Tail Vein Transection Bleeding Model in Fully Anesthetized Hemophilia A Mice

Summary

The refined tail vein transection (TVT) bleeding model in anesthetized mice is a sensitive in vivo method for the assessment of hemophilic bleeding. This optimized TVT bleeding model uses blood loss and bleeding time as endpoints, refining other models and avoiding death as an endpoint.

Abstract

Tail bleeding models are important tools in hemophilia research, specifically for the assessment of procoagulant effects. The tail vein transection (TVT) survival model has been preferred in many settings due to sensitivity to clinically relevant doses of FVIII, whereas other established models, such as the tail clip model, require higher levels of procoagulant compounds. To avoid using survival as an endpoint, we developed a TVT model establishing blood loss and bleeding time as endpoints and full anesthesia during the entire experiment. Briefly, anesthetized mice are positioned with the tail submerged in temperate saline (37°C) and dosed with the test compound in the right lateral tail vein. After 5 min, the left lateral tail vein is transected using a template guide, the tail is returned to the saline, and all bleeding episodes are monitored and recorded for 40 min while collecting the blood. If no bleeding occurs at 10 min, 20 min, or 30 min post-injury, the clot is challenged gently by wiping the cut twice with a wet gauze swab. After 40 min, blood loss is quantified by the amount of hemoglobin bled into the saline. This fast and relatively simple procedure results in consistent and reproducible bleeds. Compared to the TVT survival model, it uses a more humane procedure without compromising sensitivity to pharmacological intervention. Furthermore, it is possible to use both genders, reducing the total number of animals that need to be bred, in adherence with the principles of 3R's. A potential limitation in bleeding models is the stochastic nature of hemostasis, which can reduce the reproducibility of the model. To counter this, manual clot disruption ensures that the clot is challenged during monitoring, preventing primary (platelet) hemostasis from stopping bleeding. This addition to the catalog of bleeding injury models provides an option to characterize procoagulant effects in a standardized and humane manner.

Introduction

Animal models are essential for understanding the pathogenesis of hemophilia and developing and testing treatment regimens and therapies. The Factor VIII knock-out mouse (F8-KO) is a widely used model for the study of hemophilia A^1,2. These mice recapitulate key features of the disease and have been widely used for development of treatments, such as recombinant FVIII products^3,4,5 and gene therapy strategies^6,7.

There are various bl....

Protocol

All procedures described in this protocol have been approved by the Animal Welfare Body at Novo Nordisk A/S, and the Danish Animal Experiments Inspectorate, The Danish Ministry of Food, Agriculture, and Fisheries. The optimized 40 minutes method includes anesthesia and dosing time in the design (Figure 1). Hemophilic mice of both genders between 10-16 weeks of age are required for this procedure.

1. Preparations before the study

1. Prepare the dosing solutions in the correct concentrations.
2. Start the water bath and heat to 37 °C. Fill the 15 mL centrifuge tubes for blood collection with ....

Results

To assess the applicability of the optimized model, a study was performed in F8-KO (C57BL genetic background) mice administered with a commercially available recombinant factor VIII replacement therapy (rFVIII); four different doses were tested: 1 IU/kg, 5 IU/kg, 10 IU/kg, and 20 IU/kg. Furthermore, we tested the corresponding vehicle (negative) control in F8-KO mice and wild-type (WT) group using C57BL mice as a positive control group to assess the response range in the model.

Following the o.......

Discussion

This optimized method of tail vein transection (TVT) has several advantages compared to the TVT survival method. The animals are fully anesthetized for the entire study duration, which makes mouse handling easier and increases animal wellbeing. Further, unlike the TVT survival model, overnight observation is not required, and this optimized model offers the possibility of measuring blood loss and observing the exact bleeding time over 40 min. Also, longer periods of bleeding in conscious animals can cause death by exsang.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
#11 Scalpel blade	Swann-Morton	503
15 mL centrifuge tubes	Greiner Bio-One, Austria	188271
30 G needles connected to 300 µL precision (insulin) syringes for dosing	BD Micro-Fine + U-100 insulin syringe	320830
Advate	Takeda, Japan		Recombinant factor VIII replacement therapy (rFVIII)
Alcohol pads 70% ethanol	Hartmann, Soft-Zellin	999 979
Centrifuge	Omnifuge 2.0 RS, Heraus Sepatech
Cutting template (Stainless steel)	Self produced, you are welcomed to contact the authors for the exact drawings		Supplementary Figure 2: Size specifications: 20 mm x 40 mm x 10 mm (L x B x H). Groove: 3 mm depth and 3 mm width; radius 1.5 mm
Erythrocytes (RBC) lysing solution	Lysebio, ABX Diagnostics	906012
Gauze
Haematological analyser	Sysmex	CT-2000iv
Heating lamp on stand	Phillips	IR250
Heating pad with thermostat	CMA	model 150
Hemoglobin standards and controls - 8.81 mmol / l batch dependent	HemoCue, Denmark	HemoCue calibrator, 707037	Standards and controls are made from 2 different glasses of HemoCue calibrator. The value is determined against the International Reference Method for Hemoglobin (ICSH).
Isofluorane anaesthesia system complete with tubes, masks and induction box	Sigma Delta Dameca
Isoflurane	Baxter	26675-46-7
Magnifier with lights	Eschenbach
Measuring template (Aluminum)	Self produced, you are welcomed to contact the authors for the exact drawings		Supplementary Figure 1: Size specifications: 20 mm x 40 mm x 10 mm (L x B x H). Groove: 2.5 mm depth and 2.5 mm width; radius 1.25 mm
Micropipettes + tips	Finnpipette
Photometer	Molecular Devices Corporation, CA, USA	SpectraMax 340 photometer
Prism Software	GraphPad, San Diego, CA, USA	Version 9.0.1
Saline 0.9% NaCl	Fresenius Kabi, Sweden	883264
Special tail marker block for TVT tail cut
Tail holder
Vacuum liquid suction	Vacusafe comfort, IBS
Waterbath and thermostat	TYP 3/8 Julabo