Real-Time Platelet Monitoring Techniques To Reduce And Refine Animal Use In Cardiovascular Research

Introduction: We have developed an animal model to study platelet aggregation in vivo which overcomes the use of mortality techniques known to inflict suffering and pain since they are conducted in conscious animals and require high numbers. Indeed these techniques use death or hind limb paralysis as endpoints following the induction of thromboembolism through the intravenous injection of platelet agonist.

Methods: In our refined thromboembolic model blood is collected from anaesthetised donor mice by cardiac puncture and platelets are isolated and then radio labelled with 111Indium chloride. Radio labelled platelets are infused via the femoral vein into anaesthetised recipient mice and platelet aggregation is measured as increases in platelet-associated counts in the pulmonary vasculature following intravenous injections of platelet agonists. Data are collected via a “Single Point Extended Area Radiation” detector positioned over the pulmonary vascular bed and recorded on a UCS-20 spectrometer using custom made software. Furthermore, we developed a simplistic alternative to our model which entails the measurement of circulating platelet counts at different time points following agonist stimulation. This model has been validated by treating platelets in the presence or absence of anti-platelet drugs.

Results: Data show that increases in platelet counts in the pulmonary vasculature following platelet stimulation is associated with a measurable drop in circulating platelet counts. Additionally, we identified three published studies which have investigated the cardio protective effects of different drugs (i.e. desmolaris, aegyptin and sulforaphane) on platelet aggregation in vivo using thromboembolic mortality as an end-point. We established collaborations with these groups to promote the use of our refined model, which holds the potential of delivering more informative data by recording the complete time-course of the platelet response and reduces the mouse use by 23% (aegyptin) to 40% (sulforaphane) and avoids the use of painful procedures by conducting experiments under general anaesthesia. The data obtained with the platelet monitoring technique did not show a significant effect of the drugs on platelet aggregation in vivo in contrast to the observed increase in animal survival when using the mortality model.

Conclusions: These findings suggest that caution should be used when using thromboembolic mortality models to predict platelet function in vivo since a reduction in mortality is shown here to not necessarily reflect a change in platelet aggregation. In addition, the development of more simplisticassays of thromboembolism will drive uptake of our refined methods by making them easier to implement in other research groups.