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>

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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于血压的研究。线 myograph 技术是研究小阻力动脉生理功能和血管疾病发病机制的最佳方法之一。
小血管线 myograph 系统已在其他报告中得到很好的记录, 用于测量大鼠肠系膜动脉8和小鼠动脉 (如主动脉9) 的收缩。利用遗传操作、多种疾病模型和药物筛选模型, 鼠标已成为许多领域广泛使用的模型动物。因此, 在这里, 我们提供了一个改进的协议, 该方法测量小鼠肠系膜动脉收缩。在本报告中, 我们成功地测量了小鼠肠系膜动脉的收缩力和基本缓冲和安装步骤的修改。许多研究在体外 vasocontractility 测量使用了包含 NaHCO3的解决方案, 如克雷布斯解决方案, 以模拟生理盐溶液。然而, 这种缓冲器需要2的调节 pH 值在整个测量, 导致生产的碳酸钙3。我们选择了 h-T 溶液作为缓冲系统, 发现它工作良好。由于温度对 HEPES 的 pK值影响不大, 因此溶液的 pH 值在室温下方便调节, 在37°c 17时不变。此外, 我们使用 ca2 +免费的 H T 解决方案时, 引导导线通过血管腔, 以防止船舶收缩由 Ca2 +。此协议中的另一项修改是安装过程。一些报告8,9和设备手册5建议在修复下颌第一根导线后, 引导第二根导线。我们发现, 当两根导线通过容器管腔在安装容器之前被引导时, 它的效果更好, 因为这种方法可能会由于有限的腔室空间而减少传感器可能造成的损坏。
尽管这种方法具有很高的重现性, 但我们还是要注意一些关键步骤。最重要的是避免损坏的船只由镊子和剪刀。在血管解剖过程中, 操作者应在拉伸脂肪组织时轻轻地使用镊子, 并在切割结缔组织时小心使用剪刀。此外, 应轻轻夹紧血管, 在引导导线时应避免对内皮的损伤, 因为内皮损伤的血管会引起异常反应,例如 g。,损伤后的血管在乙酰胆碱刺激后显示出明显的力张力, 而正常血管显示松弛效应。这种现象的解释是, 受损的内皮不能正确地产生一氧化氮。注意, 在涉及内皮相关收缩的实验中, 应在力测量前检测内皮状态。此外, 我们也应该小心地安装在颚上的船只, 因为传感器是容易损坏, 如果使用硬力。最后, 在执行规范化时, 我们通常不会在千分尺上使用常量递增值。增量的价值是30或20µm 最初和10µm 在有效的压力以后到达11-12 帕。该方法可减少规范化时间, 并可防止过度, 从而减轻船舶损坏。
虽然我们的研究集中在小鼠肠系膜动脉, 这种方法也可以用于主动脉, 支气管, 和其他小血管, 包括肾, 脑和肺动脉。该系统包括四通道, 同时便于测量四平行采样。此外, 整个肠系膜血管床可以提供至少四个动脉段, 因此很容易设计不同的实验组。根据我们的经验, 每个动脉段存活至少4小时, 并保持对高钾+解决方案至少6重复的良好反应。这一属性对于测量多种候选药物的几种添加效果非常有用。然而, 电线 myograph 系统也有局限性。前体线 myograph 实验仅能测量等距 vasocontractility, 但通常应与其它测量相结合, 进行复杂的血管分析。
总之, 我们描述了一种用多线 myograph 系统测量小鼠肠系膜动脉中的异构收缩力的方法。该方法可用于评价血管平滑肌的功能和松弛剂平滑肌的筛查。

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&#183;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, 两根电线被调整为平行, 关闭, 但不接触对方 (图 2B)。在力测量前, 用高 K+溶液对容器段进行规范化和增强两次, 以稳定容器。在规范化的过程中, 该船只被拉伸数次, 直到达到 IC 100 的价值,每个拉伸周期包括强劲的收缩, 快速松弛和部队维护在六十年代 (图 3)。高钾溶液诱导的血管平滑肌收缩通常表现为两个阶段, 一个健壮的阶段和一个持续的阶段 (图 3)。只有当高钾诱发收缩出现正常且重现性时, 才可将该血管段用于进一步实验。neoliensinine 的典型测量在图 4中表示。当高 K+引起的力张力达到一个持续阶段时, 我们通过腔盖中的孔增加了 neoliensinine (1、2、4、6、8和10µM) 的累积剂量。随着剂量的增加, 力以剂量依赖性的方式减少。结果表明, neoliensinine 是一种血管平滑肌松弛物质, 可能作为候选抗高血压药物14。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58064/58064fig1.jpg"> 图 1: 动脉安装程序示意图.蓝线代表电线, 红色矩形代表动脉。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58064/58064fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58064/58064fig2.jpg"> 图 2: 在 myograph 室安装的小鼠肠系膜动脉段.(a)用两根钢丝在两颚上安装小鼠肠系膜动脉段。白色的酒吧 = 2 毫米(B)在面板 (A) 中的小鼠肠系膜动脉段的显微图像。黑酒吧 = 0.5 毫米.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58064/58064fig2large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58064/58064fig3.jpg"> 图 3: 代表原始追查显示规范化程序和增强的高 K+解决方案。第二次高 K+刺激后, 可以进行常规实验。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58064/58064fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58064/58064fig4.jpg"> 图 4: 由高钾溶液收缩的小鼠肠系膜动脉的代表追踪, 然后通过增加累计剂量的 neoliensinine 来放松.随着剂量的增加, 力减少的剂量依赖性的方式,<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58064/58064fig4large.jpg" target="_blank">请点击这里查看一个更大的版本这个数字.</a>

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

Nanyang Technological University

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

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 ...

科研

医学

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 ...

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

在体测量小鼠肺血管内皮表面层

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

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 ...

Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that system. Changes in blood flow to the small intestine can result in effects on the absorptive functions of the organ. Particular interest in toxins liberated from feedstuffs through fermentative and digestive processes has developed in ruminants as an area where productive efficiencies could be improved. The video associated with this article describes an in vitro bioassay developed to screen compounds for vasoactivity in isolated cross-sections of bovine mesenteric artery and vein using a multimyograph. Once the blood vessels are mounted and equilibrated in the myograph, the bioassay itself can be used: as a screening tool to evaluate the contractile response or vasoactivity of compounds of interest; determine the presence of receptor types by pharmacologically targeting receptors with specific agonists; determine the role of a receptor with the presence of one or more antagonists; or determine potential interactions of compounds of interest with antagonists. Through all of this, data are collected real-time, tissue collected from a single animal can be exposed to a large number of different experimental treatments (an in vitro advantage), and represents vasculature on either side of the capillary bed to provide an accurate picture of what could be happening in the afferent and efferent blood supply supporting the small intestine.

The small intestine is frequently exposed to toxins that can influence blood flow and negatively impact nutrient absorption. Using a multimyograph and mesenteric artery and vein isolates, compounds or toxins of interest can be screened for vasoactivity.

隔离和使用的牛肠系膜动脉和静脉部分，作为生物测定在小肠测试的Vasoactivity

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that ...

Measurement of the Pressure-volume Curve in Mouse Lungs

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential phenotypic changes are best assessed by measurement of the changes in lung elasticity. To best understand specific mechanisms underlying such pathologies in mice, it is essential to make functional measurements that can reflect the developing pathology. Although there are many ways to measure elasticity, the classical method is that of the total lung pressure-volume (PV) curve done over the whole range of lung volumes. This measurement has been made on adult lungs from nearly all mammalian species dating back almost 100 years, and such PV curves also played a major role in the discovery and understanding of the function of pulmonary surfactant in fetal lung development. Unfortunately, such total PV curves have not been widely reported in the mouse, despite the fact that they can provide useful information on the macroscopic effects of structural changes in the lung. Although partial PV curves measuring just the changes in lung volume are sometimes reported, without a measure of absolute volume, the nonlinear nature of the total PV curve makes these partial ones very difficult to interpret. In the present study, we describe a standardized way to measure the total PV curve. We have then tested the ability of these curves to detect changes in mouse lung structure in two common lung pathologies, emphysema and fibrosis. Results showed significant changes in several variables consistent with expected structural changes with these pathologies. This measurement of the lung PV curve in mice thus provides a straightforward means to monitor the progression of the pathophysiologic changes over time and the potential effect of therapeutic procedures.

Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung&#8217;s structural changes with developing pathology.

在小鼠肺压力容积曲线的测量

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential ...

Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. While these therapies lead to the rapid restoration of blood flow, these technologies remain limited by restenosis in the case of bare metal stents and angioplasty, or reduced healing and possibly enhanced risk of thrombosis in the case of drug eluting stents. A key pathophysiological mechanism in the formation of restenosis is intimal hyperplasia caused by the activation of vascular smooth muscle cells and inflammation due to arterial stretch and injury. Surgeries that induce arterial injury in genetically modified mice are useful for the mechanistic study of the vascular response to injury but are often technically challenging to perform in mouse models due to the their small size and lack of appropriate sized devices. We describe two approaches for a surgical technique that induces endothelial denudation and arterial stretch in the femoral artery of mice to produce robust neointimal hyperplasia. The first approach creates an arteriotomy in the muscular branch of the femoral artery to obtain vascular access. Following wire injury this arterial branch is ligated to close the arteriotomy. A second approach creates an arteriotomy in the main femoral artery that is later closed through localized cautery. This method allows for vascular access through a larger vessel and, consequently, provides a less technically demanding procedure that can be used in smaller mice. Following either method of arterial injury, a degradable drug delivery patch can be placed over or around the injured artery to deliver therapeutic agents.

We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis.

股动脉线损伤的小鼠模型的血管周围给药补丁植入

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. ...

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

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

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. ...