New approaches in tail-bleeding assay in mice: improving an important method for designing new anti-thrombotic agents

Abstract

This report describes a modified, simple, low-cost and more sensitive method to determine bleeding patterns and haemoglobin concentration in a tail-bleeding assay using BALB/c mice and tail tip amputation. The cut tail was immersed in Drabkin's reagent to promote erythrocyte lysis and haemoglobin release, which was monitored over 30 min. The operator was blinded to individual conditions of the mice, which were treated with either saline (NaCl 0.15m), DMSO (0.5%) or clinical anti-thrombotic drugs. Our experimental protocols showed good reproducibility and repeatability of results when using Drabkin's reagent than water. Thus, the use of Drabkin's reagent offered a simple and low-cost method to observe and quantify the bleeding and rebleeding episodes. We also observed the bleeding pattern and total haemoglobin loss using untreated animals or those under anti-coagulant therapy in order to validate the new Drabkin method and thus confirm that it is a useful protocol to quantify haemoglobin concentrations in tail-bleeding assay. This modified method provided a more accurate results for bleeding patterns in mice and for identifying new anti-thrombotic drugs.

Keywords: anti-thrombotic agents; bleeding time; tail-bleeding assay.