作者感谢希拉 Monfort, 布伦达. 马丁内斯, 卡洛斯 Micó, 阿尔贝托ñ oz 和曼努埃尔-莫林在收集数据时提供了出色的支持, 而卡洛斯布埃诺提供了 EGFP (+) HEK-293 细胞。这项工作得到了部分支持: 基金会 Séneca、75,000 de Ciencia Tecnología、西班牙穆尔西亚 Región (JT) (赠款号: 11935/PI/09);红 de Terapia 细胞, ISCIII-潜艇gral.Redes, VI PN i + D + i 2008-2011 (格兰特号。RD12/0019/0001) (JMM), 由欧洲联盟 (菲德) (JMM) 的结构筹资共同出资;并且, 英国的读书大学 (AG, GB) (中央资助)。资助者在研究设计、数据收集和分析、决定出版或准备原稿方面没有作用。

本文所述的实验得到了西班牙穆尔西亚大学伦理研究委员会的批准, 并按照欧洲委员会 2010/63/欧盟的指令进行。所描述的步骤是在标准操作协议下执行的, 这些程序是工作计划的一部分, 没有仅为拍摄所附视频而执行。
1. 细胞和哺乳动物表达载体的制备
注意: 在这里, 我们简要描述了一个细胞系的制备和转染的协议 (人类胚胎肾脏 293 (HEK-293));但是, 应优化单元格类型的适当的特定单元格协议 (例如、干细胞)。
<ol><li>维持 HEK-293 细胞在高葡萄糖 Dulbecco 的改良鹰的培养基 (DMEM) 中补充10% 胎小牛血清 (FCS), 1% 丙酮酸钠, 2 毫米谷氨酰胺, 1% 青霉素/链霉素, 并在37° c 的湿润气氛中孵育, 含 5% CO2.一旦细胞是次汇合, 分裂的比率为1:3。</li>
	<li>开始分裂细胞, 通过吸气介质, 然后用无菌磷酸缓冲盐 (pbs) 清洗一次, 除去过量的 pbs, 然后孵育2.5 毫升的0.5x 胰蛋白酶-乙酸酸 (EDTA) (5 分钟, 37 ° c)。<ol>
<li>添加一卷 DMEM 介质 (如上所述) 以停止反应, 然后通过缓慢、轻柔地用电子吸管将细胞分离。</li>
	</ol>
</li>
	<li>然后, 将电池悬浮液转移到适当的容器 (例如, 15-50 毫升圆锥离心管)。离心机在摆动桶 (100 x g) 中, 丢弃上清液, 用无菌 PBS 清洗两次颗粒。</li>
	<li>重在新鲜的 DMEM 培养基和种子中的颗粒在适当的细胞密度 (例如, 1 x 106细胞在 75 cm2烧瓶) 在新鲜的文化烧瓶或大盘子根据地方实验室做法。<ol>
<li>每2天用新媒体替换现有媒体。 注意: 表达式向量是从 pIRES1hyg 派生的, 并根据制造商的说明使用, 如前面所描述的21。p (EGFP) IRES1hyg 是由亚 EGFP cDNA 生成 BamHI + NotI 插入 pEGFP-N1 入 BamHI + NotI 消化 pIRES1hyg21。</li>
	</ol>
</li>
	<li>1天前转染, 种子 HEK-293 细胞在密度为 0.5 x 105细胞/cm2在 12-或 24-良好的组织培养板。</li>
	<li>在转染的当天, 在单独的1.5 毫升离心管 (1 管/井) 中制备 DNA 脂质转染试剂配合物。<ol>
<li>首先, 在每管中加入250µL 的降低血清培养基, 然后加入4µg DNA (轻轻混合)。</li>
		<li>随后, 添加250µL 从以前准备的混合10µL 脂质转染试剂和250µL 的降低血清培养基, 每管 (再次轻轻混合)。</li>
		<li>最后, 进行染, 通过孵化 HEK-293 细胞与 DNA 脂质转染试剂配合物4小时, 然后替换为 DMEM 培养基补充如上所述 (步骤 1.1), 并孵化另 48 h. 执行药物选择的稳定转100µg/毫升霉 B。</li>
	</ol>
</li>
	<li>将 HEK-293 细胞与胰蛋白酶分离, 如上所述 (步骤 1.2)。在无菌 pbs 中清洗细胞后, 重在适当的车辆中 (例如, 在 pbs 中为10% 伏/v), 以达到 5 x 106/毫升的最终细胞浓度。</li>
</ol>2. 兔的制备
注: 家兔的定位和换能器对动物心脏的形态和功能评价不理想。因此, 它是可取的执行一个完整的超声心动图检查20之前, 在该中心 (见下文), 并在随后的时间点, 由实验设计的定义。这将目的是评估心脏的基线解剖和功能特征的动物, 将接受注射, 并评估的效果, 在心脏功能。
<ol><li>以盲目的方式进行这项考试, 遵循美国兽医内科医学院和美国超声心动图协会的超声心动图委员会的指导方针/欧洲心血管成像学会22,23,24。</li>
	<li>在整个超声心动图研究中同时获得1导联心电图 (ECG) 追踪。</li>
	<li>麻醉兔与氯胺酮10毫克/千克, 结合 medetomidine 200 µg/千克, 肌肉注射 (贝聿铭)。</li>
	<li>检查麻醉后10-20 分钟的麻醉水平。</li>
	<li>使用理发钳广泛删除胸部的头发 (如, 在颈部以下的子剑区域) (图 1A)。</li>
	<li>在右侧前肢 (mediocubital 区域) 的内部面和后肢 (mediotibial 区域) (图 1B) 中, 剃须1-2 厘米2的额外区域。</li>
	<li>在四肢的剃光部位放置粘连心电图电极, 在手术过程中同步监测心脏节律 (图 1C)。</li>
	<li>将动物放在褥疮仰卧位上的热毯上, 四肢伸展, 使用附在表上的外科胶带 (图 1C)。<ol>
<li>确保耳朵向后弯曲后, 在兔子的头部/背部, 并在一个位置, 低于其前肢, 因为这有助于保持正确定位的动物胸部的整个过程。</li>
	</ol>
</li>
	<li>在整个手术过程中 (100%、2-3 升/分), 允许动物自发呼吸, 同时用面罩管理氧气。</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56699/56699fig1.jpg"> 图1。家兔的制备.(A) 从胸腔剪发;(B) 从四肢修剪毛发;(C) 连接电极, 将双腿伸出的兔子放在热毯上。<a href="//cloudflare.jove.com/files/ftp_upload/56699/56699fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
3. 经皮超声心动图在家兔中的应用
<ol><li>用洗必泰溶液清洗和消毒胸腔的皮肤。<ol>
<li>根据目前的最佳做法, 在整个过程中使用无菌技术。</li>
		<li>如有可能, 并在合理可行的情况下, 执行完全不育的程序, 包括但不限于使用无菌材料, 如长袍、手套、外科伤口窗帘、餐桌无菌敷料材料以及无菌超声波换能器盖和无菌超声凝胶。这将降低到最低的风险, 引入病原体的动物接受的, 是标准的做法, 在临床设置 (例如, 在心脏穿刺)。 注: 建议始终按照地方和国家的动物研究条例, 适用于当地的机构和国家的实践。</li>
	</ol>
</li>
	<li>应用超声波传输凝胶到胸部和/或传感器, 并与传感器线周围的实验者的颈部, 执行快速窗口扫描的动物的心脏, 这往往是有益的可视化解剖和计划的。</li>
	<li>将传感器手动放置在 4th-6 的th肋间间隙, 2-3 厘米远离右侧的胸骨线, 其入射角与胸壁右侧 (图 2A) 有关。</li>
	<li>调整换能器相对于肋间间隙的位置, 以及它的前后和 dorsoventral 角, 以优化在乳突肌水平上的改良短轴视图。在此视图中确定右心室 (RV)、左心室 (LV)、室隔膜 (IVS)、后壁 (密码) 以及前外侧 (AL) 和内侧 (PM) 的乳突肌 (图 2B)。<ol>
<li>通过在系统上使用适当的控件 (例如、按钮、拨号) 显著增加深度, 具有广泛的视野。</li>
		<li>请特别注意在这个视图中获得对称图像, 以及适当地区分心内膜和心外膜轮廓, 并在必要时通过图像优化控制 (例如, 增益) 进行调整。</li>
	</ol>
</li>
	<li>一旦获得了最佳的超声心动图 (图形 2B), 在整个过程的其余部分保持这个位置, 而第二个操作者执行的是 (见下文)。<ol>
<li>在持有传感器的同时, 避免隐藏传感器方向标记, 它应该始终朝向前方, 从而允许在随后的步骤 (图 2A, C) 中与针对齐。</li>
	</ol>
</li>
	<li>用24克针连接到1毫升注射器, 将针靠近左侧 hemithorax 的皮肤, 在一个对称的镜像位置相对于传感器。然后, 手动将针与传感器方向标记对准 ~ 90 ° (图 2C) 的角度, 并缓慢地将针穿过皮肤并进入胸腔。 注: 经皮穿刺针插入在这个位置和方向有助于在超声束的平面上的针的可视化 (图 2D, E), 从而允许实时监测, 必要时, 调整针相对于心肌靶区的位置 (图 2G, H)。</li>
	<li>与尖端在目标位置, 缓慢地交付 injectate (高达0.25 毫升每个注射站点) 在10-30 秒内 (图 2E), 同时缓慢和轻轻地缩回针在注射期间增加心肌的治疗程度。<ol>
<li>使用在 PBS 中稀释的 InI 10% (v/v) 来实现技术的标准化, 并在获得能力的同时作为原位示踪剂, 并通过大体病理和组织病理学确认在心肌内所有四个区的成功靶点 (见代表性结果)。一旦达到能力, InI 可以被一个合适的商业超声造影剂, 如果需要的替代。 注: 在 PBS 中使用或不带细胞的 10% (v/v) InI 的传递在注射靶点 (图 2E, F) 的壁 hyperechogenicity (即, 回声明亮的外观)。瞬变减速或加速心率, 与过早心室收缩 (例如, 孤立, 对联和三胞胎) 经常被观察甚而从针的第一接触与心外膜并且在和/或后不久。然而, 没有危及生命的心律失常的发展, 和急性不良反应是很少观察使用高达0.25 毫升 (1.25 x 106细胞) 的 injectate 每一个以上的注射站点 (见代表性结果和讨论)。</li>
	</ol>
</li>
	<li>在针的入射角度进行细微的变化, 必要时完成0.25 毫升的注射, 每四靶点的位置 (三在左心室游离壁 (LVFW) 和一个在 IVS)。</li>
	<li>在完成经皮超声心动图引导下, 评价心脏节律 (如, 通过串口心电图跟踪和/或24小时动态心电图), 并进行序列窗口超声扫描, 以验证没有并发症, 直到该动物完全恢复从麻醉, 然后才转移到一个轻周期的房间。<ol>
<li>在这里, 我们使用24小时动态心电图监测的影响, 与 InI 的心脏节律24小时。为此, 我们比较了一组6只正常表型 (正常组) 和6只接受静脉注射阿霉素的家兔 (一种心脏蒽药物, 通常用于治疗癌症;DOX 组) 的剂量为2毫克/千克/周8周, 然后两个队列收到了与 InI。</li>
	</ol>
</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56699/56699fig2.jpg"> 图 2.经皮超声心动图引导下心肌注射液在家兔中的比较.(A) 传感器在右 hemithorax 的位置 (以90°为角)。(B) 代表胸骨短轴观 (PSSX) 的心脏在兔的乳突肌水平的代表性图像。(C) 针在90°角上相对于传感器方向标记的对准。(D) 针在目标站点的位置, 以 PSSX 的角度观察 (注意, 针在超声束的平面上很容易被可视化)。(E和F) 在目标站点上的 hyperechogenicity 心肌注入印度墨水 (箭头突出显示壁 hyperechogenicity) 的演示。(G) 针在 LV 室中的意外位置 (箭头突出针轴)。(H) 将针重新定位到 LV 自由墙 (箭头突出了针轴)。RV = 右心室;LV = 左心室;IVS = 室隔膜;后壁;前外侧的乳突肌;PM = 内侧的乳突肌。<a href="//cloudflare.jove.com/files/ftp_upload/56699/56699fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
4. 后的分析
<ol><li>对兔心脏组织标本进行病理组织学分析。<ol>
<li>在10% 甲醛中修复 24 h 的组织, 其次是脱水, 乙醇浓度增加如下: ·1x 在 70% (60 分钟) · 1x 95% ethanol/5%methanol (60 分钟) ·1x 在 100% (60 分钟) ·1x 在 100% (90 分钟) ·1x 在 100% (120 分钟) 注: 以上孵化均以室温 (RT) 进行。然后, 以100% 二甲苯 (1 h, RT) 两次替代, 最后在石蜡中嵌入两个步骤 (60 分钟, 58 ° c)25。</li>
		<li>使用切片25执行4-5 µm 组织切片。在幻灯片上装载节。</li>
		<li>用苏木精-曙红和马尾松的三方法进行染色25,26,27。</li>
	</ol>
</li>
	<li>在移植心脏组织切片, 执行免疫组化检测 EGFP (+) HEK-293 细胞 (例如, 使用素生物素复合物 (ABC) 方法), 简要:<ol>
<li>De 蜡4-5 µm 厚的心脏部分在100% 二甲苯 (10 分钟, 在 RT)。用降低乙醇浓度溶液洗涤水化组织 (2x 在 100% (2 分钟); 2x 在 95% (2 分钟); 1x 在 70% (2 分钟); 1x 在 50% (2 min); 1x 在 30% (2 分钟); 1x ddH2O (2 min)) 在 RT。</li>
		<li>通过覆盖100µL 3% h2O2稀释的甲醇 (用 5 ml 30% 毫升的 h2O2, 并加入甲醇达50毫升的总容积) 来进行内源性过氧化物酶抑制 (孵育30分钟, RT), 然后用三缓冲盐水浸泡 (TBS; pH 值 7.6)。</li>
		<li>通过酶处理进行抗原揭露, 通过覆盖100µL 0.1% 蛋白酶 (0.01 g 蛋白酶稀释10毫升 tbs) (孵育12分钟, rt), 然后用 tbs 洗涤 (5 分钟, rt) 的部分。</li>
		<li>用100µL 每张幻灯片 (30 分钟, rt), 用 tbs 冲洗 (5 分钟, rt), 在阻断溶液中孵育 (正常的山羊血清, 10% 在 tbs)。</li>
		<li>孵化与鸡抗绿色荧光灯蛋白 (GFP) 作为主要抗体 (1:500 在 tbs) (1 小时, 30 ° c) 和洗涤与 TBS (5 min, RT)。</li>
		<li>用二次抗体孵育化山羊抗鸡 IgG (1:250 在 tbs) (1 h, 30 ° c), 然后洗涤与 TBS (5 min, RT)。</li>
		<li>孵育与素-生物素复合体 (20 分钟, 30 ° c), 然后标签使用 330-diaminobenzidine tetrahydrochloride (民建联) (RT, 5-10 min)。</li>
		<li>最后, 在增加乙醇浓度的洗涤中脱水 (1x 在 30% (2 分钟); 1x 在 50% (2 分钟); 1x 在 70% (2 分钟); 2x 在 95% (2 分钟); 2x 在 100% (2 分钟)) 在 RT, counterstain 部分使用苏木精-曙红方法25, 然后装入封面幻灯片。包括正的、负的以及同种的控制。 注: 上述简短的协议并不打算用于一般免疫组化使用;最优化的组织的利益和条件是必要的。</li>
	</ol>
</li>
</ol>

作者没有什么可透露的。

主要目的是开发一种微创技术, 可用于将干细胞输送到家兔的心肌 (一个大型的临床前动物模型)17,18, 同时利用相对低廉的成像系统在许多临床和研究中心随时可用。在这里, 我们表明, 使用临床超声心动图系统, 并辅以 InI, 一个广泛可用的代理, 既具有原位跟踪能力和回声的性质, 成功的经皮超声造影引导下, 是非常有效的把 injectate 送到兔子心脏的...

细胞和基因治疗是令人兴奋和不断发展的策略, 以再生/修复损伤心肌 HFrEF。有几项研究比较了不同的细胞传递途径的有效性 (例如、细胞保留率), 这一结果始终如一地证明了在冠状动脉或静脉通路上的优越性,1,2,3,4,5. 因此, 对受损心肌干细胞治疗的转化模型研究的很大比例, 并不令人惊讶, 在一个开放的胸部手术中直接观察到的 injectate 通过在一个直视下进行,6,7.但是, 这种方法有几个限制, 包括程序的侵入性, 这会带来围手术期死亡的风险 (通常是报告的)8。此外, 直接视野下的一个人也不能排除无意中射入心室腔的可能性。在临床实践中, 在开胸手术中, 在冠状动脉旁路移植术 (搭桥) 过程中, 可以采用一种合适的治疗细胞分娩方法, 即例如。然而, 这种方法可能不适合在全球非缺血性心肌病 (如, HFrEF 继发于蒽诱发的心肌病 (AICM)) 的细胞分娩。
毫无疑问, 缺血性心脏病 (IHD) 是最常见的原因 HFrEF (约 66%)9,10; 然而, 非缺血性心肌病, 包括 AICM, 仍然影响了相当比例的患者与 HFrEF (33%)9.事实上, 最近在临床肿瘤学的进展已经导致超过1000万的癌症幸存者在美国单独11, 与一个类似的数字在欧洲的估计, 符合整体趋势, 以改善癌症患者的生存率12 ,13。因此, 探索新疗法的好处, 如干细胞移植治疗非缺血性心肌病, 以及法的有效和微创途径的干细胞传递是至关重要的, 鉴于越来越多的病人受抗癌药物心脏的影响。
值得注意的是, 假设测试的研究采用干细胞疗法, 目的是修复/再生受伤的心肌, 经常涉及使用小啮齿动物 (例如, 老鼠和老鼠)。这些模型通常需要昂贵的高频超声系统来评价心肌功能, 通常配有线性阵列传感器, 它们有一些固有的相关限制 (例如, 混响)14。然而, 其他模型, 如家兔, 代表一个大型的前临床模型, 有一定的优势, 假设测试的干细胞治疗在 HFrEF。因此, 与老鼠和老鼠相比, 兔子保持一个 Ca+2传输系统和细胞电生理学类似的人和其他大型动物 (如如, 狗和猪)15,16,17 ,18,19。另一个优势, 是他们的可的心脏超声成像使用相对廉价和广泛可用的临床超声心动图系统配备了相对高频率相控阵传感器,例如, 12 MHz, 如常用于新生儿和儿科心脏病。这些系统允许优秀的超声心动图成像技术的状态, 他们利用谐波成像的优越性20。此外, 广泛的假设测试的潜力心脏再生疗法 (如, 干细胞治疗), 其安全性, 疗效, 心肌的潜力, 以及评估的命运, injectate 一旦交付到心肌, 是强制性的, 才可以考虑为人类使用, 他们需要使用大型的前临床动物模型, 如兔17,19。在这里, 我们描述了一种微创技术, 通过经皮超声心动图引导下的细胞分娩的临床超声心动图系统, 这是针对干细胞移植治疗非缺血性心肌病20.我们还描述了印度墨水的好处 (InI, 也称为中国墨水) 作为一个超声造影剂和原位示踪的 injectate 在兔心脏。

<table><tbody><tr><td>HD11 XE Ultrasound System</td><td>Philips</td><td>10670267</td><td>Echocardiography system.</td></tr><tr><td>S12-4</td><td>Philips</td><td>B01YgG</td><td>4-12 MHz phase array transducer</td></tr><tr><td>Ultrasound Transmision Gel (Aquasone)</td><td>Parket laboratories Inc</td><td>N 01-08</td><td></td></tr><tr><td>Vasovet 24G</td><td>Braun</td><td>REF 381212</td><td>&amp;nbsp;over-the-needle catheter</td></tr><tr><td>Omnifix-F 1 ml syringe</td><td>Braun</td><td>9161406V</td><td></td></tr><tr><td>Imalgene (Ketamine)</td><td>Merial</td><td>RN 9767</td><td>Veterinary prescription is necessary</td></tr><tr><td>Domtor (Medetomidine)</td><td>Esteve</td><td>CN 570686.3</td><td>Veterinary prescription is necessary</td></tr><tr><td>Heating Pad</td><td></td><td></td><td></td></tr><tr><td>Faber-Castel TG1</td><td>Faber-Castel</td><td>16 33 99</td><td>India (China) Ink</td></tr><tr><td>Holter Syneflash</td><td>Ela medical</td><td>SF0003044S</td><td>24 h Holter ECG system.</td></tr><tr><td>Electrodes Blue Sensor&amp;reg;</td><td>Ambu (NUMED)</td><td>VLC-00-S</td><td>Holter ECG electrodes.</td></tr><tr><td>Microtome</td><td>Leica Biosystems</td><td>RM2155</td><td></td></tr><tr><td>Microscope</td><td>Olimpus</td><td>CO11</td><td></td></tr><tr><td>ABC Vector Elite</td><td>Vector Laboratories</td><td>PK-6200</td><td>Avidin Biotin Complex Kit.</td></tr><tr><td>Chicken anti-GFP antibody</td><td>Invitrogen</td><td>A10262</td><td>Primary antibody.</td></tr><tr><td>Biotinylated goat-anti-chicken IgG Antibody</td><td>Vector Laboratories</td><td>BA-9010</td><td>Secondary Antibody.</td></tr><tr><td>3,30-diaminobenzidine tetrahydrochloride (DAB)</td><td>DAKO (Agilent)</td><td>S3000</td><td></td></tr><tr><td>Fluorescence Microscope</td><td>Carl Zeiss&lt;br/&gt; MicroImaging</td><td>Zeiss AX10 Axioskop</td><td></td></tr><tr><td>Holter ECG</td><td>Elamedical</td><td>Syneflash SF0003044S</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle medium (DMEM)&amp;nbsp;</td><td>Fisher Scientific</td><td>11965084</td><td></td></tr><tr><td>10% fetal calf serum (FCS)</td><td>Fisher Scientific</td><td>11573397</td><td></td></tr><tr><td>0.05% Trypsin-Ethylenediaminetetraacetic acid (EDTA)</td><td>Fisher Scientific</td><td>25300054</td><td></td></tr><tr><td>Lipofectamine 2000 (Lipid transfection reagent)</td><td>Fisher Scientific</td><td>11668019</td><td></td></tr><tr><td>Reduced serum medium (Opti-MEM)</td><td>Fisher Scientific</td><td>31985070</td><td></td></tr><tr><td>Hygromycin B</td><td>Calbiochem (MERCK)</td><td>400051</td><td></td></tr><tr><td>Xylene (histological)</td><td>Fisher Scientific</td><td>X3S-4</td><td></td></tr><tr><td>Hydrogen Peroxide Solution (H2O2)</td><td>Sigma</td><td>H1009</td><td></td></tr><tr><td>Pronase</td><td>Fisher Scientific</td><td>53-702-250KU</td><td></td></tr></tbody></table>

percutaneous contrast echocardiography-guided intramyocardial injection and cell delivery in a large preclinical model

细胞和基因治疗是令人振奋的和有希望的策略, 以心脏再生的目的在设置心力衰竭与减少射血分数 (HFrEF)。在人们可以考虑使用和实施之前, 在大型动物模型中需要进行广泛的临床前研究, 以评估 injectate (如干细胞) 一旦被送到心肌的安全性、有效性和命运。小啮齿类动物模型提供好处 (例如, 成本效益, 可为基因操纵);然而, 鉴于这些模型的固有局限性, 这些发现很少转化为临床。相反, 大型动物模型, 如兔子, 有优势 (例如, 与人类和其他大型动物相比, 类似的心脏电生理), 同时保持一个良好的成本效益平衡。在这里, 我们演示了如何执行经皮超声心动图引导心肌注射技术, 这是微创, 安全, 耐受性好, 并非常有效的靶向交付 injectates, 包括细胞,在兔子模型的心肌内的几个位置。为了实现这一技术, 我们也利用了广泛使用的临床超声心动图系统。在实践了这里描述的协议之后, 一个具有基本的超声知识的研究人员将会胜任在实验中常规使用的这一多才多艺和微创技术的表现, 目的是假设测试家兔模型的心脏再生疗法的能力。一旦能力达到, 整个程序可以在25分钟内完成麻醉兔。

经皮超声心动图引导下与 InI 对照:
使用上述协议, 一旦针尖的最佳定位通过超声心动图和注射启动, 壁 hyperechogenicity 观察在交付的 InI (10% 伏/v 在 PBS) (图 2E), 以及后不久, 到目标区域 (图 2F)。当他紧接着被安乐死和心脏移除后, 在心脏外部检查时很容易看到有 InI 的沉积?...

Watch this Scientific Journal Video about Percutaneous Contrast Echocardiography-guided Intramyocardial Injection and Cell Delivery in a Large Preclinical Model at JoVE.com

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

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

在心脏再生医学新的治疗策略需要广泛和详细的研究在大型前动物模型, 才可以考虑使用在人类。在这里, 我们演示了经皮超声心动图引导心肌注射技术在兔, 这是有价值的假说测试这种新疗法的功效。

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

percutaneous-contrast-echocardiography-guided-intramyocardial

Research

JoVE Journal

Medicine

11.6K 次观看.  University of Reading. 在心脏再生医学新的治疗策略需要广泛和详细的研究在大型前动物模型, 才可以考虑使用在人类。在这里, 我们演示了经皮超声心动图引导心肌注射技术在兔, 这是有价值的假说测试这种新疗法的功效。

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

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

An Injectable and Drug-loaded Supramolecular Hydrogel for Local Catheter Injection into the Pig Heart

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the limited access to donor hearts. An example of a treatment to recover the function of the heart consists of the local delivery of drugs and bioactives from a hydrogel. In this paper a method is introduced to formulate and inject a drug-loaded hydrogel non-invasively and side-specific into the pig heart using a long, flexible catheter. The use of 3-D electromechanical mapping and injection via a catheter allows side-specific treatment of the myocardium. To provide a hydrogel compatible with this catheter, a supramolecular hydrogel is used because of the convenient switching from a gel to a solution state using environmental triggers. At basic pH this ureido-pyrimidinone modified poly(ethylene glycol) acts as a Newtonian fluid which can be easily injected, but at physiological pH the solution rapidly switches into a gel. These mild switching conditions allow for the incorporation of bioactive drugs and bioactive species, such as growth factors and exosomes as we present here in both in vitro and in vivo experiments. The in vitro experiments give an on forehand indication of the gel stability and drug release, which allows for tuning of the gel and release properties before the subsequent application in vivo. This combination allows for the optimal tuning of the gel to the used bioactive compounds and species, and the injection system.

Supramolecular hydrogelators based on ureido-pyrimidinones allow full control over the macroscopic gel properties and the sol&#8211;gel switching behavior using pH. Here, we present a protocol for formulating and injecting such a supramolecular hydrogelator via a catheter delivery system for local delivery directly in relevant areas in the pig heart.

可注射药物加载超分子凝胶局部导管注入猪心

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the ...

生物工程

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 ...

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

基于微注射的免疫胚胎心肌细胞体内植入系统

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

发育生物学

Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. Experiments involving myocardial injection are currently performed by direct surgical access through a thoracotomy. While convenient when performed at the time of another experimental manipulation such as coronary artery ligation, the need for an invasive procedure for intramyocardial delivery limits potential experimental designs. With ever improving ultrasound resolution and advanced noninvasive imaging modalities, it is now feasible to routinely perform ultrasound-guided, percutaneous intramyocardial injection. This modality efficiently and reliably delivers agents to a targeted region of myocardium. Advantages of this technique include the avoidance of surgical morbidity, the facility to target regions of myocardium selectively under ultrasound guidance, and the opportunity to deliver injectate to the myocardium at multiple, predetermined time intervals. With practiced technique, complications from intramyocardial injection are rare, and mice quickly return to normal activity on recovery from anesthetic. Following the steps outlined in this protocol, the operator with basic echocardiography experience can quickly become competent in this versatile, minimally invasive technique.

Echocardiography-guided percutaneous intramyocardial injection represents an efficient, reliable, and targetable modality for the delivery of gene transfer agents or cells into the murine heart. Following the steps outlined in this protocol, the operator can quickly become competent in this versatile, minimally invasive technique.

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. ...

3D Whole-heart Myocardial Tissue Analysis

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other biologicals that enhance angio- or vasculogenesis into the infarct border zone (IBZ), tissue perfusion is improved, and the myocardium can be protected from further damage. For maximum therapeutic effect, it is hypothesized that the regenerative substance is best delivered to the IBZ. This requires accurate injections and has led to the development of new injection techniques. To validate these new techniques, we have designed a validation protocol based on myocardial tissue analysis. This protocol includes whole-heart myocardial tissue processing that enables detailed two-dimensional (2D) and three-dimensional (3D) analysis of the cardiac anatomy and intramyocardial injections. In a pig, myocardial infarction was created by a 90-min occlusion of the left anterior descending coronary artery. Four weeks later, a mixture of a hydrogel with superparamagnetic iron oxide particles (SPIOs) and fluorescent beads was injected in the IBZ using a minimally-invasive endocardial approach. 1 h after the injection procedure, the pig was euthanized, and the heart was excised and embedded in agarose (agar). After the solidification of the agar, magnetic resonance imaging (MRI), slicing of the heart, and fluorescence imaging were performed. After image post-processing, 3D analysis was performed to assess the IBZ targeting accuracy. This protocol provides a structured and reproducible method for the assessment of the targeting accuracy of intramyocardial injections into the IBZ. The protocol can be easily used when the processing of scar tissue and/or validation of the injection accuracy of the whole heart is desired.

This protocol describes a novel method for the 3D comparison of whole-heart myocardial tissue with MRI. This is designed for the accurate assessment of intramyocardial injections in the infarct border zone of a chronic porcine model of myocardial infarction.

3D全心脏心肌组织分析

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other ...

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 ...

一种减轻组织损伤的新型微创慢注射方法

A Novel Cell Injection Method with Minimum Invasion

Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient amount of suspension solution to allow the cells to enter the tissue. The volume of the suspension solution affects the tissue, and this can cause major invasive injury as a result of the cell injection. This paper reports on a novel cell injection method, called slow injection, that aims to avoid this injury. However, pushing out the cells from the needle tip requires a sufficiently high injection speed according to Newton's law of shear force. To solve the above contradiction, a non-Newtonian fluid, such as gelatin solution, was used as the cell suspension solution in this work. Gelatins solution have temperature sensitivity, as their form changes from gel to sol at approximately 20 &#176;C. Therefore, to maintain the cell suspension solution in the gel form, the syringe was kept cooled in this protocol; however, once the solution was injected into the body, the body temperature converted it to a sol. The interstitial tissue fluid flow can absorb excess solution. In this work, the slow injection technique allowed cardiomyocyte balls to enter the host myocardium and engraft without surrounding fibrosis. This study employed a slow injection method to inject purified and ball-formed neonatal rat cardiomyocytes into a remote area of myocardial infarction in the adult rat heart. At 2 months following the injection, the hearts of the transplanted groups showed significantly improved contractile function. Furthermore, histological analyses of the slow-injected hearts revealed seamless connections between the host and graft cardiomyocytes via intercalated disks containing gap junction connections. This method could contribute to next-generation cell therapies, particularly in cardiac regenerative medicine.

作者聚焦:一种侵入性最小的新型细胞注射方法  

This method eliminates any major invasion during cell injections caused by the cell suspension solution.

一种侵袭性最小的新型细胞注射方法

Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient ...

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

摘要

探索更多视频

此视频中的章节

结扎冠状动脉内注射在小鼠模型梗死

可注射药物加载超分子凝胶局部导管注入猪心

心肌内细胞交货：观察在小鼠心脏

基于微注射的免疫胚胎心肌细胞体内植入系统

超声引导下经皮内注射小鼠

3D全心脏心肌组织分析

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

一种侵袭性最小的新型细胞注射方法

一种侵袭性最小的新型细胞注射方法

一种侵袭性最小的新型细胞注射方法