这项研究由韩国教育部资助的韩国国家研究基金会（NRF-2018R1D1A1A02049346）基础科学研究计划支持。

所有动物研究程序都是根据《实验室动物福利法》、《实验室动物照料和使用指南》以及韩国天主教大学医学院机构动物护理和使用委员会（IACUC）提供的啮齿动物实验指南和政策提供的。
1. MSC 和注射明胶水凝胶的准备
<ol><li>100 毫米培养皿中的培养 MSC，37 °C 和 5% CO2。当 MSC 增长达到 80% 的汇合时，用 DPBS 洗两次菜，并在 37 °C 下加入 1 mL 的三蛋白替代剂，持续 3 分钟。 注:MSC在常规程序16后从骨髓中分离出，在杜尔贝科的改良鹰介质（DMEM）中培养，含有10%的胎儿牛血清（FBS）和1%的抗生素-抗霉菌溶液，并在7-u20129通道之间用于这项研究。</li>
	<li>加入9 mL的文化介质和离心机在500 x g 3分钟。接下来，丢弃由此产生的超常剂，在PBS的1mL中重新悬浮细胞，并在冰上保持细胞悬浮。</li>
	<li>用 10 μL 的 Trypan 蓝色稀释 10 μL 的细胞悬架，并使用自动电池计数器获得细胞浓度。</li>
	<li>以 1 x 107 细胞/mL 的密度将 MSC 重新悬浮并传输到 1 mL 管中。</li>
	<li>在PBS中准备 6.25 wt% 的 GH 结合解决方案，并分离成 2 瓶。接下来，将 GH 解决方案与 HRP （GH 解决方案 A） 的 6 微克/mL 或 H2O 2（GH 溶液 B） 的 0.07 wt% 混合。 注:根据已公布的协议12、15，准备明胶-羟基丙烯酸（GH）结合。<ol>
<li>将GH结合溶液与HRP（GH溶液A）的容积比保持在9:1，将GH结合溶液分别保持在H 2 O2（GH溶液B）。</li>
	</ol>
</li>
	<li>在将 MSC 与 GH 溶液 A 混合之前，将电池悬架短暂离心至 1，000 x g， 并小心地吸气由此产生的超纳坦。随后，将含有MSC的颗粒与GH溶液A混合。</li>
</ol>2. 原位MSC加载和三维体外培养
<ol><li>将GH溶液A（包含MSC）和GH溶液B加载到双注射器的两侧。板 300μL 的组合 GH 解决方案与 MSC 在最终密度为 5 x 106 细胞/mL 到八井室幻灯片。</li>
	<li>通过酶交叉连接进行原位水凝胶形成和随后的MSC封装后，加入含有10%FBS和1%抗生素-抗霉溶液的700μL DMEM。</li>
	<li>在 37 °C 和 5% CO2 下孵化幻灯片，每 2+u20123 天更换一次培养介质。</li>
</ol>3. 确认GH水凝胶内MSC的体外增殖和生存
<ol><li>要确定 GH 水凝胶中 3D 培养的 MSC 的可行性，请在预定的潜伏时间后使用活/死细胞染色检测。</li>
	<li>在 GH 水凝胶中孵化封装的 MSC 3、5、7 或 14 天后，吸气介质，用 PBS 两次清洗油井。</li>
	<li>在 10 mL 的 DPBS 中准备含有 5 μL 钙蛋白 AM 和 20 μL 同质-1 （EthD-1） 的染色溶液。</li>
	<li>将 200μL 的染色溶液添加到井中，并在室温下在黑暗中孵育 30 分钟。</li>
	<li>吸气染色溶液，用PBS洗两次井。</li>
	<li>小心地将腔室与滑梯分开，并在 GH 水凝胶上放置一个完整的盖片。使用共生显微镜可视化封装 MSC 的增殖程度和形态变化。 注:荧光图像是在 200 倍放大倍数下获得的，图像的激发/发射波长为 470/540 nm 用于钙素，EthD-1 获得 516/607 nm。</li>
</ol>4. 小鼠心肌梗塞的诱导
<ol><li>麻醉7周大的雄性C57BL/6小鼠（20+u201222g），在盐水中注射Zoletil（30毫克/千克）和龙朋（10毫克/千克）的混合物。</li>
	<li>手术前，使用脱毛霜去除小鼠胸部，用碘对皮肤进行消毒。</li>
	<li>将鼠标放在手术台上，通过将导管插入气管以通过机械通风提供补充氧气进行插管。</li>
	<li>用手术剪刀轻轻切开皮肤，然后用微剪刀穿透间切肌肉。使用 5-0 丝缝将第二根和第三根左肋分开，以保持胸腔开放。</li>
	<li>使用 8 - 0 的针架小心地解放左前下降 （LAD） 冠状动脉聚丙烯缝合，并使用电宫切割缝合。</li>
	<li>观察前左心室壁的即时颜色变化。</li>
</ol>5. MSC装载GH水凝胶的心肌内移植
<ol><li>在通过 LAD 结扎诱导心肌梗塞后，使用配备 26G 针头的双注射器将 10μL 的 MSC 加载 GH 溶液注入到梗塞边界区的两个不同点（总计:2 x10 5 MSC/20 μL）。<ol>
<li>按照步骤 1 中描述的相同程序，准备并将 MSC 加载 GH 解决方案转移到双注射器中。 注:为了评估在梗塞区域内雕刻的MSC装载GH水凝胶，MSC和GH结合体分别用PHK26和荧光异氰酸酯（FITC）预先标记。</li>
	</ol>
</li>
	<li>恢复开放的胸腔，并关闭肌肉和皮肤使用5-0缝合。 注:在关闭胸部之前，使用导管注射器取出空气。</li>
	<li>取出气管，在恢复过程中将鼠标放在红外灯下的笼子里。</li>
	<li>对于术后镇痛，至少72小时进行皮下酮洛芬注射（每天5毫克/千克）。所有小鼠都应在适当时间受到密切监测，以确保手术后恢复良好，并进行适当的疼痛治疗。</li>
</ol>6. 回声心动图
<ol><li>移植后四周，先用5%的异氟化麻醉小鼠，然后将异氟浓度调整到1%。</li>
	<li>使用脱毛霜脱毛胸部，并将鼠标放在加热垫上。将超声波传感器凝胶涂在胸前。</li>
	<li>获取二维寄生短轴视图，并在肌水平记录 M 模式跟踪。 注:将线性阵列传感器（7+u201215 MHz）放在左副星线中，查看解剖结构。</li>
	<li>测量LVAW、LVID和LVPW的相应线条，以获得心脏壁厚度、腔室尺寸和部分缩短。 注:比较心脏功能，包括弹出分数（ EF ）、分数缩短（ FS ）和肌水平的末代收缩体积（ ESV ），以确保在同一解剖位置进行适当的评估。</li>
</ol>7. 病理学评价
<ol><li>在将MSC装载的GH水凝胶移植到梗塞的心脏后预定时间，在CO2室中对小鼠实施安乐死，并收集心脏进行组织学分析15。</li>
	<li>对于血氧素和欧辛（H&E）和马森的三叶虫（MT）染色，修复解剖的心脏组织在4%的甲醛（PFA），并嵌入石蜡。接下来，根据标准协议17，将石蜡嵌入的心脏块切割成4个μm串行部分，并用微切除物和MT污渍染色。</li>
	<li>以 20 倍的放大率在幻灯片扫描仪上获取图像，并计算治疗组的梗死大小。 梗死大小（%）=总梗塞周长/总LV周长x 100</li>
	<li>按中线长度测量计算两个周长。对于LV中线周长，测量心内膜和震中表面之间的中线长度。对于中线梗塞周长，测量梗塞的长度，包括超过50%的肌心18的整体厚度。 注:所有图像分析都是使用 ImageJ 软件进行的。</li>
	<li>测量肌肉水平疤痕的壁厚度。</li>
	<li>计算胶原蛋白面积的分数。 胶原蛋白面积 （%） = 间质纤维化/肌细胞面积总面积 x 100</li>
</ol>

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作者对这部作品没有利益冲突。

可注射的GH水凝胶具有巨大的潜力，在体内应用，因为他们的能力，均匀地纳入不同的治疗剂就地。此外，它们的物理和生化特性很容易根据疾病依赖性要求进行操纵。在这方面，已建议注射水凝胶，以解决目前心脏干细胞治疗的主要局限性，阻碍不良生存和细胞保留（即+10%在24小时内移植后）在受伤的心脏19，20。为了克服这一不良结果，本文所述的?...

心脏干细胞治疗已成为心肌修复和再生的潜在方法1，2。尽管最近动物模型和临床试验取得了积极成果，但由于在梗塞的心脏组织3、4的留存率低和注射细胞存活率低，干细胞治疗心肌修复的应用有限。因此，对细胞组织工程的使用进行了深入的研究，包括注射生物材料5、心脏贴片6和细胞片7，以改善宿主心肌内的细胞保留和整合。
在生物工程心脏组织修复的各种潜在方法中，注射式水凝胶与适当的细胞类型相结合，如间质干细胞（MSCs）、胚胎干细胞（ESCs）和诱导多能干细胞（iPSCs），是有效将细胞输送到心肌区域8、9的一个有吸引力的选择。明胶是一种众所周知的天然聚合物，与用于生物医学应用的各种生物材料相比，由于其巨大的生物相容性、可显著的生物降解性和免疫原性降低，可以用作注射基质。虽然基于明胶的注射平台有很大的潜力，但其在体内的适用性仍然有限，因为其机械刚度低，在生理环境中容易降解。
为了克服这些局限性，提出了一种由羟基丙酸组成的明胶基水凝胶的全新简单设计，用于体内应用。明胶-羟基丙烯酸（GH）结合物可在酶、马萝卜过氧化酶（HRP）存在的情况下就地交叉连接，随后在水凝胶内封装各种药物、生物分子或细胞，表明在组织工程应用10、11、12、13、14方面具有巨大潜力。此外，我们最近调查了含有封装MSC的GH水凝胶的治疗效果，并证明它们在MI后在Murine模型15中成功用于心脏修复和再生。在此协议中，我们描述了在 GH 水凝胶内封装和体外三维 （3D） 增殖的简单技术。我们还引入了一个外科手术，旨在通过冠状动脉结结和MSC加载GH水凝胶的心内移植到梗塞的心脏产生一个Murine MI模型。

<table>
	<tbody>
		<tr>
			<td>4 % paraformaldehyde (PFA)</td>
			<td>Intron</td>
			<td>IBS-BP031-2</td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 silk suture</td>
			<td>AILEE</td>
			<td>SK534</td>
			<td></td>
		</tr>
		<tr>
			<td>8-0 polypropylene suture</td>
			<td>ETHICON</td>
			<td>M8732H</td>
			<td></td>
		</tr>
		<tr>
			<td>8-well chamber slide</td>
			<td>Nunc LAB-TEK</td>
			<td>154534</td>
			<td></td>
		</tr>
		<tr>
			<td>Angiocath Plus (22GA) catheter</td>
			<td>BD Angiocath Plus</td>
			<td>REF382423</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-antimyocotic</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>GYROGEN</td>
			<td>1582MGR</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM 510</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slipe</td>
			<td>MARIENFELD</td>
			<td>101242</td>
			<td></td>
		</tr>
		<tr>
			<td>Deluxe High Temperature Cautery kit</td>
			<td>Bovie</td>
			<td>QTY1</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11995-065</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14040-133</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual-syringe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EOSIN</td>
			<td>SIGMA-ALDRICH</td>
			<td>HT110116</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>EMSURE</td>
			<td>K49350783 739</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Fechtner conjunctiva forceps titanium</td>
			<td>WORLD PRECISISON INSTRUMENTS</td>
			<td>WP1820</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate isomer I (FITC)</td>
			<td>SIGMA-ALDRICH</td>
			<td>F7250</td>
			<td></td>
		</tr>
		<tr>
			<td>Forcep</td>
			<td>HEBU</td>
			<td>HB0458</td>
			<td></td>
		</tr>
		<tr>
			<td>Hair removal cream</td>
			<td>Ildong Pharmaceutical</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Stoelting</td>
			<td>50300</td>
			<td>Homeothermic Blanket System</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>50301</td>
			<td>Replacement Heating Pad for 50300 (10 X 12.5cm)</td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>SIGMA-ALDRICH</td>
			<td>HHS80</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish peroxide (HRP; 250-330 U/mg)</td>
			<td>SIGMA-ALDRICH</td>
			<td>P8375</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide (H2O2; 30 wt % in H2O)</td>
			<td>SIGMA-ALDRICH</td>
			<td>216763</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodine</td>
			<td>Green Pharmaceutical</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD cell staining kit</td>
			<td>Thermo Fisher</td>
			<td>R37601</td>
			<td></td>
		</tr>
		<tr>
			<td>Mechanical ventilator</td>
			<td>Harvard Apparatus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro centrifuge</td>
			<td>HANIL</td>
			<td>Micro 12</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro needle holder</td>
			<td>KASCO</td>
			<td>37-1452</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro scissor</td>
			<td>HEBU</td>
			<td>HB7381</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>OLYMPUS</td>
			<td>SZ61</td>
			<td></td>
		</tr>
		<tr>
			<td>MT staining kit</td>
			<td>SIGMA-ALDRICH</td>
			<td>HT1079-1SET</td>
			<td>Weigert&#8217;s iron hematoxylin solution</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>HT15-1KT</td>
			<td>Trichrome Stain (Masson) Kit</td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>LK LABKOREA</td>
			<td>H06-660-107</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS buffer</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>PHK26 staining kit</td>
			<td>SIGMA-ALDRICH</td>
			<td>MINI26</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide scanner</td>
			<td>Leica</td>
			<td>SCN400</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissor</td>
			<td>HEBU</td>
			<td>HB7454</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical tape</td>
			<td>3M micopore</td>
			<td>1530-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue cassette</td>
			<td>Scilab Korea</td>
			<td>Cas3003</td>
			<td></td>
		</tr>
		<tr>
			<td>Transducer gel</td>
			<td>SUNGHEUNG</td>
			<td>SH102</td>
			<td></td>
		</tr>
		<tr>
			<td>Trout-Barraquer needle holder curved</td>
			<td>KASCO</td>
			<td>50-3710c</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound system</td>
			<td>Philips</td>
			<td>Affiniti 50</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>JUNSEI</td>
			<td>25175-0430</td>
			<td></td>
		</tr>
	</tbody>
</table>

intramyocardial transplantation of msc-loading injectable hydrogels after myocardial infarction in a murine model

目前用于预防后发性心力衰竭的心脏干细胞疗法面临的主要问题之一是受伤心肌内移植细胞的保留率和存活率低，限制了其治疗效果。最近，脚手架生物材料的使用已引起人们的关注，以改善和最大限度地提高干细胞治疗。该协议的目的是引入一种简单明了的技术，使用注射羟基丙酸（GH）水凝胶移植骨髓源性间质干细胞（MSCs）:水凝胶是心脏组织工程应用的细胞输送平台，具有现场交叉连接和高生物相容性的能力。我们提出了一个简单的方法来制造MSC加载GH水凝胶（MSC/水凝胶），并评估其生存和增殖的三维（3D）体外培养。此外，我们演示了在小鼠体内移植MSC/水凝胶的技术，描述了通过左前下降（LAD）冠状动脉结结和随后的MSC/水凝胶移植诱导心肌梗塞（MI）的外科手术。

为了有效地将 MSC 输送到梗死的心肌，本协议中使用了 图 1 中描述的 MSC 现场装载的可交叉链接水凝胶。在体内移植之前，GH水凝胶中MSC的增殖和存活通过3D体外活/死细胞染色检测（活:绿色;死:红色）证实。如图 2所示，具有代表性的图像显示了足够的 MSC 增殖，显示了 GH 水凝胶内的分支网络。此外，在第14天清楚地观察到MSC广泛的多细胞3D结构，表明GH?...

Watch this Scientific Journal Video about Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model at JoVE.com

Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

干细胞治疗已成为修复心肌梗塞后受伤的心脏组织的有效策略。我们使用明胶水凝胶为干细胞移植提供最佳的体内应用，这些水凝胶能够酶交叉连接。

Murine 模型心肌梗塞后 MSC 加载注射水凝胶的心内移植

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of ...

intramyocardial-transplantation-msc-loading-injectable-hydrogels

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

4.3K 次观看.  The Catholic University of Korea. 干细胞治疗已成为修复心肌梗塞后受伤的心脏组织的有效策略。我们使用明胶水凝胶为干细胞移植提供最佳的体内应用，这些水凝胶能够酶交叉连接。

视频: Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

Acute Myocardial Infarction in Rats

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular diseases. Animal models that mimic human cardiac disease, such as myocardial infarction (MI) and ischemia-reperfusion (IR) that induces heart failure as well as pressure-overload (transverse aortic constriction) that induces cardiac hypertrophy and heart failure (Goldman and Tarnavski), are useful models to study cardiovascular disease. In particular, myocardial ischemia (MI) is a leading cause for cardiovascular morbidity and mortality despite controlling certain risk factors such as arteriosclerosis and treatments via surgical intervention (Thygesen). Furthermore, an acute loss of the myocardium following myocardial ischemia (MI) results in increased loading conditions that induces ventricular remodeling of the infarcted border zone and the remote non-infarcted myocardium. Myocyte apoptosis, necrosis and the resultant increased hemodynamic load activate multiple biochemical intracellular signaling that initiates LV dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. This pathological remodeling and failure to normalize the increased wall stresses results in progressive dilatation, recruitment of the border zone myocardium into the scar, and eventually deterioration in myocardial contractile function (i.e. heart failure). The progression of LV dysfunction and heart failure in rats is similar to that observed in patients who sustain a large myocardial infarction, survive and subsequently develops heart failure (Goldman). The acute myocardial infarction (AMI) model in rats has been used to mimic human cardiovascular disease; specifically used to study cardiac signaling mechanisms associated with heart failure as well as to assess the contribution of therapeutic strategies for the treatment of heart failure. The method described in this report is the rat model of acute myocardial infarction (AMI). This model is also referred to as an acute ischemic cardiomyopathy or ischemia followed by reperfusion (IR); which is induced by an acute 30-minute period of ischemia by ligation of the left anterior descending artery (LAD) followed by reperfusion of the tissue by releasing the LAD ligation (Vasilyev and McConnell). This protocol will focus on assessment of the infarct size and the area-at-risk (AAR) by Evan's blue dye and triphenyl tetrazolium chloride (TTC) following 4-hours of reperfusion; additional comments toward the evaluation of cardiac function and remodeling by modifying the duration of reperfusion, is also presented. Overall, this AMI rat animal model is useful for studying the consequence of a myocardial infarction on cardiac pathophysiological and physiological function.

The rat model of acute myocardial infarction (AMI) is useful to study the consequence of a MI on cardiac pathophysiological and physiological function.

大鼠急性心肌梗死

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular ...

科研

医学

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

结扎冠状动脉内注射在小鼠模型梗死

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

心肌梗死和功能预后评估的猪

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of treatments in vivo1. The traditional, established protocol has been further fine-tuned to minimize the damage to the animal. Notably, the pectoral muscle layers are teased away rather than simply cut, and the thoracotomy is approached intercostally as opposed to breaking the ribs in a sternotomy, preserving the integrity of the ribcage. With these changes, the overall stress on the animal is decreased. 
Stem cell therapies aimed to alleviate the damage caused by MIs have shown promise over the years for their pro-angiogenic and anti-apoptotic benefits. Current approaches of delivering cells to the heart surface typically involve the injection of the cells either near the damaged site, within a coronary artery, or into the peripheral blood stream2-4. While the cells have proven to home to the damaged myocardium, functionality is limited by their poor engraftment at the site of injury, resulting in diffusion into the blood stream5. This manuscript highlights a procedure that overcomes this obstacle with the use of a cell-encapsulated hydrogel patch. The patch is fabricated prior to the surgical procedure and is placed on the injured myocardium immediately following the occlusion of the left coronary artery. To adhere the patch in place, biocompatible external fibrin glue is placed directly on top of the patch, allowing for it to dry to both the patch and the heart surface. This approach provides a novel adhesion method for the application of a delicate cell-encapsulating therapeutic construct.

This protocol aims to alleviate the limitation of poor cell engraftment for stem cell treatment of myocardial infarctions through the use of a hydrogel system and a fibrin-based glue. With this approach, cell-to-tissue contact post-infarction can be maintained, increasing the therapeutic potential of beneficial agents at the site of injury.

水凝胶构造和纤维蛋白基胶方法来提供治疗的小鼠心肌梗死模型。

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of ...

Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are therefore required to confine cardiac damage, promote survival and reduce the disease burden of heart failure. Large animal experiments are an essential part in the translational process from experimental to clinical therapies. To optimize clinical translation, robust and representative outcome measures are mandatory. The present manuscript aims to address this need by describing the assessment of three clinically relevant outcome modalities in a pig acute myocardial infarction (AMI) model: infarct size in relation to area at risk (IS/AAR) staining, 3-dimensional transesophageal echocardiography (TEE) and admittance-based pressure-volume (PV) loops. Infarct size is the main determinant driving the transition from AMI to heart failure and can be quantified by IS/AAR staining. Echocardiography is a reliable and robust tool in the assessment of global and regional cardiac function in clinical cardiology. Here, a method for three-dimensional transesophageal echocardiography (3D-TEE) in pigs is provided. Extensive insight into cardiac performance can be obtained by admittance-based pressure-volume (PV) loops, including intrinsic parameters of myocardial function that are pre- and afterload independent. Combined with a clinically feasible experimental study protocol, these outcome measures provide researchers with essential information to determine whether novel therapeutic strategies could yield promising targets for future testing in clinical studies.

Reliable and accurate outcome assessment is the key for translation of preclinical therapies into clinical treatment. The current paper describes how to assess three clinically relevant primary outcome parameters of cardiac performance and damage in a pig acute myocardial infarction model.

急性心肌梗死的猪模型主要成果评估

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

心肌梗死新生小鼠，心肌再生的模型

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

使用左前部降冠动脉的永久结扎的月心肌梗死模型

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we demonstrate a murine cryoinjury infarct model that generates precise infarct sizes with high reproducibility and replicability. In brief, after intubation and sternotomy of the animal, the heart is lifted from the thorax. The probe of a handheld liquid nitrogen delivery system is applied onto the myocardial wall to induce cryoinjury. Impaired ventricular function and electrical conduction can be monitored with echocardiography or optical mapping. Transmural myocardial remodeling of the infarcted area is characterized by collagen deposition and loss of cardiomyocytes. Compared to other models (e.g., LAD-ligation), this model utilizes a handheld liquid nitrogen delivery system to generate more uniform infarct sizes.

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

研究小鼠心肌梗死的低温损伤模型

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...

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

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

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, ...

A Modified Simple Method for Induction of Myocardial Infarction in Mice

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI remodeling and evaluation of novel therapeutics. Different methods (e.g., isoproterenol treatment, cryoinjury, coronary artery ligation, etc.) have been used to induce MI. Compared with isoproterenol treatment and cryoinjury, coronary artery ligation may better reflect the ischemic response and chronic remodeling after MI. However, traditional methods for coronary ligation in mice are technically challenging. The current study describes a simple and efficient process for induction of MI in mice with readily available materials. The mouse chest skin was cut open under stable anesthesia. The heart was immediately externalized through the intercostal space after blunt separation of the pectoralis major and pectoralis minor. The left anterior descending branch (LAD) was ligated with a 6-0 suture 3 mm from its origin. Following LAD ligation, staining with 2,3,5-Triphenyltetrazolium chloride (TTC) indicated successful induction of MI and temporal changes of post-MI scar size. Meanwhile, survival analysis results showed overt mortality within 7 days after MI, mainly due to cardiac rupture. Moreover, post-MI echocardiographic assessment demonstrated successful induction of contractile dysfunction and ventricular remodeling. Once mastered, an MI model can be established in mice within 2-3 min with readily available materials.

Under adequate anesthesia, the mouse heart was externalized through the intercostal space, and myocardial infarction was successfully induced by ligating the left anterior descending artery (LAD) using materials readily available in most laboratories.

一种改良的小鼠心肌梗死诱导简易方法

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI ...