TWP was funded by the Irish Cardiac Society Brian McGovern Travelling Fellowship. WTP was funded by R01 HL095712 and an AHA Established Investigator Award.

根据批准的波士顿儿童医院的机构动物照顾及使用委员会的协议进行所有的描述的步骤。 1，准备工作<ol><li>开始注射方案之前进行超声心动图基线心脏解剖和功能评估，为心肌内注射的最佳固定传感器位置可能不会产生划定解剖和功能评估的最佳标准的意见。 </li><li>注入设置示于图1A-1C。放置带护套的针的空注射器，斜面朝上，在针筒夹持件（参见图1C），并固定超声换能器探头的扫描头夹紧装置（参见图1B）。松开扫描头夹紧球锁关节（ 图1B）和操作传感器的方向，使其平行于针的轴线。固定扫描拧紧扫描头夹球锁关节头位置。 注:对于注射成年小鼠中，地下30针用1 / 2.5厘米长是最优的。 1ml注射器可用于较大的体积，而以气密注射器可用于更精确地控制较小的体积（5-10微升）。 </li><li>自由地应用于超声凝胶至所述换能器头用刮刀以覆盖沿着其全部的头部。小心出鞘针并使用针安装控制前进直属换能器和用于可视化的超声凝胶内的针。使用针安装控制，使针清楚地显现沿其在超声波图像上的长度小的调整。如果所述换能器被适当地对准平行于针在步骤1.4中，然后将针应保持在成像平面内，因为它是前进和撤回与喷射控制旋钮（ 图1C）。 </li><li>在随后的步骤中，请勿打扰，需要通过移动或者在横轴LE /换能器的水平取向。相反，通过改变针的垂直（y轴）的位置安装并通过移动动物平台定位的心脏注射的特定区域。 </li><li>从使用扫描高度控制（ 图1B），以允许将麻醉小鼠随后放置到动物平台的动物平台移动所述换能器优。这不会破坏传感器的x轴对准到针的长轴。 </li><li>除去，是使用用于对准从针筒夹持并小心丢弃的注射器。加载新的针头和注射器与注射液的最终目标体积，从而允许在该注射器尖端死空间。请注意，以去除气泡。将注射器插入针筒夹持件，而无需调整其x轴对准。完全缩回使用喷射器上的注射器。 注:对于初次训练的目的，采用伊文思蓝的染料（1％），台盼蓝染色（0.4％）或荧光微球注射剂的悬浮液可以帮助运营商在确认能力和有针对性的注射成功。 </li></ol> 2，注射<ol><li>打开加热平台的集成暖和，并将其设置为37℃。将动物平台180°从通常的摄像方向，在麻醉的软管夹最靠近操作者的动物和头部。这使得心脏（在胸部的左侧）为同侧的针筒夹持件和针。注意:附加次要顺时针旋转调整动物的平台可能有必要正确地定向所述心脏对于将用于注射（ 图2A）胸骨旁短轴像平面。 </li><li>准备老鼠超声心动图如先前报道9。麻醉的小鼠中的感应室，用2％异氟醚。删除胸毛与脱毛膏的ð申请润滑凝胶到双眼，以防止巩膜的干燥。 </li><li>提高利用扫描头高度控制（ 图1B）的换能器。将麻醉的小鼠仰卧上盖的加热动物平台内的鼻锥体提供1-3％的异氟烷（ 图2A-2B）的口鼻部。轻轻插入直肠探头，并用胶带将四只爪子的心电电极，施加电极凝胶的电接触。 注:麻醉的适当水平，必须确保人道待遇的动物。应在心脏速率没有变化，并通过胸壁到针的放置没有响应。动物平台的集成稳态温度控制应该被用于维持正常体温（37℃±0.5℃），因为低温会导致相对的心动过缓，心室扩张，并且可能不适。 </li><li>一旦鼠标是动物平台上的安全，降低换能器到depila泰德胸部用扫描头高度控制（ 图1B）。最佳超声设置注射是对心脏进行可视化在胸骨旁短轴方向，按照标准的超声心动图技术。旋转动物平台20-30°顺时针旋转，以获得最佳的声窗口用于注射在短轴成像平面（ 图2A-2B）。注意:可替换地，喷射可以从胸骨旁长轴方向由动物平台（ 图2C）的逆时针旋转执行。 </li><li>使用动物平台调整控制以调整视场，并针对任何所需的注射部位在左心室心肌。潘回的往复从顶点到心脏的基部为目标的左心室心肌（ 图3A-3C）的所需注射位置。注:midpapillary胸骨旁短轴观（ 图3A）提供reproduciblê地标允许注射部位的后续成像。注意:可替换地，胸骨旁长轴视图可用于靶向预定的注射部位（ 图3D）。 </li><li>与在完全缩回针筒夹持的注射器开始，慢慢转动喷射控制顺时针方向（ 图4A）前进朝向动物的胸注射器。以使两者的心脏和针尖清晰超声可视化，因为它接近胸部，使用大量超声凝胶的胸部以上的左侧并通过在超声心动图的控制设置的宽视场优化声学窗口。在目标部位注射设定焦点/区。微调针安装控件可以优化沿其长度的指针的图像。 注意:某些超声波机器具有针导向的软件功能，通过与靶心肌以数字方式延伸沿针的长轴的线（ 图4B）。这样的软件工具可以是有益的，但​​不是必须的。 </li><li>与动物适当镇静剂（1-3％异氟醚混合0.5-0.8升/分钟100％氧气），通过胸壁的鼠标和进入心肌推进针头，仔细观察斜口针尖端的位置在所有次。停止行进时，针尖是目标心肌（ 图4C）内。整个斜尖应该是安全范围内的心肌，以避免注射液漏入心包腔。 </li><li>当尖端处于所需的位置，由推压注射器柱塞，提供注射剂。提供的注射液慢慢地，在5-30秒（取决于体积交付）。截至50微升注射液的可交付不影响心功能。瞬态echobright外观注入心肌区域可能会成功注射后明显。一个简短的（秒）期间相对bradycard的IA偶尔会注意到，注射到心肌并迅速解决。 </li><li>一旦注射液被给药，迅速拔出针头的注射控制旋钮逆时针旋转。鼠标应保持在麻醉下进行的超声心动图观察几分钟，确认保存心室功能并没有任何的术后并发症。如果指明，心肌的多个区域可以被串行地通过调节动物平台重新定位的方式将针的角度的注入。心肌内注射后，将小鼠放置在其自己笼中，并使其从麻醉下的观察来恢复。 </li><li>不要让动物无人看管，直到它已经恢复了足够的意识来维持胸骨斜卧。不返回已经历心肌内注射到其他动物的公司，直到完全恢复的动物。网箱应放在与真是个温控垫安以轩提供水和鼠标的饮食。操作后的不适并不预期干预失败后不久，适当地恢复正常的行为提出了一个潜在的并发症（见讨论）。 </li><li>小心地丢弃针头心肌内注射完成后，立即以减少锐器伤害到操作者或旁观者的风险。重复使用的针可导致针尖钝化使它更具挑战刺穿心肌和导致并发症的风险较高。 </li></ol>

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The authors have nothing to disclose.

生物可以通过血液输送到心肌的直接心肌内注射，心包内注射，或间接施用。在心肌梗死模型最近基于细胞的疗法试验已述的开胸术的方法来注射液12-14的输送。在心肌治疗干预的成功的重要因素取决于传递路线的选择。生物的最高局部剂量是通过心肌内递送15,16来实现的。心肌内注射在直视下是最直接的方法，并允许有针对性给药的心肌17内。然而，这种方法也是最侵入?...

心脏疾病是导致死亡的男性和女性在美国的首要原因，占每年60万人死亡1。心血管疾病的小鼠模型是研究病理生理机制，并探索潜在的治疗非常重要。心肌递送基因治疗载体，干细胞，修饰RNA，以及它们的治疗潜力的心脏疾病2-7其它治疗剂允许调查。目前，有用于在小鼠模型6心肌递送治疗剂的选择有限。心肌内注射在直视下被普遍使用，但需要胸骨切开或开胸并仅限于心脏的暴露区域。而方便的时候在另外一个实验操作，如法援署结扎时进行，需要进行侵入性手术的心肌内交付限制了潜在的实验设计，并引入了从过程dditional影响（ 例如 ，纤维化由于开胸手术）。经皮心包递送病毒载体已被报道，但在现场和治疗剂的分布是不均匀的，而且很难控制8。经皮冠状动脉注射导致注射的材料，但有效的和可重复的冠状动脉提供更多的均匀分布是具有挑战性的小鼠模型。 在这里，我们描述一个胸外心肌内注射技术，使微创，操作员控制的超声引导下治疗药物靶向。该技术是简单易学，避免了需要开胸或胸骨切开术和随之而来的实验性的并发症，并且提供了关于定时和心肌内注射的位点具有更大的灵活性。因此，超声心动图辅助心肌内注射代表了技术上的简单而高效的操纵方法心肌细胞在小鼠实验模型。

<table><tbody><tr><td>Vevo 2100 ultrasound imaging system&amp;nbsp;</td><td>Visualsonics</td><td></td><td></td></tr><tr><td>Vevo Integrated Rail System III</td><td>Visualsonics</td><td></td><td></td></tr><tr><td>Microscan MS400 transducer</td><td>Visualsonics</td><td></td><td></td></tr><tr><td>Microscan MS550D transducer</td><td>Visualsonics</td><td></td><td></td></tr><tr><td>PrecisionGlide needles</td><td>BD</td><td>305128</td><td>30 G x 1 in/2.5 cm</td></tr><tr><td>1 cc slip tip syringe</td><td>Exel International&amp;nbsp;</td><td>26048</td><td>or equivalent</td></tr><tr><td>Gastight 50 &amp;#956;l glass syringes</td><td>Hamilton</td><td>1705</td><td></td></tr><tr><td>Trypan blue stain (0.4%)</td><td>Gibco&amp;nbsp;</td><td>15250</td><td>or equivalent</td></tr><tr><td>Isoflurane</td><td>Baxter</td><td>AHN3640</td><td>or equivalent</td></tr><tr><td>Aquasonic 100</td><td>Parker Laboratories</td><td>(01-08)</td><td>or equivalent</td></tr><tr><td>Polystyrene microspheres (red fluorescent)</td><td>Life Technologies</td><td>F-8842</td><td>or equivalent</td></tr></tbody></table>

ultrasound-guided transthoracic intramyocardial injection in mice

心血管疾病的小鼠模型是研究病理生理机制，探索潜在的再生疗法很重要。累及心肌注射实验，通过开胸手术直接进入正在执行。同时方便在另一个实验操作时，如冠状动脉结扎时执行的，需要进行侵入性手术的心肌内交付限制了潜在的实验设计。随着不断提高超声的分辨率，先进的无创成像方式，现在是可行的执行例行超声引导下经皮内注射。这种方式有效，可靠地提供代理心肌的目标区域。这种技术的优点包括:避免手术并发症的，该设施为目标选择性地在超声引导下心肌的区域，并有机会提供注射液的myocardium在多个预定的时间间隔。随着实践的技术，从内注射的并发症是罕见的，老鼠迅速从麻醉恢复正常活动的恢复。按照本协议所列的步骤，基本经验的超声心动图的操作者可以快速在这种通用的，微创技术成为称职。

小鼠心肌内注射蓝色染料或荧光微球注射伊文思蓝染料是用于培训目的。一旦注射后，安乐死的小鼠并取出心脏以可视化的注入蓝色染料的位置， 图5示出了一个成功的注射液，例如，用蓝色染料渗入心肌在中期乳头肌水平（ 图5A，区域包围由虚线）。伊文思蓝染料将洗出组织的几个小时之内。为更持久的注射部位的标记，可使用该无限期地保留在注射...

Watch this Scientific Journal Video about Ultrasound-guided Transthoracic Intramyocardial Injection in Mice at JoVE.com

Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

超声引导经皮内注射表示一个高效，可靠和可靶向形式的递送基因转移剂或细胞进入小鼠心脏。按照本协议所列的步骤，操作者可以在这个多才多艺，微创技术迅速成为主管。

超声引导下经皮内注射小鼠

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. ...

ultrasound-guided-transthoracic-intramyocardial-injection-in-mice

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

14.8K 次观看.  Boston Children's Hospital. 超声引导经皮内注射表示一个高效，可靠和可靶向形式的递送基因转移剂或细胞进入小鼠心脏。按照本协议所列的步骤，操作者可以在这个多才多艺，微创技术迅速成为主管。 

视频: Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

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 ...

科研

医学

Intramyocardial Cell Delivery: Observations in Murine Hearts

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue revascularization and cell survival. In addition, further observations revealed that specific stem cells, such as cardiac stem cells, mesenchymal stem cells and cardiospheres have the ability to integrate within the surrounding myocardium by differentiating into cardiomyocytes, smooth muscle cells and endothelial cells.
Here, we present the materials and methods to reliably deliver noncontractile cells into the left ventricular wall of immunodepleted mice. The salient steps of this microsurgical procedure involve anesthesia and analgesia injection, intratracheal intubation, incision to open the chest and expose the heart and delivery of cells by a sterile 30-gauge needle and a precision microliter syringe.
Tissue processing consisting of heart harvesting, embedding, sectioning and histological staining showed that intramyocardial cell injection produced a small damage in the epicardial area, as well as in the ventricular wall. Noncontractile cells were retained into the myocardial wall of immunocompromised mice and were surrounded by a layer of fibrotic tissue, likely to protect from cardiac pressure and mechanical load.

Intramyocardial cell delivery in murine models of cardiovascular diseases, such as hypertension or myocardial infarction, is widely used to test the therapeutic potential of different cell types in regenerative studies. Therefore, a detailed description and a clear visualization of this surgical procedure will help to define the limits and advantages of cardiovascular cell therapeutic analyses in small rodents.

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 ...

Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 months. It is critical to have suitable animal models to mimic the hemodynamic spread of the metastatic cells in the clinical scenario. Here, we are introducing the use of small animal ultrasound imaging to guide an accurate injection of brain tropical breast cancer cells into the left ventricle of athymic nude mice. Longitudinal MRI is used to assessing intracranial initiation and growth of brain metastases. Ultrasound-guided intracardiac injection ensures not only an accurate injection and hereby a higher successful rate but also significantly decreased mortality rate, as compared to our previous manual procedure. In vivo high resolution MRI allows the visualization of hyperintense multifocal lesions, as small as 310 &#181;m in diameter on T2-weighted images at 3 weeks post injection. Follow-up MRI reveals intracranial tumor growth and increased number of metastases that distribute throughout the whole brain.

A breast cancer brain metastasis mouse model is established with ultrasound imaging-guided intracardiac injection of MDA-MB231/Br-GFP cells. Development of multifocal intracranial metastases has been monitored longitudinally using high-resolution 9.4 T MRI.

超声成像引导下腔内注射到患乳腺癌脑转移其次是纵向的MRI的小鼠模型

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 ...

Intramyocardial Injection for the Study of Cardiac Lymphatic Function in Zebrafish

Zebrafish have proved to be an important model for studying cardiovascular formation and function during postembryonic development and regeneration. The present protocol describes a method for injecting fluorescent tracers into the zebrafish myocardium to study interstitial fluid and debris uptake into cardiac lymphatic vessels. To do so, microspheres (200 nm diameter) and quantum dots (&#60;10 nm diameter) are introduced into the myocardium of live zebrafish, which can be tracked using ex vivo confocal microscopy. These tracers are then tracked intermittently over several hours to follow clearance from the myocardium into cardiac lymphatic vessels. Quantum dots&#160;are transported through cardiac lymphatic vessels away from the heart, while larger microspheres&#160;remain at the injection site for over three weeks. This method of intramyocardial injection can be extended to other uses, including the injection of encapsulated MS or hydrogels to locally release cells, proteins, or compounds of interest to a targeted region of the heart.

The present protocol allows efficient and stable delivery of fluorescent microspheres (MS) and quantum dots (QD) into the myocardium of live fish that can be tracked (traced) over time.

Zebrafish have proved to be an important model for studying cardiovascular formation and function during postembryonic development and regeneration. The ...

发育生物学

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have immunosuppressive potential and have been shown to suppress inflammation in mouse models of intestinal inflammation, even though the route of administration can limit their homing and effectiveness 1,3,4,5. Local application of MSCs to colonic injury models has shown greater efficacy at ameliorating inflammation in the colon. However, there is paucity of data on techniques to enhance the localization of human bone marrow-derived MSCs (hMSCs) to the small intestine, the site of inflammation in the SAMP-1/YitFc (SAMP) model of experimental Crohn&#39;s disease. This work describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in SAMP mice, a well-characterized spontaneous model of chronic intestinal inflammation. Sex- and age-matched, inflammation-free AKR/J (AKR) mice were used as controls. To analyze the biodistribution and the localization, hMSCs were transduced with a lentivirus containing a triple reporter. The triple reporter consisted of firefly luciferase (fl), for bioluminescent imaging; monomeric red fluorescent protein (mrfp), for cell sorting; and truncated herpes simplex virus thymidine kinase (ttk), for positron emission tomography (PET) imaging. The results of this study show that 24 h after the intracardiac administration, hMSCs localize in the small intestine of SAMP mice as opposed to inflammation-free AKR mice. This novel, ultrasound-guided injection of hMSCs in the left ventricle of SAMP mice ensures a high success rate of cell delivery, allowing for the rapid recovery of mice with minimal morbidity and mortality. This technique could be a useful method for the enhanced localization of MSCs in other models of small-intestinal inflammation, such as TNF&#916;RE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Murine studies in models of colonic inflammation have demonstrated that a small percentage (1 - 5%) of mesenchymal stem cells (MSC) injected intravenously or intraperitoneally home to the inflamed colon1,2. This study shows that ultrasound-guided intracardiac injections of MSCs result in increased localization to the intestine.

超声引导下人骨髓间充质干细胞在实验性肠炎小鼠模型中的应用

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have ...

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 ...

A Minimally Invasive, Accurate, and Efficient Technique for Intrathymic Injection in Mice

Intrathymic injection in mouse models is an important technique for studying thymic and immune function, including genetic and acquired T cell disorders. This requires methods for the direct deposition of reagents and/or cells into the thymus of living mice. Traditional methods of intrathymic injection include thoracic surgery or minimally invasive percutaneous blind injections, both of which have significant limitations. Ultra-high frequency ultrasound imaging devices have made image-guided percutaneous injections possible in mice, greatly improving the injection accuracy of the percutaneous injection approach and enabling the injection of smaller targets. However, image-guided injections rely on the utilization of an integrated rail system, making this a rigid and time-consuming procedure. A unique, safe, and efficient method for percutaneous intrathymic injections in mice is presented here, eliminating reliance on the rail system for injections. The technique relies on using a high-resolution micro-ultrasound unit to image the mouse thymus noninvasively. Using a free-hand technique, a radiologist can place a needle tip directly into the mouse thymus under sonographic guidance. Mice are cleaned and anesthetized before imaging. For an experienced radiologist adept at ultrasound-guided procedures, the learning period for the stated technique is quite short, typically within one session. The method has a low morbidity and mortality rate for the mice and is much faster than current mechanically assisted techniques for percutaneous injection. It allows the investigator to efficiently perform precise and reliable percutaneous injections of thymuses of any size (including very small organs such as the thymus of aged or immunodeficient mice) with minimal stress on the animal. This method enables the injection of individual lobes if desired and facilitates large-scale experiments due to the time-saving nature of the procedure.

The present protocol describes an interventional radiology procedure established for intrathymic injection in mice to avoid the risk of open surgery and improve the accuracy of blind percutaneous injections.

一种微创、准确、高效的小鼠胸腺内注射技术

Intrathymic injection in mouse models is an important technique for studying thymic and immune function, including genetic and acquired T cell disorders. ...

免疫与感染

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 ...

Ultrasound-Guided Induced Pluripotent Stem Cell-Derived Cardiomyocyte Implantation in Myocardial Infarcted Mice

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a promising cell source for cardiac repair after ischemic damage. However, a low grafted rate is a significant obstacle for effective cardiac tissue regeneration after transplantation. This protocol shows that multiple hiPSC-CM ultrasound-guided percutaneous injections into an MI area effectively increase cell transplantation rates. The study also describes the entire hiPSC-CM culture process, pretreatment, and ultrasound-guided percutaneous delivery methods. In addition, the use of human mitochondrial DNA help detect the absence of hiPSC-CMs in other mouse organs. Lastly, this paper describes the changes in cardiac function, angiogenesis, cell size, and apoptosis at the infarcted border zone in mice 4 weeks after cell delivery. It can be concluded that echocardiography-guided percutaneous injection of the left ventricular myocardium is a feasible, relatively invasive, satisfactory, repeatable, and effective cellular therapy.

Ultrasound-guided cell delivery around the site of myocardial infarction in mice is a safe, effective, and convenient way of cell transplantation.

超声引导诱导多能干细胞来源心肌细胞植入心肌梗死小鼠

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem ...

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. In vivo cardiac-specific gene delivery is commonly achieved by either systemic or local delivery. Systemic injection via tail vein is easy and efficient in manipulating cardiac gene expression by using recombinant adeno-associated virus 9 (AAV9). However, this method requires a relatively high amount of vector for efficient transduction, and may result in nontarget organ gene transduction. Here, we describe a simple, efficient, and time-saving method of intramyocardial injection for in vivo cardiac-specific gene manipulation in mice. Under anesthesia (without ventilation), the pectoral major and minor muscles were bluntly dissected, and the mouse heart was quickly exposed by manual externalization through a small incision at the fourth intercostal space. Subsequently, adenovirus encoding luciferase (Luc) and vitamin D receptor (VDR), or short hairpin RNA (shRNA) targeting VDR, was injected with a Hamilton syringe into the myocardium. Subsequent in vivo imaging demonstrated that luciferase was successfully overexpressed specifically in the heart. Moreover, Western blot analysis confirmed the successful overexpression or silencing of VDR in the mouse heart. Once mastered, this technique can be used for gene manipulation, as well as injection of cells or other materials such as nanogels in the mouse heart.