这项研究由 Additional Ventures - 单心室研究基金 （SVRF） 和单心室扩展基金（至 I.F.）以及 OeAD-GmbH 的 Marietta Blau 奖学金资助，该奖学金来自奥地利联邦教育、科学和研究部 BMBWFC（至 G.G.）。

所有动物程序均按照国家研究委员会进行。2011. 实验动物护理和使用指南:第八版。动物方案由波士顿儿童医院的机构动物护理和使用委员会审查和批准。
手术前，将所有手术器械进行蒸汽高压灭菌，并制备终浓度为22 mmol/L KCl的改良Krebs-Henseleit缓冲液作为心脏麻痹溶液（表1）。将溶液过滤灭菌并在4°C下储存过夜。异位新生大鼠心脏移植手术需要手术显微镜（12.5x）。
1.准备和麻醉
<ol><li>使用体重约为150g（5-6周龄）的雄性/雌性Lewis大鼠作为受体。</li>
	<li>首先，用剃须刀慷慨地剃掉老鼠的腹部。</li>
	<li>将大鼠放入异氟烷室中，并用2%异氟烷以2L / min打开氧气流量，直到动物被适当镇静但仍自主呼吸。腹膜内注射 45 mg/kg 氯胺酮和 5 mg/kg 甲苯噻嗪，以及 300 U/kg 肝素。通过脚趾捏试验确认麻醉是否正确。 注意:通过触诊胸部仔细监测自主呼吸和心率，以确保整个过程中稳定的血流动力学状态。</li>
	<li>插管时，将大鼠放在斜架上（图1），用绳子固定门牙，并将头部朝向外科医生。</li>
	<li>将颈部外侧的灯放在声带区域，用两根手指抓住舌头，然后轻轻向上和向左推动，为插管提供最佳视野。使用18 G，2套管用于100-150 g大鼠。用胶带固定气管插管。 注意:建议使用放大倍率为 3.5 倍的手术耳罩进行插管。</li>
	<li>将插管插管连接到小动物呼吸机上，并根据动物大小根据制造商的说明调整设置。 注意:对 150 g 大鼠使用以下设置:体积模式;呼吸频率，55/min;潮气量，1.3 mL 50 % I/E 比，但可以根据需要适当调整。确保适当的双侧和平等的胸部运动，并通过呼吸机以 0.5%-2% 的浓度连续给予异氟醚。</li>
	<li>将大鼠放在加热垫上（以保持正常体温），仰卧位，尾巴朝向外科医生。用betadine溶液和70%乙醇交替对腹部进行消毒三次。施用眼部润滑剂，并用无菌手术布覆盖大鼠，使腹部暴露在外。</li>
</ol>2.受者大鼠新生供体心脏的手术准备及异位移植
<ol><li>使用 15 刀片手术刀进行中线剖腹手术，切开皮肤，用剪刀打开前腹壁，然后用棉尖涂抹器钝暴露腹膜后腹主动脉和下腔静脉 （IVC）。</li>
	<li>活动肠道（包括降结肠），并将它们朝向右上腹。用温热的盐水浸泡的纱布覆盖肠道。使用牵开器确保IVC和腹主动脉的最佳暴露。</li>
	<li>对肾下IVC和腹主动脉向分叉处进行钝性解剖。用 10-0 尼龙缝合线结扎所有肾下分支动脉和静脉（例如肠系膜下动脉和淋巴结动脉）。 注意:这些侧枝的解剖结构存在很大差异。在没有其他血流动力学监测的情况下，目视监测主动脉的脉搏和心率。通过脚趾捏试验每 15 分钟评估一次适当的麻醉深度。相应地调整异氟烷浓度。</li>
	<li>从新生大鼠身上采集供体心脏后，在无菌条件下将切除的心脏在含有Krebs-Henseleit缓冲液的手术盆中输送到手术区域。用冰冷的心脏麻痹溶液间歇性地冲洗供体心脏。 注意:当有第二位外科医生可用时，心脏应同时准备，因为第二位外科医生会减少受体动物的总麻醉时间和供体心脏的缺血时间。当没有第二位外科医生时，用温盐水覆盖接受者的腹部，并在收获过程中监测动物。</li>
	<li>将四个小的无创伤血管钳夹应用于肾下主动脉和IVC的远端和近端节段。如果需要，用 7-0 丝线缝合线暂时闭塞不利的肾血管，并在手术后松开缝合线。将 10-0 尼龙缝合线垂直放置在主动脉前壁上，以方便主动脉切开术。用微剪刀轻轻拉动缝合线，用两个小的水平切口（楔形）进行主动脉切开术。 注意:要去除任何血凝块，建议用肝素盐水冲洗主动脉腔。</li>
	<li>将供体心脏放在主动脉的左侧（从动物的角度），并用缝合线将受体的肾下主动脉和供体的升主动脉首尾固定在主动脉切开术的 12 点钟和 6 点钟位置。继续在 3 点钟和 9 点钟位置进行第三次和第四次缝合，在第三次缝合后轻轻地将心脏翻转到主动脉右侧。通过在每个间隙添加一到两条缝合线来完成动脉吻合。 注意:在进行吻合时，应注意避免用镊子接触供体的升主动脉或受体的腹主动脉，以避免组织损伤。</li>
	<li>逆时针旋转大鼠，头部朝向外科医生的左手。将供体的主动脉移至腹主动脉的左侧，以使IVC获得最佳视野。</li>
	<li>在主动脉吻合口稍近端的IVC上进行静脉切开术，使用11刀片进行穿刺，并使用微型剪刀根据供体肺干的直径进行适当的大小调整。再次，用肝素化盐水冲洗腔内腔。</li>
	<li>从受体的 IVC 和供体肺干之间的静脉吻合开始，最好方法是在血管后壁上放置中断的 11-0 尼龙缝合线，从 12 点钟和 6 点钟位置开始（与 IVC 相关），然后在前壁上放置连续的 11-0 尼龙缝合线（从 6 点钟方向到 12 点钟位置）。</li>
	<li>用可吸收明胶海绵的小条覆盖吻合口，并从远端开始移除微血管夹。使用棉头涂抹器轻轻压缩海绵以获得最佳止血效果。</li>
	<li>观察移植物的冠状动脉血管在远端微血管钳夹松开时充盈，并确保近端钳夹松开时供体心脏立即开始跳动。 注意:根据修改后的斯坦福评分19 ，可以在术中对移植物的活力进行 0 到 4 分的评分，以确认足够的移植物功能。</li>
	<li>将肠道放回腹部，确保不扭曲动脉和静脉吻合口。</li>
	<li>在动物完全麻醉以确定术后镇痛的同时，皮下给予美洛昔康（1mg / kg）和ethiqa XR（0.65mg / kg）。然后，用连续的 5-0 可吸收 vicryl 缝合线闭合腹壁，然后用皮内 6-0 可吸收 vicryl 缝合线闭合皮肤。 注意: 表 2 中提供了有关常见故障和故障排除的指南。</li>
</ol>3. 新生儿供体心脏的采集
<ol><li>将新生供体大鼠置于充有异氟烷（2%）的腔室中用于镇静。腹膜内给予氯胺酮 （75 mg/kg） 和甲苯噻嗪 （5 mg/kg） 以及肝素 （300 U/kg）。</li>
	<li>通过捏脚趾确认麻醉深度，并将大鼠置于仰卧位，尾巴朝向您。用倍他丁和70%乙醇交替消毒整个胸壁和腹壁三次。用无菌手术布覆盖大鼠。</li>
	<li>使用 12.5 倍手术显微镜，首先在木脑肌上使用 15 刀片手术刀进行水平切口，然后用剪刀横向切开垂直切口，直至两侧的腋窝，从而切除整个前胸壁。然后可以通过继续在颈部下方的另一个水平切口来切除前胸壁。</li>
	<li>用剪刀解剖IVC、左右上腔静脉和肺血管，然后用7-0丝线缝合并结扎所有血管。用 30 G 针刺穿 IVC 并用镊子轻轻向下推隔膜，将 3 mL 冰冷、高钾改良的 Krebs-Henseleit 溶液施用到右心房。</li>
	<li>用剪刀剪开 IVC、SVC、肺血管和主动脉。尽可能横断肺动脉和头臂干远端的主动脉，以确保使用 11 刀片手术刀的适当长度。</li>
	<li>用微剪刀分离肺干和升主动脉，并使用 3 mL 注射器用冰冷的心脏麻痹溶液冲洗心脏。</li>
</ol>4. 受体的恢复和移植物监测
<ol><li>手术后，给大鼠足够的时间醒来，这通常发生在15分钟的时间窗口内，并让它在加热垫上恢复。 注意:由于感染风险非常低，并且为了不损害实验模型，因此不需要抗生素，并且对食物或水没有限制。</li>
	<li>移植后，每天通过触诊移植心脏来监测移植功能，但考虑到由于肠道覆盖，有时难以评估。 注意:腹部超声心动图可以更准确地测量移植物的活力。对于超声心动图，用通过鼻锥吸入的异氟醚（1-2%）稍微镇静大鼠，并将其放置在加热垫上。超声心动图通常在术后第 1 天 （POD） 第 1 天、第 7 天和第 14 天进行。为了评估心率和收缩力，可以很容易地获得长轴和短轴视图（图2A，B）。为了评估吻合口，使用多普勒超声心动图（图3A），并确认EFE组织的形成，如左心室内的回声明亮心内膜层（图3B，C）。</li>
</ol>

体内心脏移植研究内皮到间充质转化

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这种将新生供体大鼠心脏异位移植到受体腹部的动物模型为通过详细的组织学组织评估、确定调节信号通路和测试治疗方案来研究 EndMT 衍生的纤维化创造了可能性。由于EndMT是心脏纤维化疾病的潜在机制，因此该模型在小儿心脏外科及其他领域具有重要价值。在此模型中，许多因素会对手术结果产生负面影响。因此，正确处理由于供体心脏不成熟而导致的非常脆弱的组织，在麻醉期间正确处理动?...

心内膜弹性纤维增生症 （EFE） 定义为心内膜下组织中胶原和弹性纤维的积聚，表现为珍珠状或不透明的心内膜增厚;EFE 在胎儿期和婴儿早期经历最活跃的生长1.在一项尸检研究中，70% 的左心发育不良综合征 （HLHS） 病例与 EFE2 的存在有关。
表达成纤维细胞标志物的细胞是 EFE 中的主要细胞群，但这些细胞也同时表达心内膜内皮标志物，这是这些 EFE 细胞起源的迹象。我们小组先前确定，EFE 形成的潜在机制涉及心内膜内皮细胞通过内皮间充质转化 （EndMT）3 向成纤维细胞的表型变化。EndMT 可以使用免疫组化双染色检测内皮标志物，例如分化簇 （CD） 31 或血管内皮 （VE）-钙粘蛋白 （CD144） 和成纤维细胞标志物（例如 α-平滑肌肌动蛋白、α-SMA）。此外，我们之前还通过激活转录因子 SLUG、SNAIL 和 TWIST3 确定了 TGF-ß 通路在此过程中的调节作用。
EndMT 是胚胎心脏发育过程中发生的生理过程，导致心内膜垫形成隔膜和瓣膜4，但它也会导致心力衰竭、肾纤维化或癌症的器官纤维化，并在血管动脉粥样硬化中起关键作用 5,6,7,8。心脏纤维化中的 EndMT 主要通过 TGF-β 通路调节，正如我们和其他人报道的那样 3,9.已描述各种刺激诱发 EndMT:炎症 10、缺氧 11、机械改变12 和血流障碍，包括腔内血流改变13，EndMT 也可能是遗传疾病的结果14。
该动物模型是使用心脏 EFE 发育的关键组成部分开发的，这些组成部分是腔内血流的不成熟和改变，特别是血流停滞。通过使用新生大鼠心脏作为供体来实现不成熟，因为已知新生大鼠在出生后立即发育不成熟。异位心脏移植提供了腔内血流限制15。
从临床角度来看，该动物模型可以更好地研究EndMT对左心室（LV）生长的影响。通过 EndMT 诱导的 EFE 形成对胎儿和新生儿心脏施加的生长限制16 排除了患有左心室流出道梗阻 （LVOTO） 的患者，例如先天性严重主动脉瓣狭窄和左心发育不良综合征 （HLHS） 的治愈性解剖学双心室手术修复17。该动物模型有助于通过EndMT研究细胞机制和组织形成的调节，并允许测试药物治疗方案3,18。
经腹超声心动图用于监测移植物的活力、收缩力和吻合口的通畅性。安乐死后，可以在移植后 3 天内宏观观察 EFE 的形成。EFE 组织显示出与 LVOTO 患者的人 EFE 组织相同的组织病理学特征。
因此，这种动物模型虽然是为HLHS谱系中的儿科使用而开发的，但可以应用于基于EndMT分子机制的各种疾病。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Advanced Ventilator System For Rodents, SAR-1000</td>
			<td>CWE, Inc.</td>
			<td>12-03100</td>
			<td>small animal ventilator</td>
		</tr>
		<tr>
			<td>aSMA</td>
			<td>Sigma</td>
			<td>A2547</td>
			<td>Antibody for Immunohistochemistry</td>
		</tr>
		<tr>
			<td>Axio observer Z1&#160;</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>inverted microscope</td>
		</tr>
		<tr>
			<td>Betadine&#160;Solution</td>
			<td>Avrio Health L.P.</td>
			<td>367618150092</td>
			<td></td>
		</tr>
		<tr>
			<td>CD31</td>
			<td>Invitrogen</td>
			<td>MA1-80069</td>
			<td>Antibody for Immunohistochemistry</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td>Antibody for Immunohistochemistry</td>
		</tr>
		<tr>
			<td>DemeLON Nylon black 10-0</td>
			<td>DemeTECH</td>
			<td>NL76100065F0P</td>
			<td>10-0 Nylon suture</td>
		</tr>
		<tr>
			<td>ETFE IV Catheter, 18G x 2</td>
			<td>TERUMO SURFLO</td>
			<td>SR-OX1851CA</td>
			<td>intubation cannula</td>
		</tr>
		<tr>
			<td>Micro Clip 8mm</td>
			<td>Roboz Surgical Instrument Co.</td>
			<td>RS-6471</td>
			<td>microvascular clamps</td>
		</tr>
		<tr>
			<td>Nylon black monofilament 11-0</td>
			<td>SURGICAL SPECIALTIES CORP</td>
			<td>AA0130</td>
			<td>11-0 Nylon</td>
		</tr>
		<tr>
			<td>O.C.T. Compound</td>
			<td>Tissue-Tek</td>
			<td>4583</td>
			<td>Embedding medium for frozen tissue specimen</td>
		</tr>
		<tr>
			<td>p-SMAD2/3</td>
			<td>Invitrogen</td>
			<td>PA5-110155</td>
			<td>Antibody for Immunohistochemistry</td>
		</tr>
		<tr>
			<td>Rodent, Tilting WorkStand</td>
			<td>Hallowell EMC.</td>
			<td>000A3467</td>
			<td>oblique shelf for intubation</td>
		</tr>
		<tr>
			<td>Silk Sutures, Non-absorbable, 7-0</td>
			<td>Braintree Scientific</td>
			<td>NC9201231</td>
			<td>Silk suture</td>
		</tr>
		<tr>
			<td>Slug/Snail</td>
			<td>Abcam</td>
			<td>ab180714</td>
			<td>Antibody for Immunohistochemistry</td>
		</tr>
		<tr>
			<td>Undyed Coated Vicryl 5-0 P-3 18&#34;</td>
			<td>Ethicon</td>
			<td>J493G</td>
			<td>5-0 Vicryl</td>
		</tr>
		<tr>
			<td>Undyed Coated Vicryl 6-0 P-3 18&#34;</td>
			<td>Ethicon</td>
			<td>J492G</td>
			<td>6-0 Vicryl</td>
		</tr>
		<tr>
			<td>VE-Cadherin</td>
			<td>Abcam</td>
			<td>ab231227</td>
			<td>Antibody for Immunohistochemistry</td>
		</tr>
		<tr>
			<td>Zeiss OPMI 6-SFR</td>
			<td>Zeiss</td>
			<td></td>
			<td>Surgical microscope</td>
		</tr>
		<tr>
			<td>Zen, Blue Edition, 3.6</td>
			<td>Zen&#160;</td>
			<td></td>
			<td>inverted microscope software</td>
		</tr>
	</tbody>
</table>

a neonatal heterotopic rat heart transplantation model for the study of endothelial-to-mesenchymal transition

心内膜弹性纤维增生症 （EFE） 定义为心内膜下组织积聚，对左心室 （LV） 的发育有重大影响，并排除先天性危重主动脉瓣狭窄和左心发育不良综合征 （HLHS） 患者进行根治性解剖双心室手术修复。手术切除是目前唯一可用的治疗选择，但 EFE 经常复发，有时甚至伴有更浸润的生长模式进入邻近心肌。
为了更好地了解 EFE 的潜在机制并探索治疗策略，开发了一种适合临床前测试的动物模型。动物模型考虑到 EFE 是一种未成熟心脏的疾病，并且与血流障碍有关，这得到了临床观察的支持。因此，新生大鼠供体心脏的异位心脏移植是该模型的基础。
将新生大鼠心脏移植到青春期大鼠的腹部，并与受体的肾下主动脉和下腔静脉相连。虽然冠状动脉的灌注保留了供体心脏的活力，但左心室内的血流停滞会诱导非常不成熟的心脏的 EFE 生长。EFE 形成的潜在机制是心内膜内皮细胞向间充质细胞 （EndMT） 的转变，这是瓣膜和隔膜早期胚胎发育的良好机制，也是心力衰竭纤维化的主要原因。移植后几天内可以宏观观察EFE的形成。经腹超声心动图用于监测移植物的活力、收缩力和吻合口的通畅性。安乐死后，采集 EFE 组织，其组织病理学特征与 HLHS 患者的人类 EFE 组织相同。
这种 体内 模型允许研究心脏中EFE发育的机制，并测试治疗方案以防止这种病理性组织形成，并为EndMT诱导的纤维化提供更普遍的检查机会。

移植物存活率和跳动 在这项工作中，在移除所有夹子后目视评估移植物的活力，并允许在开腹观察移植物的情况下进行大约 10-15 分钟的再灌注时间。手术结束时的视觉评估以及 POD 1、POD 7 和 POD 14 的超声心动图使用相同的评分系统客观地验证移植物活力。
0 = 无器官功能;1=（静止）器官功能，只有最小的收缩;2 = 器官功能弱或部分;3 = 收缩率或强度降低，但器官...

Watch this Scientific Journal Video about A Neonatal Heterotopic Rat Heart Transplantation Model for the Study of Endothelial-to-Mesenchymal Transition at JoVE.com

作者聚焦:用于研究内皮-间充质转化的新生儿异位大鼠心脏移植模型  

这项工作提出了一种内皮到间充质转化诱导的纤维化的动物模型，如先天性心脏缺陷，如严重主动脉瓣狭窄或左心发育不良综合征，可以进行详细的组织学组织评估、调节信号通路的鉴定和治疗方案的测试。

新生儿异位大鼠心脏移植模型在内皮间充质转化研究中的应用

Endocardial fibroelastosis (EFE), defined by subendocardial tissue accumulation, has major impacts on the development of the left ventricle (LV) and ...

心内膜纤维弹性变性阻碍了左心室生长，并阻碍了先天性危重主动脉瓣狭窄和左心发育不良综合征患者的双心室手术修复。虽然手术切除是目前唯一可用的治疗选择，但 EFE 经常复发。因此，我们旨在研究 EFE 背后的机制，并使用动物模型开发新的治疗策略。EndMT研究领域的主要发展仅限于具有固有局限性的细胞培养模型，并且由于缺乏体内模型，研究受到阻碍。我们小组先前确定，EFE形成的潜在机制涉及心内膜内皮细胞向成纤维细胞的表型变化，这一过程称为内皮到间充质转化。该动物模型允许对EndMT诱导的心脏纤维化进行更全面的检查，而其他模型则旨在通过基因修饰，高血压或饮食限制来诱导EndMT。由于 EFE 经常在初次切除后复发，有时甚至具有更浸润的生长模式，因此我们对与潜在心肌的分子相互作用特别感兴趣。

a-neonatal-heterotopic-rat-heart-transplantation-model-for-study

Research

JoVE Journal

Medicine

A Neonatal Heterotopic Rat Heart Transplantation Model for the Study of Endothelial-to-Mesenchymal Transition

1.1K 次观看.  Harvard Medical School. 心内膜纤维弹性变性阻碍了左心室生长，并阻碍了先天性危重主动脉瓣狭窄和左心发育不良综合征患者的双心室手术修复。虽然手术切除是目前唯一可用的治疗选择，但 EFE 经常复发。因此，我们旨在研究 EFE 背后的机制，并使用动物模型开发新的治疗策略。EndMT研究领域的主要发展仅限于具有固有局限性的细胞培养模型，并且由于缺乏体内模型，研究受到阻碍。我们小?...

视频: 作者聚焦:用于研究内皮-间充质转化的新生儿异位大鼠心脏移植模型  

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

主动脉原位移植大鼠模型，研究慢性血管病变的发展

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

科研

医学

Murine Heterotopic Heart Transplant Technique

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique was developed and reported in 2001 by Niimi. Described here are the techniques that have evolved over more than a decade in the hands of three surgeons (Plenter, Grazia, Pietra) in our center. These techniques are now being passed on to a younger generation of surgeons and researchers.
Based largely on the Niimi experience, the procedures used have evolved in the fine details - details which we will endeavor to relate here in such a way that others may be able to use this very useful model. Like Niimi, we have found that a video aid to learning is a priceless resource for the beginner.

The manuscript describes the steps required to perform the heterotopic heart transplant in the mouse.

小鼠异位心脏移植技术

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become ...

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

肺动脉瓣使用异位心脏移植小鼠模型移植

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

的隔离方法<em&gt;离体</em&gt;肺灌注的大鼠模型:教训制定鼠EVLP课程中学

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

Rat Heterotopic Abdominal Heart/Single-lung Transplantation in a Volume-loaded Configuration

Herein, we describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The configuration of the transplant graft involves anastomosis of donor inferior vena cava (IVC) to recipient IVC, and donor ascending aorta (Ao) to recipient abdominal Ao. The right upper and middle lung lobes are preserved and function as conduits for blood flow from right heart to left heart.
There are several advantages to using this technique, and it lends itself to a broad range of applications. Because the graft is transplanted in a configuration that allows for dyamic volume-loading, cardiac function may be directly assessed in vivo. The use of pressure-volume conductance catheters permits characterization of load-dependent and load-independent hemodynamic parameters. The graft may be converted to a loaded configuration by applying a clamp to the recipient&#8217;s infra-hepatic IVC. We describe modified surgical techniques for both donor and recipient operations, and an ideal myocardial protection strategy. Depending on the experimental aim, this model may be adapted for use in both acute and chronic studies of graft function, immunologic status, and variable ventricular loading conditions. The conducting airways to the transplanted lung are preserved, and allow for acute lung re-ventilation. This facilitates analysis of the effects of the mixed venous and arterial blood providing coronary perfusion to the graft.
A limitation of this model is its technical complexity. There is a significant learning curve for new operators, who should ideally be mentored in the technique. A surgical training background is advantageous for those wishing to apply this model. Despite its complexity, we aim to present the model in a clear and easily applicable format. Because of the physiologic similarity of this model to orthotopic transplantation, and its broad range of study applications, the effort invested in learning the technique is likely to be worthwhile.

We describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The transplant configuration results in a partially loaded graft circulation, allowing direct functional assessment. This model may be employed for acute or chronic studies of function and immunologic status of the transplanted graft.

大鼠异位心脏腹部/在卷装配置单肺移植

Herein, we describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The configuration of the transplant graft ...

Heterotopic Cervical Heart Transplantation in Mice

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies this model by avoiding challenging suture anastomoses of small vessels thereby reducing warm ischemia time. In comparison to abdominal graft implantation the cervical model is less invasive and the implanted graft is easily accessible for further follow-up examinations. Anastomoses are performed by pulling the ascending aorta of the graft over the cuff with the recipient&#8217;s common carotid artery and by pulling the main pulmonary artery over the cuff with the external jugular vein. Selection of appropriate cuff size and complete mobilization of the vessels are important for successful revascularization. Ischemia-reperfusion (I/R) injury can be minimized by perfusing the graft with a cardioplegic solution and by hypothermia. In this article, we provide technical details for a simplified and improved cuff technique, which should allow surgeons with basic microsurgical skills to perform the procedure with a high success rate.

The cuff technique shortens and facilitates the model of heterotopic cervical heart transplantation in mice by avoiding technically challenging suture anastomoses of small vessels. The technical details shown in this video paper should allow researches to establish this model in their laboratories.

颈椎异位心脏移植小鼠

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies ...

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression must be pursued. Employing vascularized bone marrow transplantation and co-transplantation of the thymus have shown promise in this regard in various animal models.1-11 Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism ultimately playing a supportive role in the induction of immune regulation. The combination of solid organ and vascularized composite allotransplantation can uniquely combine these strategies in the form of a novel transplant model. Murine models serve as an excellent paradigm to explore the mechanisms of acute and chronic rejection, chimerism, and tolerance induction, thus providing the foundation to propagate superior allograft survival strategies for larger animal models and future clinical application. Herein, we developed a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique. The experience in syngeneic and allogeneic transplant settings is described for future broader immunological investigations via an instructional manuscript and video supplement.

To study combined solid organ and vascularized composite allotransplantation, we describe a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique.

一种新的显微模型异位，整块胸壁，胸腺，和心脏移植小鼠

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

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

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

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

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

A Simplified Model for Heterotopic Heart Valve Transplantation in Rodents

There is an urgent clinical need for heart valve replacements that can grow in children. Heart valve transplantation is proposed as a new type of transplant with the potential to deliver durable heart valves capable of somatic growth with no requirement for anticoagulation. However, the immunobiology of heart valve transplants remains unexplored, highlighting the need for animal models to study this new type of transplant. Previous rat models for heterotopic aortic valve transplantation into the abdominal aorta have been described, though they are technically challenging and costly. For addressing this challenge, a renal subcapsular transplant model was developed in rodents as a practical and more straightforward method for studying heart valve transplant immunobiology. In this model, a single aortic valve leaflet is harvested and inserted into the renal subcapsular space. The kidney is easily accessible, and the transplanted tissue is securely contained in a subcapsular space that is well vascularized and can accommodate a variety of tissue sizes. Furthermore, because a single rat can provide three donor aortic leaflets and a single kidney can provide multiple sites for transplanted tissue, fewer rats are required for a given study. Here, the transplantation technique is described, providing a significant step forward in studying the transplant immunology of heart valve transplantation.

This protocol describes a simple and efficient method for the transplantation of aortic valve leaflets under the renal capsule to allow for the study of alloreactivity of heart valves.

啮齿动物异位心脏瓣膜移植的简化模型

There is an urgent clinical need for heart valve replacements that can grow in children. Heart valve transplantation is proposed as a new type of ...

A Porcine Heterotopic Heart Transplantation Protocol for Delivery of Therapeutics to a Cardiac Allograft

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts and complications such as primary graft dysfunction and graft rejection. The recent clinical use of an ex vivo perfusion device in cardiac transplantation introduces a unique opportunity for treating cardiac allografts with therapeutic interventions to improve function and avoid deleterious recipient responses. Establishing a translational, large-animal model for therapeutic delivery to the entire allograft is essential for testing novel therapeutic approaches in cardiac transplantation. The porcine, heterotopic heart transplantation model in the intraabdominal position serves as an excellent model for assessing the effects of novel interventions and the immunopathology of graft rejection. This model additionally offers long-term survival for the pig, given that the graft is not required to maintain the recipient's circulation. The aim of this protocol is to provide a reproducible and robust approach for achieving ex vivo delivery of a therapeutic to the entire cardiac allograft prior to transplantation and provide technical details to perform a survival heterotopic transplant of the ex vivo perfused heart.

We present a protocol for utilizing a normothermic ex vivo sanguinous perfusion system for the delivery of therapeutics to an entire cardiac allograft in a porcine heterotopic heart transplant model.

一种猪异位心脏移植方案，用于向心脏同种异体移植物提供治疗药物

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts ...