Name: tail vein transection bleeding model in fully anesthetized hemophilia a mice
Uploaded: 2021-09-30T14:00:00
Description: Watch this Scientific Journal Video about Tail Vein Transection Bleeding Model in Fully Anesthetized Hemophilia A Mice at JoVE.com

Esther Bloem and Thomas Nygaard are acknowledged for support with measurements of FVIII in plasma. Bo Alsted is acknowledged for drawing and machining the template and cutting blocks.

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
<ol>
	<li>Prepare the dosing solu...

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...

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 A1,2. These mice recapitulate key features of the disease and have been widely used for development of treatments, such as recombinant FVIII products3,4,5 and gene therapy strategies6,7.
There are various bleeding injury models for evaluating the pharmacological effects of different hemostatic compounds in vivo. One of these coagulation models is the tail vein transection survival model in mice8,9,10,11,12,13,14, measuring the ability of hemophilic mice to survive exsanguination after tail transection. This method was introduced more than four decades ago15 and is still used9,16,17. However, the model utilizes survival as an endpoint and requires observation of the animals over a period of up to 24 h, during which the animals are conscious and hence can experience pain and distress.
Bleeding models of shorter duration and under full anesthesia have been described previously, such as the tail clip model (also known as the tail tip)8,18,19,20,21,22,23,24,25,26,27,28. Nevertheless, for a complete normalization of blood loss after the bleeding challenge, these models require doses of procoagulant compounds (e.g., FVIII) far higher than those administered clinically29. A different injury model under anesthesia, the vena saphena bleeding method, is sensitive to lower doses of procoagulant compounds30 but requires a high level of experimenter intervention since the clots must be disrupted frequently (as opposed to 3 times in the presented model).
Standardization towards a common protocol to test new procoagulant compounds would greatly&#160;facilitate data comparison between laboratories31,32,33. In TVT models, there is not yet a common agreement on studied endpoints (blood loss7,26, bleeding time9,34, and survival rate35,36), and experimental length varies between studies13.
Our primary objective is to describe and characterize an optimized model with high reproducibility, the possibility to study on-demand as well as a prophylactic treatment, sensitivity to pharmacological intervention equivalent to the survival model, yet not using death or near-death as endpoints. In order to reduce pain and distress, the animals should not be conscious during bleeding and a more ethical endpoint needs to be implemented37.
Tail clip models are generally conducted in one of two variants, either amputating the tip of the tail, e.g., amputation of 1-5 mm18,19,20,21,23,24 or, in a more severe variant, transected at a tail diameter around 1-3 mm8,22,25. This causes a combined arteriovenous bleed, as the lateral and dorsal veins and ventral artery are usually severed, and in general, the larger the amputation, the lower the sensitivity to a procoagulant compound. Furthermore, since the tail tip is amputated, the arteriovenous injury is exposed without any opposing tissue; thus, at least in theory, it is dissimilar to the most common hemophilic bleeds.
As the name implies, only the vein is injured in tail vein transection models such as described in this paper, thus resulting in an exclusively&#160;venous bleed. Since the vessel is not fully severed, the injury is expected to be smaller than in the amputation models, and the tissue around the cut, which a clot may adhere to, is retained. In addition, there is lower blood pressure in the vein as opposed to the artery. These factors contribute to an increased sensitivity relative to amputation models, such that normalization of bleeding can be achieved with clinically relevant doses of replacement therapy, e.g., with rFVIII in hemophilia A, which is useful for evaluating the magnitude and durability of effects of procoagulant treatment26,38,39.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>#11 Scalpel blade</td>
			<td>Swann-Morton</td>
			<td>503</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tubes</td>
			<td>Greiner Bio-One, Austria</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr>
			<td>30 G needles connected to 300 &#181;L precision (insulin) syringes for dosing</td>
			<td>BD Micro-Fine + U-100 insulin syringe</td>
			<td>320830</td>
			<td></td>
		</tr>
		<tr>
			<td>Advate</td>
			<td>Takeda, Japan</td>
			<td></td>
			<td>Recombinant factor VIII replacement therapy (rFVIII)</td>
		</tr>
		<tr>
			<td>Alcohol pads 70% ethanol</td>
			<td>Hartmann, Soft-Zellin</td>
			<td>999 979</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Omnifuge 2.0 RS, Heraus Sepatech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cutting template (Stainless steel)</td>
			<td>Self produced, you are welcomed to contact the authors for the exact drawings</td>
			<td></td>
			<td>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</td>
		</tr>
		<tr>
			<td>Erythrocytes (RBC) lysing solution</td>
			<td>Lysebio, ABX Diagnostics</td>
			<td>906012</td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Haematological analyser</td>
			<td>Sysmex</td>
			<td>CT-2000iv</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating lamp on stand</td>
			<td>Phillips</td>
			<td>IR250</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad with thermostat</td>
			<td>CMA</td>
			<td>model 150</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemoglobin standards and controls - 8.81 mmol / l batch dependent</td>
			<td>HemoCue, Denmark</td>
			<td>HemoCue calibrator, 707037</td>
			<td>Standards and controls are made from 2 different glasses of HemoCue calibrator. The value is determined against the International Reference Method for Hemoglobin (ICSH).</td>
		</tr>
		<tr>
			<td>Isofluorane anaesthesia system complete with tubes, masks and induction box</td>
			<td>Sigma Delta Dameca</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter</td>
			<td>26675-46-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnifier with lights</td>
			<td>Eschenbach</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Measuring template (Aluminum)</td>
			<td>Self produced, you are welcomed to contact the authors for the exact drawings</td>
			<td></td>
			<td>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</td>
		</tr>
		<tr>
			<td>Micropipettes + tips</td>
			<td>Finnpipette</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Photometer</td>
			<td>Molecular Devices Corporation, CA, USA</td>
			<td>SpectraMax 340 photometer</td>
			<td></td>
		</tr>
		<tr>
			<td>Prism Software</td>
			<td>GraphPad, San Diego, CA, USA</td>
			<td>Version 9.0.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline 0.9% NaCl</td>
			<td>Fresenius Kabi, Sweden</td>
			<td>883264</td>
			<td></td>
		</tr>
		<tr>
			<td>Special tail marker block for TVT tail cut</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tail holder</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum liquid suction</td>
			<td>Vacusafe comfort, IBS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Waterbath and thermostat</td>
			<td>TYP 3/8 Julabo</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

tail vein transection bleeding model in fully anesthetized hemophilia a mice

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&#176;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&#39;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.

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...

Watch this Scientific Journal Video about Tail Vein Transection Bleeding Model in Fully Anesthetized Hemophilia A Mice at JoVE.com

Tail Vein Transection Bleeding Model in Fully Anesthetized Hemophilia A Mice

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.

Tail bleeding models are important tools in hemophilia research, specifically for the assessment of procoagulant effects. The tail vein transection (TVT) ...

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