Standardizing a simpler, more sensitive and accurate tail bleeding assay in mice

AIM: To optimize the experimental protocols for a simple, sensitive and accurate bleeding assay.METHODS: Bleeding assay was performed in mice by tail tip amputation, immersing the tail in saline at 37 ℃, continuously monitoring bleeding patterns and measuring bleeding volume from changes in the body weight. Sensitivity and extent of variation of bleeding time and bleeding volume were compared in mice treated with the P2 Y receptor inhibitor prasugrel at various doses or in mice deficient of Fc Rγ, a signaling protein of the glycoprotein VI receptor.RESULTS: We described details of the bleeding assay with the aim of standardizing this commonly used assay. The bleeding assay detailed here was simple to operate and permitted continuous monitoring of bleedingpattern and detection of re-bleeding. We also reported a simple and accurate way of quantifying bleeding volume from changes in the body weight, which correlated well with chemical assay of hemoglobin levels(r2 = 0.990, P < 0.0001). We determined by tail bleeding assay the dose-effect relation of the anti-platelet drug prasugrel from 0.015 to 5 mg/kg. Our results showed that the correlation of bleeding time and volume was unsatisfactory and that compared with the bleeding time, bleeding volume was more sensitive in detecting a partial inhibition of platelet's haemostatic activity(P < 0.01). Similarly, in mice with genetic disruption of Fc Rγ as a signaling molecule of P-selectin glycoprotein ligand-1 leading to platelet dysfunction, both increased bleeding volume and repeated bleeding pattern defined the phenotype of the knockout mice better than that of a prolonged bleeding time.CONCLUSION: Determination of bleeding pattern and bleeding volume, in addition to bleeding time, improved the sensitivity and accuracy of this assay, particularly when platelet function is partially inhibited.

Post-infarct cardiac injury, protection and repair:roles of non-cardiomyocyte multicellular and acellular components

Following myocardial infarction(MI), cardiomyocytes and infarct size are the focus of our attention when evaluating the extent of cardiac injury, efficacy of therapies or success in repairing the damaged heart by stem cell therapy. Numerous interventions have been shown by pre-clinical studies to be effective in limiting infarct size, and yet clinical trials designed accordingly have yielded disappointing outcomes. The ultimate goal of cardiac protection is to limit the adverse cardiac remodeling. Accumulating studies have revealed that post-infarct remodeling can be attenuated without infarct size limitation. To reconcile this, one needs to appreciate the significance of various cellular and acellular myocardial components that, like cardiomyocytes, undergo significant damage and dysfunction, which impact the ultimate cardiac injury and remodelling. Microvascular injury following ischemia-reperfusion may influence infarct size and promote inflammation. Myocardial injury evokes innate immunity with massive inflammatory infiltration that, although essential for the healing process, exacerbates myocardial injury and damage to extracellular matrix leading to dilative remodeling. It is also important to consider the multiple non-cardiomyocyte components in evaluating therapeutic efficacy. Current research indicates the pivotal role of these components in achieving cardiac regeneration by cell therapy. This review summarizes findings in this field, highlights a broad consideration of therapeutic targets,and recommends cardiac remodeling as the ultimate target.