我们感谢马里兰大学医学院电子显微镜核心设备的谢汝清博士在技术支持下处理扫描电子显微镜样品。

道德声明:动物处理方案由马里兰大学机构动物保护和使用委员会（IACUC）批准（协议号0416009），并按照AAALAC-International标准进行。 手术程序<ol><li> 麻醉技术 <ol><li>在121°C蒸汽灭菌30分钟后，将所有用于生存手术的仪器灭菌。 </li><li> 通过经精密蒸发器输送的100％O 2和2.5％异氟烷的感应箱诱导麻醉。后感应，中止异氟烷并用O 2冲洗室。 通过面罩和吸入1 L / min O 2维持1.5-2％异氟烷麻醉。 </li><li>将诱导室和面罩连接到用于废气吸附的炭清除剂中，以保护人员。通过证明确保充足的麻醉机表示没有对有毒刺激（脚趾捏）的反应。 </li><li>创建由具有等温垫的手术托盘组成的手术区域，以在手术期间提供热支撑。额外的等温垫将为其恢复笼中的动物提供热支持。 </li></ol></li><li> 动物准备 <ol><li>对10-12周龄的雄性C57BL / 6小鼠进行以下调查。 </li><li>用脱毛剂或具有40号刀片的电动剪刀从头骨上取下动物腹侧腹部的头发至腹股沟区域。 <ol><li>在脱毛剂的情况下，将该化合物施用于手术区2-3分钟，然后用棉花去除。我们使用市售的氢氧化钙和氢氧化钠的化合物。 </li></ol></li><li>用70％酒精制备肩膀上的区域，并用25号或更小的针孔皮下注射卡洛芬（5 mg / kg）。这个治疗将为动物提供术后止痛。 </li><li>将动物转移到外科手术区域，并且位于背侧卧位。 </li><li>用干净的棉花涂布器或棉纱布擦洗8-12％的碘 - 碘来准备手术部位。然后用70％酒精冲洗皮肤两次。 </li><li>在双眼中放置眼部润滑剂以减少角膜干燥的发生率。用无菌悬垂覆盖手术部位。 </li></ol></li><li> 操作技术 <ol><li>做一个中间腹部剖腹手术切口大约2-2.5厘米长度与手术刀开始立即尾端剑突过程并延伸到骨盆。 </li><li>调动小肠和十二指肠，并向右侧向反射。卷起一条无菌棉布，并用无菌盐水进行注射，以便包装内脏以改善暴露。 </li><li>随着小肠动员到右侧腹部暴露后腹膜并将腹主动脉从左肾静脉暴露于主动脉分叉（ 图1 ）。 </li><li>使用无菌棉花涂抹器，每次持续5秒，连续进行粉碎30次。 </li><li>取出包装，并允许内脏返回原来的位置。 </li><li>使用运行中的4-0可吸收单丝缝合（聚二恶烷酮）封闭筋膜。用运行中的6-0不可吸收的单丝缝合线（尼龙）封闭皮肤。 </li></ol></li><li> 恢复和后期护理 <ol><li>在手术之后，将动物置于具有干净的床垫的恢复笼中，以等温垫以继续热支持，直到动物能够正常行走。不要让动物无人看管，直到它显示出维持胸骨躺卧的能力。 </li><li>术后第4 h每小时监测动物。一旦动物正在行走正常返回我<html>到指定的畜牧室。在完全恢复之前，动物不会被安置在另一只动物的公司内。 </li><li>在手术后的第一个72小时内每天监测动物两次，每周至少3次。监测包括每周三次称重动物。 </li><li>皮下注射卡波芬（5 mg / kg），术后第一个72 h，每日两次。 </li></ol></li></ol> 2.采购组织<ol><li> 安乐死方法 <ol><li>在预定的时间点安乐死动物。 </li><li>通过经精密蒸发器输送的100％O 2和2.5％异氟烷的感应箱诱导麻醉。 <ol><li>研究内皮细胞的完整性，将伊文思蓝染料给予动物。 注意:Evans Blue是一种带负电荷的偶氮染料，对白蛋白具有很高的结合亲和力，只能在没有完整内皮的情况下染色血管s ="xref"&gt; 6。 </li><li>对于内皮完整性研究，在安乐死时，用5 mL 0.3％伊文思蓝染料，然后用生理压力5 mL PBS灌注动物5 min。在灌注过程中，将动物维持在麻醉手术平面中。 </li></ol></li><li>达到深度麻醉平面后，用胸骨切开术打开胸部。在右心房撕裂以允许从动物排出血液，并用21G针头进入左心室，并注射磷酸盐缓冲盐水（PBS），直到来自右心房的流出物清除。 </li><li>用PBS灌注后，通过中线切口进入腹部。 </li><li>再次，将小肠移动到暴露肾下主动脉的腹部右侧，从相邻组织中急剧解剖主动脉，并将其从左肾静脉切除至主动脉分叉。 </li><li>将切除的主动脉存放在4％的多形态中直到组织处理发生。 <ol><li>对于内皮完整性研究，纵向打开主动脉并将其固定到暴露整个管腔表面6的蜡片上。用Evans Blue染料6进行染色程度，对内皮完整性进行定性评估。 </li><li>对于其他组织学检查，横断面主动脉，并将其嵌入最佳切割温度（OCT）化合物中，如所使用的组织学方法所决定。 </li></ol></li></ol></li></ol>

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这项工作由退伍军人事务部职业发展部（1IK2BX001553-01）（TSM）和Vascular Cures EJ Wylie奖学金（TSM）资助。

我们已经描述了导致内侧增生和内皮屏障功能障碍的鼠主动脉损伤模型的影响。沿主动脉内膜的部分EC分离伴随细胞细胞接触的丧失和细胞突起的增强。相应地，内皮屏障功能受到显着损害，刺激了丝裂原敏感信号传导途径，导致VSMC的增殖和血管壁的增厚。这种模式的优点是在技术上比其他主动脉疾病模型更容易学习和执行，并允许同时评估对损伤和内皮功能的增殖反应。该协议的关键步骤是实?...

再狭窄 遵循心血管手术（旁路手术，血管成形术或支架置入术）是降低这些手术的耐久性的重大问题。所有血运重建手术都受到再狭窄的困扰。目前防止再狭窄的策略（药物洗脱支架和药物包衣球囊）抑制血管平滑肌细胞（VSMC）和内皮细胞增殖（EC）。因此，这些干预措施可以预防VSMC介导的再狭窄，而且可以防止内皮细胞的再生。没有完整的内皮，患者需要使用有效的抗血小板药物，以降低出血并发症风险的原位血栓形成的风险。理想的治疗方法是抑制VSMC增殖，同时促进内皮再生。因此，需要同时研究VSMC增殖和内皮屏障功能。 目前来看，再狭窄的小鼠模型1 。这些模型包括颈动脉结扎和股动脉线损伤2 。主动脉模型包括支架置入3 ，球囊损伤4和主动脉同种异体移植5 。所有目前的型号都是有限的。颈动脉结扎产生流动介导的新生内膜损伤并且不具有内皮损伤。此外，颈动脉和股动脉与人血管相比，细胞层的折叠多少，限制了它们的平移值。直径约1.3毫米的小鼠主动脉是接近临床相关（冠状动脉）人动脉（3）的唯一血管。 尽管鼠主动脉疾病模型具有翻译潜力，但目前的模型有局限性。这些模型需要先进的显微外科技术和专门的设备，如血管成形术气球和支架。在这里，我们现在一种新颖的，可重复的技术，以同时诱导VSMC增殖并破坏内皮屏障功能。

<table border="0" cellpadding="0" cellspacing="0" width="920">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Ocular lubricant</td>
			<td style="width:160px;">Dechra</td>
			<td style="width:163px;">17033-211-38</td>
			<td style="width:247px;">Pharmaceutical agents</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Isoflurane</td>
			<td>VetOne</td>
			<td>502017</td>
			<td>Pharmaceutical agents</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Carprofen</td>
			<td>Zoetis</td>
			<td>26357</td>
			<td>Pharmaceutical agents</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Precision vaporizer</td>
			<td>Summit Medical</td>
			<td>10675</td>
			<td>Surgical supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Charcoal scavenger</td>
			<td>Bickford Inc.</td>
			<td>80120</td>
			<td>Surgical supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Isothermal pad</td>
			<td>Harvard Apparatus</td>
			<td>50-7053-R</td>
			<td>Surgical supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Sterile cotton-tipped applicator</td>
			<td>Fisher Scientific</td>
			<td>23-400-124</td>
			<td>Surgical supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">4-0 absorbable monofilament suture&#160;</td>
			<td>Ethicon, Inc</td>
			<td>J310</td>
			<td>Surgical supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">5-0 non-absorbable monofilament suture</td>
			<td>Ethicon,Inc</td>
			<td>1666</td>
			<td>Surgical supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">21-gauge x 1 inch needle</td>
			<td>BD Biosciences</td>
			<td>305165</td>
			<td>Surgical supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">25-gauge x 1 inch&#160; needle</td>
			<td>BD Biosciences</td>
			<td>305125</td>
			<td>Surgical supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dry sterilizer</td>
			<td>Cellpoint&#160;</td>
			<td>7770</td>
			<td>Surgical supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fine scissors</td>
			<td>Fine Science Tools</td>
			<td>14058-09</td>
			<td>Surgical instruments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Adson forceps</td>
			<td>Fine Science Tools</td>
			<td>11006-12</td>
			<td>Surgical instruments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Dumont #5 fine forceps</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td>Surgical instruments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Vannas Spring Scissors 3mm cutting edge</td>
			<td>Fine Science Tools</td>
			<td>15000-00</td>
			<td>Surgical instruments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Needle driver</td>
			<td>Fine Science Tools</td>
			<td>91201-13</td>
			<td>Surgical instruments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scalpel handle #4</td>
			<td>Fine Science Tools</td>
			<td>10004-13</td>
			<td>Surgical instruments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scalpel blades #10</td>
			<td>Fine Science Tools</td>
			<td>10010-00</td>
			<td>Surgical instruments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS&#160;</td>
			<td>Lonza</td>
			<td>17-516F</td>
			<td>Reagents for tissue processing</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Evans Blue</td>
			<td>Sigma-Aldrich</td>
			<td>E2129</td>
			<td>Reagents for tissue processing</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>Reagents for tissue processing</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Modeling wax</td>
			<td>Bego</td>
			<td>40001</td>
			<td>Reagents for tissue processing</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OCT compound</td>
			<td>Tissue-Tek Sakura</td>
			<td>4583</td>
			<td>Reagents for tissue processing</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mayer&#39;s hematoxylin solution</td>
			<td>Sigma-Aldrich</td>
			<td>MHS16</td>
			<td>Reagents for immunohistological analysis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eosin Y solution alcoholic&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>HT110316</td>
			<td>Reagents for immunohistological analysis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Elastin stain kit</td>
			<td>Sigma-Aldrich</td>
			<td style="width:163px;">HT25A</td>
			<td>Reagents for immunohistological analysis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Click-it Edu Alexa-488 Imaging Kit</td>
			<td>Invitrogen</td>
			<td>C10337</td>
			<td>Reagents for immunohistological analysis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Anti-Erk1/2 antibody</td>
			<td>Cell Signaling Technology</td>
			<td>4695</td>
			<td>Reagents for immunohistological analysis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Anti-phospho-Erk1/2 antibody</td>
			<td>Cell Signaling Technology</td>
			<td>4370</td>
			<td>Reagents for immunohistological analysis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Anti-p27kip1 antibody</td>
			<td>Cell Signaling Technology</td>
			<td>3698</td>
			<td>Reagents for immunohistological analysis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Trichloroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>T9159</td>
			<td>Reagents for immunohistological analysis</td>
		</tr>
	</tbody>
</table>

murine aortic crush injury: an efficient in vivo model of smooth muscle cell proliferation and endothelial function

动脉重建，无论是血管成形术还是旁路手术，都涉及医源性创伤，引起内皮细胞破裂和血管平滑肌细胞（VSMC）增殖。常见的小鼠模型研究小血管如颈动脉和股动脉。这里我们描述一种体内系统，其中VSMC增殖和内皮屏障功能可以在大型血管中同时进行评估。我们研究了C57BL / 6小鼠肾下主动脉对损伤的反应。主动脉从左肾静脉到主动脉分叉，用棉花头施放器以5秒持续时间进行30次透壁压迫。用常规组织学评估形态学变化。从腔表面到外膜测量主动脉壁厚度。用DAPI和α-肌动蛋白进行EdU整合和反染色以证明VSMC增殖。激活ERK1 / 2，一种已知的内膜增生形成调节剂是阻止的由Western Blot分析开采。通过B细胞，T细胞和巨噬细胞的免疫组织化学测定炎症的作用。用扫描电子显微镜（SEM）观察内皮的内表面部分。用伊文思蓝染色测定内皮屏障功能。透壁性损伤导致主动脉壁增厚。这种损伤引起VSMC增殖，损伤后3天最突出，ERK1 / 2的早期激活和p27 kip1表达降低。损伤不会导致血管壁中增加的B细胞，T细胞或巨噬细胞浸润。损伤导致部分内皮细胞剥蚀和细胞 - 细胞接触丧失。损伤导致内皮屏障功能的显着损失，七天后恢复到基线。小鼠透壁性钝性主动脉损伤模型提供了一种同时研究大血管中VSMC增殖和内皮屏障功能的有效系统。

切片嵌入OCT的横断面主动脉切片，用苏木精和伊红染色，然后用Verhoeff-Van Gieson（VVG）染色法染色，鉴定内外弹性片7 。与用假手术治疗的动物的主动脉相比，剖腹术和单独的小肠动员相比，粉碎性损伤导致主动脉壁增厚。损伤后3天（42.2±1.7μm，单独用于剖腹手术时为22.1±1.1μm），外膜厚度与外膜距离为止（ 图2A-B ）。损伤导致细胞具有更圆?...

Watch this Scientific Journal Video about Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function at JoVE.com

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

心血管手术后的再狭窄（旁路手术，血管成形术或支架置入术）是降低这些手术的耐久性的重大问题。理想的治疗方法是抑制平滑肌细胞（VSMC）的增殖，同时促进内皮的再生。我们描述了同时评估体内VSMC增殖和内皮功能的模型。

小鼠主动脉夹层损伤:高效<em&gt;体内</em&gt;平滑肌细胞增殖和内皮功能模型

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell ...

murine-aortic-crush-injury-an-efficient-vivo-model-smooth-muscle-cell

Research

JoVE Journal

Biology

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

8.3K 次观看.  Baltimore Veterans Affairs Medical Center. 心血管手术后的再狭窄（旁路手术，血管成形术或支架置入术）是降低这些手术的耐久性的重大问题。理想的治疗方法是抑制平滑肌细胞（VSMC）的增殖，同时促进内皮的再生。我们描述了同时评估体内VSMC增殖和内皮功能的模型。 

视频: Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Focal Ca2+ Transient Detection in Smooth Muscle

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ from internal stores, and allows multiple cells to be monitored simultaneously to assess, for example, coupling in syncytial tissue. Subcellular Ca2+ transients are common in smooth muscle, yet are difficult to measure accurately because of the problems caused by their stochastic occurrence, over an often wide field of view, in an organ that it prone to contract. To overcome this problem, we've developed a series of imaging protocols and analysis routines to acquire and then analyse, in an automated fashion, the frequency, location and amplitude of such events. While this approach may be applied in other contexts, our own work involves the detection of local purinergic Ca2+ transients for locating transmitter release with submicron resolution. 
ATP is released as a cotransmitter from autonomic nerves, where it binds to P2X1 receptors on the smooth muscle of the detrusor and vas deferens. Ca2+ enters the smooth muscle, resulting in purinergic neuroeffector Ca2+ transients (NCTs). The focal Ca2+ transients allow the optical monitoring of neurotransmitter release in a manner that has many advantages over electrophysiology. Apart from the greatly improved spatial resolution, optical recording has the additional advantage of allowing the recording of transmitter release from many distinguishable sites simultaneously. Furthermore, the optical plane of focus is easier to maintain or correct during long recording series than is the repositioning of an intracellular sharp microelectrode. 
In summary, a method for imaging of Ca2+ fluorescence is outlined which details the preparation of tissue, and the acquisition and analysis of data. We outline the use of several scripts for the analysis of such Ca2+ transients.

Focal Ca2+ Transient Detection in Smooth Muscle

Details methods for high-resolution Ca2+ imaging of smooth muscle within isolated organs, including: preparation of the tissue, image acquisition and data analysis.

焦距钙 2 +</sup瞬态检测平滑肌

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ ...

科研

生物学

Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

Contraction or relaxation of smooth muscle cells within the walls of resistance arteries determines the artery diameter and thereby controls flow of blood through the vessel and contributes to systemic blood pressure. The contraction process is regulated primarily by cytosolic calcium concentration ([Ca2+]cyt), which is in turn controlled by a variety of ion transporters and channels. Ion channels are common intermediates in signal transduction pathways activated by vasoactive hormones to effect vasoconstriction or vasodilation. And ion channels are often targeted by therapeutic agents either intentionally (e.g. calcium channel blockers used to induce vasodilation and lower blood pressure) or unintentionally (e.g. to induce unwanted cardiovascular side effects).
Kv7 (KCNQ) voltage-activated potassium channels have recently been implicated as important physiological and therapeutic targets for regulation of smooth muscle contraction. To elucidate the specific roles of Kv7 channels in both physiological signal transduction and in the actions of therapeutic agents, we need to study how their activity is modulated at the cellular level as well as evaluate their contribution in the context of the intact artery.
The rat mesenteric arteries provide a useful model system. The arteries can be easily dissected, cleaned of connective tissue, and used to prepare isolated arterial myocytes for patch clamp electrophysiology, or cannulated and pressurized for measurements of vasoconstrictor/vasodilator responses under relatively physiological conditions. Here we describe the methods used for both types of measurements and provide some examples of how the experimental design can be integrated to provide a clearer understanding of the roles of these ion channels in the regulation of vascular tone.

Measurements of Kv7 (KCNQ) potassium channel activity in isolated arterial myocytes (using patch clamp electrophysiological techniques) in parallel with measurements of constrictor/dilator responses (using pressure myography) can reveal important information about the roles of Kv7 channels in vascular smooth muscle physiology and pharmacology.

探讨动脉平滑肌的KV7钾离子通道功能使用膜片钳电生理和压力Myography

Contraction  or relaxation of smooth muscle cells within the walls of resistance arteries  determines the artery diameter and thereby controls flow of ...

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Body movements are mainly provided by mechanical function of skeletal muscle. Skeletal muscle is composed of numerous bundles of myofibers that are sheathed by intramuscular connective tissues. Each myofiber contains many myofibrils that run longitudinally along the length of the myofiber. Myofibrils are the contractile apparatus of muscle and they are composed of repeated contractile units known as sarcomeres. A sarcomere unit contains actin and myosin filaments that are spaced by the Z discs and titin protein. Mechanical function of skeletal muscle is defined by the contractile and passive properties of muscle. The contractile properties are used to characterize the amount of force generated during muscle contraction, time of force generation and time of muscle relaxation. Any factor that affects muscle contraction (such as interaction between actin and myosin filaments, homeostasis of calcium, ATP/ADP ratio, etc.) influences the contractile properties. The passive properties refer to the elastic and viscous properties (stiffness and viscosity) of the muscle in the absence of contraction. These properties are determined by the extracellular and the intracellular structural components (such as titin) and connective tissues (mainly collagen) 1-2. The contractile and passive properties are two inseparable aspects of muscle function. For example, elbow flexion is accomplished by contraction of muscles in the anterior compartment of the upper arm and passive stretch of muscles in the posterior compartment of the upper arm. To truly understand muscle function, both contractile and passive properties should be studied.
The contractile and/or passive mechanical properties of muscle are often compromised in muscle diseases. A good example is Duchenne muscular dystrophy (DMD), a severe muscle wasting disease caused by dystrophin deficiency 3. Dystrophin is a cytoskeletal protein that stabilizes the muscle cell membrane (sarcolemma) during muscle contraction 4. In the absence of dystrophin, the sarcolemma is damaged by the shearing force generated during force transmission. This membrane tearing initiates a chain reaction which leads to muscle cell death and loss of contractile machinery. As a consequence, muscle force is reduced and dead myofibers are replaced by fibrotic tissues 5. This later change increases muscle stiffness 6. Accurate measurement of these changes provides important guide to evaluate disease progression and to determine therapeutic efficacy of novel gene/cell/pharmacological interventions. Here, we present two methods to evaluate both contractile and passive mechanical properties of the extensor digitorum longus (EDL) muscle and the contractile properties of the tibialis anterior (TA) muscle.

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Changes in limb muscle contractile and passive mechanical properties are important biomarkers for muscle diseases. This manuscript describes physiological assays to measure these properties in the murine extensor digitorum longus and tibialis anterior muscles.

伸趾长肌的肌肉功能评价<em&gt;体外</em和胫骨前肌<em在原位</em在小鼠

Body  movements are mainly provided by mechanical function of skeletal muscle. Skeletal  muscle is composed of numerous bundles of myofibers that are ...

Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues. While this technique has been implemented for over 100 years, the versatility, simplicity and reproducibility of this assay helps it to remain an indispensable tool for pharmacologists and physiologists alike. Tissue bath systems are available in a wide array of shapes and sizes, allowing a scientist to evaluate samples as small as murine mesenteric arteries and as large as porcine ileum &#8211; if not larger. Central to the isolated tissue bath assay is the ability to measure concentration-dependent changes to isometric contraction, and how the efficacy and potency of contractile agonists can be manipulated by increasing concentrations of antagonists or inhibitors. Even though the general principles remain relatively similar, recent technological advances allow even more versatility to the tissue bath assay by incorporating computer-based data recording and analysis software. This video will demonstrate the function of the isolated tissue bath to measure the isometric contraction of an isolated smooth muscle (in this case rat thoracic aorta rings), and share the types of knowledge that can be created with this technique. Included are detailed descriptions of aortic tissue dissection and preparation, placement of aortic rings in the tissue bath and proper tissue equilibration prior to experimentation, tests of tissue viability, experimental design and implementation, and data quantitation. Aorta will be connected to isometric force transducers, the data from which will be captured using a commercially available analog-to-digital converter and bridge amplifier specifically designed for use in these experiments. The accompanying software to this system will be used to visualize the experiment and analyze captured data.

This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition.

平滑肌功能中分离出来的组织浴，应用药理学研究测量

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues.  ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

细胞内钙的双光子成像<sup&gt; 2+</sup&gt;的离体大鼠主动脉的处理和一氧化氮的内皮细胞和平滑肌细胞

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

隔离和鼠标主动脉内皮细胞原代培养

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

鼠冠状动脉血管平滑肌细胞的分离

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Measuring Proliferation of Vascular Smooth Muscle Cells Using Click Chemistry

The ability of a cell to proliferate is integral to the normal function of most cells, and dysregulation of proliferation is at the heart of many disease processes. For these reasons, measuring proliferation is a common tool used to assess cell function. Cell proliferation can be measured simply by counting; however, this is an indirect means of measuring proliferation. One common means of directly detecting cells preparing to divide is by incorporation of labeled nucleoside analogs. These include the radioactive nucleoside analog 3H-thymidine plus non-radioactive nucleoside analogs such as 5-bromo-2&#8217; deoxyuridine (BrdU) and 5-ethynyl-2&#8242;-deoxyuridine (EdU). Incorporation of EdU is detected by click chemistry, which has several advantages when compared to BrdU. In this report, we provide a protocol for measuring proliferation by the incorporation of EdU. This protocol includes options for various readouts, along with the advantages and disadvantages of each. We also discuss places where the protocol can be optimized or altered to meet the specific needs of the experiment planned. Finally, we touch on the ways that this basic protocol can be modified for measuring other cell metabolites.

Proliferation is a critical part of cellular function, and a common readout used to assess potential toxicity of new drugs. Measuring proliferation is, therefore, a frequently used assay in cell biology. Here we present a simple, versatile method of measuring proliferation that can be used in adherent and non-adherent cells.

使用点击化学测量血管平滑肌细胞增殖

The ability of a cell to proliferate is integral to the normal function of most cells, and dysregulation of proliferation is at the heart of many disease ...

In vivo Measurement of Knee Extensor Muscle Function in Mice

Skeletal muscle plasticity in response to countless conditions and stimuli mediates concurrent functional adaptation, both negative and positive. In the clinic and the research laboratory, maximal muscular strength is widely measured longitudinally in humans, with knee extensor musculature the most reported functional outcome. Pathology of the knee extensor muscle complex is well documented in aging, orthopedic injury, disease, and disuse; knee extensor strength is closely related to functional capacity and injury risk, underscoring the importance of reliable measurement of knee extensor strength. Repeatable, in vivo assessment of knee extensor strength in pre-clinical rodent studies offers valuable functional endpoints for studies exploring osteoarthritis or knee injury. We report an in vivo and non-invasive protocol to repeatedly measure isometric peak tetanic torque of the knee extensors in mice across time. We demonstrate consistency using this novel method to measure knee extensor strength with repeated assessment in multiple mice producing similar results.

Quantification of knee extensor maximal strength is imperative to understand functional adaptations to aging, disease, injury, and rehabilitation. We present a novel method to repeatedly measure in vivo knee extension isometric peak tetanic torque.

小鼠膝盖外展肌肉功能的体内测量

Skeletal muscle plasticity in response to countless conditions and stimuli mediates concurrent functional adaptation, both negative and positive. In the ...

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in the aorta and regulates vasoconstriction and vasodilation. Mutations in genes encoding for the SMC contractile apparatus proteins are associated with aortic diseases, such as thoracic aortic aneurysms. Measuring SMC contraction in vitro is challenging, especially in a high-throughput manner, which is essential for screening patient material. Currently available methods are not suitable for this purpose. This paper presents a novel method based on electric cell-substrate impedance sensing (ECIS). First, an explant protocol is described to isolate patient-specific human primary SMCs from aortic biopsies and patient-specific human primary dermal fibroblasts for the study of aortic aneurysms. Next, a detailed description of a new contraction method is given to measure the contractile response of these cells, including the subsequent analysis and suggestion for comparing different groups. This method can be used to study the contraction of adherent cells in the context of translational (cardiovascular) studies and patient and drug screening studies.

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro

This paper describes an explant culture-based method for the isolation and culturing of primary, patient-specific human aortic smooth muscle cells and dermal fibroblasts. Furthermore, a novel method is presented for measuring cell contraction and subsequent analysis, which can be used to study patient-specific differences in these cells.

原发性患者特异性主动脉平滑肌细胞的分离和体外半定量实时收缩测量

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in ...

小鼠主动脉夹层损伤:高效

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