禽胚胎左心房结扎作为早期血管发育期间血流动力学负荷改变的模型
Summary
在这里，我们提出了一个详细的视觉方案，用于在禽类胚胎中执行左心房结扎（LAL）模型。LAL模型改变了心内血流，从而改变了壁剪切应力载荷，模仿左心发育不良综合征。提出了一种克服这种困难的显微手术模型挑战的方法。
Abstract
由于其四腔成熟心室结构、易于培养、成像可达性和效率，禽胚胎是研究心血管发育的首选脊椎动物模型。旨在了解先天性心脏缺陷正常发育和预后的研究广泛采用该模型。引入显微手术技术来改变特定胚胎时间点的正常机械负荷模式，并跟踪下游分子和遗传级联。最常见的机械干预是左卵黄静脉结扎术、头丛束带术和左心房结扎术 （LAL），通过血流调节壁内血管压和壁剪切应力。鲎试剂，特别是 卵产中进行的鲎试剂，是最具挑战性的干预措施，由于极其精细的连续显微外科手术，样本产量非常小。尽管风险很高，但 卵 巢鲎试剂在科学上非常有价值，因为它模仿左心发育不良综合征 （HLHS） 的发病机制。HLHS 是一种在人类新生儿中观察到的临床相关的复杂先天性心脏病。本文记录了 卵内 LAL的详细方案。简而言之，受精的禽胚胎通常在37.5°C和60%恒定湿度下孵育，直到它们达到汉堡-汉密尔顿（HH）阶段20至21。将蛋壳裂开，去除外膜和内膜。轻轻旋转胚胎以露出心房的左心房球。将 10-0 尼龙缝合线的预组装微结轻轻定位并系在左心耳芽周围。最后，胚胎回到原来的位置，LAL完成。正常心室和鲎试剂器械化心室在组织压实方面表现出统计学上的显着差异。高效的LAL模型生成管道将有助于研究心血管成分胚胎发育过程中的同步机械和遗传操作。同样，该模型将为组织培养研究和血管生物学提供扰动的细胞源。
Introduction
先天性心脏缺陷 （CHD） 是由于胚胎发育异常而发生的结构性疾病1.除遗传条件外，发病机制还受到机械负荷改变的影响2,3。左心发育不良综合征 （HLHS） 是一种先天性心脏病，导致出生时心室/主动脉发育不全4 死亡率高 5,6。尽管HLHS的临床管理取得了最新进展，但HLHS的血管生长和发育动态仍不清楚7。在正常胚胎发育中，随着早期胚胎心管形成的进展，左心室 （LV） 心内膜和心肌起源于心脏祖细胞。据报道，心肌小梁逐渐形成、增厚层和心肌细胞增生2.对于 HLHS，观察到小梁重塑改变和左心室扁平化，进一步导致心肌细胞异常迁移导致心肌发育不全 2,8,9,10
在广泛用于研究心脏发育和了解血流动力学状况的模式生物中 11，禽胚胎因其四腔成熟心脏和易于培养而受到青睐11,12,13,14。另一方面，斑马鱼胚胎和转基因/敲除小鼠的先进成像通道具有明显的优势11,12。已经对鸟类胚胎进行了各种机械干预测试，这些干预改变了发育心血管成分的壁内压力和壁剪切应力。这些模型包括左卵黄结扎术、髂状结扎术15 和左心房结扎术 （LAL）11、12、16。在以早期预后为重点的研究中，可以在手术干预后约 24-48 小时观察到由于机械负荷改变而产生的表型11,13。鲎试剂介入治疗是一种流行的技术，通过在房室开口周围放置缝合环来缩小左心房 （LA） 的功能容积。同样，还进行了针对右心房结扎 （RAL） 的显微外科干预17,18。同样，一些研究人员使用微夹靶向左心耳 （LAA） 以减少 LA19,20 的体积。在一些研究中，将手术尼龙线应用于房室结19,21。使用的干预措施之一是鲎试剂，它可以模仿HLHS，但也是最难执行的模型，由于需要极其精细的显微外科手术，样本产量非常小。在我们的实验室中，鲎试剂在汉堡-汉密尔顿 （HH） 第 20 期和第 21 期之间以卵形进行，在公共心房完全隔膜6、14、22、23 之前。在LA周围放置手术缝合线，从而改变心内血流。在HLHS的LAL模型中，观察到心室壁硬度增加，肌纤维角度改变和左心室腔大小减小14,24。
在本视频文章中，提供了 卵内 LAL的详细协议和方法。简而言之，将受精的禽胚胎孵育进行显微手术，将蛋壳裂开，并清除外膜和内膜。然后缓慢旋转胚胎，以便可以进入LA。将 10-0 尼龙手术缝合线绑在心房芽上，胚胎恢复到原来的方向，完成 LAL 程序25。 通过 光学相干断层扫描和基础组织学比较鲎试剂和正常心室的组织压实度和心室容积。
如本文所述，成功执行的LAL模型管道将有助于专注于心血管成分胚胎发育的基础研究。该模型还可以与遗传操作和高级成像模式一起使用。同样，急性鲎试剂模型是组织培养实验中病变血管细胞的稳定来源。
Protocol
受精的白色 Leghorn 卵是从值得信赖的供应商处获得的，并根据大学批准的指南进行孵化。雏鸡胚胎，第 18 阶段（第 3 天）至 24 阶段（第 4 天）（本文介绍的阶段）不被视为欧盟 （EU） 指令 2010/63/EU 和美国机构动物护理和使用委员会 （IACUC） 指南的活脊椎动物。根据美国法律，雏鸡胚胎在孵化第 19 天后被视为“活体动物”，但不适用于欧盟。每个鸡蛋都标有孵化开始日期，并计划在孵化的第…
Representative Results
先进的时间分辨成像技术可用于观察由于LAL干预引起的结构和形态变化10。此外，鲎试剂样品也适用于分子和生物学方法19,28。在表1中，提供了采用LAL模型结果的样本研究。在这种情况下，对达到 HH20-21 的雏鸡胚胎进行鲎试剂干预。对照（健康）和鲎试尔组织心脏均在HH25-26时从胚胎中取出。然后，将样品在4°…
Discussion
在 HLHS 中，由于结构缺陷导致血流改变，导致左侧形态异常 4,6。本模型提供了一个实用的实验系统，以更好地了解HLHS的进展，甚至可以模拟其发病机制8。然而，建立一个完全临床等效的HLHS动物模型是一项具有挑战性的任务。除了本文介绍的禽鲎模型外，最近对小鼠、胎羊和青蛙的研究还试图复制HLHS预后的形态学、血流动力学和病理…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢 Tubitak 2247A 首席研究员奖 120C139 提供资金。作者还要感谢 PakTavuk Gıda。A. S.，土耳其伊斯坦布尔，提供受精卵并支持心血管研究。
Materials
| 10-0 nylon surgical suture | Ethicon | ||
| Elastica van Gieson staining kit | Sigma-Aldrich | 115974 | For staining connective tissues in histological sections |
| Ethanol absolute | Interlab | 64-17-5 | For the sterilization step, 70% ethanol was obtained by diluting absolute ethanol with distilled water. |
| Incubator | KUHL, Flemington, New Jersey-U.S.A | AZYSS600-110 | |
| Kimwipes | Interlab | 080.65.002 | |
| Microscissors | World Precision Instruments (WPI), Sarasota FL | 555640S | Vannas STR 82 mm |
| Parafilm M | Sigma-Aldrich | P7793-1EA | Sealing stage for egg reincubation |
| Paraplast Bulk | Leica Biosystems | 39602012 | Tissue embedding medium |
| Stereo Microscope | Zeiss Stemi 508 | Stemi 508 | Used at station 1 |
| Stereo Microscope | Zeiss Stemi 2000-C | Stemi 2000-C | Used at station 2 |
| Tweezer (Dumont 4 INOX #F4) | Adumont & Fils, Switzerland | Used to return the embryo | |
| Tweezer (Super Fine Dumont #5SF) | World Precision Instruments (WPI), Sarasota FL | 501985 | Used to remove the membranes on the embryo |
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Cite This Article
Sevgin, B., Coban, M. N., Karatas, F., Pekkan, K. Left Atrial Ligation in the Avian Embryo as a Model for Altered Hemodynamic Loading During Early Vascular Development. J. Vis. Exp. (196), e65330, doi:10.3791/65330 (2023).