The 3Rs of Animal research

If you work with animals in the European Union you have to think about and apply the 3Rs (reduction refinement and replacement) principles to animal research. 

MS research in animals is considered to be one of the more “severe” procedures that nasty researchers do to animals. Therefore the regulators watch out for things that may make the EAE procedure less severe.

Many years ago, the way to induce disease in beasties was to inject animals in the feet with nerve proteins mixed with an immune stimulator. This used to make an immune response in the feet of the animals and so it became swollen, and sensitive to pressure and so the poor animals could not walk properly, because it was painful.  I hear you say Uck!. I say Uck too!

So not surprisingly this procedure was banned.

This occurred in the UK many, many years ago. The EU was a bit slower when it came to banning this and some countries don’t really give a stuff about ethics of use of animals and still do this practice.

I never accept any paper that does this, as it is unethical, and have urged other people to do the same. 

I was lambasted by some person for saying this. However, my retort was that there are ways to achieve sensitization without causing the pain to the animals, so time to change rather that doing nothing.

Some groups have been looking in how to avoid the injection reactions and one approach is 

Saul L, Besusso D, Mellanby RJ.LPS-matured CD11c+ bone marrow-derived dendritic cells can initiate autoimmune pathology with minimal injection site inflammation.Lab Anim. 2016 Aug 3. pii: 0023677216663584. [Epub ahead of print]

The pathogenesis of human autoimmune disorders is incompletely understood. This has led to the development of numerous mouse models in which the pathogenesis of autoimmunity can be probed and the efficacy of novel therapies can be tested. One of the most widely-used mouse models of autoimmunity is experimental autoimmune encephalomyelitis (EAE). To induce autoimmune pathology, mice are often immunized with an autoantigen alongside an adjuvant, typically complete Freund’s adjuvant (CFA). Unfortunately, CFA causes significant inflammation at the site of administration. Despite the well-recognized complication of injection site inflammation, CFA with autoantigen immunization is widely used to induce central nervous system autoimmunity. We performed a literature review which allowed us to estimate that over 10,000 mice were immunized with CFA in published EAE studies in 2013. In this study, we demonstrated that subcutaneously administered myelin basic protein (MBP)-pulsed CD11c+ bone marrow-derived dendritic cells (BMDC) were as effective at inducing EAE as subcutaneously administered MBP plus CFA. Importantly, we also discovered that the CD11c+ BMDC caused significantly less injection site inflammation than MBP plus CFA immunization. This study demonstrated that the use of CD11c+ BMDC can enable the development of autopathogenic T-cells to be studied in vivo without the unwanted side-effects of long-lasting injection site inflammation. This model represents a significant refinement to existing EAE models and may lead to the improvement of the welfare of experimental mice used to study the development of autoimmunity in vivo.

So in this study they look at C57BL/10.PLx C57BL/6 mice which are not that susceptible to EAE and show that if you transfer dendritic cells (an immune stimulator immune cell) that have been bound to myelin basic protein that you can induce disease that is as good as the EAE induced by injecting mice with a nasty immune stimulating adjuvant. 

The Home Office in the UK, waters its lips and sees a way to reduce the severity of animal research because the study is published in an animal care rag.  

However, the problem is that most animal strains do not respond to myelin basic protein and there are no transgenic animals for all antigens and for all strains and species, to do all the background work

You can also induce disease using myelin specific T cells, rather than doing the procedures done in the current study.

Importantly, nobody in Europe injects into the legs and feet anymore or nobody should be injecting into feet, so the revelation is so much not a revelation but a misnoma. 

However this is never mentioned in the manuscript. The nasty adjuvant is injected under the skin in the flank and so does not cause the distress that this manuscript aims to suggest.

Most people now inject the immune stimulator under loose skin in the flank. It is certainly less painful there and one may ask if it is painful at all? Certainly the injections site do not appear painful to touch in mice, based on pain faces and the lack of appreciable responses to the injection sites being touched.

In this study they first inject normal animals with T cells that respond to the myelin antigen and then they inject the animals with antigen on dendritic cells.

They get good disease, but the injection site reaction is better compared to the nasty adjuvant, which is never now used in the way reported.

Now I am waiting for the Home office Inspector to come and say why not use this 3Rs approach, and we will spend weeks and thousands investigating this.

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1 comment

  • Thank you for your compassion in dealing with the animals. Thank you, too, for breaking up the text into manageable reading chunks.

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