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Biología

小鼠肺动脉收缩引起的右心室衰竭及右心室功能评价

Published: May 13, 2019
doi:
10.3791/59431
, , , , , ,
1Department of Cardiology, State Key Laboratory of Organ Failure Research, Nanfang Hospital,Southern Medical University, 2The Third Clinical Medical College,Guangzhou Medical University

Summary

本文为研究右心室故障机制提供了一种有用的方法。利用内部制造的手术器械,建立了一种更方便、更高效的肺动脉收缩方法。此外,还提供了通过超声心动图和导管法来评估该方法质量的方法。

Abstract

右心室衰竭(RVF)的机制需要澄清,因为裂谷热的独特性、高发病率、高死亡率和耐火性。以前的老鼠模型模仿裂谷热进展已经描述。与大鼠相比,老鼠更容易接触、经济、广泛地用于动物实验。我们开发了肺动脉收缩(PAC)方法,该方法包括将小鼠的肺干带以诱导右心室(RV)肥大。设计了一种特殊的手术闩锁针,便于主动脉和肺干分离。在我们的实验中,使用这种制造的闩锁针降低了动脉瘤的风险,并将手术成功率提高到90%。我们使用不同的填充针直径来精确创建定量收缩,从而引起不同程度的RV肥大。我们通过评估PA的血流速度来量化收缩的程度,这是通过非侵入性转胸超声心动图测量的。RV功能在手术后8周内通过右心导管精确评估。室内制造的手术器械由常用材料组成,使用易于掌握的简单工艺。因此,此处描述的 PAC 方法很容易使用实验室中制造的仪器进行模仿,并可广泛应用于其他实验室。本研究提出了一种经过改进的PAC方法,其成功率高于其他型号,8周手术后存活率为97.8%。这种PAC方法为研究裂谷热机制提供了一种有用的技术,有助于增加对裂谷热的理解。

Introduction

RV 功能障碍 (RVD), 定义为异常 RV 结构或功能的证据, 与不良的临床结果相关.裂谷热作为RV功能的终点,是一种临床综合征,其征期RVD1会导致心力衰竭的体征和症状。左心室(LV)衰竭和裂谷热在结构和生理功能上存在差异,病理生理机制不同。已报告裂谷热中的一些独立病理生理学机制,包括过度表达β2-肾上腺素受体信号2、炎症3、横向小管重塑和Ca2+处理功能障碍4.

裂谷热可能由 RV 的体积或压力过载引起。以前的动物模型已经使用SU5416(血管内皮生长因子受体的一种强效和选择性抑制剂)结合缺氧(SuHx)5,6或单克罗塔线7诱导肺高血压,这导致裂谷热继发于肺血管疾病2。进行这些研究的研究人员专注于血管,而不是裂谷热的病理进展。此外,单克罗酮具有心脏外效应,不能精确代表心脏病。其他型号已经使用动脉分流诱导体积过载和裂谷热8。然而,这种手术很难进行,不适合小鼠,他们需要较长的诱导期来生产裂谷热。

使用带状夹的鼠PAC模型也存在9,10。与大鼠相比,小鼠作为心脏病的动物模型具有许多优点,如繁殖方便、使用范围更广、成本降低、基因修饰等优点。然而,带带夹的直径通常从0.5毫米到1.0毫米,这对小鼠9来说太大了。此外,带带夹很难在其他实验室中产生、模仿和普及。

我们提供一个协议,以开发一个基于报告的研究的修改生殖裂谷热小鼠模型,它使用PAC来模仿法洛特和努南综合征或其他肺动脉高血压疾病的四科病12,13, 14,15,16,17,18,19。这种PAC方法是通过使用内部制成的闩锁和填充针对小鼠的肺干进行粘接,以控制收缩程度而创建的。闩锁针由 90° 弯曲的注射注射器制成,其编织的丝线缝合通过注射器。针头由常用材料制成,使用易于掌握的工艺。衬垫针与仪表针弯曲 120°。根据小鼠的重量(20-35 克),使用不同直径(0.6-0.8 mm)的填充针。此外,我们还建立了评估标准,通过超声心动图和右心导管测定来确定裂谷热模型的稳定性和质量。我们使用老鼠作为模型动物,因为它们在其他实验中广泛使用。实验室生产的针头易于复制,可广泛应用于其他实验室。本研究为研究人员研究裂谷热机理提供了一种很好的方法。

Protocol

所有程序均按照动物研究机构准则进行,该指南符合美国国家卫生研究院出版的《实验室动物护理和使用指南》(NIH出版物第85-23号,1996年修订)。C57BL/6雄性小鼠(8-10周大,重20-25克)由南方医科大学动物中心提供。到达后,老鼠被安置在12/12小时的暗/光周期下,有足够的食物和水。 1. 手术器械的准备和针头的制造 准备无菌手术器械(图1A),6-0编织丝缝合线(图1B[a])</…

Representative Results

在这项研究中,小鼠被随机分配到PAC组(n = 9)或假操作组(n = 10)。在手术后1、4和8周进行超声心动图。手术后八周,在最后一次超声心动图和导管化评估后,小鼠被安乐死,他们的心脏被收获进行形态学和组织学评估。 肺躯干收缩引起RV肥大(图2)。与假组相比,从半身长轴视图中,更高的峰值速度(<stro…

Discussion

RV填充压力的病理增加导致隔膜向左移动,从而改变LV几何形状21。这些变化导致心脏输出和LV弹出分数(LVEF)的减少,这可能导致循环系统22的血液动力学紊乱。因此,为研究裂谷热机制提供一个高效、稳定、经济的模型是有价值的。

我们使用内部制造的闩锁和衬垫针,开发出一种更有效、更可重复的 PAC 方法。内部制造的闩锁可以更容易地分离主?…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

这项工作得到了国家自然科学基金(81570464,81770271;廖博士)和广州市科技规划项目(201804020083)(向廖博士)的资助。

Materials

ALC-V8S ventilator SHANGHAI  ALCOTT  BIOTECH  CO ALC-V8S Assist ventilation
Animal Mini Ventilator Haverd Type 845 Assist ventilation
Animal ultrasound system VEVO2100 Visual Sonic  VEVO2100 Echocardiography
Cold light illuminator Olympus ILD-2 Light
Heat pad- thermostatic surgical system (ALC-HTP-S1) SHANGHAI  ALCOTT  BIOTECH  CO ALC-HTP-S1 Heating
Isoflurane RWD life science R510-22 Inhalant anaesthesia
Matrx VIP 3000 Isofurane Vaporizer Midmark Corporation VIP 3000 Anesthetization
Medical braided silk suture (6-0) Shanghai Pudong Jinhuan Medical Supplies Co. 6-0 Ligation
Medical nylon suture (5-0) Ningbo Medical Needle Co. 5-0 Suture
Millar Catheter (1.0 F) AD instruments 1.0F For right heart catheterization
Pentobarbital sodium salt Merck 25MG Anesthetization
PowerLab multi-Directional physiological Recording System AD instruments 4/35 Record the result of right heart catheterization
Precision electronic balance Denver Instrument TB-114 Weighing sensor
Self-made latch needle Separate the aorta and pulmonary trunk
Self-made padding needle  Constriction
Self-made tracheal intubation Tracheal intubation 
Small animal microsurgery equipment Napox MA-65 Surgical instruments
Transmission Gel Guang Gong pai 250ML Echocardiography
Veet hair removal cream Reckitt Benchiser RQ/B 33 Type 2 Remove hair of mice
Vertical automatic electrothermal pressure steam sterilizer Hefei Huatai Medical Equipment Co. LX-B50L Auto clean the surgical instruments
Vertical small animal surgery microscope Yihua Optical Instrument Y-HX-4A For right heart catheterization
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Tags

Keywords: Right Ventricular FailurePulmonary Artery ConstrictionRight Ventricular FunctionMouse ModelPulmonate Pressure OverloadTransverse Aortic ConstrictionPost Heart Transplantation ComplicationsCongenital Heart DiseaseSurgical ProcedureTracheal CannulationMechanical VentilationThoracotomyPulmonary Artery IsolationPulmonary Artery Constriction
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Wang, Q., Chen, K., Lin, H., He, M., Huang, X., Zhu, H., Liao, Y. Induction of Right Ventricular Failure by Pulmonary Artery Constriction and Evaluation of Right Ventricular Function in Mice. J. Vis. Exp. (147), e59431, doi:10.3791/59431 (2019).
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