Real-time monitoring techniques and simplistic platelet assays to reduce and refine animal use in cardiovascular and respiratory biomedical research

Platelets are blood cells which are important in clotting to prevent blood loss and in the formation of clots during conditions such as heart attack. Studying platelets to better understand disease and to develop new treatments involves many experiments in mice since the formation of clots is regulated by numerous tissues and cannot be replicated outside an animal. A number of research groups conduct studies in conscious animals by inducing blood clots which lead to the death of the animal following blockage of blood vessels in the lungs. Anaesthesia is not used so the animals suffer considerably. We have developed techniques that allow clot formation to be measured in a single mouse under general anaesthesia. This means that fewer mice are used and painful procedures are avoided. We now wish to work with scientists who currently conduct experiments that involve death in concious mice to develop and implement alternatives. Instead of causing death, we shall measure platelet numbers in circulating blood to predict clot formation. This works because when platelets form a clot in a tissue, there are fewer of them in the general circulation. It may also be possible to collect more than one blood sample from each mouse to further reduce the numbers of mice needed for an experiment. This technique may be used instead of models involving death. The technique is simple to use because blood is easy to collect and cells can be counted under a microscope. The method will be checked using existing drugs that are used to treat heart attack and newer test drugs from research groups who are presently dependent upon models involving death. Groups have already agreed to take part in our study and we shall increase the numbers taking part. This approach will encourage scientists to switch studies using death to very simple alternative techniques that use fewer mice and avoid painful procedures because the mice are anaesthetised for the entire experiment.
Platelets are also involved in defending us against germs and infection. To attack germs platelets need to leave the blood stream and travel through tissue to reach the infection. The body does this through a protective mechanism called the inflammatory response. In diseases such as asthma, platelets enter the lungs from the bloodstream. Better understanding of how platelets and other cells enter the lung will lead to new treatments. To study this process scientists cause inflammation in the lungs of mice to replicate conditions such as asthma. Mice are then killed at different times after the start of inflammation and the severity of inflammation assessed by microscopic examination to determine the numbers of cells in the tissue. This involves the use of large numbers of mice because a mouse has to be killed at every time point that needs to be studied. We aim to measure cell movement into the lungs by making cells such as platelets radioactive and tracking their movement in an individual anaesthetised animal. We shall also assess inflammation by measuring the fall in platelets in circulating blood by taking blood samples from individual anaesthetised mice. New therapies can be assessed by measuring their effect on cell movement. This will involve the use of fewer mice and we predict reducing mouse use by 70 to 80%.
Overall, we aim to develop techniques that can be used to determine the effects of drugs that may be useful in treating diseases of both the heart and lungs. The applicants have experience in both of these areas. The techniques that will be developed are cheap and easy to use which will increase their uptake by other scientists.

Platelets are critical participants in haemostasis, thrombosis and inflammation. We have developed technology for assessing radiolabelled platelet aggregation responses in real-time in anaesthetised mice. Our model is a refinement of thromboembolic mortality models that operates at a lower severity level and uses considerably fewer mice. Mortality models continue to be used by a number of research groups and by initiating dialogue with users we have identified a need for more simplistic alternatives and proof of principle using current test reagents to facilitate uptake. We shall therefore evaluate systemic platelet counting as an alternative to thromboembolic mortality models using conventional antithrombotics and reagents from groups currently using mortality techniques. Our approach will refine procedures currently being used to reduce pain and suffering and will also reduce animal use by participating groups by 50%.
We shall also develop techniques for assessing platelet recruitment to the lung during allergic inflammation in real-time in anaesthetised mice. This will provide an alternative to killing of mice at numerous time points for histological determination of cell infiltration. We anticipate a reduction in mouse use of 70-80%. Antithrombotic drugs will be used to distinguish the inflammatory platelet response from thrombotic platelet activation. The model will ultimately be established in a laboratory that is active in the field of inflammatory lung disease to facilitate immediate application and subsequent dissemination to other research groups.
We therefore propose to develop low cost and easily applied models for assessing platelet activation during cardiovascular and respiratory diseases that refine and reduce procedures involving experimental mice.

Who might benefit from this research?
The most predictable impact of this research will be on our academic beneficiaries including the targeted beneficiaries for whom the cardiovascular elements of the proposal were designed.
Pharmaceutical and biotechnology companies are very active in the development of either anti-inflammatory drugs or, conversely, drugs to boost the immune system to fight infections. Pharmaceutical companies therefore believe that pursuing novel therapeutic strategies for the treatment of infections or inflammatory disorders are economically attractive research strategies. Creating models that facilitate this therefore delivers benefit.
Educational establishments and pupils interested in a career in science will benefit from this research, as will the wider public that has an interest in following scientific discoveries and the issue of animal welfare and the 3Rs in science. Imperial College is particularly well placed to engage with the public on this issue due to our links with the Science Museum and our longstanding and successful open days which would provide opportunities to deliver benefits in the form of increased understanding and engagement with the issue of the use of animals in medical research.
We also believe that public policy makers in government and associated organisations will benefit from the research since the scientific and ethical value of this study can be used as an example for the promotion of the broader objectives of NC3Rs.

How will they benefit from this research?
Our academic beneficiaries will benefit because the project is designed to deliver publishable data both within the timeframe of the study and beyond. The models we develop and promote in this proposal have a strong scientific as well as an ethical rationale. The use of mortality as an end-point is highly flawed, it is not a specific end-point in relation to the diseases of interest and, obvious to say, is not a treatable component of the disease. It makes much more sense scientifically to model diseases at an end-point that is directly relevant to the disease and which is reversible or preventable e.g. platelet aggregation or recruitment to sites of inflammation. Our academic beneficiaries will therefore benefit by generating data that is on a stronger scientific footing and which would allow authors to comply with the ARRIVE guidelines for conducting animal research. This would facilitate publication of data in high quality scientific journals.
Pharmaceutical companies would benefit from this research, as we develop and validate technologies that could be used for evaluation of putative anti-platelet or anti-inflammatory therapies on a relatively low cost basis and in models which considerably reduce animal use allowing companies to implement their policies in relation to the 3Rs in a practical way. In the long term, assisting in the development of strategies that effectively combat platelet-driven disorders and infections or inflammatory disorders will enhance the quality of life, and lessen the economic burden of the UK.
The wider public, educational establishments and pupils that are interested in science or are recipients of the various outreach activities that we conduct benefit by becoming more knowledgeable of why appropriate and 3Rs focussed use of animals in science is important and how it can change society for the better. Increasing public support through knowledge of research activities may therefore in the longer term increase funds available for further scientific research. Most importantly, we will hopefully encourage pupils to go to university and study science. Gaining a positive view of the efforts of scientists to work appropriately with animals can be really important in persuading young people to enter the profession rather than being put off by more negative portrayals of the use of animals in research.