Name: 用于缺血再灌注研究的左前降冠状动脉结扎术:通过技术修改和质量控制改进模型 (视频) | JoVE Journal
Uploaded: 2022-12-16T12:40:00.000+00:00
Duration: 5 min 41 s
Description: 学习左前降冠状动脉结扎技术用于大鼠心肌缺血再灌注研究。该标准化方案通过质量控制改进传统手术方法，提高结扎准确性与实验可重复性，为心血管疾病研究提供可靠模型。

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5 minutes

该模型的开发获得了中国台湾科技部（MOST 109-2320-B-030-006-MY3）的经费支持。

所有动物实验均按照美国国立卫生研究院（NIH出版物编号85-23，1996年修订版）发布的《实验动物护理与使用指南》进行。本研究方案已获得辅仁大学机构动物护理与使用委员会的批准，并遵循其相关指南。
1. 手术前准备
<ol>
	<li>制备生理盐水湿棉球
	<ol>
		<li>戴上外科口罩和手套。</li>
		<li>取少量无菌棉，揉搓成球状。重复此操作。</li>
		<li>将棉球浸入无菌0.9%生理盐水中，挤去多余液体。</li>
		<li>将棉球存放于经75%乙醇消毒的洁净盒内。</li>
	</ol>
	</li>
	<li>制备固定钩
	<ol>
		<li>戴上外科口罩和手套。</li>
		<li>用75%乙醇对夹子和橡胶圈进行消毒。</li>
		<li>将夹子弯曲成钩状，用于固定胸壁和组织。</li>
		<li>用一个、两个或三个橡胶圈连接弯曲的夹子，确保手术窗切口的张力足够大，以便进行左前降支（LAD）结扎。</li>
		<li>在经75%乙醇消毒的洁净盒中准备并储存至少五个自制固定钩。</li>
	</ol>
	</li>
	<li>制备结扎环
	<ol>
		<li>将7-0丝线的中点置于锥形无针眼手术针（1/2圆，3号）的弹簧眼中。</li>
	</ol>
	</li>
	<li>制备套扎环控制器
	<ol>
		<li>用剪刀截取一段5 mm长的聚乙烯（PE）-10管。</li>
		<li>在火焰下加热并软化管子，使其两端边缘光滑。</li>
	</ol>
	</li>
	<li>大鼠的准备
	<ol>
		<li>选择8周龄雄性Sprague-Dawley大鼠，体重不低于250 g。</li>
		<li>将大鼠饲养在受控温度（21 °C ± 2 °C）下，维持12小时光照/12小时黑暗循环，并可自由摄取食物、标准小鼠颗粒饲料和自来水。</li>
		<li>通过腹腔注射戊巴比妥钠（50 mg/kg）对大鼠进行麻醉。 
		注：每小时应追加一次麻醉剂（戊巴比妥钠，30 mg/kg）。</li>
		<li>通过夹捏尾巴和后肢足部检查大鼠的反射，以确认其已充分麻醉。</li>
		<li>用剪刀剪开声门下方两个弹夹环之间的组织，插入一段3 cm长的PE-10管作为气管插管25。</li>
		<li>手动将气管插管连接至呼吸机。</li>
		<li>观察动物胸廓运动是否与呼吸周期同步，以确认肺部通气充分。</li>
		<li>切开颈部区域，进行颈静脉插管26。</li>
	</ol>
	</li>
</ol>
2. 左前降支结扎
<ol>
	<li>戴上外科口罩和手套。</li>
	<li>触摸胸部，找到胸骨柄和胸骨角（胸骨柄与胸骨体的连接处）。</li>
	<li>通过手动触摸，确定与胸骨角相连的左侧肋骨（肋骨A）。</li>
	<li>确定位于肋骨A正下方的肋间隙。使用尖头镊子轻轻提起靠近肋间隙处的皮肤，然后使用带刀片的外科手术刀，沿皮肤张力线从距胸骨体左侧约5 mm处开始，做一条长约1 cm的斜切口。</li>
	<li>使用弯头镊子轻轻分离切口处的皮肤和肌肉层。用弯夹钩住左前胸壁外侧的肌肉层并向下牵拉，以暴露下方的肋骨。</li>
	<li>确定位于肋骨A下方的肋骨（肋骨B）。用钝头剪刀从肋软骨中部（距胸骨体约2–3 mm）剪断肋骨B。若出现出血，可用生理盐水浸湿的棉球轻轻按压伤口数秒。</li>
	<li>利用四个弯夹从肋骨B的切口处小心打开胸腔。每个弯夹应钩住肋间肌和肋骨，向四个方向（即左上方、右上方、左下方和右下方）轻柔撑开胸壁，形成一个矩形手术窗口。</li>
	<li>用另一个弯夹轻轻钩住覆盖在心包上的左肺及其他邻近组织，以防止操作过程中意外损伤组织。</li>
	<li>用镊子轻轻去除薄层心包膜，暴露心脏。辨认左冠状动脉主干（LMCA）的第一分支，通常位于肺动脉与左心耳之间。左冠状动脉主干及其前降支（LAD）表现为一条浅表的鲜红色线条，从左心耳边缘向心尖方向走行。</li>
	<li>使用预先准备好的手术针，在左前降支（LAD）上位于左冠状动脉主干（LMCA）第一分支远端紧邻处，从LAD左侧向右侧方向穿入并穿过丝线，形成一个开放结扎环，以避免意外刺破左心耳。通过单次缝合形成开放环。若因液体或血液覆盖导致LAD不可见，可轻轻擦拭心脏表面以显露冠状动脉。</li>
	<li>握住缝线一端，使用持针器轻轻将针从缝线中分离。</li>
	<li>将开放环一侧的丝线两端穿入另一侧的环中，形成一个套索环。</li>
	<li>在闭合套索环之前，将套索环上丝线的两端插入预先准备好的套索控制器中。</li>
	<li>沿丝线滑动套索控制器，同时轻柔拉紧丝线以闭合套索环，阻断LAD的冠状动脉血流，诱导暂时性心肌缺血，持续1小时。</li>
	<li>当套索环牢固结扎后，用Kelly止血钳夹住丝线以固定套索控制器的位置。在LAD结扎期间，将Kelly止血钳的另一端放置于手术台上。</li>
	<li>在LAD结扎期间，用生理盐水浸湿的棉球覆盖手术窗口。</li>
	<li>松开Kelly止血钳。</li>
	<li>释放套索控制器，恢复冠状动脉血流，进行再灌注，持续2小时。</li>
	<li>沿心脏基底部和血管边界小心切除心脏，避免夹持组织。 
	注：以每分钟40%笼体体积的流速使用CO2&#160;处死大鼠&#160;。</li>
</ol>

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<li>De Villiers, C., Riley, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+models+of+myocardial+infarction%3A+Comparing+permanent+ligation+and+ischemia-reperfusion%5BTitle%5D)+AND+%22Disease+Models+%26+Mechanisms%22%5BJournal%5D)">Mouse models of myocardial infarction: Comparing permanent ligation and ischemia-reperfusion.</a> Disease Models & Mechanisms. 13 (11), (2020).</li>
<li>Reichert, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Murine+left+anterior+descending+%28LAD%29+coronary+artery+ligation%3A+An+improved+and+simplified+model+for+myocardial+infarction%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Murine left anterior descending (LAD) coronary artery ligation: An improved and simplified model for myocardial infarction.</a> Journal of Visualized Experiments. (122), e55353(2017).</li>
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作者声明本文的发表不存在利益冲突。

本方案具有多个显著特点，例如可确定左前降支结扎的精确位置，设计一种装置以单根缝线控制套索环，并采用改良的外科操作手法以减少组织损伤，从而帮助研究人员准确、牢固且一致地结扎左前降支，同时能够即时调控套索环的状态，用于急性心肌缺血-再灌注研究。
左前降支结扎的位置会影响心肌梗死的范围和大小。通常建议在左前降支近端的某一特定距离处进行结扎27,28。忽略冠状动脉分支模式的个体差异可能会增加心肌梗死面积的变异性23,24。在本研究中，左前降支结扎位置选择在左主冠状动脉第一分支的远端紧邻处，从而避免意外结扎左回旋支或室间隔支，有助于获得稳定的心肌梗死面积，并降低致死性心律失常的发生概率29,30,<sup class="x...

缺血性心脏病是全球主要的死亡原因1,2。除了控制可改变的危险因素以预防冠心病的发生外，急性冠状动脉综合征的治疗策略也至关重要3,4。研究发现，急性ST段抬高型心肌梗死（STEMI）中的心源性休克和致死性心律失常会增加院内死亡的风险5,6,7,8。经皮冠状动脉介入治疗（PCI）是STEMI的首选治疗方法9,10,11；然而，当门球时间（door-to-balloon time）&#60;90分钟时，治疗效果已接近上限12,13。因此，需要其他辅助策略以进一步改善该疾病的临床预后14,15,16,17,18,19。
通过结扎大鼠左前降支动脉（LAD）建立急性心肌缺血-再灌注（IR）实验，是模拟临床情境的一种动物模型，该情境要求ST段抬高型心肌梗死（STEMI）患者在极短时间内实现从入院到球囊扩张的治疗，以挽救心肌免受缺血性损伤。然而，在小型动物中实施手术诱导的STEMI常面临较大的技术挑战，因其属于复杂手术操作，常伴随较高的死亡率以及梗死面积的高度变异性20,21,22,23,24。为克服这些技术难题，本研究建立了一种全面且高效的适用于大鼠（因其体型大于小鼠）的动物模型，通过技术改进，制定出一套可靠且可重复的急性心肌IR研究方案。该方案可显著减少手术并发症、减轻组织损伤，并降低术中死亡风险。此外，本研究还采用了测定梗死面积及风险区域（AAR）的方法，以验证研究结果的质量。该方案可用于探究急性心肌IR应激的病理生理过程，进而为开发针对心肌损伤的新型治疗策略提供支持。

<table><tbody><tr><td>Evan&amp;rsquo;蓝色</td><td>Sigma Aldrich</td><td>E2129</td><td></td></tr><tr><td>镊子</td><td>Shinva</td><td></td><td></td></tr><tr><td>戊巴比妥</td><td>Sigma Aldrich</td><td>1507002</td><td></td></tr><tr><td>手术刀片</td><td>Shinva</td><td>s2646</td><td></td></tr><tr><td>手术刀柄</td><td>Shinva</td><td></td><td></td></tr><tr><td>丝线缝合线</td><td>SharpointTM</td><td>DC-2150N</td><td></td></tr><tr><td>外科缝合针</td><td>AnchorTM</td><td></td><td></td></tr><tr><td>氯化三苯基四氮唑（TTC）溶液</td><td>Solarbio</td><td>T8170-1</td><td></td></tr><tr><td>呼吸机</td><td>哈佛小鼠呼吸机</td><td></td><td></td></tr></tbody></table>

左前降支冠状动脉结扎用于缺血-再灌注研究：通过技术改进与质量控制优化模型

Left Anterior Descending Coronary Artery Ligation for Ischemia-Reperfusion Research: Model Improvement via Technical Modifications and Quality Control

冠心病是全球死亡的首要原因。冠状动脉血流完全中断会导致ST段抬高型心肌梗死（STEMI），进而引发心源性休克和致死性心律失常，这些并发症与高死亡率密切相关。通过经皮冠状动脉介入治疗（PCI）实现冠状动脉再通可显著改善STEMI患者的预后，但尽管在缩短门球时间方面已取得进展，院内死亡率并未随之降低，提示需要额外的治疗策略。在大鼠中进行左前降支冠状动脉（LAD）结扎是一种急性心肌缺血-再灌注（IR）研究的动物模型，其临床对应情况为通过PCI快速实现冠状动脉再通以治疗STEMI；然而，PCI诱导的STEMI是一种技术要求高且操作复杂的手术，常伴随较高的死亡率以及梗死面积的巨大变异性。我们确定了LAD结扎的最佳位置，设计了一种用于控制套索环的装置，并改进了外科操作手法，从而减少组织损伤，建立了一种可靠且可重复的大鼠急性心肌缺血-再灌注（IR）研究方案。该手术为非存活性手术。我们还提出了一种用于验证研究结果质量的方法，这是确保后续生化分析准确性的关键步骤。

在心肌缺血和再灌注结束时，应在进行进一步的生物化学或分子分析之前评估LAD结扎的质量。
通过中心静脉导管注入1 mL的2%伊文思蓝染料，以评估结扎法阻断左前降支（LAD）的充分性。有冠状动脉灌注的心肌被染成蓝色，而无灌注区域仍呈红色（图1A）。红色区域即为心肌梗死的缺血风险区（AAR）。
通过量化实验动物间AAR百分比的变异程度，进一步评估了LAD结扎位置的准确性。心脏水平切片后，AAR百分比通过将AAR除以整个心肌质量来确定（图1B）。实验动物间AAR百分比变异程度较低，表明LAD结扎位置准确。
心肌梗死面积是急性心肌缺血再灌注（IR）研究中的主要结果指标。为定量该参数，将切片的心脏组织切片在37 °C条件下用1% 2,3,5-三苯基四氮唑氯化物（TTC）生理盐水溶液孵育30分钟，随后用10%甲醛固定3天。梗死区域呈白色。梗死面积百分比计算为梗死区面积...

本文介绍了一种针对左前降支冠状动脉结扎术质量控制的实验方案，通过在技术上改进大鼠急性心肌缺血-再灌注研究中的传统操作步骤，以提高实验的稳定性和可重复性。

冠心病是全球死亡的首要原因。冠状动脉血流完全中断会导致ST段抬高……

缺血性心脏病是全球主要的死亡原因。本实验可用来在心脏中诱导缺血-再灌注损伤。该方案旨在准确、牢固且一致地结扎前降支动脉（LAD）。制备生理盐水湿润棉球时，取少量无菌棉，反复揉搓成球状。将棉球浸入无菌0.9%生理盐水中，挤去多余液体。将棉球存放于经75%乙醇消毒的洁净容器中。制备持钩时，用75%乙醇对夹子和橡胶圈进行灭菌。制备结扎环时，将7-0丝线缝合线的中点置于锥形无孔外科缝合针的3号半圆形针眼中。制备套环控制器时，使用剪刀截取一段5毫米长的聚乙烯10号管。接下来，进行实验时，麻醉一只八周龄的雄性Sprague Dawley大鼠，并通过夹捏尾巴和后肢足部检查反射。然后用剪刀剪开声门下方两个软骨环之间的组织，插入一根3厘米长的聚乙烯50导管作为气管内插管。将气管内插管连接至呼吸机。触摸胸部，找到胸骨柄和胸骨角。然后确定与胸骨角相连的左侧肋骨。确定肋骨A下方的肋间隙。使用尖头镊子轻轻提起靠近肋间隙的皮肤。然后使用剪刀从胸骨体左侧约五毫米处开始，沿皮肤张力线做一厘米长的斜切口。用剪刀分离切口处的皮肤和肌肉层。接着用弯夹将左前胸壁外侧的肌肉层向下钩起，以暴露下方的肋骨。在确定肋骨A下方的肋骨B后，使用钝头剪刀从肋软骨中部剪断肋骨B。如果出现出血，可用生理盐水浸湿的棉球轻轻接触并压迫伤口数秒钟。接着，将四个弯钩夹分别钩住肋间肌和肋骨，向四个方向轻轻牵拉胸壁，形成一个矩形手术窗口，从而从肋骨B的切口处小心打开胸腔。再用另一个弯钩夹轻轻钩住左肺及覆盖心包的邻近组织，以防止手术过程中意外损伤组织。用镊子去除薄层心包膜，暴露心脏。然后在肺动脉和左心耳之间辨认左主冠状动脉（LMCA）的第一个分支。使用外科针，在LMCA第一分支远端紧邻处，从左向右穿过左前降支（LAD）下方，将丝线缝合穿过LAD下方形成一个开放的结扎环，以避免意外刺破左心耳。如果由于液体或血液覆盖心脏表面而无法看到左前降支（LAD），可轻轻擦拭心脏表面以显露冠状动脉。将开口环一侧的丝线两端穿入另一侧的环圈内，形成套索环。然后在闭合环圈之前，将套索环的丝线两端插入预先准备好的套索控制器中。在轻轻拉伸丝线以闭合套索环的同时，将套索环控制器沿丝线滑动。阻断左前降支（LAD）的冠状动脉血流，以诱导心肌缺血持续一小时。待套索环牢固结扎后，用血管钳夹住丝线，固定套索环控制器的位置。将Kelly止血钳的另一端置于手术台上。在结扎LAD期间，用生理盐水浸湿的棉球覆盖手术窗口。打开Kelly止血钳，然后松开套线环控制器，以恢复冠状动脉血流灌注两小时。通过伊文思蓝染色验证LAD结扎质量。有冠状动脉灌注的心肌被染成蓝色，而无灌注区域则保持红色。实验动物中风险区域面积（AAA百分比）变异程度较低，表明LAD结扎位置准确。此外，通过计算梗死面积占对照组和治疗组AAR的比值来评估梗死大小。治疗组的梗死面积百分比低于对照的LAD结扎组。该技术

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用于缺血再灌注研究的左前降冠状动脉结扎术:通过技术修改和质量控制改进模型

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大鼠急性心肌梗死模型

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