全麻醉血友病A小鼠尾静脉横断出血模型
1Global Drug Discovery,Novo Nordisk A/S, 2Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences,University of Copenhagen, 3Department of Biomedical Sciences, Faculty of Health and Medical Sciences,University of Copenhagen, 4Independent consultant, 5Catalyst Biosciences, 6Værløse Dyreklinik
Summary
麻醉小鼠的精制尾静脉横断（TVT）出血模型是评估血友病出血的灵敏 体内 方法。这种优化的TVT出血模型使用失血和出血时间作为终点，完善其他模型并避免死亡作为终点。
Abstract
尾出血模型是血友病研究的重要工具，专门用于评估促凝剂效应。由于对临床相关剂量的FVIII敏感，尾静脉横断（TVT）生存模型在许多地区是首选，而其他已建立的模型（如尾夹模型）需要更高水平的促凝剂化合物。为了避免使用生存作为终点，我们开发了一个TVT模型，将失血和出血时间确定为终点，并在整个实验期间进行全麻醉。简而言之，将麻醉的小鼠定位，将尾巴浸没在温带盐水（37°C）中，并在右侧尾静脉中与测试化合物一起给药。5分钟后，使用模板导轨切除左外侧尾静脉，将尾巴返回到盐水，并在收集血液的同时监测并记录所有出血发作40分钟。如果在受伤后10分钟，20分钟或30分钟没有发生出血，则用湿纱布拭子拭子两次，轻轻地挑战凝块。40分钟后，通过出血到盐水中的血红蛋白量来量化失血量。这种快速且相对简单的过程导致一致且可重复的出血。与TVT生存模型相比，它使用更人道的程序，而不会影响对药物干预的敏感性。此外，可以使用两种性别，减少需要繁殖的动物总数，符合3R的原则。出血模型的潜在局限性是止血的随机性，这会降低模型的可重复性。为了解决这个问题，手动破坏凝块可确保在监测期间对凝块进行挑战，防止原发性（血小板）止血。对出血损伤模型目录的这一补充提供了一种选择，以标准化和人道的方式表征促凝剂效应。
Introduction
动物模型对于了解血友病的发病机制以及开发和测试治疗方案和疗法至关重要。因子VIII敲除小鼠（F8-KO）是研究血友病A1，2的广泛使用的模型。这些小鼠概括了该疾病的关键特征，并已被广泛用于开发治疗，例如重组FVIII产品3，4，5 和基因治疗策略6，7。
有多种出血损伤模型用于评估不同止血化合物 在体内的药理作用。这些凝血模型之一是尾静脉横断术存活模型8，9，10，11，12，13，14，测量血友病小鼠在尾部横断后血血后存活的能力。这种方法是在四十多年前15 年引入的，至今仍在使用9，16，17。然而，该模型利用生存作为终点，需要在长达24小时的时间内观察动物，在此期间动物是有意识的，因此可以经历疼痛和痛苦。
以前已经描述过持续时间较短且完全麻醉下的出血模型，例如尾夹模型（也称为尾尖）8，18，19，20，21，22，23，24，25，26，27，28.然而，为了在出血激发后使失血完全正常化，这些模型需要远高于临床施用的促凝剂化合物（例如FVIII）的剂量29。麻醉下的另一种损伤模型，即沙芬纳静脉出血法，对较低剂量的促凝剂化合物30敏感，但需要高水平的实验者干预，因为凝块必须经常破坏（而不是所提出的模型中的3次）。
标准化为测试新的促凝剂化合物的通用方案将大大促进实验室之间的数据比较31，32，33。在TVT模型中，关于研究的终点（失血量7，26，出血时间9，34和存活率35，36）尚未达成共识，并且实验长度因研究而异13。
我们的主要目标是描述和表征具有高可重复性的优化模型，按需研究的可能性以及预防性治疗，对相当于生存模型的药物干预的敏感性，但不使用死亡或濒临死亡作为终点。为了减轻疼痛和痛苦，动物在出血期间不应该有意识，需要实施更合乎道德的终点37。
尾夹模型通常以两种变体之一进行，要么截肢尾尖，例如截肢1-5毫米18，19，20，21，23，24，或者在更严重的变体中，在尾部直径约1-3毫米8，22，25处截断.这会导致联合动静脉出血，因为侧静脉和背静脉以及腹动脉通常被切断，并且通常截肢越大，对促凝化合物的敏感性越低。此外，由于尾尖被截肢，动静脉损伤暴露在没有任何对立组织的情况下;因此，至少在理论上，它与最常见的血友病出血不同。
顾名思义，在如本文所述的尾静脉横断模型中，只有静脉受伤，从而导致完全静脉出血。由于血管未完全切断，因此预计损伤将小于截肢模型，并且保留切口周围的组织（凝块可能粘附）。此外，与动脉相比，静脉中的血压较低。这些因素有助于相对于截肢模型增加敏感性，因此可以通过临床相关剂量的替代治疗（例如，在血友病A中使用rFVIII）实现出血正常化，这有助于评估促凝治疗效果的大小和持久性26，38，39。
Protocol
本议定书中描述的所有程序均已获得诺和诺德公司动物福利机构以及丹麦食品、农业和渔业部丹麦动物实验监察局的批准。优化的 40 分钟方法在设计中包括麻醉和给药时间（图 1）。该手术需要10-16周龄的两性血友病小鼠。 1. 研究前的准备工作 以正确的浓度制备加液。 开始水浴并加热至37°C。 用盐水（0.9%NaCl）填充15mL离心管以收集血液?…
Representative Results
为了评估优化模型的适用性，在用市售重组因子VIII替代疗法（rFVIII）施用的F8-KO（C57BL遗传背景）小鼠中进行了研究;测试了四种不同的剂量：1 IU / kg，5 IU / kg，10 IU / kg和20 IU / kg。此外，我们使用C57BL小鼠作为阳性对照组，在F8-KO小鼠和野生型（WT）组中测试了相应的载体（阴性）对照，以评估模型中的反应范围。 在优化方案之后，与载体组相比，rFVIII治疗组的失血量显着减少…
Discussion
与TVT生存方法相比，这种优化的尾静脉横断（TVT）方法具有几个优点。在整个研究期间，动物被完全麻醉，这使得小鼠处理更容易，并增加了动物的健康。此外，与TVT生存模型不同，不需要过夜观察，并且这种优化的模型提供了测量失血量和观察40分钟内确切出血时间的可能性。此外，意识动物出血时间较长会导致血液出血，导致动物疼痛和痛苦，并可能产生压力，可能导致变异增加<sup class="xref"…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Esther Bloem和Thomas Nygaard因支持等离子体中FVIII的测量而受到认可。Bo Alsted因绘制和加工模板和切割块而获得认可。
Materials
| #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 |
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Cite This Article
Carol Illa, A., Baumgarten, S., Danielsen, D., Larsen, K., Elm, T., Johansen, P. B., Knudsen, T., Lauritzen, B., Tranholm, M., Ley, C. D. Tail Vein Transection Bleeding Model in Fully Anesthetized Hemophilia A Mice. J. Vis. Exp. (175), e62952, doi:10.3791/62952 (2021).