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>

<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>

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><tbody><tr><td>Ocular lubricant</td><td>Dechra</td><td>17033-211-38</td><td>Pharmaceutical agents</td></tr><tr><td>Isoflurane</td><td>VetOne</td><td>502017</td><td>Pharmaceutical agents</td></tr><tr><td>Carprofen</td><td>Zoetis</td><td>26357</td><td>Pharmaceutical agents</td></tr><tr><td>Precision vaporizer</td><td>Summit Medical</td><td>10675</td><td>Surgical supplies</td></tr><tr><td>Charcoal scavenger</td><td>Bickford Inc.</td><td>80120</td><td>Surgical supplies</td></tr><tr><td>Isothermal pad</td><td>Harvard Apparatus</td><td>50-7053-R</td><td>Surgical supplies</td></tr><tr><td>Sterile cotton-tipped applicator</td><td>Fisher Scientific</td><td>23-400-124</td><td>Surgical supplies</td></tr><tr><td>4-0 absorbable monofilament suture&amp;nbsp;</td><td>Ethicon, Inc</td><td>J310</td><td>Surgical supplies</td></tr><tr><td>5-0 non-absorbable monofilament suture</td><td>Ethicon,Inc</td><td>1666</td><td>Surgical supplies</td></tr><tr><td>21-gauge x 1 inch needle</td><td>BD Biosciences</td><td>305165</td><td>Surgical supplies</td></tr><tr><td>25-gauge x 1 inch&amp;nbsp; needle</td><td>BD Biosciences</td><td>305125</td><td>Surgical supplies</td></tr><tr><td>Dry sterilizer</td><td>Cellpoint&amp;nbsp;</td><td>7770</td><td>Surgical supplies</td></tr><tr><td>Fine scissors</td><td>Fine Science Tools</td><td>14058-09</td><td>Surgical instruments</td></tr><tr><td>Adson forceps</td><td>Fine Science Tools</td><td>11006-12</td><td>Surgical instruments</td></tr><tr><td>Dumont #5 fine forceps</td><td>Fine Science Tools</td><td>11254-20</td><td>Surgical instruments</td></tr><tr><td>Vannas Spring Scissors 3 mm cutting edge</td><td>Fine Science Tools</td><td>15000-00</td><td>Surgical instruments</td></tr><tr><td>Needle driver</td><td>Fine Science Tools</td><td>91201-13</td><td>Surgical instruments</td></tr><tr><td>Scalpel handle #4</td><td>Fine Science Tools</td><td>10004-13</td><td>Surgical instruments</td></tr><tr><td>Scalpel blades #10</td><td>Fine Science Tools</td><td>10010-00</td><td>Surgical instruments</td></tr><tr><td>PBS&amp;nbsp;</td><td>Lonza</td><td>17-516F</td><td>Reagents for tissue processing</td></tr><tr><td>Evans Blue</td><td>Sigma-Aldrich</td><td>E2129</td><td>Reagents for tissue processing</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td>Reagents for tissue processing</td></tr><tr><td>Modeling wax</td><td>Bego</td><td>40001</td><td>Reagents for tissue processing</td></tr><tr><td>OCT compound</td><td>Tissue-Tek Sakura</td><td>4583</td><td>Reagents for tissue processing</td></tr><tr><td>Mayer&#039;s hematoxylin solution</td><td>Sigma-Aldrich</td><td>MHS16</td><td>Reagents for immunohistological analysis</td></tr><tr><td>Eosin Y solution alcoholic&amp;nbsp;</td><td>Sigma-Aldrich</td><td>HT110316</td><td>Reagents for immunohistological analysis</td></tr><tr><td>Elastin stain kit</td><td>Sigma-Aldrich</td><td>HT25A</td><td>Reagents for immunohistological analysis</td></tr><tr><td>Click-it Edu Alexa-488 Imaging Kit</td><td>Invitrogen</td><td>C10337</td><td>Reagents for immunohistological analysis</td></tr><tr><td>Anti-Erk1/2 antibody</td><td>Cell Signaling Technology</td><td>4695</td><td>Reagents for immunohistological analysis</td></tr><tr><td>Anti-phospho-Erk1/2 antibody</td><td>Cell Signaling Technology</td><td>4370</td><td>Reagents for immunohistological analysis</td></tr><tr><td>Anti-p27kip1 antibody</td><td>Cell Signaling Technology</td><td>3698</td><td>Reagents for immunohistological analysis</td></tr><tr><td>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

Universidad de Cartagena UDEC

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

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic lamellae and smooth muscle cells (SMCs), and an outer layer of fibroblasts and extracellular matrix. In contrast to the widespread study of pathological models (e.g., atherosclerosis) in the adult aorta, much less is known about the embryonic and perinatal aorta. Here, we focus on SMCs and provide protocols for the analysis of the morphogenesis and pathogenesis of embryonic and perinatal aortic SMCs in normal development and disease. Specifically, the four protocols included are: i) in vivo embryonic fate mapping and clonal analysis; ii) explant embryonic aorta culture; iii) SMC isolation from the perinatal aorta; and iv) subcutaneous osmotic mini-pump placement in pregnant (or non-pregnant) mice. Thus, these approaches facilitate the investigation of the origin(s), fate, and clonal architecture of SMCs in the aorta in vivo. They allow for modulating embryonic aorta morphogenesis in utero by continuous exposure to pharmacological agents. In addition, isolated aortic tissue explants or aortic SMCs can be used to gain insights into the role of specific gene targets during fundamental processes such as muscularization, proliferation, and migration. These hypothesis-generating experiments on isolated SMCs and the explanted aorta can then be assessed in the in vivo context through pharmacological and genetic approaches.

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

Protocols for studying the embryonic and perinatal murine aorta using in vivo clonal analysis and fate mapping, aortic explants, and isolated smooth muscle cells are detailed here. These diverse approaches facilitate the investigation of the morphogenesis of the embryonic and perinatal aorta in normal development and the pathogenesis in disease.

使用体内和组织和细胞外植体方法研究胚胎和围产期主动脉的形态和发病机制

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic ...

科研

发育生物学

Murine Spinotrapezius Model to Assess the Impact of Arteriolar Ligation on Microvascular Function and Remodeling

The murine spinotrapezius is a thin, superficial skeletal support muscle that extends from T3 to L4, and is easily accessible via dorsal skin incision. Its unique anatomy makes the spinotrapezius useful for investigation of ischemic injury and subsequent microvascular remodeling. Here, we demonstrate an arteriolar ligation model in the murine spinotrapezius muscle that was developed by our research team and previously published1-3. For certain vulnerable mouse strains, such as the Balb/c mouse, this ligation surgery reliably creates skeletal muscle ischemia and serves as a platform for investigating therapies that stimulate revascularization. Methods of assessment are also demonstrated, including the use of intravital and confocal microscopy. The spinotrapezius is well suited to such imaging studies due to its accessibility (superficial dorsal anatomy) and relative thinness (60-200 &mu;m). The spinotrapezius muscle can be mounted en face, facilitating imaging of whole-muscle microvascular networks without histological sectioning. We describe the use of intravital microscopy to acquire metrics following a functional vasodilation procedure; specifically, the increase in arterilar diameter as a result of muscle contraction. We also demonstrate the procedures for harvesting and fixing the tissues, a necessary precursor to immunostaining studies and the use of confocal microscopy.

We demonstrate a novel arterial ligation model in murine spinotrapezius muscle, including a step-by-step procedure and description of required instrumentation. We describe the surgery and relevant outcome measurements relating to vascular network remodeling and functional vasodilation using intravital and confocal microscopy.

小鼠Spinotrapezius模型来评估的影响小动脉结扎​​术，微血管功能与重塑

The murine spinotrapezius is a thin, superficial skeletal support muscle that extends from T3 to L4, and is easily accessible via dorsal skin incision. ...

医学

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

颈动脉支架植入再狭窄的研究的小鼠模型

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

A New Murine Model of Endovascular Aortic Aneurysm Repair

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of Abdominal aortic aneurysm (AAA) pathophysiology. There is no abdominal aortic aneurysm endograft exclusion model cheap and reproducible, which would allow deep investigations of AAA before and after treatment. We hereby describe how to induce, and then to exclude with a covered coronary stentgraft an abdominal aortic aneurysm in a rat. The well known elastase induced AAA model was first reported in 19901 in a rat, then described in mice2. Elastin degradation leads to dilation of the aorta with inflammatory infiltration of the abdominal wall and intra luminal thrombus, matching with human AAA. Endovascular exclusion with small covered stentgraft is then performed, excluding any interactions between circulating blood and the aneurysm thrombus. Appropriate exclusion and stentgraft patency is confirmed before euthanasia by an angiography thought the left carotid artery. Partial control of elastase diffusion makes aneurysm shape different for each animal. It is difficult to create an aneurysm, which will allow an appropriate length of aorta below the aneurysm for an easy stentgraft introduction, and with adequate proximal and distal neck to prevent endoleaks. Lots of failure can result to stentgraft introduction which sometimes lead to aorta tear with pain and troubles to stitch it, and endothelial damage with post op aorta thrombosis. Giving aspirin to rats before stentgraft implantation decreases failure rate without major hemorrhage. Clamping time activates neutrophils, endothelium and platelets, and may interfere with biological analysis.

Histological and biochemical modifications after aneurysm endograft exclusion are still unclear. We describe a new model of endograft implantation on a murine aneurysm. Through thrombus analysis with and without persistant circulating blood stream interface, and new endovascular biomaterials evaluation, this model will help to better understand endovascular aneurysm exclusion pathobiology.

一种新的小鼠模型主动脉瘤腔内修复

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of ...

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These complications can be attributed to impairments in re-endothelialization within the wound margins. Yet, the cellular and molecular mechanisms of re-endothelialization remain to be defined. While several animal models to study re-endothelialization after arterial denudation are available, few are performed in the mouse because of surgical limitations. This undermines the opportunity to exploit transgenic mouse lines and investigate the contribution of specific genes to the process of re-endothelialization. Here, we present a step-by-step protocol for creating a highly reproducible murine model of arterial denudation injury in the infrarenal abdominal aorta using external vascular clamping. Immunocytochemical staining of injured aortas for fibrinogen and &#946;-catenin demonstrate the exposure of a pro-thrombotic surface and the border of intact endothelium, respectively. The method presented here has the advantages of speed, excellent overall survival rate, and relative technical ease, creating a uniquely practical tool for imposing arterial denudation injury in transgenic mouse models. Using this method, investigators may elucidate the mechanisms of re-endothelialization under normal or pathological conditions.

Understanding the cellular and molecular mechanisms of re-endothelialization following arterial denudation injury is of paramount importance in preventing thrombosis and restenosis of arteries. Here we describe a protocol for reproducible arterial denudation injury of the infrarenal abdominal aorta. The procedure was developed to investigate the underlying mechanisms that regulate endothelial regeneration using mouse models.

重复性的动脉剥脱损伤腹主动脉阻断在小鼠模型

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These ...

Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine balloon denudation model, which is comparable with established vessel injury models in large animals. The aorta denudation model with balloon catheters mimics the clinical setting and leads to comparable pathobiological and physiological changes. Briefly, after performing a horizontal incision in the aorta abdominalis, a balloon catheter will be inserted into the vessel, inflated, and introduced retrogradely. Inflation of the balloon will lead to intima injury and overdistension of the vessel. After removing the catheter, the aortic incision will be closed with single stiches. The model shown in this article is reproducible, easy to perform, and can be established quickly and reliably. It is especially suitable for evaluating expensive experimental therapeutic agents, which can be applied in an economical fashion. By using different knockout-mouse strains, the impact of different genes on MH development can be assessed.

This article demonstrates a murine model to study the development of myointimal hyperplasia (MH) after aortic balloon injury.

球囊损伤诱发小鼠腹主动脉 Myointimal 增生的研究

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine ...

Construction of a Human Aorta Smooth Muscle Cell Organ-On-A-Chip Model for Recapitulating Biomechanical Strain in the Aortic Wall

Conventional two-dimensional cell culture techniques and animal models have been used in the study of human thoracic aortic aneurysm and dissection (TAAD). However, human TAAD sometimes cannot be characterized by animal models. There is an apparent species gap between clinical human studies and animal experiments that may hinder the discovery of therapeutic drugs. In contrast, the conventional cell culture model is unable to simulate in vivo biomechanical stimuli. To this end, microfabrication and microfluidic techniques have developed greatly in recent years, providing novel techniques for establishing organoids-on-a-chip models that replicate the biomechanical microenvironment. In this study, a human aorta smooth muscle cell organ-on-a-chip (HASMC-OOC) model was developed to simulate the pathophysiological parameters of aortic biomechanics, including the amplitude and frequency of cyclic strain experienced by human aortic smooth muscle cells (HASMCs) that play a vital role in TAAD. In this model, the morphology of HASMCs became elongated in shape, aligned perpendicularly to the strain direction, and presented a more contractile phenotype under strain conditions than under static conventional conditions. This was consistent with the cell orientation and phenotype in native human aortic walls. Additionally, using bicuspid aortic valve-related TAAD (BAV-TAAD) and tricuspid aortic valve-related TAAD (TAV-TAAD) patient-derived primary HASMCs, we established BAV-TAAD and TAV-TAAD disease models, which replicate HASMC characteristics in TAAD. The HASMC-OOC model provides a novel in vitro platform that is complementary to animal models for further exploring the pathogenesis of TAAD and discovering therapeutic targets.

Here, we developed a human aorta smooth muscle cell organ-on-a-chip model to replicate the in vivo biomechanical strain of smooth muscle cells in the human aortic wall.

构建人主动脉平滑肌细胞器官芯片模型，用于概括主动脉壁中的生物力学应变

Conventional two-dimensional cell culture techniques and animal models have been used in the study of human thoracic aortic aneurysm and dissection ...

生物工程

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of &#946;-aminopropionitrile-induced Aortic Aneurysm and Dissection

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of aortic disease exist, in vivo imaging of the vasculature has been limited. In recent years, micro-computerized tomography (micro-CT) has emerged as a preferred modality for imaging both large and small vessels both in vivo and ex vivo. In conjunction with a method of vascular casting, we have successfully used micro-CT to characterize the frequency and distribution of aortic pathology in &#946;-aminopropionitrile-treated C57/Bl6 mice. Technical limitations of this method include variations in the quality of the perfusion introduced by poor animal preparation, the application of proper methodologies for vessel size quantification, and the non-survivability of this procedure. This article details a methodology for the intravascular perfusion of a lead-based radiopaque silicone rubber for the quantitative characterization of aortopathy in a mouse model of aneurysm and dissection. In addition to visualizing aortic pathology, this method may be used for examining other vascular beds in vivo or vascular beds removed post-mortem.

This article describes a detailed methodology of using a radiopaque lead-based silicone rubber to perfuse the murine vasculature for aortic diameter quantification in a mouse model of aortic aneurysm and dissection.

β-aminopropionitrile 诱发主动脉瘤和解剖小鼠模型 Aortopathy 的定量显微 CT 分析

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of ...

Murine Cervical Aortic Transplantation Model using a Modified Non-Suture Cuff Technique

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. However, only minor improvement in the long-term results of transplanted solid organs could be observed over the past decades. In this context, chronic allograft vasculopathy (CAV) still represents the leading cause of late organ failure in cardiac, renal and pulmonary transplantation.
Thus far, the underlying pathogenesis of CAV development remains unclear, explaining why effective treatment strategies are presently missing and emphasizing a need for relevant experimental models in order to study the underlying pathophysiology leading to CAV formation. The following protocol describes a murine heterotopic cervical aortic transplantation model using a modified non-suture cuff technique. In this technique, a segment of the thoracic aorta is interpositioned in the right common carotid artery. With the use of the non-suture cuff technique, an easy to learn and reproducible model can be established, minimizing the possible heterogeneity of sutured vascular micro anastomoses.

Here, we present a protocol of heterotopic aortic transplantation in mice using the non-suture cuff technique in a cervical murine model. This model can be used to study the underlying pathology of chronic allograft vasculopathy (CAV) and can help evaluate new therapeutic agents in order to prevent its formation.

使用改性非缝合铐技术的宫颈主动脉移植模型

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. ...

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

摘要

探索更多视频

此视频中的章节

使用体内和组织和细胞外植体方法研究胚胎和围产期主动脉的形态和发病机制

小鼠Spinotrapezius模型来评估的影响小动脉结扎术，微血管功能与重塑

颈动脉支架植入再狭窄的研究的小鼠模型

一种新的小鼠模型主动脉瘤腔内修复

重复性的动脉剥脱损伤腹主动脉阻断在小鼠模型

球囊损伤诱发小鼠腹主动脉 Myointimal 增生的研究

构建人主动脉平滑肌细胞器官芯片模型，用于概括主动脉壁中的生物力学应变

β-aminopropionitrile 诱发主动脉瘤和解剖小鼠模型 Aortopathy 的定量显微 CT 分析

使用改性非缝合铐技术的宫颈主动脉移植模型

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

小鼠主动脉夹层损伤:高效

摘要

探索更多视频

此视频中的章节

使用体内和组织和细胞外植体方法研究胚胎和围产期主动脉的形态和发病机制

小鼠Spinotrapezius模型来评估的影响小动脉结扎​​术，微血管功能与重塑

颈动脉支架植入再狭窄的研究的小鼠模型

一种新的小鼠模型主动脉瘤腔内修复

重复性的动脉剥脱损伤腹主动脉阻断在小鼠模型

球囊损伤诱发小鼠腹主动脉 Myointimal 增生的研究

构建人主动脉平滑肌细胞器官芯片模型，用于概括主动脉壁中的生物力学应变

β-aminopropionitrile 诱发主动脉瘤和解剖小鼠模型 Aortopathy 的定量显微 CT 分析

使用改性非缝合铐技术的宫颈主动脉移植模型

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

小鼠Spinotrapezius模型来评估的影响小动脉结扎术，微血管功能与重塑