我们感谢马格迪雅各布研究所（MYI），用于支持显微镜分析，涉及心肌修复，技术人员和我们的动物设施的管理器中的项目。这项工作已经由英国心脏基金会（BHF），项目补助PG/10/019支持。 MPS是由MYI和BHF支撑。 TP是BHF-卓越研究研究员。 NR是NH＆MRC澳大利亚研究员。

所有动物实验均符合国际（欧洲议会指令2010/63/EU）和国家（英国内政部，1986法案）的规定执行。本文所述的方法是我们在英国的许可证主管部门的工作计划的一部分，并没有被进行记录的目的。 1。细胞的制备本协议描述了用于演示目的编制特定的细胞系（人类胚胎肾，HEK293细胞）。小区特定的协议必须采用用于生长并最终分化多能性或成体干细胞分化成特定细胞谱系。 <ol><li>生长在DMEM培养基（高葡萄糖，4500毫克/升）含1％丙酮酸钠，2mM谷氨酰胺，1％抗生素青霉素/链霉素和10％胎牛血清（FBS）的细胞。一个10cm平板应能正常分裂为1:3和媒体补充新鲜每两天。 </li><li>轻度治疗细胞tryps作为在1:1的描述1X和重悬在DMEM培养基。 </li><li>计数细胞在血细胞计数器室中，并收集3×10 6细胞到一个单独的试管中。 </li><li>离心机在摆动斗转子，在100×g离心5分钟。 </li><li>弃去上清液并用无菌磷酸盐缓冲液2倍洗涤细胞沉淀。 </li><li>重悬在PBS 1x在的10 6/50微升注射入immunodepleted小鼠的左心室的浓度。 </li></ol> 2。手术前的条件<ol><li>每次手术前，保持immunodepleted小鼠（NOD.CB17-Prkdscid/JHliHSD）用无菌的食物颗粒和水在温度控制隔离器（ 表1 22℃）。 </li><li>在层流罩进行immunodepleted小鼠的手术。用70％乙醇清洁工作区和高压灭菌手术工具（镊子和剪刀）。 </li><li>打开加热垫的手术和恢复。加热垫是很重要的阻止发生体温的过程中减少和手术后。 </li><li>将干净和蒸压笼上的恢复加热垫。 </li><li>运输老鼠在原来的笼子从隔离到手术室到无菌高压灭菌袋。 </li><li>称量小鼠并根据体重注射皮下（SC），其中包含美托咪定和盐酸氯胺酮稀释在0.9％无菌生理盐水溶液来麻醉小鼠的溶液，以及放松的肌肉（美托咪定1毫克/千克;盐酸氯胺酮75毫克/千克）。 </li><li>从笼中取出鼠标时完全麻醉（在1-2分钟，刺激后无脚趾捏反射） </li><li>取适量涂抹于胸部左侧脱毛膏和咽喉区。经过一段时间（约2-5分钟）适量，擦干头发松散与水的湿纸巾。 </li><li>把鼠标在手术面板上的仰卧位置。从外科医生的角度来看的位置是垂直的，尾巴向下的头。 </li><li>注入镇痛溶液在0.1-0.2微克/克稀释在无菌盐水中的溶液（丁丙诺啡）插入后肢皮下注射，与使用棉尖涂药无菌溶液聚维酮碘覆盖剃的区域。 </li><li>聚焦显微镜（2.5X物镜）的咽喉地带。 </li></ol> 3。手术条件<ol><li>用钝的剪刀做出0.5-1厘米长度略低于环状软骨的腹面正中线切开皮肤。皮肤结缔组织分开，以可视化的唾液腺。 </li><li>通过同时拉动各部分侧面带有钳子和磁胸部拉钩各执其自然中线划分唾液腺和暴露气管。 </li><li>轻轻一拉舌的一侧，露出喉。在插管小心滑入进气管。管尖端可见通过显示升arynx和气管。重要的是，如果管中，但不是清晰可见的，它是在食道。中取出，并改变它的管前端的位置。 </li><li>管连接到通风的机器。在200微升和150冲程/分钟设定的每搏输出量。固定手术面板用胶带上的通风管道。 </li><li>用钝的剪刀和镊子，使垂直尾翼头1 - 1.5厘米长的皮肤切口在左胸部区域。 </li><li>拧松结缔组织/肌肉层和主要的和次要的胸肌皮肤而不做切口。 </li><li>如下所述第三和第四肋之间进行开胸： <ol><li>通过捏和小，圆形钳刮穿孔肋间肌肉层。 </li><li>一旦肋间肌层穿孔，放置胸腔牵开器，最大限度地打开胸腔。左心室（LV），其上覆耳廓（心房）的上部和中间部分是现在可见。 </li></ol></li><li>准备一个精密注射用细胞。注射器应能提供的10微升体积的量为每次注射。投了30号针头的注射器的尖端。 </li><li>固定用胶带的小塑料套管（从introcan-W 20 G）上的针，留下暴露的仅1毫米，包括针的开口顶端。这将防止针穿透整个左心室壁和注射细胞到左心室腔。 </li><li>用5X物镜的帮助下，可视化在此放大率左心室和注入的细胞进入左心室壁在5个不同的位置（每个注射将提供10微升，总共106个细胞的注射）。如果注射是成功的，白色的区域和不存在细胞的主要反冲洗将是可见的（如果注入不成功，该动物将被排除的功能分析）。 </li><li>取出拉钩。关闭胸腔B墙Ÿ内附两个分离的肋骨6-0丝线缝合两针。 </li><li>滋润胸肌和皮肤用棉尖湿透的PBS 1x和轻轻把肌肉回到与镊子原来的位置。 </li><li>关闭3-4针6-0丝线缝合皮肤。缝合切口喉咙用2-3缝线关闭皮肤。 </li><li>注射阿替美唑（1毫克/千克终浓度）恢复的麻醉作用。 </li><li>只要鼠标开始自主呼吸，取出管从气管，把鼠标在它的右侧。鼠标预计将在几分钟之内恢复。 </li><li>当鼠标开始转身，将其放置在温暖的回收笼（IVC小笼子封闭过滤顶部）一个小时。 </li><li>离开隔夜动物在受控制的加热箱（37℃）。 </li><li>装满水和食物颗粒菜在手术后第2天，以支持复苏。 </li></ol>十大心中注入细胞和10个控制未注射的心是组织学分析如下： <ol><li> 1周喷射（ 图1和3）后进行移植的细胞的分析。 <ol><li>安乐死异氟醚的过量后，灌注的心用20毫升4％多聚甲醛（PFA）的修复组织。脱水的固定的组织中增加的乙醇百分比（从70-100％），并嵌入石蜡块。 </li><li>节的心在与石蜡切片机在5微米和染色用苏木精和曙红的形态分析。 </li><li>通过使用马松三色染色可视化结缔组织。 </li><li>分析的滑动扫描器显微镜（ 图1A，2A，3A，3B和3C）下的图像，并使用数字幻灯片扫描仪显微镜（FIGU可视化整个心脏中短轴视图水库1B和1C）。 </li></ol></li><li>经过以上分析，deparaffinize的心脏组织中的二甲苯，再水合浸没标本在减少乙醇的百分比（从100％到水中）和过程为扫描电子显微镜（SEM），如下所示： <ol><li>孵育脱石蜡块，用1％单宁酸在1小时的0.1M磷酸盐缓冲液中。 </li><li>在增加的百分比乙醇（25-100％）脱水标本。 </li><li>干样品HMDS（六甲基二）。 </li><li>安装在拔标本与艾奇逊银达格和外套，用金 - 钯。 </li><li>视图样品中的分析扫描电子显微镜（ 图2B）。 </li></ol></li></ol>

作者什么都没有透露。

在这个手稿中，我们已经展示了如何在小鼠心脏进行心肌内注射细胞。作为这种方法的一个证据，我们已经使用HEK293细胞。需要强调的是HEK293细胞中的任何细胞治疗研究中没有使用，因此该手稿的发现并不适合直接翻译为一种治疗方法是很重要的。然而，事实是HEK293细胞不收缩细胞，不要在其他细胞类型转分化侧重于技术方面，例如要传送的单元的属性和传送路线的注意。 ?...

各种细胞输送协议已经在心血管疾病的小鼠和大鼠模型进行了测试与翻译这个实验过程在人类患者的效率，有效性和安全性的目的。在小型啮齿动物的心，心肌内细胞传递是传递细胞1,2的最可行的方法，而在大鼠心脏顺行3和4逆行冠脉内细胞输注，也可以使用。这两种方法有很大的局限性和优势。经冠状动脉内途径细胞递送拥有注射在促进全球细胞传播3直接肌肉注射理论上的优势，但它也有引起冠状动脉栓塞3,5的风险。在心肌内交付限制与机械性损伤，急性炎症，心肌损害6,7关联。在人类中，细胞是心脏修复是通过心肌内注射，通过心内膜或心外膜的手术递送接近或冠状动脉内的动脉途径8。注射液经血管途径适合于治疗急性脑梗死和心肌再灌注，但可能无法进行的情况下完全闭塞或受影响的领土9的血管里流动不畅的。直接注射到左心室壁通过经心或transepicardial注射在技术上是可行取决于病人的健康状况。事实上，已经表明，这种技术是安全的10,11，尽管对于transepicardial注射是必要的开胸手术和经心方法是必需的，以区分存活缺血或心肌疤痕9的部位的电生理标测对每个病人。 重要的是，在细胞治疗研究的最佳细胞移植的选择仍在进一步调查中。短期的分析（4周）表明，注射定义为cardiospheres心脏干细胞的<s向上&gt; 12或从骨髓13侧群细胞诱导的心功能恢复的小鼠14和大鼠模型15心肌梗塞通过减少瘢痕大小和细胞死亡。在没有免疫抑制大鼠心肌梗死模型cardiospheres同种异体移植被认为是安全的，促进心肌再生，并通过内源性修复机制15刺激改善心脏功能。在心脏Lin-/c-kit +能成体祖细胞被证明是自我更新，克隆，和多能在体外和体内 ，并且当注射到缺血大鼠心脏重构受伤的心肌壁16的大的部分和有能力，形成导电性和中等大小的冠状动脉17。这些可喜的数据引发的I期和人类II期临床试验：注射自体和异体骨髓间充质干细胞（MSC）18，cardiospheres 19，或c-Kit阳性心脏干细胞（CSC）20在缺血人类的心灵每个显示，在长期的研究心脏功能的有益作用。然而，大量的长期随访和回顾性荟萃分析表明，干细胞疗法提供了显著的好处有些病人，而不是在别人与一系列不可预知的结果21。这是可能的，这些限制将需要的细胞递送的每个个人和每一种疾病的具体协议的设计。 在小鼠和大鼠模型中，长期的研究表明，细胞注射未进一步改善心脏功能（12个月）。的确，人胚胎干细胞衍生的心肌细胞（hESC细胞-CM）的移植物主要是由一层纤维组织22,23的分离自宿主的心肌。骨骼肌成肌细胞心肌内移植到梗死小鼠24的心脏后，类似的结果也被观察到。 Furthermo重，同种异体干细胞的保存功能中的梗塞心脏的长期能力已经通过从immunoprivileged到免疫原性的状态转移分化25后受到限制。 考虑到上述挑战和前景，我们在这里展示如何通过心肌内注射小鼠，提供细胞。我们观察到细胞无心肌收缩性能不与宿主心肌相连形成一个粘合体具有薄纤维化障碍。尽管在某些情况下，这种结果可能是有利的，下面的分析可能是有用的，以了解细胞植入可被调制，以产生功能性连接的心肌的结构为好。

<table border="0" cellpadding="0" cellspacing="0" width="560">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:103px;">Isolator</td>
			<td style="width:115px;">Pfi systems</td>
			<td style="width:88px;">-</td>
			<td style="width:255px;">Quotation needed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:103px;">Heating Pad</td>
			<td style="width:115px;">Vet Tech Solutions</td>
			<td style="width:88px;">HE006</td>
			<td style="width:255px;">For small animals</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:103px;">medetomidine</td>
			<td style="width:115px;">National veterinary Service</td>
			<td style="width:88px;">-</td>
			<td style="width:255px;">Veterinary prescription is necessary</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:103px;">ketamine hydrochloride</td>
			<td style="width:115px;">National veterinary Service</td>
			<td style="width:88px;">-</td>
			<td style="width:255px;">Veterinary prescription is necessary</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:103px;">atipamezole</td>
			<td style="width:115px;">National veterinary Service</td>
			<td style="width:88px;">-</td>
			<td style="width:255px;">Veterinary prescription is necessary</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:103px;">Hair removal cream</td>
			<td style="width:115px;">Commercial shops</td>
			<td style="width:88px;">-</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:103px;">buprenorphine</td>
			<td style="width:115px;">NVS</td>
			<td style="width:88px;">-</td>
			<td style="width:255px;">Veterinary prescription is necessary</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:103px;">Leica MZFLIII microscope</td>
			<td style="width:115px;">Leica</td>
			<td style="width:88px;">Model S6E</td>
			<td style="width:255px;">With swing arm stand TS0</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:103px;">Hamamatsu Nanozoomer digital slide scanner</td>
			<td style="width:115px;">Hamamatsu</td>
			<td style="width:88px;">RS series</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:103px;">Scanning Electron Microscope</td>
			<td style="width:115px;">Jeol</td>
			<td style="width:88px;">JSM-6610</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:103px;">Blunt scissors</td>
			<td style="width:115px;">FST</td>
			<td style="width:88px;">14084-09</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:103px;">Minivent</td>
			<td style="width:115px;">Harvard apparatus</td>
			<td style="width:88px;">73-0043</td>
			<td style="width:255px;">Including small Y adapter (73-0027) and intubation cannula (73-2844)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:103px;">Forceps</td>
			<td style="width:115px;">FST</td>
			<td style="width:88px;">11052-10</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:103px;">Retraction system</td>
			<td style="width:115px;">FST</td>
			<td style="width:88px;">18200-20</td>
			<td style="width:255px;">Kit for animals up to 200grams</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:103px;">30G 12mm; &#189; inch</td>
			<td style="width:115px;">BBraun</td>
			<td style="width:88px;">A210</td>
			<td style="width:255px;">Fine yellow</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:103px;">microliter syringe</td>
			<td style="width:115px;">ESSLAB</td>
			<td style="width:88px;">81201</td>
			<td style="width:255px;">Also include a Hamilton repeating dispenser PB 600-1 Catalogue number 83700</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:103px;">6-0 silk suture</td>
			<td style="width:115px;">Ethicon</td>
			<td style="width:88px;">W1614T</td>
			<td style="width:255px;"></td>
		</tr>
	</tbody>
</table>

intramyocardial cell delivery: observations in murine hearts

以往的研究表明，细胞传递促进心功能改善细胞因子和增加心肌组织再血管化和细胞生存因子的释放。此外，进一步的观察发现，特定的干细胞，如心肌干细胞，间充质干细胞和cardiospheres有通过分化成心肌细胞，平滑肌细胞和内皮细胞周围的心肌内整合的能力。 这里，我们提出的材料和方法，以可靠地提供noncontractile细胞进入immunodepleted小鼠的左心室壁。其显着的步骤此显微程序涉及麻醉和镇痛注射液，气管插管，切开打开胸部和无菌30号针头和精密微升注射器暴露细胞的心脏和交付。 包括心脏收获，embeddi组织处理毫微克，切片及组织学染色显示心肌内注射细胞产生一小的伤害的心外膜区域，以及在心室壁上。 Noncontractile细胞被保留到免疫缺陷小鼠的心肌壁并分别由一层纤维组织，有可能从心脏压力和机械负载，以保护所包围。

我们注入的HEK293细胞中，这是由它们的不同的形态可区分从心脏细胞（ 图1），用鹅卵石形状较细长的心肌细胞（ 图1A）。相比，心肌细胞（粉红色），有可能的，因为它们的增加的核含量（ 图1A）的HEK293细胞，更具反应性，以苏木精染料（蓝色）。为了进一步区分注射的细胞从宿主组织中，将HEK293细胞用DAPI标记前2小时注射。标记的细胞是可见的心肌组织，...

Watch this Scientific Journal Video about Intramyocardial Cell Delivery: Observations in Murine Hearts at JoVE.com

Intramyocardial Cell Delivery: Observations in Murine Hearts

心血管疾病，如高血压或心肌梗死的小鼠模型心肌内细胞递送，被广泛用于测试在再生研究不同细胞类型的治疗潜力。因此，有详细说明，该手术过程有一个清晰的可视化将有助于确定在小型啮齿动物心血管细胞治疗分析的局限性和优势。

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 ...

intramyocardial-cell-delivery-observations-in-murine-hearts

Research

JoVE Journal

Medicine

13.4K 次观看.  Imperial College London. 心血管疾病，如高血压或心肌梗死的小鼠模型心肌内细胞递送，被广泛用于测试在再生研究不同细胞类型的治疗潜力。因此，有详细说明，该手术过程有一个清晰的可视化将有助于确定在小型啮齿动物心血管细胞治疗分析的局限性和优势。 

视频: Intramyocardial Cell Delivery: Observations in Murine Hearts

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

Reduction in Left Ventricular Wall Stress and Improvement in Function in Failing Hearts using Algisyl-LVR

Injection of Algisyl-LVR, a treatment under clinical development, is intended to treat patients with dilated cardiomyopathy. This treatment was recently used for the first time in patients who had symptomatic heart failure. In all patients, cardiac function of the left ventricle (LV) improved significantly, as manifested by consistent reduction of the LV volume and wall stress. Here we describe this novel treatment procedure and the methods used to quantify its effects on LV wall stress and function.
Algisyl-LVR is a biopolymer gel consisting of Na+-Alginate and Ca2+-Alginate. The treatment procedure was carried out by mixing these two components and then combining them into one syringe for intramyocardial injections. This mixture was injected at 10 to 19 locations mid-way between the base and apex of the LV free wall in patients.
Magnetic resonance imaging (MRI), together with mathematical modeling, was used to quantify the effects of this treatment in patients before treatment and at various time points during recovery. The epicardial and endocardial surfaces were first digitized from the MR images to reconstruct the LV geometry at end-systole and at end-diastole. Left ventricular cavity volumes were then measured from these reconstructed surfaces.
Mathematical models of the LV were created from these MRI-reconstructed surfaces to calculate regional myofiber stress. Each LV model was constructed so that 1) it deforms according to a previously validated stress-strain relationship of the myocardium, and 2) the predicted LV cavity volume from these models matches the corresponding MRI-measured volume at end-diastole and end-systole. Diastolic filling was simulated by loading the LV endocardial surface with a prescribed end-diastolic pressure. Systolic contraction was simulated by concurrently loading the endocardial surface with a prescribed end-systolic pressure and adding active contraction in the myofiber direction. Regional myofiber stress at end-diastole and end-systole was computed from the deformed LV based on the stress-strain relationship.

This article describes procedures for implanting a novel hydrogel in failing hearts and quantifying its effect on left ventricular wall stress and function. These procedures have been successfully applied in dogs and humans.

左室壁应力和改进功能衰竭心脏使用Algisyl LVR减少

Injection of  Algisyl-LVR, a treatment under clinical development, is intended to treat  patients with dilated cardiomyopathy. This treatment was recently ...

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. ...

Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. While these therapies lead to the rapid restoration of blood flow, these technologies remain limited by restenosis in the case of bare metal stents and angioplasty, or reduced healing and possibly enhanced risk of thrombosis in the case of drug eluting stents. A key pathophysiological mechanism in the formation of restenosis is intimal hyperplasia caused by the activation of vascular smooth muscle cells and inflammation due to arterial stretch and injury. Surgeries that induce arterial injury in genetically modified mice are useful for the mechanistic study of the vascular response to injury but are often technically challenging to perform in mouse models due to the their small size and lack of appropriate sized devices. We describe two approaches for a surgical technique that induces endothelial denudation and arterial stretch in the femoral artery of mice to produce robust neointimal hyperplasia. The first approach creates an arteriotomy in the muscular branch of the femoral artery to obtain vascular access. Following wire injury this arterial branch is ligated to close the arteriotomy. A second approach creates an arteriotomy in the main femoral artery that is later closed through localized cautery. This method allows for vascular access through a larger vessel and, consequently, provides a less technically demanding procedure that can be used in smaller mice. Following either method of arterial injury, a degradable drug delivery patch can be placed over or around the injured artery to deliver therapeutic agents.

We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis.

股动脉线损伤的小鼠模型的血管周围给药补丁植入

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. ...

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

诱导和缺血再灌注损伤评估中的Langendorff灌注鼠心

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for ...

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 Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.

大鼠胡萝卜动脉压力控制段气球损伤与腹腔ventit 治疗应用

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...

Slicing and Culturing Pig Hearts under Physiological Conditions

Many novel drugs fail in clinical studies due to cardiotoxic side effects as the currently available in vitro assays and in vivo animal models poorly predict human cardiac liabilities, posing a multi-billion-dollar burden on the pharmaceutical industry. Hence, there is a worldwide unmet medical need for better approaches to identify drug cardiotoxicity before undertaking costly and time consuming &#39;first in man&#39; trials. Currently, only immature cardiac cells (human induced pluripotent stem cell-derived cardiomyocytes [hiPSC-CMs]) are used to test therapeutic efficiency and drug toxicity as they are the only human cardiac cells that can be cultured for prolonged periods required to test drug efficacy and toxicity. However, a single cell type cannot replicate the phenotype of the complex 3D heart tissue which is formed of multiple cell types. Importantly, the effect of drugs needs to be tested on adult cardiomyocytes, which have different characteristics and toxicity responses compared to immature hiPSC-CMs. Culturing human heart slices is a promising model of intact human myocardium. This technology provides access to a complete multicellular system that mimics the human heart tissue and reflects the physiological or pathological conditions of the human myocardium. Recently, through optimization of the culture media components and the culture conditions to include continuous electrical stimulation at 1.2 Hz and intermittent oxygenation of the culture medium, we developed a new culture system setup that preserves viability and functionality of human and pig heart slices for 6 days in culture. In the current protocol, we are detailing the method for slicing and culturing pig heart as an example. The same protocol is used to culture slices from human, dog, sheep, or cat hearts. This culture system has the potential to become a powerful predictive human in situ model for acute cardiotoxicity testing that closes the gap between preclinical and clinical testing results.

This protocol describes how to slice and culture heart tissue under physiological conditions for 6 days. This culture system could be used as a platform for testing the efficacy of novel heart failure therapeutics as well as reliable testing of acute cardiotoxicity in a 3D heart model.

生理条件下猪心的切片和培育

Many novel drugs fail in clinical studies due to cardiotoxic side effects as the currently available in vitro assays and in vivo animal models poorly ...

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 ...