我想感谢提供资金，以支持他们的警惕和援助我的研究和比较医学部的研究和研究生部。我也想生理学教研室的支持和指导以及在我的实验室技术人员和学生认识到他们的帮助。最后，我要感谢我的博士后导师，博士查尔斯E.默里，培训机会，在此期间，我学到了小鼠显微。

<ol><li>无菌手术器械（表1）和3“棉倾斜涂药放在无菌underpad珠灭菌器（Germinator 500）打开。 </li><li>小鼠（年龄：6个星期; WT：&gt;18克）20μL/ g的tribromoethanol BW（250毫克;持续时间 - 约40分钟）腹腔注射麻醉。 </li><li>脱毛（例如奈尔），当老鼠的反应迟钝脚趾捏，无菌润滑剂（泪续签）是适用于眼睛干燥和胸部的左侧，以保护他们的是涂有从皮肤移除的皮毛。 </li><li>脱毛是温暖的自来水和优碘/酒精擦拭是用来消毒手术区冲走。 </li><li>鼠标被放置在一个温暖的deltaphase温垫是固定在一个有机玻璃表。每个肢体固定用胶带和水平厚厚的线程被放置在牙顶举行的地方上颚。 </li><li>该表是垂直放置和纤维的光线是直接照耀到颈部食管照明。这就需要开放的咽喉，是一个光线充足的孔口的精确位置等，从而使气管，可视化，以方便插入PE管材。 </li><li>管理恒定的正压通气（TOPO的呼吸;率125次/分;高峰吸气压力10-12 CMH 2; *注油管是连接到呼吸机（连接到95％O 2 / 5％的CO 2 ）设置与应变和性别1-3而异）。一旦确认同步胸部运动，通风，连接固定用胶带垫，以避免在手术拔管。 </li><li>使用齿钳拉皮肤远离胸部，10＃无菌手术刀刀片连接到＃3手术刀的处理是用来在皮肤平行的胸骨1.5厘米切口。 </li><li>弯曲万纳microscissors用于切割胸大肌肌肉，使肋间肌的小孔。 </li><li>直，钝microscissors用于切割通过3肋骨。 </li><li>采用9mm小儿眼科镜是用于收回的左肋。 </li><li>用弯钳，拉离心脏和心包用有齿镊轻轻撕裂它打开。 </li><li>下方的左前降冠状动脉前（沿心脏的长轴）缝合8-0聚乙烯使用的Castroviejo持针器，采用6mm锥形点3 / 8针线程垂直于它。 <ol><li>定时再灌注，可用于删除一个临时结扎，消毒0.5 - 1CM PE90片是对心脏冠状动脉平行。缝合，首先根据冠状动脉循环，然后绑在油管。当时它被释放，放松结扎。这可以反复进行，根据需要和闭塞/再闭塞的时间可以修改4。根据协议和使用麻醉类型的长度，补充可能是必要的。 </li><li>一个永久性闭塞，股价下冠状动脉结扎是简单的并列。漂白和运动障碍是显而易见的，长的缝合结束削减5-10。 </li><li>对于内注射（S），汉密尔顿30号无菌针头斜面无菌注射器引入上述损伤结扎右侧区域的核心基地。到损伤区，然后将针先进撤回轻微斜角可以约在边境区。一些在注射器（2 - 3μl）溶液注入心针是在地方举行。撤回另1 - 3mm的注射器和解决方案的其余部分被注入。注射器举行，直至形成的解决方案是由气泡消散。然后取出针。如果有任何出血，棉签轻轻压入针插入部位，直到出血停止 5-7 。 </li></ol></li><li>一旦心肌操作完成后，肋骨拉钩将被删除，使用6-0 surgipro缝合2-3床垫缝线关闭胸腔。 </li><li>两三个床垫缝合，然后关闭胸大肌肌肉，0.25％marcaine 1:10在无菌生理盐水（0.1ml/25g鼠标）1-3滴，适用于肌肉，然后2-3床垫缝线作出关闭的皮肤。 </li><li>鼠标是从呼吸中删除。一旦有节奏，快速，呼吸浅验证，鼠标就可以拔管。 </li><li> 0.5毫升温暖的无菌生理盐水注入背侧皮下空间和鼠标放置在一个笼子里的变暖垫，直到它恢复流动性（1小时最低）。 </li><li>生存实验中，小鼠被放置到他们的笼子，直到牺牲时返回到动植物公园。在第2天，蘸食品放在笼地板上，以方便饲养（所以他们没有达到这可能会导致疼痛）和丁丙诺啡，应每6 - 12小时。手术后的护理，还包括验证充足的流动性，疏导和饮食习惯的第一个星期的日常监测。 </li><li>手术器械用乙醇擦拭干净，并插入珠未来手术前灭菌。 </li><li>牺牲时，小鼠的戊巴比妥钠（65mg/ml; 55-65毫克/公斤）麻醉。当达到足够的麻醉平面，打开胸腔。 </li><li>虽然心脏还在跳动，注射器与含冷氯化钾的23号针（氯化钾，30MM）或2,3 - 丁二酮肟（BDM; 10MM）用于穿刺后脑室底部区域和解决方案慢慢注入室，直到心脏在舒张被捕。 </li><li>心一旦被删除，使用注射器含PBS逆行灌注冲洗，以消除任何血液，仍然心。对于急性研究，在再灌注期的结束，法援署重新结扎在闭塞的原始点。主动脉注入含有1％的埃文蓝色，是一个解决方案。一旦心脏中提取，这是横向切割同等厚度分为3个部分，在1％2,3,5 -三苯基氯化孵育，和11个形态分析成像。对于慢性的研究，心是那么沉浸在固定液，然后按常规程序处理和嵌入式。幻灯片可以染色组织学和形态分析（使用接穗，美国国立卫生研究院图像J或图像临加）9,10,12成像。 </li></ol>代表性的成果： 当操作正确，在小鼠的存活率（男：年龄8-10个星期，22 - 28G，女性年龄在10至12周，20 - 26G）：超过90％的急性缺血/再灌注和缺血预处理实验，超过永久动脉结扎术的研究，85％和心肌内注射约80％。由于早期损伤是更容易代谢的变化，而不是结构，在缺血/再灌注心肌梗死大小的决心和缺血预处理实验执行注入到的主动脉这将灌注心，是不是由法援署提供1％的埃文的蓝色染料可见（图1A）。一旦心脏被删除，横向切一半，组织培养在1％的2,3,5 - 三苯基氯化测量梗死面积（图1B）的解决方案。该地区使用接穗或美国国立卫生研究院图像处理软件可以使用在相同的放大倍率成像千分尺进行校准。这些数字是用来计算风险/左心室心肌梗死面积/风险11区的区。应变的差异可能会导致体重和心脏的大小，所以应采取这些措施规范化，心脏重量，体重，或胫骨的长度为便于比较的变化。 常驻动脉结扎总值的结构性变化，如坏死，管壁变薄，腔扩张。治疗和/或梗死面积和左心室，室面积，室间隔和左室游离壁的厚度在永久性闭塞模型（图2A）坏死相对时间的影响比较，也可以使用接穗或美国国立卫生研究院成像测量软件。 picrosirius红/快绿（图2B）胶原染色可以用来衡量insterstitial纤维化与功能指数壁变硬8-10。图3中的形象代表6ul溶液注射到心脏的边境地带，永久动脉结扎后（Evan的蓝色）的分布。请注意，它伤害的方向进行，以及向基地也transmurally。 <img src="/files/ftp_upload/2581/2581fig1.jpg" alt="图1" /> 图1。 A.埃文的蓝色注入主动脉前切除 ，此图像显示了心（染色）和闭塞的区 ​​域（未染色）灌注地区 。B.埃文的蓝和TTC染色急性缺血/再灌注损伤。这是一个形象代表（20倍），显示的蓝色染料染色unoccluded地区以及代谢可行的组织TTC染色（红色）的分布。坏死区不染色，所以他们仍然苍白（概述）。 <img src="/files/ftp_upload/2581/2581fig2.jpg" alt="图2" /> 图2。 A.苏木精和伊红染色，这是一个形象代表的H＆E染色削减横在4天的梗死区域的心肌梗死后（20倍）通过一个小鼠心脏（20X）。 *为组织坏死，箭头指向肉芽组织，RV =右心室和LV =左心室。 Picrosirius红色和绿色快速染色，这是一个picrosirius红/快速绿色染色心肌梗死后4周的小鼠心脏截面的形象代表（20X）。细胞质内渍绿色和胶原纤维是红色的。 <img src="/files/ftp_upload/2581/2581fig3.jpg" alt="图3" /> 图3。埃文的蓝色染料染色分布6ul内注射。显示全球的埃文蓝色染料和透心整个6ul在边境地带内注射后立即结扎冠状动脉（12X）的分布，这是一个形象代表。

这种动物协议的批准和机构在东卡罗来纳大学的动物护理和使用委员会提出的准则和法规的规定。

冠心病仍然是一个流行病学和财政重大的公共卫生问题。大量的基础研究仍然需要了解的机制，其中损伤和重塑进行潜在疗法可以调节这些过程，如果他们将被开发为临床使用。鼠害是最常用的和广泛的转基因小鼠可使得这个物种更具吸引力的模式。 虽然有老鼠和其他物种之间的差异，也有一个小鼠模型的诸多优点。使用一个简单的解剖范围或放大镜，光线充足的条件下，使血管容易看出（详细的大体解剖，血管， 看到萨尔托Tellez等，2004年13）。为了减少手术后的死亡率的风险，这是非常重要，以避免切断大血管，因为一个25克鼠标的总血量低于2毫升14。在事件发生出血过多，温柔的压力或查明烧灼的应用可用于止血。 此过程也可以在多种方式进行修改。例如，老鼠可以使用异氟醚，氯胺酮/甲苯噻嗪，或戊巴比妥钠和适当的选择是由确定的协议的期限15-18的麻醉。脚趾捏反射是最常用的麻醉深度的指数。此外，为了改善长期生存的可能性，一些研究人员使用抗心律失常药物，如利多卡因，以减少致命性心律失常 19,20然而，它必须考虑到这最近已被证明具有抗细胞凋亡在急性属性的发生率模型21。此外，为减少手术后的疼痛，止痛药，包括丁丙诺啡可管理的第一个48小时的手术后 3,16,17,22,23 。为了保持身体在手术过程中的温度（尤其是较长的协议），在加热垫系列直肠探头通常用于恒温垫。对于缺血/再灌注和/或缺血前或后处理：可以改变闭塞（S）和再灌注（S）的持续时间;永久性闭塞，梗死面积可能通过调整结扎的位置修改;心肌内注射（如细胞，蛋白质等），可以有1-3注射地点和每次注射量可高达15μL24 。如果细胞被注入，用于衡量针（通常26-30）5,25,26选择应根据细胞的大小，使针的内部直径足够大，以避免sheering。为了避免混淆由于手术引发的炎症过程，一些研究人员报告说，使用一个圈套，操纵体内闭塞和reperfuse在一个封闭的胸部鼠标的心，在任何手术后27-29点。最近，高30 等。表明，可以进行临时和永久性闭塞不通风的需要，一些实验室已经开始使用超声波来执行封闭胸部心肌内注射 25,31 。 由于由约翰和奥尔森发表于1954年32的第一项研究中，证明在小鼠冠状动脉结扎的可行性，许多人都采用这种模式，并修改它来研究各方面的心肌损伤和重塑3,33-45。小鼠的大小，生殖能力，并购买和维护费用相对较少方面的性质，使这一物种的生理和病理生理研究的广泛吸引力的工具。由于在体内的进步46-49成像的小型化技术，以及执行和分析大型基因组学和蛋白组学，药物筛选，以及基于细胞和/或蛋白质疗法的疗效的生物材料50-64，结合心肌梗死的小鼠模型的范围越来越广，无处不在的或组织的特定的基因或突变体/基因敲除小鼠所提供的遗传操作，无疑将继续在评估急性心肌损伤和长期重塑的宝贵工具。因此，有不容置疑的价值能够可靠和可重复执行这些实验。

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		<th>Catalogue Number</th>
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<tr>
<td>Long Vanna Scissors</td>
<td>&nbsp;</td>
<td>George Tiemann</td>
<td>160-159</td>
<td>&nbsp;</td>
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<td>Micro Dissecting Scissors</td>
<td>&nbsp;</td>
<td>George Tiemann</td>
<td>160-161</td>
<td>&nbsp;</td>
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<td>Forceps &#x2013; straight, 1x2 teeth</td>
<td>&nbsp;</td>
<td>George Tiemann</td>
<td>105-205</td>
<td>&nbsp;</td>
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<td>Scalpel handle #3</td>
<td>&nbsp;</td>
<td>George Tiemann</td>
<td>105-64</td>
<td>#10 sterile blade</td>
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<td>Forceps &#x2013; half curved serrated</td>
<td>&nbsp;</td>
<td>George Tiemann</td>
<td>160-19</td>
<td>&nbsp;</td>
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<td>Tissue Scissors</td>
<td>&nbsp;</td>
<td>George Tiemann</td>
<td>105-410</td>
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<td>Miltex</td>
<td>18-1828</td>
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<td>Cook Eye Speculum</td>
<td>&nbsp;</td>
<td>Miltex</td>
<td>18-63</td>
<td>&nbsp;</td>
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<td>Surgipro II 8-0</td>
<td>&nbsp;</td>
<td>Suture Express</td>
<td>VP-900-X</td>
<td>&nbsp;</td>
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<td>Prolene 6-0</td>
<td>&nbsp;</td>
<td>Suture Express</td>
<td>8776</td>
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<td>Germinator 500 Bead Sterilizer</td>
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<td>Cellpoint Scientific</td>
<td>65369-1</td>
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<td>Deltaphase isothermal pad</td>
<td>&nbsp;</td>
<td>Braintree Scientific</td>
<td>39DP</td>
<td>&nbsp;</td>
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<td>Hamilton syringe - 25&mu;l</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>80430</td>
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<td>30 gauge beveled needle</td>
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<td>Hamilton</td>
<td>7803-07</td>
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<td>Ventilator</td>
<td>&nbsp;</td>
<td>Kent Scientific</td>
<td>TOPO</td>
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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.

Watch this Scientific Journal Video about Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction at JoVE.com

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

众多的遗传操作和/或心肌内注射基因，蛋白质，细胞，和/或生物材料叠加后的时间在急性缺血/再灌注损伤和小鼠慢性重塑的研究维度。该视频演示了缺血/再灌注损伤的显微外科程序，永久结扎冠状动脉内注射研究。

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 ...

coronary-artery-ligation-intramyocardial-injection-murine-model

Research

JoVE Journal

Medicine

51.7K 次观看.  East Carolina University. 众多的遗传操作和/或心肌内注射基因，蛋白质，细胞，和/或生物材料叠加后的时间在急性缺血/再灌注损伤和小鼠慢性重塑的研究维度。该视频演示了缺血/再灌注损伤的显微外科程序，永久结扎冠状动脉内注射研究。

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

Modified Technique for Coronary Artery Ligation in Mice

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI we need better knowledge about pathophysiology of myocardial ischemia. This knowledge may be valuable to define new therapeutic targets for innovative cardiovascular therapies4. Experimental MI model in mice is an increasingly popular small-animal model in preclinical research in which MI is induced by means of permanent or temporary ligation of left coronary artery (LCA)5. In this video, we describe the step-by-step method of how to induce experimental MI in mice.
The animal is first anesthetized with 2% isoflurane. The unconscious mouse is then intubated and connected to a ventilator for artificial ventilation. The left chest is shaved and 1.5 cm incision along mid-axillary line is made in the skin. The left pectoralis major muscle is bluntly dissociated until the ribs are exposed. The muscle layers are pulled aside and fixed with an eyelid-retractor. After these preparations, left thoracotomy is performed between the third and fourth ribs in order to visualize the anterior surface of the heart and left lung. The proximal segment of LCA artery is then ligated with a 7-0 ethilon suture which typically induces an infarct size ~40% of left ventricle. At the end, the chest is closed and the animals receive postoperative analgesia (Temgesic, 0.3 mg/50 ml, ip). The animals are kept in a warm cage until spontaneous recovery.

The surgical procedure used to induce experimental myocardial infarction in mice begins with left thoracotomy between the third and the fourth ribs in order to visualize the anterior surface of the heart and left lung. The left coronary artery is ligated, the chest is closed and the mouse is allowed to recover spontaneously.

改性技术在小鼠冠状动脉结扎

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI ...

科研

医学

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

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

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 ...

Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It is characterized by fluid retention, shortness of breath, and fatigue, in particular on exertion. Heart failure is a growing public health problem, the leading cause of hospitalization, and a major cause of mortality. Ischemic heart disease is the main cause of heart failure.
Ventricular remodelling refers to changes in structure, size, and shape of the left ventricle. This architectural remodelling of the left ventricle is induced by injury (e.g., myocardial infarction), by pressure overload (e.g., systemic arterial hypertension or aortic stenosis), or by volume overload. Since ventricular remodelling affects wall stress, it has a profound impact on cardiac function and on the development of heart failure. A model of permanent ligation of the left anterior descending coronary artery in mice is used to investigate ventricular remodelling and cardiac function post-myocardial infarction. This model is fundamentally different in terms of objectives and pathophysiological relevance compared to the model of transient ligation of the left anterior descending coronary artery. In this latter model of ischemia/reperfusion injury, the initial extent of the infarct may be modulated by factors that affect myocardial salvage following reperfusion. In contrast, the infarct area at 24 hr after permanent ligation of the left anterior descending coronary artery is fixed. Cardiac function in this model will be affected by 1) the process of infarct expansion, infarct healing, and scar formation; and 2) the concomitant development of left ventricular dilatation, cardiac hypertrophy, and ventricular remodelling.
Besides the model of permanent ligation of the left anterior descending coronary artery, the technique of invasive hemodynamic measurements in mice is presented in detail.

Heart failure is the leading cause of hospitalization and a major cause of mortality. A model of permanent ligation of the left anterior descending coronary artery in mice is applied to investigate ventricular remodelling and cardiac dysfunction post-myocardial infarction. The technique of invasive hemodynamic measurements in mice is presented.

左冠状动脉前降支的小鼠结扎：对心肌梗死后重塑和心衰模型

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

冠脉流量和冠状动脉血流储备用小鼠压力超负荷基于模型的超声评估

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

Murine Left Anterior Descending (LAD) Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction

Ischemic heart disease (IHD), or acute coronary syndrome (ACS), is one of the leading causes of death in the United States. IHD is characterized by reduced blood supply to the heart, resulting in the loss of oxygen to and the ensuing necrosis of the heart muscle. The MI model has gained popularity for its use as a short-term ischemia-reperfusion model and a long-term permanent ligation model. Below, we describe a reliable method for the permanent ligation of the LAD. With mouse genetic engineering technology becoming more advanced, and with an increasing availability of quality murine surgical instruments, the mouse has become a popular model for MI surgeries. Our surgical model incorporates the use of an easily reversible anesthetic for the rapid recovery of the mouse; a minimally invasive endotracheal intubation without involving a tracheotomy; and a thoracentesis through the original thoracotomy site without creating an additional incision in the chest, as is done in some other methods, to effectively remove excess blood and air from the chest cavity. This method is comparatively less invasive than other methods, which dramatically reduces surgical and post-surgical complications and mortality and improves reproducibility.

Murine Left Anterior Descending LAD Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction

We provide a reliable method for left anterior descending artery (LAD) ligation in a mouse model. This method is comparatively less invasive than other methods, involving endotracheal intubation, a left-sided thoracotomy approach, and thoracentesis. This method can be used as a model for both acute and chronic myocardial infarction (MI).

鼠左前降支（LAD）冠状动脉结扎：改进和简化模型的心肌梗死

Ischemic heart disease (IHD), or acute coronary syndrome (ACS), is one of the leading causes of death in the United States. IHD is characterized by ...

Percutaneous Contrast Echocardiography-guided Intramyocardial Injection and Cell Delivery in a Large Preclinical Model

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection fraction (HFrEF). Before they can be considered for use, and implemented in humans, extensive preclinical studies are required in large animal models to evaluate the safety, efficacy, and fate of the injectate (e.g., stem cells) once delivered into the myocardium. Small rodent models offer advantages (e.g., cost effectiveness, amenability for genetic manipulation); however, given inherent limitations of these models, the findings in these rarely translate into the clinic. Conversely, large animal models such as rabbits, have advantages (e.g., similar cardiac electrophysiology compared to humans and other large animals), whilst retaining a good cost-effective balance. Here, we demonstrate how to perform a percutaneous contrast echocardiography-guided intramyocardial injection (IMI) technique, which is minimally invasive, safe, well tolerated, and very effective in the targeted delivery of injectates, including cells, into several locations within the myocardium of a rabbit model. For the implementation of this technique, we also have taken advantage of a widely available clinical echocardiography system. After putting in practice the protocol described here, a researcher with basic ultrasound knowledge will become competent in the performance of this versatile and minimally invasive technique for routine use in experiments, aimed at hypothesis testing of the capabilities of cardiac regenerative therapeutics in the rabbit model. Once competency is achieved, the whole procedure can be performed within 25 min after anaesthetizing the rabbit.

Novel therapeutic strategies in cardiac regenerative medicine require extensive and detailed studies in large preclinical animal models before they can be considered for use in humans. Here, we demonstrate a percutaneous contrast echocardiography-guided intramyocardial injection technique in rabbits, which is valuable for hypothesis testing the efficacy of such novel therapies.

经皮超声心动图引导心肌注射液与大鼠前体细胞的临床研究

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors 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 ...

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, ...

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

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of transplanted cells within the injured myocardium, limiting their therapeutic efficacy. Recently, the use of scaffolding biomaterials has gained attention for improving and maximizing stem cell therapy. The objective of this protocol is to introduce a simple and straightforward technique to transplant bone marrow-derived mesenchymal stem cells (MSCs) using injectable hydroxyphenyl propionic acid (GH) hydrogels; the hydrogels are favorable as a cell delivery platform for cardiac tissue engineering applications due to their ability to be cross-linked in situ and high biocompatibility. We present a simple method to fabricate MSC-loading GH hydrogels (MSC/hydrogels) and evaluate their survival and proliferation in three-dimensional (3D) in vitro culture. In addition, we demonstrate a technique for intramyocardial transplantation of MSC/hydrogels in mice, describing a surgical procedure to induce myocardial infarction (MI) via left anterior descending (LAD) coronary artery ligation and subsequent MSC/hydrogels transplantation.

Stem cell-based therapy has emerged as an efficient strategy to repair injured cardiac tissues after myocardial infarction. We provide an optimal in vivo application for stem cell transplantation using gelatin hydrogels that are able to be enzymatically cross-linked.

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 ...