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.

Differential virulence of Trypanosoma brucei rhodesiense isolates does not influence the outcome of treatment with anti-trypanosomal drugs in the mouse model

“…Blood for estimation of parasitaemia levels was collected daily for the first 14 days and thereafter three times in a week from each mouse using the tail tip amputation method [20]. The PP and parasitaemia progression were determined using the rapid matching method of [16] [21].…”

Assessing Blood Clotting and Coagulation Factors in Mice

“…The tail bleeding assay detailed here is conceptually similar to the human template bleeding time in that the amount of bleeding from an induced injury and time to cessation of bleeding is quantitated. However, the mouse tail bleeding assay has not been completely standardized, and thus “mouse tail bleeding assay” is presently a blanket term for all assays involving the measurement of bleeding from an induced injury to the tail of a mouse (Greene et al., ; Saito et al., ). The nonstandardization of this assay causes varying results between laboratories and makes communication between the laboratories difficult with regard to agreeing on what exactly has been assayed when a mouse tail bleeding assay has been performed.…”

“…The mouse tail clip assay is the most commonly used bleeding model to measure bleeding time and the amount of blood loss, and it is one of only a few in vivo murine assays. This assay has proven useful to compare the differences between mouse models of hemostatic and thrombotic disorders, especially for evaluating hemostasis in genetically deficient mice (Broze, Yin, & Lasky, 2001;Sambrano et al, 2001) and for testing antihemostatic (Bi et al, 1995;Broze et al, 2001) or antithrombotic (Saito et al, 2016) compounds or drugs. Both total blood loss and time to cessation of blood flow are used as measures of the hemostatic system.…”

“…Current Protocols in Mouse Biology from an induced injury to the tail of a mouse (Greene et al, 2010;Saito et al, 2016). The nonstandardization of this assay causes varying results between laboratories and makes communication between the laboratories difficult with regard to agreeing on what exactly has been assayed when a mouse tail bleeding assay has been performed.…”

“…We assayed the undiluted samples, and these were read using a spectrophotometer at 416-nm wavelength. Recently, Saito et al (2016) described a potentially more sensitive method to determine hemoglobin concentration by immersing the cut tail directly into 2.5 ml of a solution containing Drabkin's reagent (Sigma-Aldrich, cat. no.…”