer required for profiling experiments, thereby reducing the numbers of mice used in the study very substantially.
Publication of this new profiling method has gleaned significant interest from the community and Dr Kelsey’s team plan to work to implement this method with national and international collaborators.
Project reference: MR/K011332/1
The National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs)
The MRC is a major funder of The National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), a scientific organisation which leads the discovery, development and promotion of new ways to replace, reduce and refine the use of animals in research and testing (the 3Rs). The NC3Rs is the UK’s major sponsor of 3Rs research. The NC3Rs have issued various guidance documents, and provide an extensive library of 3Rs resources. These can be found here.
3Rs impact in MRC funded research
Currently, about one third of MRC–funded research programmes involve the use of animals under the Animals (Scientific Procedures) Act. Each year, the MRC collects information from its researchers on how they have implemented 3Rs in their work via the evaluation tool, Researchfish. Some examples of how researchers implement the 3Rs in practice include:
- Replaced some animal use with alternative technique
- Reduced number of animals required (e.g. improved experimental design or statistical analysis)
- Refinement or development of experimental techniques or procedures to improve animal welfare
- Improved housing, including environmental enrichment
- Substitution by a species of lower neurophysiological sensitivity
- Shared use of tissues, organs or other material.
- Changes resulting in downgrading of severity limits for procedures/protocols
- Avoidance of specific procedures or adverse effects
Most awards (59%) implement more than one of these measures. The most frequently reported implementation measure is ‘Reduced number of animals required’ (34%) followed by ‘Replaced some animal use with alternative technique’ (21%) and ‘Avoidance of specific procedures or adverse effects’ (15%).
Around 17% of MRC-funded researchers proposing to use animals in their research reported making additional changes to further reduce, refine or replace animal use during the course of their project. Furthermore, around 12% were able to refine or develop methodology with 3Rs impact that could be shared/adopted by others.