Name: isometric contractility measurement of the mouse mesenteric artery using wire myography
Uploaded: 2018-08-20T12:30:00.000+00:00
Duration: 9 min 42 s
Description: Watch this Scientific Journal Video about Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography at JoVE.com

我们感谢魏琪博士 (苏州大学, 苏州市, 中国) 和闫宁桥 (陕西师范大学, 西安, 中国) 的技术援助。这项工作得到了中国国家自然科学基金 (赠款31272311、81373295和 81473420) 的支持, 并由江苏省高等教育机构优先学术项目开发资助的项目 (批准号:ysxk-2016)。

动物操作由南京大学动物研究中心的动物保育和使用委员会 (IACUC) 批准。
1. 解决方案准备
<ol><li>用137.0 毫米氯化钠, 2.7 毫米氯化钾, 1.8 毫米 CaCl2, 1 毫米氯化镁2∙6H2O, 5.6 毫米 d-葡萄糖, 10 毫米 HEPES, pH 7.3-7.4, 制备 HEPES Tyrode 溶液 (h-T)。</li>
	<li>用140.6 毫米氯化钠, 2.7 毫米氯化钾, 1 毫米氯化镁2∙6H2O, 5.6 毫米 d-葡萄糖, 10 毫米 HEPES, pH 7.3-7.4, 制备 HEPES-Tyrode 溶液, 不含钙 (Ca2 +自由 H T)。</li>
	<li>用15.7 毫米氯化钠, 124.0 毫米氯化钾, 1.8 毫米 CaCl2, 1 毫米氯化镁2∙6H2O, 5.6 毫米 d-葡萄糖, 10 毫米 HEPES, pH 7.3-7.4, 制备 HEPES-Tyrode 溶液, 使用124毫米氯化钾 (高 K+)。</li>
</ol>2. 实验准备
<ol><li>使用37摄氏度水浴预热 H T 和高钾+溶液。</li>
	<li>打开 myograph 系统、数据采集硬件和计算机。</li>
	<li>用5毫升的 H T 溶液将每个 myograph 腔仔细地填满。</li>
	<li>分别用20毫升4°c 的 h t 和 Ca2 +免费的 h t 溶液填充两个培养皿, 并储存在冰上。</li>
	<li>用20毫升的 H T 溶液填充10厘米涂层培养皿, 并在室温下保持。</li>
</ol>3. 小鼠肠系膜动脉夹层
<ol><li>弄死8-12 周大的 C57BL/6J 雌或雄性小鼠颈椎脱位。把鼠标钉在腹部朝上。</li>
	<li>用70% 乙醇滋润腹部。然后, 沿腹中线从腹股沟切开皮肤与剪刀, 并使切口从第一切口开始向下到两侧的腿。将皮肤拉回两侧;做类似的切口打开腹膜。</li>
	<li>使用剪刀, 切断食道, 结肠和其他结缔组织, 完全隔离胃肠道与喂养血管从身体。</li>
	<li>用镊子, 将隔离段移入含有在步骤2.4 中制备的冷 H t 的盘中, 并在 h t 溶液中轻轻冲洗组织几次以冲洗血液。</li>
	<li>将隔离段转移到2.5 步制备的涂层培养皿中, 并在室温下进行肠系膜动脉夹层。</li>
	<li>顺时针方向平滑胃、空肠、回肠和盲肠, 分别将胃和盲肠固定在左右侧。</li>
	<li>伸展肠系膜血管床, 用针固定肠道以暴露肠系膜动脉的解剖。 注意: 在这些条件下, 动脉在血管的顶端。</li>
	<li>打开立体显微镜的透射光源, 在显微镜下解剖动脉。确保整个组织沉浸在溶液中。</li>
	<li>用镊子钳住动脉周围的脂肪组织, 用解剖剪刀切断所有结缔组织, 隔离动脉。避免损伤动脉。</li>
</ol>4. 动脉安装
<ol><li>将肠系膜动脉树转移并浸入冷钙2 +游离的 H T 溶液 (在步骤2.4 中制备), 方法是用镊子夹紧多余的动脉。</li>
	<li>切除肠系膜拱廊肠道壁近1.4 毫米的动脉部分, 并使用两个镊子仔细打开这段动脉的两侧。</li>
	<li>准备两段不锈钢丝长度为2.5 厘米, 并将它们放入同一盘中。</li>
	<li>用镊子轻轻夹住动脉的一端, 在另一钳的帮助下, 小心地将两根导线插入动脉的腔内。确保导线保持直, 不要接触内皮。</li>
	<li>使用两个钳, 同时夹紧螺纹容器外的两根钢丝, 并小心地将容器从培养皿中转移到以前用 h-T 溶液填充的 myograph 室 (步骤 2.3)。</li>
	<li>把下巴拧紧, 使空间安装。用两个钳夹住两根插入导线之一的两侧, 并将该容器置于颚间隙 (图 1A)。</li>
	<li>将夹紧导线的两侧缠绕在与千分尺连接的颚螺钉上 (图 1B)。</li>
	<li>用顺时针方向拧紧固定左手螺钉。用右手钳伸直导线, 然后顺时针旋转 (图 1C) 固定右手螺钉。确保该容器始终在颚间隙内, 但请勿触摸下颚以避免损坏。</li>
	<li>使用千分尺关闭两个颚 (图 1D)。确保两个下巴足够近, 但他们不接触对方, 不固定的电线是在电线的顶部。</li>
	<li>使用右手钳, 小心地折叠在下巴的角落连接到力传感器的拧开的电线, 并按顺时针方向绕右螺钉 (图 1E)。然后, 固定螺丝。在导线的左侧重复此步骤, 并固定左侧螺钉 (图 1F)。</li>
	<li>仔细旋转千分尺 (图 1G), 将颚稍微分开。避免伸展船只。使用镊子将在千分尺侧的导线移动到换能器一侧导线的水平面上。小心地旋转千分尺, 使两只颚之间的缝隙能容纳两条线。</li>
	<li>重复步骤4.2 至 4.11, 将动脉安装到其他房间。将所有房间连接到设备, 盖住房间, 连接100% 氧气供应和一个温度探头, 并开始加热到37摄氏度。打开绘图软件, 然后按 "图表视图" 窗口上的 "开始" 按钮开始录制。</li>
	<li>平衡约20分钟。</li>
</ol>5. 正常化
注意: 为了规范实验条件, 获得可靠的血管生理响应, 规范化的程序是必要的15。根据该容器的主动力与内圆周之间的关系, myograph 系统有一个标准的规范化程序来评估装入的容器5、8的内部周长 (IC), 9。简而言之, 计算 IC (µm), 读取千分尺和输入值作为 X 值和传感器输出力,即静止壁张力 (锰/毫米), 作为 Y 值。该程序将返回一个拟合曲线 (X, Y) 和计算的 IC 对应的壁压力 100 mmHg (IC100)。当主动响应最大时, 该容器被设置为规范化的内部圆周 (IC1)。
<ol><li>对设备上的所有通道设置强制为零, 并平衡另一个1-2 分钟。</li>
	<li>从 "DMT" 菜单中选择规范化设置, 并按如下方式设置参数: 目镜校准 (毫米/div): 0.36;目标压力 (人民军): 13.3;ic1/ic100: 0.9;在线平均时间 (秒): 2;延迟时间 (秒):60。单击"确定" 按钮关闭 " DMT 规范化设置" 窗口。</li>
	<li>从dmt 菜单中选择感兴趣的通道, 打开相应通道的 dmt 规范化窗口。在窗口中输入常量值, 如下所示: 组织终点 a1: 0.1;组织终点 a2: 4;钢丝直径 (µm):40。该窗口将计算出的容器长度显示为1.40 毫米。</li>
	<li>阅读适当的组织室的千分尺。在 "千分尺" 阅读框中输入值, 然后单击 "添加点" 按钮。此值是 X (X0) 的初始值。在六十年代延迟时间之后, 窗口显示与此千分尺值对应的力和有效压力 (ERTP)。同时,千分尺读数盒变得活跃。</li>
	<li>通过在逆时针方向转动千分尺来拉伸容器正常化。在 "千分尺" 阅读框中输入千分尺值, 然后单击 "添加点" 按钮。等待六十年代的延迟时间。</li>
	<li>重复步骤 5.5, 继续拉伸该容器, 并添加千分尺值, 直到窗口显示 "千分尺 X1" 的值, 这是将容器拉伸到其 IC1所需的计算千分尺设置。</li>
	<li>将千分尺设置为 X1值。 注: 正常化的张力通常为1-2 锰。</li>
</ol>6. 动脉收缩记录
注意: 在步骤2.1 中编写了所有解决方案, 包括本节中使用的 h-T 和高 K+解决方案。
<ol><li>规范化后, 将容器平衡在腔内15-20 分钟。 注意: 这些不需要在这个步骤中更改解决方案。</li>
	<li>用高 K+溶液对容器进行两次挑战。<ol>
<li>对该容器进行挑战, 用5毫升高钾溶液取代 h-t 溶液,以诱导收缩10分钟, 接着用5毫升的 h t 溶液洗涤3-4 次。 注: 典型收缩有最大力超过3锰和恒定持续力大约2.5 锰12。如果第一个挑战产生的最大力低于2.5 锰或持续力下降的时间或第二个挑战产生比第一次剂量更低的力量, 该船舶被丢弃, 不会被用于进一步调查。</li>
	</ol>
</li>
	<li>用5毫升的高钾溶液来挑战容器 , 以诱导收缩。5分钟后, 将 neoliensinine 库存溶液的0.5 µL (10 毫米在亚砜)14放入室内, 以使容器在最后浓度为1µM neoliensinine 时松弛。</li>
	<li>当力稳定 (这通常需要几分钟), 添加另外0.5 µL 的 neoliensinine 库存溶液进入会议厅, 以增加浓度为2µM. 每次添加1µL 的库存解决方案, 增加浓度为 4, 6, 8 和10µM 产生 t他的剂量反应曲线。 注意: 药品的库存和工作浓度各不相同。</li>
</ol>

<ol>
	<li>Bevan, J. A., Osher, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+direct+method+for+recording+tension+changes+in+the+wall+of+small+blood+vessels+in+vitro.">A direct method for recording tension changes in the wall of small blood vessels in vitro.</a> Agents and Actions. 2 (5), 257-260 (1972).</li><li>Mulvany, M. J., Halpern, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+vascular+smooth+muscle+cells+in+situ.">Mechanical properties of vascular smooth muscle cells in situ.</a> Nature. 260 (5552), 617-619 (1976).</li><li>Mulvany, M. J., Halpern, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contractile+Properties+of+Small+Arterial+Resistance+Vessels+in+Spontaneously+Hypertensive+and+Normotensive+Rats.">Contractile Properties of Small Arterial Resistance Vessels in Spontaneously Hypertensive and Normotensive Rats.</a> Circulation Research. 41 (1), 19-26 (1977).</li><li>Mulvany, M. J., Nyborg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+increased+calcium+sensitivity+of+mesenteric+resistance+vessels+in+young+and+adult+spontaneously+hypertensive+rats.">An increased calcium sensitivity of mesenteric resistance vessels in young and adult spontaneously hypertensive rats.</a> British Journal of Pharmacology. 71 (2), 585-596 (1980).</li><li>Mulvany, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Procedures for investigation of small vessels using small vessel myograph. , (2004).</li><li>Halpern, W., Mulvany, M. J., Warshaw, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+smooth+muscle+cells+in+the+walls+of+arterial+resistance+vessels.">Mechanical properties of smooth muscle cells in the walls of arterial resistance vessels.</a> The Journal of Physiology. 275, 88-101 (1978).</li><li>Michael, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+blood+pressure+arising+from+a+defect+in+vascular+function.">High blood pressure arising from a defect in vascular function.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (18), 6702-6707 (2008).</li><li>Bridges, L. E., Williams, C. L., Pointer, M. A., Awumey, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenteric+artery+contraction+and+relaxation+studies+using+automated+wire+myography.">Mesenteric artery contraction and relaxation studies using automated wire myography.</a> Journal of Visualized Experiments. (55), (2011).</li><li>del Campo, L., Ferrer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Wire Myography to Study Vascular Tone and Vascular Structure of Isolated Mouse Arteries. , (2015).</li><li>Fisher, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+smooth+muscle+phenotypic+diversity+and+function.">Vascular smooth muscle phenotypic diversity and function.</a> Physiological Genomics. 42 (3), 169-187 (2010).</li><li>Crowley, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+roles+for+the+kidney+and+systemic+tissues+in+blood+pressure+regulation+by+the+renin-angiotensin+system.">Distinct roles for the kidney and systemic tissues in blood pressure regulation by the renin-angiotensin system.</a> The Journal of Clinical Investigation. 115 (4), 1092-1099 (2005).</li><li>Qiao, Y. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+phosphatase+target+subunit+1+(MYPT1)+regulates+the+contraction+and+relaxation+of+vascular+smooth+muscle+and+maintains+blood+pressure.">Myosin phosphatase target subunit 1 (MYPT1) regulates the contraction and relaxation of vascular smooth muscle and maintains blood pressure.</a> The Journal of Biological Chemistry. 289 (32), 22512-22523 (2014).</li><li>He, W. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+myosin+light+chain+kinase+in+regulation+of+basal+blood+pressure+and+maintenance+of+salt-induced+hypertension.">Role of myosin light chain kinase in regulation of basal blood pressure and maintenance of salt-induced hypertension.</a> American Journal of Physiology-Heart and Circulatory Physiology. 301 (2), H584-H591 (2011).</li><li>Yang, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+identification+of+a+tribenzylisoquinoline+alkaloid+from+Nelumbo+nucifera+Gaertn,+a+novel+potential+smooth+muscle+relaxant.">Isolation and identification of a tribenzylisoquinoline alkaloid from Nelumbo nucifera Gaertn, a novel potential smooth muscle relaxant.</a> Fitoterapia. 124, 58-65 (2018).</li><li>Slez&#225;k, P., Waczul&#237;kov&#225;, I., Bali&#353;, P., P&#250;zserov&#225;, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+Normalization+Factor+for+Wire+Myography+of+Rat+Femoral+Artery.">Accurate Normalization Factor for Wire Myography of Rat Femoral Artery.</a> Physiological Research. 59 (6), 1033-1036 (2010).</li><li>Kearney, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+burden+of+hypertension:+analysis+of+worldwide+data.">Global burden of hypertension: analysis of worldwide data.</a> The Lancet. 365 (9455), 217-223 (2005).</li><li>Good, N. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+Ion+Buffers+for+Biological+Research.">Hydrogen Ion Buffers for Biological Research.</a> Biochemistry. 5 (2), 467-477 (1966).</li></ol>

作者没有什么可透露的。

高血压是一个广泛的公共卫生挑战, 由于其严重并发症, 包括心血管和肾脏疾病16。了解高血压发病机制, 探索更多抗高血压药物已成为该领域的一项紧迫任务。血压是由循环的外周阻力产生和维持的。根据泊肃叶定律, 相对较小的动脉产生很大比例的循环阻力, 并作为主导的血压3,10的生产者。因此, 测量小阻力动脉而不是大动脉更适合?...

这里使用的小容器 myograph 系统是用来测量直径从100到400µm 的小电阻容器的等距收缩. 隔离的小容器 (大约2毫米长) 被两个 40-µm 直径导线插入并且然后按顺序安装在千分尺端和换能器侧颚上。这 myograph 技术在1972年第一次被建议了, 然后主要由 Mulvany 和他的同事开发了2,3,4,5,6。它现在是一个成熟的技术与稳定的设备, 容易的性能和标准规范化程序7,8,9。我们利用这种方法对小鼠肠系膜动脉的测量进行了一些修改。
血管平滑肌线几乎所有血管的墙壁。它们的基本功能是通过收缩来产生力量来响应各种刺激。血管平滑肌的正常收缩力对血压调节和营养补充10至关重要。血压异常调节导致多种疾病, 包括高血压、心力衰竭和缺血.一些研究表明, 异常的血压总是与功能失调的血管平滑肌收缩7,11,12,13。myograph 方法允许研究各种刺激, 包括血管收缩剂, 抑制剂和药物诱发的小鼠血管的等距收缩力。成功的收缩测量将有助于我们了解血压维持机制和血管平滑肌相关疾病的发病机理, 并探讨新的治疗方法。
许多中草药已广泛用于血管疾病的临床治疗;然而, 它们的有效成分通常仍不得而知。因此, 有效成分的分离和鉴定对新药的开发具有重要意义。多线 myograph 技术为筛选草药中的活性成分提供了一种简单的方法。我们报告了几项研究使用小血管 myograph 系统来调查小鼠肠系膜动脉收缩和确定的天然化合物与抗高血压活动12,13,14。在这里, 我们描述了 myograph 方法的详细协议, 并评估了 neoliensinine 从莲子的胚胎中分离出的松弛效应 (莲莲藕)。14。

<table><tbody><tr><td>Multi wire myograph system</td><td>DMT</td><td>610-M</td><td></td></tr><tr><td>Stainless steel wire</td><td>DMT</td><td>400447</td><td></td></tr><tr><td>Geuder dissection scissor</td><td>DMT</td><td>400431</td><td></td></tr><tr><td>Dumont forceps</td><td>DMT</td><td>300413</td><td></td></tr><tr><td>PowerLab/8SP</td><td>ADInstruments</td><td>ML785</td><td></td></tr><tr><td>Software</td><td>ADInstruments</td><td>LabChart 5</td><td></td></tr><tr><td>NaCl</td><td>SigmaAldrich</td><td>S5886</td><td></td></tr><tr><td>KCl</td><td>SigmaAldrich</td><td>P5405</td><td></td></tr><tr><td>CaCl2</td><td>SigmaAldrich</td><td>C4901</td><td></td></tr><tr><td>MgCl2&amp;middot;6H2O</td><td>SigmaAldrich</td><td>M2393</td><td></td></tr><tr><td>D-Glucose</td><td>SigmaAldrich</td><td>G6152</td><td></td></tr><tr><td>HEPES</td><td>Sangon Biotech</td><td>A100511-0250</td><td></td></tr><tr><td>NaOH</td><td>SigmaAldrich</td><td>S8045</td><td></td></tr><tr><td>DMSO</td><td>SigmaAldrich</td><td>D2650</td><td></td></tr></tbody></table>

isometric contractility measurement of the mouse mesenteric artery using wire myography

线 myograph 技术是用来评估血管平滑肌收缩性的反应, 对退极化, GPCR 激动剂/抑制剂和药物.它广泛应用于血管平滑肌的生理功能、高血压等血管疾病的发病机制, 以及平滑肌松驰药物的发展。老鼠是一种广泛使用的模型动物, 有大量的疾病模型和基因改良菌株。本方法对小鼠肠系膜动脉的等距收缩进行了详细的测定。1.4 毫米的小鼠肠系膜血管阻力动脉被隔离, 并安装在一个 myograph 室通过通过两个钢丝通过其流明。在平衡和规范化步骤之后, 在收缩试验前两次用高钾+溶液增强容器段。作为该方法在药物开发中应用的一个例子, 我们测量了一种新的天然物质 neoliensinine 的松弛效应, 从中草药中分离出来的, 莲子的胚胎 (莲莲莲藕). 小鼠肠系膜上动脉。在 myograph 室安装的容器段受到高 K+溶液的刺激。当力张力达到稳定的持续阶段时, neoliensinine 的累积剂量增加到腔室。我们发现 neoliensinine 对平滑肌收缩有剂量依赖性的松弛作用, 因此表明它对高血压有潜在的活性。此外, 由于在高钾溶液的安装和维持收缩力的作用下, 血管段至少能存活4小时 , 我们建议 myograph 系统可用于药物筛选的耗时过程。

我们用多线 myograph 系统测量了小鼠肠系膜动脉的等距收缩力, 并对莲子胚 neoliensinine 纯化的松驰效应进行了评估 (莲莲藕)。14. 小鼠肠系膜抗性动脉被隔离, 清除结缔组织, 并切割成1.4 毫米段。动脉段是由两根钢丝插入在 Ca2 +免费的 H T 溶液中的培养皿, 然后该段安装在两个颌骨的 myograph 室 (图 2A)。安装后, 两根电线被?...

Watch this Scientific Journal Video about Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography at JoVE.com

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

采用线 myograph 技术对血管平滑肌功能和筛查新药进行了研究。我们报告一个详细的协议, 以测量的等距收缩性的小鼠肠系膜动脉和筛选新的松弛剂血管平滑肌。

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

isometric-contractility-measurement-mouse-mesenteric-artery-using

Research

JoVE Journal

Medicine

14.6K 次观看.  Nanjing University. 采用线 myograph 技术对血管平滑肌功能和筛查新药进行了研究。我们报告一个详细的协议, 以测量的等距收缩性的小鼠肠系膜动脉和筛选新的松弛剂血管平滑肌。

视频: Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

Myo-mechanical Analysis of Isolated Skeletal Muscle

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

离体骨骼肌肌的力学分析

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

科研

医学

Mouse Model of Middle Cerebral Artery Occlusion

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2. Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3, many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4. The technique can be used to model permanent or transient occlusion 5. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.

We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

大脑中动脉闭塞的小鼠模型

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the ...

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to develop suitable animal models to study the events that unfold during ischemic insult. These techniques include transient or permanent, focal or global ischemia models using many different animal models, with the most common being rodents.
The permanent MCA ligation method which is also referred as pMCAo in the literature is used extensively as a focal ischemia model in rodents 1-6. This method was originally described for rats by Tamura et al. in 1981 7. In this protocol a craniotomy was used to access the MCA and the proximal regions were occluded by electrocoagulation. The infarcts involve mostly cortical and sometimes striatal regions depending on the location of the occlusion. This technique is now well established and used in many laboratories 8-13. Early use of this technique led to the definition and description of &#x201C;infarct core&#x201D; and &#x201C;penumbra&#x201D; 14-16, and it is often used to evaluate potential neuroprotective compounds 10, 12, 13, 17. Although the initial studies were performed in rats, permanent MCA ligation has been used successfully in mice with slight modifications 18-20 .
This model yields reproducible infarcts and increased post-survival rates. Approximately 80% of the ischemic strokes in humans happen in the MCA area 21 and thus this is a very relevant model for stroke studies. Currently, there is a paucity of effective treatments available to stroke patients, and thus there is a need for good models to test potential pharmacological compounds and evaluate physiological outcomes. This method can also be used for studying intracellular hypoxia response mechanisms in vivo. 
Here, we present the MCA ligation surgery in a C57/BL6 mouse. We describe the pre-surgical preparation, MCA ligation surgery and 2,3,5 Triphenyltetrazolium chloride (TTC) staining for quantification of infarct volumes.

Middle cerebral artery (MCA) ligation is a technique to study focal cerebral ischemia in animal models. In this method, the middle cerebral artery is exposed by craniotomy and ligated by cauterization. This method gives highly reproducible infarct volumes and increased post-operative survival rates compared to other methods available.

在小鼠永久性大脑中动脉结扎术中的应用

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to ...

Mesenteric Artery Contraction and Relaxation Studies Using Automated Wire Myography

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between 100-400 &mu;m in diameter function primarily in directing blood flow to various organs according to the overall requirements of the body. The rat mesenteric artery has a diameter greater than 100 &mu;m. The myography technique, first described by Mulvay and Halpern1, was based on the method proposed by Bevan and Osher2. The technique provides information about small vessels under isometric conditions, where substantial shortening of the muscle preparation is prevented. Since force production and sensitivity of vessels to different agonists is dependent on the extent of stretch, according to active tension-length relation, it is essential to conduct contraction studies under isometric conditions to prevent compliance of the mounting wires. Stainless steel wires are preferred to tungsten wires because of oxidation of the latter, which affects recorded responses3.The technique allows for the comparison of agonist-induced contractions of mounted vessels to obtain evidence for normal function of vascular smooth muscle cell receptors.
We have shown in several studies that isolated mesenteric arteries that are contracted with phenylyephrine relax upon addition of cumulative concentrations of extracellular calcium (Ca2+e). The findings led us to conclude that perivascular sensory nerves, which express the G protein-coupled Ca2+-sensing receptor (CaR), mediate this vasorelaxation response. Using an automated wire myography method, we show here that mesenteric arteries from Wistar, Dahl salt-sensitive(DS) and Dahl salt-resistant (DR) rats respond differently to Ca2+e. Tissues from Wistar rats showed higher Ca2+-sensitivity compared to those from DR and DS. Reduced CaR expression in mesenteric arteries from DS rats correlates with reduced Ca2+e-induced relaxation of isolated, pre-contracted arteries. The data suggest that the CaR is required for relaxation of mesenteric arteries under increased adrenergic tone, as occurs in hypertension, and indicate an inherent defect in the CaR signaling pathway in Dahl animals, which is much more severe in DS.
The method is useful in determining vascular reactivity ex vivo in mesenteric resistance arteries and similar small blood vessels and comparisons between different agonists and/or antagonists can be easily and consistently assessed side-by-side6,7,8.

An automated myography method for force measurements in isolated mesenteric arteries is described. It employs a Mulvany-Halpern Auto Dual Wire Myograph 510A to determine responses to phenylephrine and extracellular calcium. The method allows consistent determination of isometric responses to agonists in small vessels of diameters of 60 - 300 &mu;m, independently.

肠系膜动脉收缩和舒张的研究，使用自动线Myography

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between ...

Epidural Intracranial Pressure Measurement in Rats Using a Fiber-optic Pressure Transducer

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and cardiac arrest. This elevation may result in further neurological injury, in the form of transtentorial herniation1,2,3,4, midbrain compression, neurological deficit or increased cerebral infarct2,4. Current therapies are often inadequate to control elevated ICP in the clinical setting5,6,7 . Thus there is a need for accurate methods of ICP measurement in animal models to further our understanding of the basic mechanisms and to develop new treatments for elevated ICP.
In both the clinical and experimental setting ICP cannot be estimated without direct measurement. Several methods of ICP catheter insertion currently exist. Of these the intraventricular catheter has become the clinical 'gold standard' of ICP measurement in humans8. This method involves the partial removal of skull and the instrumentation of the catheter through brain tissue. Consequently, intraventricular catheters have an infection rate of 6-11%9. For this reason, subdural and epidural cannulations have become the preferred methods in animal models of ischemic injury. 
Various ICP measurement techniques have been adapted for animal models, and of these, fluid-filled telemetry catheters10 and solid state catheters are the most frequently used11,12,13,14,15. The fluid-filled systems are prone to developing air bubbles in the line, resulting in false ICP readings. Solid state probes avoid this problem (Figure 1). An additional problem is fitting catheters under the skull or into the ventricles without causing any brain injury that might alter the experimental outcomes. Therefore, we have developed a method that places an ICP catheter contiguous with the epidural space, but avoids the need to insert it between skull and brain. 
An optic fibre pressure catheter (420LP, SAMBA Sensors, Sweden) was used to measure ICP at the epidural location because the location of the pressure sensor (at the very tip of the catheter) was found to produce a high fidelity ICP signal in this model. There are other manufacturers of similar optic fibre technologies13 that may be used with our methodology. Alternative solid state catheters, which have the pressure sensor located at the side of the catheter tip, would not be appropriate for this model as the signal would be dampened by the presence of the monitoring screw. 
Here, we present a relatively simple and accurate method to measure ICP. This method can be used across a wide range of ICP related animal models.

A novel technique to record the pressures within the skull is described. The minimally invasive method uses a fibre-optic pressure sensing system to accurately measure intracranial pressure (ICP) in anaesthetized rats without causing significant brain trauma. The technique may be used in a wide range of experimental models.

硬膜外颅内压测量中使用的光纤压力传感器的大鼠

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and ...

Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic response is a key regulator of local blood flow. In isobaric conditions small resistance arteries develop sustained constriction known as myogenic tone (MT), which is a major determinant of systemic vascular resistance (SVR). Hence, ex vivo&#160;pressurized preparations of small resistance arteries are major tools to study microvascular function in near-physiological states. To achieve this, a freshly isolated intact segment of a small resistance artery (diameter ~260 &#956;m) is mounted onto two small glass cannulas and pressurized. These arterial preparations retain most in vivo characteristics and permit assessment of vascular tone in real-time. Here we provide a detailed protocol for assessing vasoactivity in pressurized small resistance mesenteric arteries from rats; these arteries develop sustained vasoconstriction - approximately 25% of maximal diameter - when pressurized at 70 mmHg. These arterial preparations may be used to study the effect of investigational compounds on relationship between intra-arterial pressure and vasoactivity and determine changes in microvascular function in animal models of various diseases.

Pressure myography is used to assess vasoactivity of small arteries that develop sustained constriction when pressurized. This manuscript provides a detailed protocol to assess in isolated segments of small mesenteric arteries from rats, vasoactivity and the effect of intraluminal pressure on vascular diameter.

评估肌反应及血管活性阻力肠系膜动脉使用压力肌动描记

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic ...

Assessing Murine Resistance Artery Function Using Pressure Myography

Pressure myograph systems are exquisitely useful in the functional assessment of small arteries, pressurized to a suitable transmural pressure. The near physiological condition achieved in pressure myography permits in-depth characterization of intrinsic responses to pharmacological and physiological stimuli, which can be extrapolated to the in vivo behavior of the vascular bed. Pressure myograph has several advantages over conventional wire myographs. For example, smaller resistance vessels can be studied at tightly controlled and physiologically relevant intraluminal pressures. Here, we study the ability of 3rd order mesenteric arteries (3-4 mm long), preconstricted with phenylephrine, to vaso-relax in response to acetylcholine. Mesenteric arteries are mounted on two cannulas connected to a pressurized and sealed system that is maintained at constant pressure of 60 mmHg. The lumen and outer diameter of the vessel are continuously recorded using a video camera, allowing real time quantification of the vasoconstriction and vasorelaxation in response to phenylephrine and acetylcholine, respectively.
	To demonstrate the applicability of pressure myography to study the etiology of cardiovascular disease, we assessed endothelium-dependent vascular function in a murine model of systemic hypertension. Mice deficient in the &alpha;1 subunit of soluble guanylate cyclase (sGC&alpha;1-/-) are hypertensive when on a 129S6 (S6) background (sGC&alpha;1-/-S6) but not when on a C57BL/6 (B6) background (sGC&alpha;1-/-B6). Using pressure myography, we demonstrate that sGC&alpha;1-deficiency results in impaired endothelium-dependent vasorelaxation. The vascular dysfunction is more pronounced in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice, likely contributing to the higher blood pressure in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice. 
	Pressure myography is a relatively simple, but sensitive and mechanistically useful technique that can be used to assess the effect of various stimuli on vascular contraction and relaxation, thereby augmenting our insight into the mechanisms underlying cardiovascular disease.

In pressure myography, an intact small segment of a vessel is mounted onto two small cannulas and pressurized to a suitable luminal pressure. Here, we describe the method to measure vasorelaxation response of the mouse 3rd order mesenteric arteries in c57 and sGC&alpha;1-/- mice using pressure myography.

评估抗小鼠动脉功能使用压力Myography

Pressure myograph systems are  exquisitely useful in the functional assessment of small arteries, pressurized  to a suitable transmural pressure. The  ...

生物学

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The contractile characteristics can be evaluated independently of external influences such as vascular tonus or neurohumoral status. It depicts a scientific approach between single cell measurements with isolated cardiac myocytes and in vivo studies like echocardiography. Thus, papillary muscle preparations serve as an excellent model to study cardiac physiology/pathophysiology and can be used for investigations like the modulation by pharmacological agents or the exploration of transgenic animal models. Here, we describe a method of isolating the murine left anterior papillary muscle to investigate cardiac contractility in an organ bath setup. In contrast to a muscle strip preparation isolated from the ventricular wall, the papillary muscle can be prepared in toto without damaging the muscle tissue severely. The organ bath setup consists of several temperature-controlled, gassed and electrode-equipped organ bath chambers. The isolated papillary muscle is fixed in the organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer and parameters such as twitch force amplitude and twitch kinetics are analyzed. Different experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents such as catecholamines or other pharmaceuticals. Additionally, pathologic conditions like acute ischemia can be simulated.

Murine left ventricular papillary muscle can be used to investigate cardiac contractility in vitro. This article describes in detail the isolation and experimental protocols to study cardiac contractile characteristics.

对离体乳头肌的心脏收缩力对小鼠的研究收缩尺寸

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

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

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic diseases. Endothelial dysfunction represents a major culprit for the reduced vasodilatation and enhanced vasoconstriction of arteries. Adipose (fat) tissues surrounding the arteries play important roles in the regulation of endothelium-dependent relaxation and/or contraction of the vascular smooth muscle cells. The cross-talks between the endothelium and perivascular adipose tissues can be assessed ex vivo using mounted blood vessels by a wire myography system. However, optimal settings should be established for arteries derived from animals of different species, ages, genetic backgrounds and/or pathophysiological conditions.

The protocol describes the use of wire myography to evaluate the transmural isometric tension of mesenteric arteries isolated from mice, with special consideration of the modulation by factors released from endothelial cells and 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

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

Measurement of Endothelium-Dependent Vasorelaxation in the Mouse Thoracic Aorta Using Tensometric Small Volume Chamber Myography

Small volume chamber tensometric myography is a commonly used technique to evaluate the vascular contractility of small and large blood vessels in laboratory animals and small arteries isolated from human tissue. The technique allows researchers to maintain isolated blood vessels in a tightly controlled and standardized (near-physiological) setting, with the option of adjusting to various environmental factors, while challenging the isolated vessels with different pharmacological agents that can induce vasoconstriction or vasodilation. The myograph chamber also provides a platform to measure vascular reactivity in response to various hormones, inhibitors, and agonists that may impact the function of smooth muscle and endothelial layers separately or simultaneously. The blood vessel wall is a complex structure consisting of three different layers: the intima (endothelial layer), media (smooth muscle and elastin fibers), and adventitia (collagen and other connective tissue). To gain a clear understanding of the functional properties of each layer, it is critical to have access to an experimental platform and system that would allow for a combinational approach to study all three layers simultaneously. Such an approach demands access to a semi-physiological condition that would mimic the in vivo environment in an ex vivo setting. Small volume chamber tensometric myography has provided an ideal environment to evaluate the impact of environmental cues, experimental variables, or pharmacological agonists and antagonists on vascular properties. For many years, scientists have used the tensometric myograph technique to measure endothelial function and smooth muscle contractility in response to different agents. In this report, a small volume chamber tensometric myograph system is used to measure endothelial function in the isolated mouse aorta. This report focuses on how small volume chamber tensometric myography can be used to evaluate the functional integrity of the endothelium in small segments of a large artery such as the thoracic aorta.

The present protocol describes the concepts and technical application of the tensometric myograph technique using a multi-chamber myograph system in the experimental ex vivo assessment of mouse aortic endothelial function.