Name: delayed intramyocardial delivery of stem cells after ischemia reperfusion injury in a murine model
Uploaded: 2020-09-03T13:00:00
Description: Watch this Scientific Journal Video about Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model at JoVE.com

实验对雌性C57BL/6小鼠进行，年龄为10-12周，体重为20-25克。所有动物程序均符合《实验室动物护理和使用指南》（美国国家科学院实验室动物资源研究所、美国医学博士所贝塞斯达）中规定的标准，并经梅奥诊所医学院机构动物护理和使用委员会（IACUC）批准。
1. 准备和插管
<ol><li>手术前将所有手术器械都去处理。如果要在一次手术中进行多次手术，请在每个动...

<ol>
	<li>Franchi, F., 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=The+Myocardial+Microenvironment+Modulates+the+Biology+of+Transplanted+Mesenchymal+Stem+Cells.">The Myocardial Microenvironment Modulates the Biology of Transplanted Mesenchymal Stem Cells.</a> Molecular Imaging Biology. , (2020).</li><li>Bergmann, O., 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=Evidence+for+cardiomyocyte+renewal+in+humans.">Evidence for cardiomyocyte renewal in humans.</a> Science. 324 (5923), 98-102 (2009).</li><li>Writing Group, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+Disease+and+Stroke+Statistics-2016+Update:+A+Report+From+the+American+Heart+Association.">Heart Disease and Stroke Statistics-2016 Update: A Report From the American Heart Association.</a> Circulation. 133 (4), 38 (2016).</li><li>Gersh, B. J., Simari, R. D., Behfar, A., Terzic, C. M., Terzic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+cell+repair+therapy:+a+clinical+perspective.">Cardiac cell repair therapy: a clinical perspective.</a> Mayo Clinic Protocol. 84 (10), 876-892 (2009).</li><li>Terzic, A., Behfar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+heart+failure+therapy+headed+for+optimization.">Regenerative heart failure therapy headed for optimization.</a> European Heart Journal. 35 (19), 1231-1234 (2014).</li><li>Beegle, 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=Hypoxic+preconditioning+of+mesenchymal+stromal+cells+induces+metabolic+changes,+enhances+survival,+and+promotes+cell+retention+in+vivo.">Hypoxic preconditioning of mesenchymal stromal cells induces metabolic changes, enhances survival, and promotes cell retention in vivo.</a> Stem Cells. 33 (6), 1818-1828 (2015).</li><li>Kubli, D. A., Gustafsson, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+and+mitophagy:+the+yin+and+yang+of+cell+death+control.">Mitochondria and mitophagy: the yin and yang of cell death control.</a> Circulation Research. 111 (9), 1208-1221 (2012).</li><li>Psaltis, P. 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=Noninvasive+monitoring+of+oxidative+stress+in+transplanted+mesenchymal+stromal+cells.">Noninvasive monitoring of oxidative stress in transplanted mesenchymal stromal cells.</a> JACC Cardiovascular Imaging. 6 (7), 795-802 (2013).</li><li>Peet, C., Ivetic, A., Bromage, D. I., Shah, A. 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全世界有8500多万人患有心血管疾病。这些缺血事件的高流行率值得进一步发展和扩大替代疗法，以促进受损组织的再生。传统方法利用缺血再灌注程序在急性环境中，随后给予治疗1。炎症反应处于3-4天之间的高峰期，在心脏缺血事件后，与嗜中性粒细胞，巨噬细胞，和增加细胞因子信号10，12,的渗透。在这一段死细?...

心血管疾病仍然是全世界发病率和死亡率的最常见原因。心脏缺血事件被发现有害于心肌和周围细胞1的整体功能。只有̴0.45-1.0%的心肌细胞在心肌损伤发生后每年再生。尽管不断增长的需求和固有的重点开发治疗，治疗帮助受伤组织再生已经很难建立，仍然需要进一步优化3，4，5。,4,5干细胞疗法已被引入，作为在缺血事件后恢复受损组织活力的替代途径;然而，这些疗法的进步已经受到有限的生存和保留细胞到受伤区域6的挑战。
缺血事件后心脏的微环境可以定性为缺氧、亲氧化和亲炎，为治疗干细胞适应生存7，8的敌对条件。由于免疫反应在受伤后触发，天真的淋巴细胞，巨噬细胞，嗜中性粒细胞和乳腺细胞试图通过去除垂死的细胞和调节组织重塑9，10，119,的过程来修复损伤。在缺血后的第一个3天内，炎症处于高峰期，在10，12区释放大量嗜中性粒细胞和单核细胞的亲炎细胞因子。7天后，大部分炎症已经消退，向修复细胞的过渡开始，一直持续到重塑级联完成，大约14天在小鼠13。我们的手术方法是将生物制剂引入心肌中，以绕过缺血再灌注损伤后的峰值先天免疫反应的一种潜在替代方法。同时，它将允许研究任何治疗在亚急性/慢性缺血的情况，其中可能有不同的变量要考虑相比，急性心肌梗死。

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			<td height="17" style="height:17px;width:322px;">0.9% NaCl Irrigation, USP</td>
			<td style="width:191px;">Baxter</td>
			<td style="width:132px;">0338-0048-04</td>
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			<td height="17" style="height:17px;">11x12&#34; Press n&#39; Seal surgical drape, autoclavable</td>
			<td>SAI Infusion Technologies</td>
			<td>PSS-SD</td>
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			<td height="17" style="height:17px;">24G 3/4&#34; IV catheter tube</td>
			<td>Jelco</td>
			<td>4053</td>
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			<td height="17" style="height:17px;">28G x 1/2&#34; 1mL allergy syringe</td>
			<td>BD</td>
			<td>305500</td>
			<td>Injection of analgesic</td>
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			<td height="17" style="height:17px;">30G x 1/2&#34; 3/10cc insulin syringe</td>
			<td>Ulticare</td>
			<td>08222.0933.56</td>
			<td>Injection of stem cells</td>
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			<td height="17" style="height:17px;">6-0 S-29, 12&#34; Vicryl suture</td>
			<td>Ethicon</td>
			<td>J556G</td>
			<td>Intercostal, superficial muscle and skin layer incision closure</td>
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			<td height="17" style="height:17px;">9-0 BV100-4, 5&#34; Ethilon suture</td>
			<td>Ethicon</td>
			<td>2829G</td>
			<td>Ligation of the LAD artery</td>
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			<td>Thermo Fischer Scientific</td>
			<td>14-206-64</td>
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			<td>Coviden</td>
			<td>6818</td>
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			<td>49235</td>
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			<td>Fine Science Tools</td>
			<td>14085-09</td>
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			<td height="17" style="height:17px;">Buprenorphine HCL SR LAB 1mg/ml, 5 ml</td>
			<td>ZooPharm Pharmacy</td>
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			<td>Buprenorphine narcotic analgesic formulated in a polymer that slows absorption extending duration of action (72 hours duration of activity).</td>
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			<td height="17" style="height:17px;">Castroviejo needle holders, curved</td>
			<td>Fine Science Tools</td>
			<td>12061-01</td>
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			<td height="17" style="height:17px;">Curity sterile gauze sponges</td>
			<td>Coviden</td>
			<td>397310</td>
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			<td height="17" style="height:17px;">Delicate suture tying forceps, 45 angle bent</td>
			<td>Fine Science Tools</td>
			<td>11063-07</td>
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			<td height="17" style="height:17px;">Electric Razor</td>
			<td>Wahl</td>
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			<td>Fur removal</td>
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			<td height="17" style="height:17px;">Isoflurane 100 ml</td>
			<td>Cardinal Health</td>
			<td>PI23238</td>
			<td>Anesthetic</td>
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			<td height="17" style="height:17px;">Lab coat</td>
			<td></td>
			<td></td>
			<td></td>
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			<td height="17" style="height:17px;">Monoject 1 mL hypodermic syringe</td>
			<td>Coviden</td>
			<td>8881501400</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;">Moria iris forceps, curved, serrated (x2)</td>
			<td>Fine Science Tools</td>
			<td>11370-31</td>
			<td></td>
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			<td height="17" style="height:17px;">Moria speculum retractor</td>
			<td>Fine Science Tools</td>
			<td>17370-53</td>
			<td></td>
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			<td height="17" style="height:17px;">Mouse endotracheal intubation kit</td>
			<td>Kent Scientific</td>
			<td></td>
			<td></td>
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			<td height="17" style="height:17px;">Nair depilatory cream</td>
			<td>Johnson &#38; Johnson</td>
			<td></td>
			<td>Fur removal</td>
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			<td height="17" style="height:17px;">Optixcare eye lube plus</td>
			<td>Aventix</td>
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			<td>Sterile ocular lubricant</td>
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			<td height="17" style="height:17px;">Physiosuite ventilator</td>
			<td>Kent Scientific</td>
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			<td height="17" style="height:17px;">PolyE Polyethylene tubing</td>
			<td>Harvard Apparatus</td>
			<td>72-0191</td>
			<td>Temporary compression of LAD artery</td>
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			<td height="17" style="height:17px;">Povidone-iodine swabs</td>
			<td>PDI</td>
			<td>S41125</td>
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			<td>Bard-Parker</td>
			<td>371610</td>
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			<td height="17" style="height:17px;">Sterile 3&#34; cotton tipped applicators</td>
			<td>Cardinal Health</td>
			<td>C15055-003</td>
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			<td>Puritan</td>
			<td>25-826-5WC</td>
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			<td>Cardinal Health</td>
			<td>N8830</td>
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			<td>Medline</td>
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			<td height="17" style="height:17px;">Surgical Microscope</td>
			<td>Leica</td>
			<td>M125</td>
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			<td height="17" style="height:17px;">Suture tying forceps, straight (x2)</td>
			<td>Fine Science Tools</td>
			<td>10825-10</td>
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			<td height="17" style="height:17px;">Transpore surgical tape</td>
			<td>3M</td>
			<td>1527-1</td>
			<td></td>
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			<td height="17" style="height:17px;">Triple antibiotic ointment</td>
			<td>G&#38;W Laboratories</td>
			<td>11-2683ILNC2</td>
			<td>Topical application to prevent infection</td>
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			<td height="17" style="height:17px;">Vannas-T&#252;bingen Spring Scissors, curved</td>
			<td>Fine Science Tools</td>
			<td>15004-08</td>
			<td></td>
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		<tr height="17">
			<td height="17" style="height:17px;">Vetflo vaporizer</td>
			<td>Kent Scientific</td>
			<td></td>
			<td></td>
		</tr>
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delayed intramyocardial delivery of stem cells after ischemia reperfusion injury in a murine model

人们对于使用干细胞（SCs）恢复心肌损伤个体的心脏功能有着浓厚的兴趣。最常见的是，心脏干细胞治疗是通过将SC同时与心肌损伤诱导同时提供来研究。然而，这种方法存在两个显著限制：早期敌对的亲炎缺血环境可能会影响移植的SC的生存，并且它并不代表可能使用SC的亚急性梗塞情景。在这里，我们描述了一个两部分的手术程序系列，用于诱导缺血-再灌注损伤和传递中质干细胞（MSCs）。这种干细胞管理方法可以通过规避最初的免疫反应，使受损组织周围的生存能力和保留时间更长。在小鼠中诱导了缺血再灌注损伤的模型，并伴之以间质干细胞（3.0 x 105），稳定地表达着记者基因萤火虫荧光酶在构成表达CMV促进剂下，7天后在心内。通过超声波和生物发光成像分别对动物进行成像，以确认损伤和细胞注射。重要的是，在执行此两程序方法进行 SC 交付时，没有增加的并发症率。这种干细胞施用方法，与利用最先进的报告基因一起，可能允许在临床上常见的慢性缺血情况下，在体内研究移植的SC的生存能力和保留，同时规避最初的亲炎反应。总之，我们建立了一个协议，延迟将干细胞传递到心肌，这可以用来作为促进受损组织再生的潜在新方法。

第0天，小鼠诱发缺血再灌注损伤，在干细胞植入前一天进行术后超声心动图和心电图。超声波和心电图分析证实梗死和心室收缩功能下降（图1A-D）。进一步检查数据显示，接受缺血性损伤的小鼠的弹射分数和分数缩短率降低，而末体舒张和收缩量增加（表1）。与正常小鼠心脏（图2A）相比，马森三色染色心肌组织7天后受伤（...

Watch this Scientific Journal Video about Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model at JoVE.com

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

干细胞作为心肌损伤患者的潜在治疗方法不断被研究，然而，干细胞在损伤组织中生存能力的降低和保留会影响其长期疗效。在这份手稿中，我们描述了一种替代干细胞传递方法，该模型采用缺血再灌注损伤的穆林模型。

在穆林模型中缺血再灌注损伤后干细胞的心肌内传递延迟

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, ...

缺血性损伤诱发一种影响移植细胞生存的敌对的亲炎环境。我们的协议提供了一种替代方法，通过规避对损伤的初始免疫反应来提供生物制剂。该协议的主要优点是，它使治疗治疗后一个星期受伤，模仿治疗在临床环境。我们主要关注的是缺血再灌注损伤模型和STEM细胞的移植。然而，这种方法也可用于涉及引入生物疗法的炎症性疾病模型。首先，对所有的手术器械进行高压灭。如果要在一次手术中进行多次手术，请在每个动物之后清洁仪器，并使用热珠消毒器重新消毒。皮下管理镇痛剂，称量动物，并输入呼吸机的重量。将胸部左侧从胸骨剃到肩部水平，并涂抹脱毛霜以去除多余的毛皮。为了防止肺衰竭，保持正端呼气压力在两厘米的水为缺血再灌注程序，并更改为三厘米的水延迟注射细胞程序。麻醉小鼠后，用20测量的气管管插管，将其转移到受控加热垫上，以保持35至37摄氏度的体温。将鼠标放在横向卧式，左端为颅端，右侧为颅端。在碘和酒精拭子之间交替擦洗手术区域三次，将眼科软膏涂抹到两只眼睛上。使用数字 10 刀片手术刀在视野中最左侧的奶嘴右侧 2.5 毫米的垂直切口，然后使用剪刀切割表面肌肉层，直到肉间肌肉和肋骨可见。抬起肋骨和周围组织时，切穿第四排和第五根肋骨之间的间空间，然后将眼睑缩回器插入开放空间。使用弯曲钳子收回百分层，将肺向上移动，并离开视图。通过LAD动脉下方2.5毫米的肌钙通过9-0尼龙缝合线，保持平稳和一致的运动，以防止组织撕裂或刺穿主要血管。系上一个松动的方结，然后把一厘米的聚乙烯管放在松动的结内。固定管子周围的缝合，通过苍白和心室心律失常确认缺血，然后在35分钟后释放。取出管子后，等待五分钟，确认重新灌注的心肌。将 24 表 IV 导管管放入开口右侧的胸腔中。一个间腔空间。使用 6-0 可吸收缝合线以简单的中断图案关闭成本间切口，以连续缝合模式关闭肌肉层。关闭表面肌肉层后，用一毫升块茎注射器从胸腔中抽出空气，取出胸管。以连续水平床垫图案的 6-0 可吸收缝合线关闭皮肤切口。完成后，施用1.5毫升的暖盐水皮，并在切口部位涂抹三重抗生素软膏，以防止感染。将鼠标转移到无床上用品的笼子或带盖被窝的笼子，放在温暖的垫子上，温度为 35 至 37 摄氏度，直到完全恢复。在准备和插管鼠标之前描述后，用剪刀和钳子从皮肤层上取下缝合线，然后使用数字 10 手术刀在与上一个手术相同的位置进行切口。继续切开疤痕组织，直到肌肉层缝合可见，然后用剪刀和钳子去除缝合并切开肌肉层。取出将肋骨放在一起的缝合线，继续从上一个切口中切开科斯特间肌肉。将眼睑缩回器放入成本间空间，并找到上一个结扎区域。将悬浮在20微升PBS中的30万个中质STEM细胞加载到30量量的胰岛素注射器中，并稍微弯曲针头，以达到正确的注射角度。从顶点向心脏底部移动，将注射器插入近外侧区域，直到针头完全进入心肌，然后缓慢地将细胞注入心肌，等待三秒钟并取出针头。仔细观察心脏三分钟，确保对细胞没有异常反应，如心室颤动。三分钟后，将一个24表IV导管插入胸腔，在开口右侧的一个间腔空间。关闭成本间肌肉和皮肤层，并删除胸管，如前所述。以皮下管理1.5毫升温暖的盐水，并在切口部位涂抹三重抗生素软膏，以防止感染。将鼠标转移到无床上用品的笼子或带盖的床上用品的笼子，直到完全恢复。术后超声心动图和心电图在STEM细胞植入当天进行，确认梗塞和降低心室收缩功能。进一步检查数据显示，接受缺血性损伤的小鼠的弹射分数和分数缩短率降低，而末体舒张和收缩量增加。心肌组织七天后的马森三铬染色显示，接受缺血损伤的小鼠心脏的胶原蛋白沉积增加，左心室壁变薄。在体内生物发光成像或BLI在中生期STEM细胞植入后的第二天进行。与引起缺血再灌注损伤但未接受MSCs的小鼠相比，BLI信号的成功传递就是例证。在尝试此协议时，请记住，在切穿成本间、连接血管和注射细胞时，小故意运动对于防止在视野中对心脏或肺部造成不当损害至关重要。这些程序提供通过超声波检查后监测心脏功能的能力，通过生物发光成像或根据所用疗法进一步采用分子或生化技术来监测移植后的心脏功能。

Research

JoVE Journal

Medicine

Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury

常温心脏骤停和心肺复苏术：缺血再灌注损伤的小鼠模型

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused ...

科研

医学

A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion

闭胸鼠心肌缺血再灌注模型

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

急性肾损伤和伤后纤维化小鼠缺血再灌注模型

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

Intramyocardial Cell Delivery: Observations in Murine Hearts

心肌内细胞交货：观察在小鼠心脏

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue ...

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

心肌缺血再灌注损伤的通过左前降支结扎小鼠模型

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

Murine Model of Intestinal Ischemia-reperfusion Injury

肠缺血再灌注损伤的小鼠模型

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure

视网膜缺血再灌注损伤的小鼠模型通过眼压升高

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, ...

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

大鼠心肌缺血再灌注后冠状动脉流动储备评价

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

重建显微外科缺血再灌注损伤研究的临床前大鼠模型

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

Tracking Superparamagnetic Iron Oxide-labeled Mesenchymal Stem Cells using MRI after Intranasal Delivery in a Traumatic Brain Injury Murine Model

跟踪超副磁性氧化铁标记的美感血干细胞使用MRI后，创伤性脑损伤Murine模型内伤分娩

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical ...