Name: murine aortic crush injury: an efficient in vivo model of smooth muscle cell proliferation and endothelial function
Uploaded: 2017-06-11T14:00:00
Description: 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

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。 ?...

<ol>
	<li>Carmeliet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+angiogenesis+and+arteriogenesis.">Mechanisms of angiogenesis and arteriogenesis.</a> Nat Med. 6 (4), 389-395 (2000).</li><li>Carmeliet, P., Moons, L., Collen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+angiogenesis,+arterial+stenosis,+atherosclerosis+and+hemostasis.">Mouse models of angiogenesis, arterial stenosis, atherosclerosis and hemostasis.</a> Cardiovasc Res. 39 (1), 8-33 (1998).</li><li>Baker, A. B., 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=Heparanase+Alters+Arterial+Structure,+Mechanics,+and+Repair+Following+Endovascular+Stenting+in+Mice.">Heparanase Alters Arterial Structure, Mechanics, and Repair Following Endovascular Stenting in Mice.</a> Circ Res. 104 (3), 380-387 (2009).</li><li>Petrov, L., Laurila, H., Hayry, P., Vamvakopoulos, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+aortic+angioplasty+for+genomic+studies+of+neointimal+hyperplasia.">A mouse model of aortic angioplasty for genomic studies of neointimal hyperplasia.</a> J Vasc Res. 42 (4), 292-300 (2005).</li><li>Li, J., 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=Vascular+smooth+muscle+cells+of+recipient+origin+mediate+intimal+expansion+after+aortic+allotransplantation+in+mice.">Vascular smooth muscle cells of recipient origin mediate intimal expansion after aortic allotransplantation in mice.</a> Am J Path. 158 (6), 1943-1947 (2001).</li><li>Radu, M., Chernoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vivo+assay+to+test+blood+vessel+permeability.">An in vivo assay to test blood vessel permeability.</a> J Vis Exp. (73), e50062 (2013).</li><li>Turbett, G. R., Sellner, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+optimal+cutting+temperature+compound+can+inhibit+amplification+by+polymerase+chain+reaction.">The use of optimal cutting temperature compound can inhibit amplification by polymerase chain reaction.</a> Diagn Mol Pathol. 6 (5), 298-303 (1997).</li><li>Puchtler, H., Waldrop, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+mechanism+of+Verhoeff's+elastica+stain:+a+convenient+stain+for+myelin+sheaths.">On the mechanism of Verhoeff's elastica stain: a convenient stain for myelin sheaths.</a> Histochem. 62 (3), 233-247 (1979).</li><li>Salic, A., Mitchison, T. 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+chemical+method+for+fast+and+sensitive+detection+of+DNA+synthesis+in+vivo.">A chemical method for fast and sensitive detection of DNA synthesis in vivo.</a> Proc Natl Acad Sci U S A. 105 (7), 2415-2420 (2008).</li><li>Nelson, P. R., Yamamura, S., Mureebe, L., Itoh, H., Kent, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smooth+muscle+cell+migration+and+proliferation+are+mediated+by+distinct+phases+of+activation+of+the+intracellular+messenger+mitogen-activated+protein+kinase.">Smooth muscle cell migration and proliferation are mediated by distinct phases of activation of the intracellular messenger mitogen-activated protein kinase.</a> J Vasc Surg. 27 (1), 117-125 (1998).</li><li>Rzucidlo, 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=Signaling+pathways+regulating+vascular+smooth+muscle+cell+differentiation.">Signaling pathways regulating vascular smooth muscle cell differentiation.</a> Vascular. 17, S15-S20 (2009).</li><li>Aoki, T., Sumii, T., Mori, T., Wang, X., Lo, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier+disruption+and+matrix+metalloproteinase-9+expression+during+reperfusion+injury:+mechanical+versus+embolic+focal+ischemia+in+spontaneously+hypertensive+rats.">Blood-brain barrier disruption and matrix metalloproteinase-9 expression during reperfusion injury: mechanical versus embolic focal ischemia in spontaneously hypertensive rats.</a> Stroke. 33 (11), 2711-2717 (2002).</li><li>Yu, 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=MARCKS+Signaling+Differentially+Regulates+Vascular+Smooth+Muscle+and+Endothelial+Cell+Proliferation+through+a+KIS-,+p27kip1-+Dependent+Mechanism.">MARCKS Signaling Differentially Regulates Vascular Smooth Muscle and Endothelial Cell Proliferation through a KIS-, p27kip1- Dependent Mechanism.</a> PLoS One. 10 (11), e0141397 (2015).</li><li>Banai, S., 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=Rabbit+ear+model+of+injury-induced+arterial+smooth-muscle+cell-proliferation+-+kinetics,+reproducibility,+and+implications.">Rabbit ear model of injury-induced arterial smooth-muscle cell-proliferation - kinetics, reproducibility, and implications.</a> Circ Res. 69 (3), 748-756 (1991).</li></ol>

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 ...

Research

JoVE Journal

Biology

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

Focal Ca2+ Transient Detection in Smooth Muscle

Focal Ca2+ Transient Detection in Smooth Muscle

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

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

人脐动脉平滑肌细胞的分离（HUASMC）

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

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

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

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

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

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

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

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

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 ...

The Murine Choline-Deficient, Ethionine-Supplemented (CDE) Diet Model of Chronic Liver Injury

The Murine Choline-Deficient, Ethionine-Supplemented CDE Diet Model of Chronic Liver Injury

慢性肝损伤小鼠胆碱缺乏、氨基补充的饮食模型

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual ...

Measuring Proliferation of Vascular Smooth Muscle Cells Using Click Chemistry

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 ...