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。

Endocardial fibroelastosis (EFE), defined by the accumulation of collagen and elastic fibers in the subendocardial tissue, presents as a pearly or opaque thickened endocardium; EFE undergoes most active growth during the fetal period and early infancy1. In an autopsy study, 70% of cases with hypoplastic left heart syndrome (HLHS) were associated with the presence of EFE2.
Cells expressing markers for fibroblasts are the main cell population in EFE, but these cells also concomitantly express endocardial endothelial markers, which is an indication of the origin of these EFE cells. Our group previously established that the underlying mechanism of EFE formation involves a phenotypical change of endocardial endothelial cells to fibroblasts through endothelial-to-mesenchymal transition (EndMT)3. EndMT can be detected using immunohistochemical double-staining for endothelial markers such as cluster of differentiation (CD) 31 or vascular endothelial (VE)-cadherin (CD144) and fibroblast markers (e.g., alpha-smooth muscle actin, &#945;-SMA). Furthermore, we also previously established the regulatory role of the TGF-&#223; pathway in this process with activation of the transcription factors SLUG, SNAIL, and TWIST3.
EndMT is a physiological process that occurs during embryonic cardiac development and leads to the formation of the septa and valves from endocardial cushions4, but it also causes organ fibrosis in heart failure, kidney fibrosis, or cancer and plays a key role in vascular atherosclerosis5,6,7,8. EndMT in cardiac fibrosis is mainly regulated through the TGF-&#946; pathway, as we and others have reported3,9. Various stimuli have been described to induce EndMT: inflammation10, hypoxia11, mechanical alterations12, and flow disturbances, including alterations of the intracavitary blood flow13, and EndMT may also be a consequence of a genetic disease14.
This animal model was developed using the key components of cardiac EFE development, which are immaturity and alterations of the intracavitary blood flow, specifically flow stagnation. Immaturity was fulfilled by using neonatal rat hearts as donors, since neonatal rats are known to be developmentally immature immediately after birth. Heterotopic heart transplantation offered the provision of intracavitary flow restriction15.
From a clinical point of view, this animal model allows for better investigating the impact of EndMT on the growing left ventricle (LV). The growth restriction imposed on the fetal and neonatal heart through EndMT-induced EFE formation16 precludes patients with left ventricular outflow tract obstructions (LVOTO) such as congenital critical aortic stenosis and hypoplastic left heart syndrome (HLHS) from curative anatomical biventricular surgical repair17. This animal model facilitates the study of the cellular mechanisms and regulation of tissue formation through EndMT and allows for the testing of pharmacological treatment options3,18.
Transabdominal echocardiography is used to monitor the graft viability, contractility, and the patency of the anastomoses. Following euthanasia, EFE formation can be macroscopically observed within 3 days after transplantation. EFE tissue shows the same histopathological characteristics as human EFE tissue from patients with LVOTO.
Hence, this animal model, though developed for pediatric use in the spectrum of HLHS, can be applied when studying various diseases based on the molecular mechanism of EndMT.

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  

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 fibro elastosis hinders left ventricular growth and prevents biventricular surgical repair for patients with congenital critical aortic stenosis and hypoplastic left heart syndrome. While surgical resection is currently the only available treatment option, EFE frequently recurs. Therefore, we aimed to investigate the mechanisms behind EFE and develop new therapeutic strategies using an animal model.The main developments in the field of EndMT research are restricted to cell culture models which have inherent limitations, and research has been hampered due to a lack of in vivo models. Our group previously established that the underlying mechanism of EFE formation involves a phenotypical change of endocardial endothelial cells to fibroblasts, a process called endothelial to mesenchymal transition. This animal model allows for a more generalized examination of EndMT induced fibrosis in the heart, whereas other models are designed to induce EndMT through genetic modifications, hypertension, or dietary restrictions.As EFE often recurs after primary resection, sometimes with an even more infiltrative growth pattern, we are especially interested in the molecular interaction with the underlying myocardium.

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

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。

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

814 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  

Endocardial fibroelastosis (EFE), defined by subendocardial tissue accumulation, has major impacts on the development of the left ventricle (LV) and precludes patients with congenital critical aortic stenosis and hypoplastic left heart syndrome (HLHS) from curative anatomical biventricular surgical repair. Surgical resection is currently the only available therapeutic option, but EFE often recurs, sometimes with an even more infiltrative growth pattern into the adjacent myocardium.
To better understand the underlying mechanisms of EFE and to explore therapeutic strategies, an animal model suitable for preclinical testing was developed. The animal model takes into consideration that EFE is a disease of the immature heart and is associated with flow disturbances, as supported by clinical observations. Thus, the heterotopic heart transplantation of neonatal rat donor hearts is the basis for this model.
A neonatal rat heart is transplanted into an adolescent rat&#39;s abdomen and connected to the recipient&#39;s infrarenal aorta and inferior vena cava. While perfusion of the coronary arteries preserves the viability of the donor heart, flow stagnation within the LV induces EFE growth in the very immature heart. The underlying mechanism of EFE formation is the transition of endocardial endothelial cells to mesenchymal cells (EndMT), which is a well-described mechanism of early embryonic development of the valves and septa but also the leading cause of fibrosis in heart failure. EFE formation can be macroscopically observed within days after transplantation. Transabdominal echocardiography is used to monitor the graft viability, contractility, and the patency of the anastomoses. Following euthanasia, the EFE tissue is harvested, and it shows the same histopathological characteristics as human EFE tissue from HLHS patients.
This in vivo model allows for studying the mechanisms of EFE development in the heart and testing treatment options to prevent this pathological tissue formation and provides the opportunity for a more generalized examination of EndMT-induced fibrosis.

This work presents an animal model of endothelial-to-mesenchymal transition-induced fibrosis, as seen in congenital cardiac defects such as critical aortic stenosis or hypoplastic left heart syndrome, which allows for detailed histological tissue evaluation, the identification of regulatory signaling pathways, and the testing of treatment options.

科研

医学

Surgical Preparation and Heterotopic Transplantation of the Neonatal Donor Heart in the Recipient Rat

Begin harvesting the neonatal donor heart by anesthetizing and sterilizing the rat, and making a horizontal incision at the xiphoid with a 15 blade scalpel under a 12.5x surgical microscope. Next, use scissors to create vertical incisions laterally up to the axillae, on both sides. Remove the anterior thoracic wall with another horizontal incision beneath the neck.Dissect the inferior vena cava, or IVC, right and left superior venae cavae, or SVC, and pulmonary vessels with scissors. Encircle and ligate dull vessels using a 7-0 silk suture. Slightly push the diaphragm down with forceps and administer three milliliters of ice-cold, high-potassium modified Krebs-Henseleit solution to the right atrium by puncturing the IVC with a 30-gauge needle.Cut the IVC, SVC, and pulmonary vessels using scissors. Ensure proper length by transecting the pulmonary arteries as far as possible, and the aorta, distal to the brachiocephalic trunk, using a number 11 blade scalpel. Flush the heart with the ice-cold cardioplegic solution using a three milliliter syringe.Place the anesthetized rat on an oblique shelf. Secure its front teeth with a string, and position the head towards the surgeon. Place the light on the outside of the neck onto the area of the vocal cords.Grasp the tongue with two fingers and gently push it upwards and to the left for optimal intubation visibility. Now, use an 18-gauge, two-inch cannula for a 100 to 150 gram rat and secure the intratracheal tube with tape. Connect the intubation cannula to the small animal ventilator, and adjust the settings based on the animal's size, following the manufacturer's instructions.After intubating the rat, perform a midline laparotomy using a 15 blade scalpel to make the skin incision. Open the anterior abdominal wall with scissors and mobilize the intestines toward the right upper quadrant. Next, expose the retroperitoneal abdominal aorta and IVC, using cotton tip applicators.Cover the intestines with warm, saline-soaked gauze and use retractors to maintain optimal exposure of the IVC and abdominal aorta. Now, perform blunt dissection of the infrarenal IVC and abdominal aorta up to the bifurcation. Ligate dull infrarenal branching arteries and veins with a 10-0 nylon suture.Transport the harvested donor's heart in a sterile surgical basin containing Krebs-Heneseleit buffer to the surgical field. Ensure to irrigate the donor heart intermittently with ice-cold cardioplegic solution. Next, separate the pulmonary trunk and descending aorta using micro scissors, and irrigate the donor heart with ice-cold cardioplegic solution.Next, apply four atraumatic vascular clamps to the distal and proximal segments of the infrarenal aorta and IVC. Perform an aortotomy using micro scissors to make two small horizontal cuts. Then, place the donor heart on the left side of the aorta.Secure the recipient's infrarenal aorta and the donor's ascending aorta and to side at the 12 and six o'clock positions of the aortotomy with sutures. Proceed with the third and fourth sutures at the three and nine o'clock positions, gently flipping the heart to the left side of the aorta after the third suture. Finalize the arterial anastomosis by adding one to two sutures to each interspace.Now, rotate the rat counterclockwise, so the head faces the surgeon's left hand. Shift the donor's heart to the left side of the abdominal aorta for an optimal view of the IVC. Perform a venotomy on the IVC, slightly proximal to the aortic anastomosis.Using an 11 blade for puncture and micro scissors to adjust the size according to the donor's pulmonary trunk diameter, flush the intracoval lumen with heparinized saline. Next, perform the venous anastomosis between the recipient's IVC and the donor's pulmonary trunk by placing interrupted 11-0 nylon sutures on the back wall of the vessel, starting at the 12 and six o'clock positions. Place a continuous 11-0 nylon suture on the front wall, place absorbable gelatin sponge strips over the anastomoses, and remove the microvascular clamps, beginning with the distal ones.Ensure that the coronary vessels fill and that the donor's heart begins beating shortly upon release of the proximal clamps. Apply light pressure using a cotton tip applicator on the sponges to achieve optimal hemostasis. Carefully place the intestines back into the abdomen, being cautious not to distort the arterial and venous anastomosis.Close the abdominal wall and allow the animal to recover. A horizontal slice of the donor graft resected at the mid ventricular level of the right and left ventricles is shown. Histological analysis indicated that the endocardial fibroelastosis tissue had high amounts of organized collagen and elastin fibers.Endocardial fibroelastosis tissue, double stained for VE-Cadherin and alpha-SMA. CD-31, phospho-SMAD2 and SMAD3, CD-31 and slug or snail indicated endothelial to mesenchymal transition.