Name: assessment of vascular tone responsiveness using isolated mesenteric arteries with a focus on modulation by perivascular adipose tissues
Uploaded: 2019-06-03T15:00:00
Description: Watch this Scientific Journal Video about Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues at JoVE.com

这项工作得到了香港研究赠款委员会 [17124718 和 17124718]、香港卫生和医学研究基金 [13142651 和 13142651]、香港合作研究基金 [C7055-14G] 和国家基金会的资助。中国研究项目 [973 项目 2015CB553603]。

所有用于以下研究的动物均由香港大学医学院实验动物组提供。获得了省实验室动物用于教学和研究委员会的道德认可 (CULATR, 编号: 4085-16)。
1. 准备工作
<ol><li>药物的制备
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<li>按照材料安全数据表 (MSDS) 中的规定, 在收到药物后立即妥善储存药物。将粉末形式的药物作为高浓度库存溶液溶解在溶剂中, 然后在-20°c 下储存。 注: 大多数药物溶解在蒸馏水中, 以...

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除了内皮细胞外, PVAT 产生的信号在调节平滑肌张力反应性 30方面发挥着重要作用。健康的 pvat 释放 no 和抗炎脂联素, 对动脉产生抗收缩作用, 在肥胖和代谢综合征等病理条件下丢失 31,32。在疾病状态下, pvat 有助于内皮功能障碍和其他心血管异常的发展33,34。据报道, 肥胖动物的动脉<sup class="x...

动脉扩张和收缩分别是通过血管平滑肌细胞的松弛和收缩来实现的。小动脉血管反应性的变化有助于自主神经和血液中存在的激素 (如儿茶酚胺、血管紧张素 II.、血清素、加压素) 对动脉血压的稳态调节。在局部一级, 平滑肌细胞的血管反应被来自内膜内皮细胞和动脉周围脂肪组织的信号所调节 (图 1)。
内皮不仅是一个被动屏障, 而且还作为一个表面, 以交换血液和潜在的血管平滑肌细胞之间的信号。通过释放各种血管活性物质, 内皮细胞在血管张力反应1的局部控制中起着至关重要的作用。例如, 在对乙酰胆碱的反应中, 内皮一氧化氮合酶 (eNOS) 在内皮细胞中被激活, 产生一氧化氮 (NO), 通过激活可溶性利尼环化酶 (sGC)诱导下血管平滑肌的松弛。2. 其他血管活性物质包括环氧合酶 (如前列腺素和血栓素 a2)、脂氧合酶 (如 12-羟基磷酸四烯酸、12-hete) 和细胞色素 p450 单胞酶 (hetes 和环氧血尿酸、Eet)、活性氧种类 (ros) 和血管活性肽 (如内皮素-1 和血管紧张素 II.) 和内皮衍生的超极化因子 (EDHF)3。内皮衍生血管扩张剂和血管收缩剂之间的微妙平衡保持局部血管运动张力4,5。
内皮功能障碍的特点是内皮依赖性血管舒张6的损伤, 血管老化的标志 7.随着年龄的发展, 内皮促进血管舒张的能力逐渐降低, 主要原因是 NO 生物利用度下降, 以及血管平滑肌细胞内皮细胞和 sGC 中 eNOS 的异常表达和功能8,9,10. no 的生物利用度降低, 可促进内皮依赖性血管收缩剂11,12的产生。在老年动脉中, 内皮功能障碍会导致介质增生, 这反映在壁厚、内侧细胞核数量的显著增加上, 这让人想起了高血压和动脉粥样硬化中观察到的动脉增厚。病人13,14。此外, 肥胖、糖尿病或高血压等病理生理条件加速内皮功能障碍的发展15,16。
血管周围脂肪组织 (PVAT) 释放大量脂肪因子, 以调节血管结构和功能17。Pvat 的抗收缩作用是由放松因子介导的, 如脂联素、no、过氧化氢和硫化氢 18,19, 20。然而, 根据位置和病理生理条件, PVAT 也可以提高不同动脉的收缩反应21。Pvat 产生的亲收缩物质包括血管紧张素 ii、瘦素、抵抗素和 ros22、23。 在大多数关于孤立血管的研究中, PVAT 被认为是血管的简单结构支撑, 因此在准备血管环段的过程中被切除。由于脂肪功能障碍是高血压和相关心血管并发症的一个独立的危险因素24,在调查血管的血管反应性时, 应考虑血管周围的 PVAT。不同的动脉。
多线肌瘤系统已被广泛用于研究各种血管的血管运动功能, 包括主动脉、肠系膜、肾、股、脑和冠状动脉 25,26。本文描述的协议将使用线断层成像来评估从转基因小鼠模型中分离出的肠系膜动脉中的血管反应性, 并特别关注 PVAT 的调节。

<table>
	<tbody>
		<tr>
			<td>Acetylcholine</td>
			<td>Sigma-Aldrich</td>
			<td>A6625</td>
			<td>Stock concentration: 10-1 M 
			Working concentration: 10-10 to 10-5 M</td>
		</tr>
		<tr>
			<td>L-NAME (N&#969;-nitro-L-arginine methyl ester)</td>
			<td>Sigma-Aldrich</td>
			<td>N5751</td>
			<td>Stock concentration: 3 x 10-2 M 
			Working concentration: 10-4 M</td>
		</tr>
		<tr>
			<td>Phenylephrine</td>
			<td>Sigma-Aldrich</td>
			<td>P6126</td>
			<td>Stock concentration: 10-2 M 
			Working concentration: 10-10 to 10-5 M</td>
		</tr>
		<tr>
			<td>U46619 (9,11-dideoxy-9&#945;,11&#945;methanoepoxy prostaglandin F2&#945;)</td>
			<td>Enzo</td>
			<td>BML-PG023-0001</td>
			<td>Stock concentration: 10-5 M 
			Working concentration: 1-3 x 10-8 M</td>
		</tr>
		<tr>
			<td>Multiwire myograph</td>
			<td>Danish MyoTechnology (DMT)</td>
			<td>620M</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerLab 4/26</td>
			<td>ADInstruments</td>
			<td>ML848</td>
			<td></td>
		</tr>
		<tr>
			<td>Labchart7</td>
			<td>ADInstruments</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Adipo-SIRT1 wild type mice</td>
			<td>Laboratory Animal Unit, The University of Hong Kong</td>
			<td>CULATR NO.: 4085-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon-coated Petri dishes</td>
			<td>Danish MyoTechnology (DMT)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten wires</td>
			<td>Danish MyoTechnology (DMT)</td>
			<td>300331</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical tools</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of vascular tone responsiveness using isolated mesenteric arteries with a focus on modulation by perivascular adipose tissues

血管张力改变对病理生理刺激的反应有助于广泛的心血管和代谢疾病的发展。内皮功能障碍是减少血管舒张和增强动脉血管收缩的主要元凶。动脉周围的脂肪组织在调节血管平滑肌细胞内皮依赖性松弛和/或收缩方面发挥着重要作用。内皮和血管周围脂肪组织之间的交叉谈话可以通过线断层成像系统在体内使用安装的血管进行评估。然而, 应建立从不同物种、年龄、遗传背景和/或病理生理条件的动物中获得的动脉的最佳设置。

检查纵向/张力关系, 以获得归一化因子k
应用于血管段的拉伸量会影响阿克林-肌球蛋白相互作用的程度, 从而产生最大的作用力。因此, 对于每一种类型的血管, 确定最大活跃力所需的拉伸量是适当的脊髓造影研究所必需的。在这里, 对从小鼠模型中分离出的肠系膜动脉进行了纵向张力关系的归一化 (<strong clas...

Watch this Scientific Journal Video about Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues at JoVE.com

Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues

以血管内炎组织调节为重点, 利用肠系膜孤立动脉评价血管张力

该协议描述了使用线状成像来评估从小鼠分离肠系膜动脉的神经间等距张力, 特别考虑的是内皮细胞和血管周围脂肪组织释放的因素的调节。

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic ...

需要非常严格的协议，以便我们可以有效比较来自同一动物或不同动物或动脉的制剂，在它们周围的脂肪存在或存在或内皮存在或不存在。这种实验技术的主要优点是，我们可以比较等轴测条件下同一动脉的环，从而对所研究的实验因素的影响作出有效的结论。该协议提供了不同转基因动物的血管脂肪组织对血管反应的调制的见解。脂肪组织功能障碍是高血压及其相关心血管并发症的独立危险因素。我们的 myograph 涉及多个复杂的步骤，每个步骤都需要格外小心，以避免损坏容器，尤其是在安装过程中，并确保容器对各种刺激做出适当反应。从文本协议中描述的中肠动脉环的准备和解剖开始这个程序。打开计算机并打开数据记录软件。将实验另存为实验室图表数据文件，并设置新名称，以避免覆盖原始设置文件。打开规范化设置窗口，将 K 因子设置为 1。接受 IP 校准、目标压力、联机平均时间和延迟时间的默认值。单击"确定"以保存设置。选择感兴趣的通道，并在规范化窗口中输入导线直径、组织端点和初始千分尺读数。要开始规范化程序，请逆时针转动千分尺螺钉，将第一个无源拉伸施加到血管上。等待三分钟后，容器稳定下来，在规范化窗口中输入新的千分尺读数。墙张张自动计算，并在图表上显示为点。每次无源拉伸后，用含有150毫摩尔氯化钾的等同渗透高钾克赖布替换控制克雷布斯缓冲液。当收缩在大约三分钟时达到高原时，通过从钾激活力中减去每段伸展的被动力来记录主动力。计算墙张力以及内部周长值。用新鲜的克赖斯缓冲液替换高钾缓冲液，从而去除高钾状况。重复洗涤五分钟三次。重复规范化步骤，诱导被动拉伸，然后交替主动收缩，直到主动张力开始减小。彻底洗掉高钾克赖布缓冲液，将准备再平衡30至45分钟。将基底张力重置为零，以便在随后的实验中只记录主动收缩响应。准备并安装配对动脉环，如每个动脉相邻部分的文本协议中所述。准备一个动脉环，PVET完好无损，另一个动脉环与PVAT去除。正常化后，通过向含有氯克的腔室添加115毫摩尔氯化钾溶液，预签约动脉段与高钾克赖布缓冲液。收缩到高原期后，洗掉高钾，用新鲜的高盐缓冲液代替缓冲液。在五分钟内重复洗涤三次。重复氯化钾刺激和洗涤三次，通过从氯化钾刺激造成的张力中减去基线张力，记录对氯化钾的最大收缩反应。在最后的收缩和洗涤后，用温暖、加注的克赖布缓冲液重新填充腔室，让动脉恢复约30分钟，然后再执行下一个任务。在每个腔室中，加入累积量的苯肾上腺素，以诱导静态制剂的浓度依赖性增加和等轴测张力。加入最后一剂激动剂后，彻底洗净药物，用新鲜的克勒布斯缓冲液重新填充腔室。将浓度依赖反应绘制为氯化钾诱导最大收缩百分比的增加。为了评估一氧化氮的贡献，在添加苯甲肾上腺素之前，用一氧化氮合成酶抑制剂L-NAME孵育制剂30分钟。要按顺序执行第二个浓度响应曲线，请完全反复清洗腔室，以去除所有先前的激动剂，直到观察到音调的进一步变化。对于从用标准周或高脂肪饮食喂养的小鼠模型中分离出的肠动脉，对长度-张力关系进行规范化。通过动脉段的增量拉伸建立被动长度-张力关系，直到获得与100毫米汞横膜压力或IC100相对应的内部周长。每次伸展后，应用115毫摩尔氯化钾刺激收缩。主动长度张力曲线是根据 Y 轴上的主动力数据和 X 轴上的微米数据计算的 IC 值绘制的。位于山顶高原内的 IC 值为 IC1。规范化因子K计算为IC1和IC100的比率，然后可应用于后续实验中同一容器类型的样品。此处显示的是血管收缩器对肠动脉中苯丙胺反应的输出记录，带或不带周围PVAT。使用苯甲酸酯的累积浓度来刺激从16周大的阿迪波塞特一只小鼠收集的肠动脉收缩。以150毫摩尔氯化钾引起的最大收缩百分比记录和计算收缩反应。绘制收缩曲线下的区域进行比较。请注意，来自阿迪波集的PVAT对苯甲肾上腺素的反应产生了抗收缩作用。当获得KCL引起的收缩时，反应必须稳定和稳定。如果 KCL 的三次增量管理后响应仍在增加，则可能需要 KCL 刺激。要达到最优条件的张力，正常化步骤是非常重要的，因为这决定了打开血管的力量。如果做得不好，就会影响整个实验。除了苯肾上腺素引起的收缩或松弛外，此协议可应用于其他设置。例如，在存在或不存在近血管脂肪组织、自主神经或内皮的情况下。通过这个协议，研究人员可以在不同的病理生理条件下检查来自患病动物模型的小血管，如肥胖、糖尿病和心血管疾病。

Research

JoVE Journal

Biology

Right Hemihepatectomy by Suprahilar Intrahepatic Transection of the Right Hemipedicle using a Vascular Stapler

右右Hemipedicle Suprahilar肝内血管吻合器横断Hemihepatectomy

Successful hepatic resection requires profound anatomical knowledge and delicate surgical technique. Hemihepatectomies are mostly performed after ...

科研

生物学

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

“体外骨骼肌微血管制备离体血管反应性的调查

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

Using Digital Image Correlation to Characterize Local Strains on Vascular Tissue Specimens

利用数字图像相关表征的血管组织标本本地菌株

Characterization of the mechanical behavior of biological and engineered soft tissues is a central component of fundamental biomedical research and ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

通过光谱流式细胞仪由固态组织中分离出悬浮细胞系的分析

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Differentiation Capacity of Human Aortic Perivascular Adipose Progenitor Cells

人主动脉周脂肪祖细胞的分化能力

Adipose tissue is a rich source of multi-potent mesenchymal stem cells (MSC) capable of differentiating into osteogenic, adipogenic, and chondrogenic ...

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

从小鼠表皮脂肪脂肪组织中制备脂肪祖细胞

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident ...

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging

使用[18F]FDG微PET / MR成像对小鼠中的棕色和米色脂肪组织进行可视化和定量

Brown and beige adipocytes are now recognized as potential therapeutic targets for obesity and metabolic syndromes. Non-invasive molecular imaging methods ...

Mouse Abdominal Aortic Aneurysm Model Induced by Perivascular Application of Elastase

血管周围应用弹性蛋白酶诱导的小鼠腹主动脉瘤模型

Abdominal aortic aneurysm (AAA), although primarily asymptomatic, is potentially life-threatening as the rupture of AAA usually has a devastating outcome. ...

Semi-Automated Isolation of Murine White Adipose Tissue

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator (Video) | JoVE 

使用组织解离器从小鼠白色脂肪组织中半自动分离基质血管部分

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their ...