Name: induction of right ventricular failure by pulmonary artery constriction and evaluation of right ventricular function in mice
Uploaded: 2019-05-13T13:30:00
Description: Watch this Scientific Journal Video about Induction of Right Ventricular Failure by Pulmonary Artery Constriction and Evaluation of Right Ventricular Function in Mice at JoVE.com

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。

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
<ol><li>准备无菌手术器械(图1A),6-0编织丝缝合线(图1B[a])</...

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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?...

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。

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		<tr height="21">
			<td height="21" style="height:21px;width:395px;">ALC-V8S ventilator</td>
			<td style="width:310px;">SHANGHAI&#160; ALCOTT&#160; BIOTECH&#160; CO</td>
			<td style="width:123px;">ALC-V8S</td>
			<td style="width:327px;">Assist ventilation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Animal Mini Ventilator</td>
			<td style="width:310px;">Haverd</td>
			<td style="width:123px;">Type 845</td>
			<td style="width:327px;">Assist ventilation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Animal ultrasound system VEVO2100</td>
			<td style="width:310px;">Visual Sonic</td>
			<td style="width:123px;">&#160;VEVO2100</td>
			<td style="width:327px;">Echocardiography</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Cold light illuminator</td>
			<td style="width:310px;">Olympus</td>
			<td style="width:123px;">ILD-2</td>
			<td style="width:327px;">Light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Heat pad- thermostatic surgical system (ALC-HTP-S1)</td>
			<td style="width:310px;">SHANGHAI&#160; ALCOTT&#160; BIOTECH&#160; CO</td>
			<td style="width:123px;">ALC-HTP-S1</td>
			<td style="width:327px;">Heating</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Isoflurane</td>
			<td style="width:310px;">RWD life science</td>
			<td style="width:123px;">R510-22</td>
			<td style="width:327px;">Inhalant anaesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Matrx VIP 3000 Isofurane Vaporizer</td>
			<td style="width:310px;">Midmark Corporation</td>
			<td style="width:123px;">VIP 3000</td>
			<td style="width:327px;">Anesthetization</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:395px;">Medical braided silk suture (6-0)</td>
			<td style="width:310px;">Shanghai Pudong Jinhuan Medical Supplies Co.</td>
			<td style="width:123px;">6-0</td>
			<td style="width:327px;">Ligation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Medical nylon suture (5-0)</td>
			<td style="width:310px;">Ningbo Medical Needle Co.</td>
			<td style="width:123px;">5-0</td>
			<td style="width:327px;">Suture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Millar Catheter (1.0 F)</td>
			<td style="width:310px;">AD instruments</td>
			<td style="width:123px;">1.0F</td>
			<td style="width:327px;">For right heart catheterization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Pentobarbital sodium salt</td>
			<td style="width:310px;">Merck</td>
			<td style="width:123px;">25MG</td>
			<td style="width:327px;">Anesthetization</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:395px;">PowerLab multi-Directional physiological Recording System</td>
			<td style="width:310px;">AD instruments</td>
			<td style="width:123px;">4/35</td>
			<td style="width:327px;">Record the result of right heart catheterization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Precision electronic balance</td>
			<td style="width:310px;">Denver Instrument</td>
			<td style="width:123px;">TB-114</td>
			<td style="width:327px;">Weighing sensor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Self-made latch needle</td>
			<td style="width:310px;"></td>
			<td style="width:123px;"></td>
			<td style="width:327px;">Separate the aorta and pulmonary trunk</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Self-made padding needle&#160;</td>
			<td style="width:310px;"></td>
			<td style="width:123px;"></td>
			<td style="width:327px;">Constriction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Self-made tracheal intubation</td>
			<td style="width:310px;"></td>
			<td style="width:123px;"></td>
			<td style="width:327px;">Tracheal intubation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Small animal microsurgery equipment</td>
			<td style="width:310px;">Napox</td>
			<td style="width:123px;">MA-65</td>
			<td style="width:327px;">Surgical instruments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Transmission Gel</td>
			<td style="width:310px;">Guang Gong pai</td>
			<td style="width:123px;">250ML</td>
			<td style="width:327px;">Echocardiography</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Veet hair removal cream</td>
			<td style="width:310px;">Reckitt Benchiser</td>
			<td style="width:123px;">RQ/B 33 Type 2</td>
			<td style="width:327px;">Remove hair of mice</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:395px;">Vertical automatic electrothermal pressure steam sterilizer</td>
			<td style="width:310px;">Hefei Huatai Medical Equipment Co.</td>
			<td style="width:123px;">LX-B50L</td>
			<td style="width:327px;">Auto clean the surgical instruments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Vertical small animal surgery microscope</td>
			<td style="width:310px;">Yihua Optical Instrument</td>
			<td style="width:123px;">Y-HX-4A</td>
			<td style="width:327px;">For right heart catheterization</td>
		</tr>
	</tbody>
</table>

induction of right ventricular failure by pulmonary artery constriction and evaluation of right ventricular function in mice

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。

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...

Watch this Scientific Journal Video about Induction of Right Ventricular Failure by Pulmonary Artery Constriction and Evaluation of Right Ventricular Function in Mice at JoVE.com

Induction of Right Ventricular Failure by Pulmonary Artery Constriction and Evaluation of Right Ventricular Function in Mice (Video) | JoVE

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。

小鼠肺动脉收缩引起的右心室衰竭及右心室功能评价

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of ...

该方法为研究右心室失效机理提供了一种有用的技术。该技术的主要优点是节省时间，提高手术成功率。这种技术可以帮助我们模仿右心肥大和失败在人类通过收缩肺动脉，以产生一个脉动压力过载。该方法可应用于横向大体收缩技术，可对心脏移植后并发症和先天性心脏病研究提供见解。手术将由我实验室的技术人员黄晓霞演示。在开始手术之前，通过从一毫升注射器中拆卸的 25 量针通过一块 6-0 编织的丝缝。使用止血钳将针头弯曲 90 度。然后弯曲 22 表针 120 度。在确认对脚趾捏没有反应后，将麻醉小鼠放在37摄氏度加热垫上的上角位置，用缝合固定切口。用胶带固定四肢，并使用脱毛霜将头发从颈部去除至西腓过程。用连续碘和75%乙醇湿巾对裸露的皮肤进行消毒，并使用尖钳稍微提取舌头。用定制的铲子提升毛骨力，以暴露球状，并使用冷光源引导小心插入定制气管管通过球状。调整迷你呼吸机参数，将呼吸速率设置为每分钟 150 次呼吸，潮汐量为 300 微升。然后连接管子和呼吸机，以确认在用胶带固定导管之前，管和呼吸机已正确插入。要打开胸部，请制作一个与第二根肋骨平行约 10 毫米的皮肤切口。使用剪刀和肘钳将胸腔和第二个间空间上方的胸腔和小肌肉分开。从胸角数一数肋骨，以识别第二个成本间空间，并使用剪刀打开此空间。然后钝分开的胸腺和胸腺，直到肺干是可见的。为了收缩肺动脉，使用肘钳钝分离肺干和上升主动脉。使用 90 度闩锁针通过肺干和上升主动脉之间的结缔组织通过缝合线。将 120 度填充针放在肺干干上。使用 6-0 编织的丝线缝合将填充针装到肺干，在观察肺锥填充后立即取出填充针。切割缝合线两端，并继续观察肺锥的填充，以评估是否存在收缩。然后再次评估动物的踏板反射，以确保结扎的成功，并关闭胸部和皮肤与5-0尼龙缝合线。手术后至少三天，在肺动脉收缩动物的胸部涂抹超声波凝胶。将 30 兆赫探头放在胸前，逆时针位置 30 度，相对于左侧副星线，同时将槽口定向到凹槽方向。在 B 模式下调节 Y 轴和 X 轴，直到斜阀、主主管和左心室清晰可见。将探头在其 Y 轴上旋转 30 到 40 度，朝向胸部旋转。调节 Y 轴和 X 轴，直到肺锥清晰可见，并将光标放在肺瓣单的尖端，以测量峰值流速。选择颜色和 PW，将脉冲波虚线平行于血流方向。调整垫的左侧，使其高于右侧，使探头沿右侧内部轴线向地平线方向保持 30 度，缺口指向凹槽方向。调节 X 轴和 Y 轴，直到右心室清晰可见。然后单击 M 模式两次以显示指标线。手术八周后，如证明，插管小鼠，并通过隔膜在西腓体下做一个1.5厘米的双边切口，以打开胸腔。切开肋骨以暴露心脏，并使用23测量针穿透右心室自由壁。用棉签轻轻按压穿刺点以止血，并使用导管尖端刺穿伤口的心室。当曲线稳定时，调节导管尖端向右心室流出。单击选择并分析右心室收缩压和舒张压、最大上升速率、指数时间常数放松和心率 10 分钟。然后将导管从心脏转移到盐水容器中。与Sham组相比，在肺动脉收缩组手术后八周内，从准星长轴视图获得更高的峰值速度、更大的压力梯度和更大的右心室壁厚。此外，与手术后八周的Sham组相比，右心室的收缩功能在肺动脉收缩组显著降低。肺动脉收缩组在囊肿和胆囊中具有较高的右心室压力，与Sham组相比，收缩指数降低。右心室Tau和最大上升速率在肺动脉收缩组也大于在沙姆组。证明右心室功能障碍在小鼠在肺动脉带状八周后诱发。右心室的侵入性血动力测试显示，在导管监测之前和之后，心率保持稳定。与沙姆组相比，右心室尺寸明显增大，右心室重量较高。此外，组织学检查表明，心肌细胞覆盖区域的心脏纤维化在肺动脉收缩组比沙姆组更大。手术中最重要的部分是确保肺动脉正确和完全分离。为了避免不正确的分离，分离点应靠近肺动脉的分支，其中动脉之间的结缔组织将松散。通过这种方法，我们提供了一种更方便、更高效的诱导右心室肥大的方法。一般来说，外科手术和评估方法是非常安全的。

Research

JoVE Journal

Biology

Induction of Right Ventricular Failure by Pulmonary Artery Constriction and Evaluation of Right Ventricular Function in Mice

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