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

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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.