Name: induction and characterization of pulmonary hypertension in mice using the hypoxia/su5416 model
Uploaded: 2020-06-03T12:30:00
Description: Watch this Scientific Journal Video about Induction and Characterization of Pulmonary Hypertension in Mice using the Hypoxia/SU5416 Model at JoVE.com

这项工作得到了美国心脏协会（AHA-17SDG333370112和18IPA34170258）的资助，以及来自国家卫生研究院NIH K01 HL135474到Y.S.O.B的资助，并得到德国赫兹斯蒂夫通的支持。

在任何动物实验之前，获得当地机构动物护理委员会的授权。目前的实验是在西奈山伊坎医学院的机构动物护理和使用委员会（IACUC）批准后进行的。
1. PH 感应
<ol><li>制备<ol>
<li>在开始研究之前，请仔细规划实验设计。确保小鼠在第一次SU5416注射的同一时间点受到缺氧。图1 A 中显示了使用缺氧/SU5416 方法诱导 PH 的实验 设计示例。对照鼠标...

<ol>
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该协议描述了如何通过结合两种病理刺激在小鼠的PH模型：慢性缺氧和SU5416注射（缺氧/SU5416）1818。为了试图将此鼠标模型与人类PH条件关联，不可避免地必须查看表1所示的当前PH分类。几乎所有形式的PH的特点是肺血管收缩和内皮细胞和平滑肌肉细胞的异常增殖。这导致肺动脉压力升高，从而增加右心室的后负荷。
每一次尝试描述PH动物模型应?...

肺高血压 （PH） 是一种病理生理状况，由平均肺动脉 （PA） 压力在休息时超过 25 mm Hg 定义，由右心导管1，2评估。有各种各样的疾病，可导致PH。为了组织与PH相关的条件，开发了几种分类系统。目前的临床分类分类的多个PH相关疾病在5个不同的组1。这种区别很重要，因为不同组的患者的临床表现、病理学、预后和治疗反应都不同。表1总结了目前的分类，并辅之以每种疾病的基本组织病理学特征。
<img alt="Table 1" src="/files/ftp_upload/59252/59252tbl1.jpg"> 表1：PH临床分类概述，以及各组内的主要组织病理学特征。低氧/SU5416 协议的适用性，用于建模 PH。此表已从19 起修改。PH： 肺高血压， PAH： 肺动脉高血压
尽管在治疗PH相关疾病方面取得了重大进展，但PH仍然无法治愈，3年死亡率在20%至80%之间。这表明迫切需要了解PH的基本机制，然后，开发新的疗法，以防止，减缓进展，并治愈疾病。动物模型对于这个范围至关重要。目前，研究PH的各种模型都存在。有兴趣的读者会参考有关这个题目2，3，4,的优秀,评论。考虑到导致PH的各种疾病，很明显，人类PH的各种条件不能完全概括为一种动物模型。可用的动物模型可以分为 i） 单击， ii） 两击， iii） 淘汰， 和 iv） 过度表达模型3。在单击模型中，PH 是由单个病理刺激诱导的，而双命中模型将两种病理刺激与诱导更严重 PH 的目标相结合，从而更密切地模仿复杂的人类疾病。除了病因差异外，几种刺激导致PH建模差异，也取决于动物的种类和遗传背景4。
最常用的经典PH啮齿动物模型之一是慢性缺氧模型2。众所周知，低氧在人类和几种动物物种中诱发PH。缺氧的好处是PH的生理刺激（表1）。然而，虽然用于诱导啮齿动物PH的缺氧程度比人类严重得多，单一的侮辱（缺氧）只导致轻微的血管重塑。这不能模仿人类疾病的严重程度。增加第二次命中，一个额外的刺激诱导PH，显示了有希望的结果：注射化合物SU5416啮齿动物结合低氧刺激诱导更严重的PH表型2，5，6。,5,6SU5416是血管内皮生长因子（VEGF）受体-2的抑制剂。它阻断VEGF受体并导致内皮细胞凋亡。在缺氧条件下，这刺激了抗凋亡内皮细胞子集的增殖。此外，SU5416导致平滑的肌肉细胞增殖。这些效果的组合导致肺循环的病理血管重塑，并导致PA压力升高和右心室重塑2，5，7。,5,7该模型首先在大鼠6中描述，后来应用于小鼠,4、5、7。547与大鼠相比，小鼠模型表现出的血管重塑不太严重。此外，当回到诺莫夏时，PH在大鼠中继续进步，而在大鼠中则部分可逆。
以下协议描述了使用缺氧/SU5416 方法（规划、时间线、执行）在小鼠中建模 PH 的所有步骤。此外，本方案还描述了该模型的特征：功能性（通过侵入性测量右心室（RV）压力使用开放胸部技术）、形态测量（通过解剖和称重右心室和左心室），以及组织学（通过评估肺血管重塑、右心室心肌细胞肥大和纤维化）。
本协议中描述的所有步骤和方法都可以在任何经验级别上由调查人员轻松实施。虽然使用开放胸腔技术（此处描述）的 RV 的功能测量不是该领域的黄金标准方法，但它的优点是，即使经验较少的实验者也可以快速了解和准确再现。

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			<td height="21" style="height:21px;width:319px;">Acetic acid glacial</td>
			<td style="width:231px;">Roth</td>
			<td style="width:119px;">3738.1</td>
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			<td height="21" style="height:21px;width:319px;">Acetone, Histology Grade</td>
			<td style="width:231px;">The Lab Depot</td>
			<td style="width:119px;">VT110D</td>
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			<td height="21" style="height:21px;width:319px;">ADVantage Pressure-Volume System</td>
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			<td height="21" style="height:21px;width:319px;">Bouin&#39;s solution</td>
			<td style="width:231px;">Sigma</td>
			<td style="width:119px;">Ht10132</td>
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			<td height="21" style="height:21px;width:319px;">Cautery System</td>
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			<td style="width:119px;">18000-00</td>
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			<td height="21" style="height:21px;width:319px;">Connection tubing and valves</td>
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			<td height="21" style="height:21px;width:319px;">Cotton-Tipped Applicators</td>
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			<td style="width:119px;">1871</td>
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			<td height="21" style="height:21px;width:319px;">Data Acquisition and Analysis</td>
			<td>Emka</td>
			<td style="width:119px;">iox2</td>
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			<td height="21" style="height:21px;width:319px;">DMSO (Dimethyl Sulfoxide)</td>
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			<td style="width:119px;">276855</td>
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			<td style="width:231px;">Fine Science Tools</td>
			<td style="width:119px;">11251-10</td>
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			<td height="21" style="height:21px;width:319px;">Dumont # 7 Fine Forceps</td>
			<td style="width:231px;">Fine Science Tools</td>
			<td style="width:119px;">11274-20</td>
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			<td height="21" style="height:21px;width:319px;">Embedding molds</td>
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			<td height="21" style="height:21px;width:319px;">Eosin Solution Aqueous</td>
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			<td height="42" style="height:42px;width:319px;">Ethanol, laboratory Grade</td>
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			<td height="21" style="height:21px;width:319px;">Fast Green FCF</td>
			<td style="width:231px;">Sigma</td>
			<td style="width:119px;">F7252-5G</td>
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			<td height="21" style="height:21px;width:319px;">Fine scissors</td>
			<td style="width:231px;">Fine Science Tools</td>
			<td style="width:119px;">14090-09</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Goat Serum</td>
			<td style="width:231px;">invitrogen</td>
			<td style="width:119px;">16210-064</td>
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			<td height="21" style="height:21px;width:319px;">Hematoxylin 2</td>
			<td style="width:231px;">Thermo Scientific</td>
			<td style="width:119px;">7231</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Hypoxic chamber</td>
			<td style="width:231px;">Biospherix</td>
			<td style="width:119px;">A30274P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Induction chamber</td>
			<td style="width:231px;">DRE Veterinary</td>
			<td>12570</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:319px;">Intubation catheter (i.v. catheter SurFlash (20 G x 1&#34;) )</td>
			<td style="width:231px;">Terumo&#160;</td>
			<td>SR*FF2025</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Iris scissors</td>
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			<td style="width:119px;">14084-08</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:319px;">Isoflurane</td>
			<td style="width:231px;">Baxter</td>
			<td style="width:119px;">NDC-10019-360-40</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Isoflurane vaporizer</td>
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			<td>12432</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Mice (C57BL/6)</td>
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			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Needles 25 G x 5/8&#34;</td>
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		</tr>
		<tr height="21">
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			<td style="width:231px;">Tissue Tek</td>
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			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">PBS (Phosphate Buffered Saline)</td>
			<td style="width:231px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
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			<td style="width:231px;">Sigma</td>
			<td style="width:119px;">P6744-1GA</td>
			<td></td>
		</tr>
		<tr height="21">
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			<td style="width:231px;">Transonic</td>
			<td style="width:119px;">FTH-1212B-4018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Retractor</td>
			<td style="width:231px;">Kent Scientific</td>
			<td style="width:119px;">SURGI-5001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Static oxygen Controller ProOx 360</td>
			<td style="width:231px;">Biospherix</td>
			<td style="width:119px;">P360</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">SU 5416</td>
			<td style="width:231px;">Sigma Aldrich</td>
			<td style="width:119px;">S8442</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Surgical Suture, black braided silk, 5.0</td>
			<td style="width:231px;">Surgical Specialties Corp.&#160;</td>
			<td style="width:119px;">SP116</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Surgical tape</td>
			<td style="width:231px;">3M</td>
			<td style="width:119px;">1527-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Syringe 10 ml</td>
			<td style="width:231px;">BD</td>
			<td style="width:119px;">303134</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Syringes with needle 1 ml</td>
			<td style="width:231px;">BD</td>
			<td style="width:119px;">309626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Sytox Green Nuclein Acid Stain</td>
			<td style="width:231px;">Thermo Scientific</td>
			<td style="width:119px;">S7020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Tenotomy scissors</td>
			<td style="width:231px;">Pricon</td>
			<td style="width:119px;">60-521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Toluol</td>
			<td style="width:231px;">Roth</td>
			<td style="width:119px;">9558.3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Ventilator&#160;</td>
			<td style="width:231px;">CWE</td>
			<td style="width:119px;">SAR-830/P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">WGA Alexa Fluor&#160;</td>
			<td style="width:231px;">Thermo Scientific</td>
			<td style="width:119px;">W11261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Xylene</td>
			<td style="width:231px;">Roth</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

induction and characterization of pulmonary hypertension in mice using the hypoxia/su5416 model

肺高血压 （PH） 是一种病理生理状况，由超过 25 mm Hg 的呼吸道动脉压力定义，由右心导管评估。广泛的疾病可导致PH，其病因、基础病理学、临床表现、预后和治疗反应各不相同。尽管近年来取得了重大进展，但PH仍然是一种未治愈的疾病。了解基本机制可以为开发新疗法铺平道路。动物模型是实现这一目标的重要研究工具。目前，有几个模型可用于重述PH。该协议描述了两击鼠标 PH 模型。PH发育的刺激是缺氧和SU5416的注射，一种血管内皮生长因子（VEGF）受体拮抗剂。低氧/SU5416开始三周后，动物发展肺血管重塑模仿在人类PH中观察到的组织病理学变化（主要是第1组）。肺循环中的血管重塑导致右心室 （RV） 的重塑。详细描述了测量RV压力（使用开放胸法）、RV的形态分析（通过解剖和称重两个心室）和重塑术（通过评估血管重塑和心脏评估肺功能评估）的过程。该协议的优点是，在野生型和转基因小鼠中应用的可能性，相对容易和低成本的实施，以及感兴趣的疾病的快速发展（3周）。这种方法的局限性是小鼠不发展严重的表型和PH是可逆的返回诺莫夏。预防以及治疗研究可以很容易地在这个模型中实施，而没有必要的高级技能（而不是手术啮齿动物模型）。

在该协议中，我们详细描述了低氧/SU5416模型的创建，该模型用于在小鼠中诱导PH。此外，我们详细介绍了在观察期结束时进行肺血管和心脏评估的所有必要步骤。
此模型的实验设计概述如图 1AA13，14,所示。 Figure 1小鼠患有正常缺氧（10%O2），并连续三周每周注射一次SU5416。图1B和1C中显示?...

Watch this Scientific Journal Video about Induction and Characterization of Pulmonary Hypertension in Mice using the Hypoxia/SU5416 Model at JoVE.com

Induction and Characterization of Pulmonary Hypertension in Mice using the Hypoxia/SU5416 Model

该协议描述了小鼠肺高血压（PH）的诱导，基于暴露于缺氧和VEGF受体拮抗剂的注射。动物在协议启动3周后出现PH和右心室（RV）肥大。介绍了模型的功能和形态特征。

使用缺氧/SU5416模型对小鼠的肺高血压进行诱导和特征

Pulmonary Hypertension (PH) is a pathophysiological condition, defined by a mean pulmonary arterial pressure exceeding 25 mm Hg at rest, as assessed by ...

该协议可用于诱导肺高血压和右心室的功能测量。Sugen低氧模型易于实施，可以在诱导后两到三周内重新概括肺高血压的一些特征。利用这个模型，我们可以确定与疾病发展有关的新途径。我们可以确定新的靶点并测试其治疗潜力。在开始感应程序之前，将氮气罐固定到缺氧室附近，并设置氧气控制器在 10% 氧气点。接下来，将新鲜准备好的SU5416溶液装入一个装有25量针的毫升注射器中，并手动约束第一个要注射的动物。抓住皮肤形成与脊柱平行的帐篷，确保紧紧抓住后背，避免被鼠标咬伤。将针头皮下插入松弛皮肤的侧翼，与皮肤平行，并交付 100 微升的注射器内容物。10秒后，取回针头，将动物返回到笼子。当所有动物都注射后，将小鼠放在通风的缺氧室中。对于缺氧暴露，确认腔室配有氧气传感器，用于测量氧气水平，每三天打开腔室不超过 20 分钟，用于清洁和添加食物和水。每天检查动物是否有压力迹象，并连续三周重复SU5416注射。在启动压力测量程序之前，准备 Y 管接头并使用手动模式检查呼吸机的功能。将吸气压力设置为不到一厘米的水，以避免巴罗风湿，并设置呼吸速率为每分钟110次呼吸。切割 20 量表血管内导管，创建气管管并打开压力体积控制单元。启动数据采集软件，将压力体积导管放在 37 摄氏度水浴中 15 毫升 PBS 离心管中。然后根据制造商的协议校准导管。将鼠标放在加热垫上，放置直肠探头以进行体温监测。接下来，确认麻醉小鼠对手趾捏缺乏反应，并剃光颈部和胸部区域。用手术胶带固定小鼠的四肢，并在宫颈皮中做一厘米的切口。使用棉尖施用器，钝化分离的帕罗蒂德和亚曼迪布唾液腺，以暴露气管背后的肌肉。小心地切割肌肉，以完全暴露气管，并使用剪刀在气管细管之间做一个小切口。插入准备好的气管管，取出血管内导管的金属导管。然后将导管连接到呼吸机，通过手动轻轻充气肺来验证气管位置。用胶带固定位置。对于右心室压力测量，在西腓过程和上腹部做一厘米的皮肤切口，从中线到西腓，并小心地在两侧横向移动，将皮肤分离到胸部和腹部壁上。打开腹腔，小心切开隔膜，注意不要伤害跳动的心脏或肺部。并使用棉尖施用器轻轻去除中压。将压力导管靠近鼠标，并使用装有针头的棉尖施用器在右心室的非典型远点部分进行刺伤。小心地用压力导管更换针头，将压力导管插入与右心室方向平行的带头，尖端朝向肺动脉。观察压力波跟踪，确保导管的正确定位。一旦压力信号稳定下来，暂停呼吸并获取至少三个测量值。记录完所有测量值后，小心地将导管返回到水浴中充满 PBS 的离心管。在这里，显示了代表性的压力曲线和观察期结束时右心室压力的定量分析。缺氧后肺动脉的组织学分析加上SU5416治疗表明，与正常动物相比，中壁厚度增加。小麦胚芽粘胶素染色评估心肌细胞肥大表明，缺氧加上SU5416暴露导致心肌细胞大小和右心室肥大显著增加。此外，通过富尔顿指数的测量对右心室肥大进行形态评估，证实低氧动物右心室肥大增加。执行功能测量对于避免出血非常重要，因为这会对研究结果产生负面影响。该协议也可用于诱导肺高血压的大鼠只有轻微的修改。值得注意的是，大鼠只有一次苏根注射足以诱发肺高血压。

Research

JoVE Journal

Medicine

Increasing Pulmonary Artery Pulsatile Flow Improves Hypoxic Pulmonary Hypertension in Piglets

增加肺动脉脉动流改善缺氧性肺动脉高压的仔猪

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular ...

科研

医学

Chronic Thromboembolic Pulmonary Hypertension and Assessment of Right Ventricular Function in the Piglet

慢性血栓栓塞性肺动脉高压和右心室功能的仔猪评估

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. ...

Hemodynamic Characterization of Rodent Models of Pulmonary Arterial Hypertension

肺动脉高压的啮齿动物模型的血流动力学特性

Pulmonary arterial hypertension (PAH) is a rare disease of the pulmonary vasculature characterized by endothelial cell apoptosis, smooth muscle ...

A Magnetic Microbead Occlusion Model to Induce Ocular Hypertension-Dependent Glaucoma in Mice

A磁条微珠缺血模型，以诱导小鼠高眼压，青光眼相关

The use of rodent models of glaucoma has been essential to understand the molecular mechanisms that underlie the pathophysiology of this multifactorial ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

小鼠加速动脉硬化的诱导："电线损伤"模型

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Shunt Surgery, Right Heart Catheterization, and Vascular Morphometry in a Rat Model for Flow-induced Pulmonary Arterial Hypertension

分流手术，右心导管，血管形态计量学在大鼠模型中的流量引起的肺动脉高压

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt ...

Induction and Phenotyping of Acute Right Heart Failure in a Large Animal Model of Chronic Thromboembolic Pulmonary Hypertension

慢性血栓栓塞性肺动脉高压大型动物模型中急性右心衰竭的诱导和表型分析

The development of acute right heart failure (ARHF) in the context of chronic pulmonary hypertension (PH) is associated with poor short-term outcomes. The ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Left Atrial Stenosis Induced Pulmonary Venous Arterialization and Group 2 Pulmonary Hypertension in Rat

左心房狭窄致大鼠肺静脉动脉化及第2组肺动脉高压

The mechanism of mitral stenosis-induced pulmonary venous arterialization and group 2 pulmonary hypertension (PH) is unclear. There is no rodent model of ...

The Left Pneumonectomy Combined with Monocrotaline or Sugen as a Model of Pulmonary Hypertension in Rats

左肺切除术联合单氯联氨酸或苏根为大鼠肺动脉高压的模型

In this protocol, we detail the correct procedural steps and necessary precautions to successfully perform a left pneumonectomy and induce PAH in rats ...