这项工作得到了NIH的OD R24拨款OD031956和NICHD R01拨款HD073104的支持。我们感谢达西·凯利（Darcy Kelly）对该协议的有益讨论和初步意见。我们还要感谢Samantha Jalbert，Jill Ralston和Wil Ratzan的帮助和支持，以及我们的三位匿名同行评审员的反馈。

所有实验均按照哈佛医学院IACUC（机构动物护理和使用委员会）（IS 00001365_3）的规则和规定进行。
注意：尽管美国兽医协会12认为所描述的主要安乐死方法是一种可接受的安乐死技术，但尚未发现它会导致心跳停止13。即使是常用的二次双皮管方法也不能阻止这种情况，也不能从动物身上取出心脏。麻醉动物的放血被认为是成功安乐死的人道和有效的方法12.由于通过安乐死维持新鲜组织是该协议的目标，因此心脏继续通过MS-222的初级安乐死跳动是有益的，并且灌注本身是通过放血的继发性安乐死方法。
1. 准备
<ol><li>确保研究机构已批准本协议中描述的安乐死和灌注技术。</li>
	<li>制备5 g / L MS-222（甲磺酸三卡因）和5 g / L碳酸氢钠的溶液。体积应大于完全覆盖被安乐死动物所需的体积。检查pH值以确保其为≥7。</li>
	<li>每乳杆菌制备 500 μL 180 U/mL 肝素的磷酸盐缓冲盐水 （PBS），或每热带乳杆菌制备 200 μL。</li>
	<li>通过将 非洲爪蟾 放入该溶液中进行初级安乐死（从步骤1.2开始）;动物将保持浸没状态总共1小时。</li>
	<li>确认 非洲爪蟾 在安乐死后 15 分钟捏住脚失去了疼痛反应。如果动物有反应，请将其返回到安乐死溶液中，直到失去这种反应。</li>
	<li>称量非洲爪蟾并在取样前进行所需的任何其他测量。</li>
	<li>使用 31 G 针头，将 250 μL 的 X. laevis 和 100 μL 180 U/mL 肝素的 PBS（来自步骤 1.3）注射到每个前肢的肌肉组织中。</li>
	<li>在PBS灌注液中制备1mL / g动物重量的54U / mL肝素溶液。对该方案更有经验的人可能会发现完成灌注所需的培养基更少。</li>
	<li>使用 22 G 皮下注射针头灌注 X. laevis，使用 25 G 皮下注射针头灌注 热带弧菌。用线切割器修剪尖端，使灌注针变钝（图1）14。 注意：如果移动，这降低了针头穿过心室的可能性。除了钝化外，针头可能会用磨刀石或锉刀稍微磨碎，但仍保持足够锋利以刺穿心室。</li>
	<li>通过连接修剪好的针头并循环 54 U/mL 肝素化 PBS 灌注物（从步骤 1.8 开始）来准备泵。确保清除管道中的所有气泡，以消除空气栓塞的可能性，这会导致灌注效率降低或失败（见 表1）。在手术过程中将灌注介质保持在冰上。</li>
	<li>如果泵不可编程，请在针头就位的情况下，测量不同设置下的泵送介质体积，以确定哪些设置最接近 5 mL/min 和 10 mL/min。无论物种如何，都将使用这些流速。如果灌注泵是可编程的，请按照制造商的说明使用针头进行校准。</li>
	<li>将解剖表面（托盘或泡沫板）放在辅助容器内的斜坡处，或将其布置以方便血液引流。</li>
	<li>一旦青蛙在溶液中放置1小时，初级安乐死就完成了。取出青蛙，并通过捏脚重新检查疼痛反应的丧失。</li>
	<li>将青蛙放在背部并固定每个肢体（图2）。如果需要保留四肢组织，可以通过四肢周围的手指或 U 形订书钉放置细针。</li>
	<li>用解剖剪刀，切开皮肤，沿着中线向上，然后横向，做两个皮瓣。（图2）</li>
	<li>使用镊子抓住白线并将其从腔腔中拉开（图3）。小心地用剪刀剪开肌肉组织。从腔壁上制作两个襟翼，并将所有襟翼切开或固定。</li>
	<li>使用解剖剪刀切开喙骨并切掉多余的组织，以更好地进入心脏（图3）。</li>
	<li>心脏应该还在跳动。如果心脏在灌注前停止跳动，请注意样品新鲜度已受到影响。</li>
</ol>2. 灌注
<ol><li>识别胃并轻轻移动它，使其位于肝脏左叶的顶部（在观察者的右侧），其脉管系统在手术过程中可见。识别肺并使用组织钳抓住它的尖端。将肺拉出腔腔外并将其固定在尖端（图4）。轻轻地这样做，因为破裂的血管不能很好地灌注。注意叶内是否可见血液，因为这会影响确定手术完成的能力。</li>
	<li>拍摄腔腔的图像，以更好地评估灌注效率，并可能在以后识别异常组织。</li>
	<li>识别薄心包并用组织钳将其拉动（图5）。使用虹膜切除剪刀的尖端轻轻穿孔心包，注意不要切割下面的组织。将心包从心脏的三个腔室剥离。</li>
	<li>用镊子轻轻抓住心室的顶点。施加有限的压力，以便在镊子的牵引表面之间有足够的空间供灌注针通过（图6）。</li>
	<li>通过镊子的闭合将针头插入心室腔室，注意不要穿过心室（图7）。使用止血器使用持针器将组织钳夹到位。 注意：此技术可稳定针的位置，该位置仍然锋利。将针头直接夹在心室上也会造成不必要的损伤，如果需要重新夹紧，则使重新夹紧更加困难（见 表1）。</li>
	<li>以大约 5 mL/min 的速度启动泵的流量。心脏和动脉干的三个腔室会充血（图8;见 表1）。</li>
	<li>用剪刀小心地用右耳廓（在观看者的左侧）;鲜血会涌出。将流速设置为 5 mL/min 或增加到 10 mL/min。</li>
	<li>继续直到胃的脉管系统变白（见 表1），然后刮起心脏的左耳廓（在观察者的右侧）。如果流速仍为 5 mL/min，请将其增加到大约 10 mL/min。</li>
	<li>使用移液管冲洗灌注介质中的腔腔，以帮助保持可见性并更好地评估从耳廓流出的灌注液的颜色。</li>
	<li>将针保持在原位，直到从耳廓流出的灌注液清除（见 表1）并且肺失去红色（见 表1; 图9）。</li>
</ol>

用于转录组学和蛋白质组学的非洲爪蛙血液灌注

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(124), e55740 (2017).</li><li>Lametschwandtner, A., Minnich, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+anatomy+of+the+brain+of+the+adult+pipid+frog,+Xenopus+laevis+(Daudin):+A+scanning+electron+microscopic+study+of+vascular+corrosion+casts.">Microvascular anatomy of the brain of the adult pipid frog, Xenopus laevis (Daudin): A scanning electron microscopic study of vascular corrosion casts.</a> Journal of Morphology. 279 (7), 950-969 (2018).</li><li>Lametschwandtner, A., Minnich, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+anatomy+of+the+urinary+bladder+in+the+adult+African+clawed+toad,+Xenopus+laevis:+A+scanning+electron+microscope+study+of+vascular+casts.">Microvascular anatomy of the urinary bladder in the adult African clawed toad, Xenopus laevis: A scanning electron microscope study of vascular casts.</a> Journal of Morphology. 282 (3), 368-377 (2021).</li><li>Lametschwandtner, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+anatomy+of+the+gallbladder+of+the+adult+South+African+clawed+toad,+Xenopus+laevis+Daudin:+A+scanning+electron+microscope+study+of+vascular+corrosion+casts.">Microvascular anatomy of the gallbladder of the adult South African clawed toad, Xenopus laevis Daudin: A scanning electron microscope study of vascular corrosion casts.</a> Microscopy and Microanalysis. 13, 492-493 (2007).</li><li>Lametschwandtner, A., Spornitz, U., Minnich, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+anatomy+of+the+non-lobulated+liver+of+adult+Xenopus+laevis:+A+scanning+electron+microscopic+study+of+vascular+casts.">Microvascular anatomy of the non-lobulated liver of adult Xenopus laevis: A scanning electron microscopic study of vascular casts.</a> Anatomical Record. 305 (2), 243-253 (2022).</li><li>Miodo&#324;ski, A. J., B&#228;r, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arterial+supply+of+the+choriocapillaris+of+anuran+amphibians+(Rana+temporaria,+Rana+esculenta).+Scanning+electron-microscopic+(SEM)+study+of+microcorrosion+casts.">Arterial supply of the choriocapillaris of anuran amphibians (Rana temporaria, Rana esculenta). Scanning electron-microscopic (SEM) study of microcorrosion casts.</a> Cell and Tissue Research. 249 (1), 101-109 (1987).</li><li>Nenni, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenbase:+Facilitating+the+use+of+Xenopus+to+model+human+disease.">Xenbase: Facilitating the use of Xenopus to model human disease.</a> Frontiers in Physiology. 10, 154 (2019).</li><li>Tandon, P., Conlon, F., Furlow, J. D., Horb, M. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+physiology+and+diseases+of+amphibians.+Veterinary+clinics+of+North+America.">Cardiovascular physiology and diseases of amphibians. Veterinary clinics of North America.</a> The Veterinary Clinics of North America. Exotic Animal Practice. 12 (1), 39-50 (2009).</li><li>Stephenson, A., Adams, J. 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作者声明没有竞争利益。

该协议描述了用于进入腔腔的传统解剖技术。其他技术也是可以接受的，只要它们对组织造成的损害最小，心脏是可接近的，肺和胃是可见的。同样，列出的大多数解剖工具都可以很容易地用类似的项目代替。
虽然已经尝试优化该程序的功效，但结果可能会因一个人的经验和个体青蛙之间的变异性而异。血液灌注的一个有趣的方面仍然不在本文的范围之内，即该程序与接受?...

为了保存和固定的目的，常规完成两栖动物的全身灌注1，2，3，4，5，6。然而，这些程序的发生速度限制了每只动物可以采集的新鲜样本数量。这项工作的目标是在非洲爪蟾中开发一种有效的血液灌注方案，优先考虑该技术的速度。对于热带X. laevis，每只动物需要不到10分钟，每只X. laevis动物需要不到15分钟。次要优先事项是易于复制和使用易于获取的设备，以便可以在非洲爪蟾实验室之间广泛共享高质量的样品。
非洲爪 蟾广泛用于生物医学研究，以研究跨物种保存的基本生物学和病理过程。这种四足动物与其他水生模型相比，与哺乳动物有更密切的进化关系，有肺、三腔心脏和带手指的四肢。国际社会有效地利用 非洲爪蟾 ，通过对疾病相关基因功能的深入疾病建模和分子分析，对人类疾病有了更深入的了解。 非洲爪蟾 作为动物模型的众多优点使其成为研究人类发育和疾病的分子基础的宝贵工具;这些优点包括：卵母细胞和胚胎大小大，繁殖力高，易于容纳，快速的外部发育以及易于基因组操作。据估计，非洲 爪蟾 共享~80%的已确定的人类疾病基因7。
与流行的哺乳动物模型相比，非洲爪蟾是一种快速、经济高效的模型，具有吗啉蛋白敲低的易用性，并且可以使用CRISPR8进行高效的转基因和靶向基因突变。定量质谱和单细胞转录组学已成功应用于非洲爪蟾胚胎9，10，但最近来自非洲爪蟾的细胞图谱显示，大多数组织的组成以血细胞11型为主。通过开发一种快速放血组织并使用冷却培养基的技术，样品新鲜度受灌注的影响最小。这对于以分析生理上不受干扰的mRNA或蛋白质表达为目标的应用尤其重要。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5x Magnifying glass with LED light and stand</td>
			<td>amazon.com</td>
			<td>B08QJ6J8P1</td>
			<td>light must not produce heat</td>
		</tr>
		<tr>
			<td>Disposable transfer pipets</td>
			<td>VWR</td>
			<td>414004-036</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting fine-pointed forceps</td>
			<td>Fisher Scinetific</td>
			<td>08-875</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissors sharp piont, straight 6.5&#34;</td>
			<td>VWR</td>
			<td>76457-374</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection tray</td>
			<td>Fisher Scinetific</td>
			<td>14-370-284</td>
			<td>styrofoam sheets are an acceptable alternative</td>
		</tr>
		<tr>
			<td>Euthanasia container</td>
			<td>US Plastic</td>
			<td>Item&#160;2860</td>
			<td>alternative opaque containers acceptable</td>
		</tr>
		<tr>
			<td>Euthanasia container lid</td>
			<td>US Plastic</td>
			<td>Item&#160;3047</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine dissection pins</td>
			<td>Living Systems Instrumentation</td>
			<td>PIN-#3</td>
			<td></td>
		</tr>
		<tr>
			<td>General use hypodermic needles, 22 G</td>
			<td>Fisher Scientific</td>
			<td>14-826-5A</td>
			<td>for X. laevis</td>
		</tr>
		<tr>
			<td>General use hypodermic needles, 25 G</td>
			<td>Fisher Scientific</td>
			<td>14-826AA</td>
			<td>for X. tropicalis</td>
		</tr>
		<tr>
			<td>Heparin, porcine intestinal mucosa</td>
			<td>MilliporeSigma</td>
			<td>37-505-410MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Iridectomy scissors 6&#34;</td>
			<td>vwr</td>
			<td>470018-938</td>
			<td>iris scissors are an acceptable alternative</td>
		</tr>
		<tr>
			<td>Luer-to-barb adapter male Luer with lock ring</td>
			<td>amazon.com</td>
			<td>B09PTX6M2Z</td>
			<td>size will be dependant on the hosing of the pump used</td>
		</tr>
		<tr>
			<td>Mayo-Hegar needle holder</td>
			<td>Fisher Scinetific</td>
			<td>08-966</td>
			<td>mosquito forceps are an acceptable alternative</td>
		</tr>
		<tr>
			<td>MS-222: Syncaine (formerly tricaine)</td>
			<td>Pentair AES</td>
			<td>TRS1</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1x</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic liquid pump dosing pump 5&#8211;100 mL/min</td>
			<td>amazon.com</td>
			<td>B07PWY4SM6</td>
			<td>any peristaltic pump capable of pumping 5-10mL/min is acceptable</td>
		</tr>
		<tr>
			<td>Sharpening stone</td>
			<td>VWR</td>
			<td>470150-112</td>
			<td>optional; for dulling needles</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate, powder, USP</td>
			<td>Fisher Scientific</td>
			<td>18-606-333</td>
			<td></td>
		</tr>
		<tr>
			<td>Specimen forceps, serrated</td>
			<td>VWR</td>
			<td>82027-442</td>
			<td></td>
		</tr>
		<tr>
			<td>T-Pins for dissecting</td>
			<td>Fisher Scinetific</td>
			<td>S99385</td>
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			<td>Ultra-fine short insulin syringes, 31 G</td>
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			<td>amazon.com</td>
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</table>

effective rapid blood perfusion in xenopus

100多年来，非洲爪蟾一直是了解脊椎动物发育和疾病的强大模式生物。在这里，定义了非洲爪蟾的快速血液灌注方案，旨在一致和急剧地减少所有组织内的血液。通过将针头直接插入心脏心室并通过血管系统泵送肝素化磷酸盐缓冲盐水（PBS）来进行灌注。每只动物可以在大约 10 分钟内完成该过程。血液由一些高度丰富的蛋白质和细胞类型主导，由于这些蛋白质掩盖了大多数其他感兴趣的分子和细胞类型，因此会产生许多问题。使用定量蛋白质组学和单细胞转录组学对成人非洲爪蟾组织的可重复表征将受益于在器官取样之前应用该协议。组织取样的方案在配套论文中定义。这些程序旨在使不同性别、年龄和健康状况的非洲爪蟾的做法标准化，特别是非洲爪蟾和热带爪蟾。

The whole body perfusion of amphibians is routinely completed for the purposes of preservation and fixation1,2,3,4,5,6. However, these procedures occur at a rate that limits the number of fresh samples that can be taken per animal. The goal of this work is to develop an effective blood perfusion protocol in Xenopus, prioritizing the speed of the technique. The protocol takes less than 10 min per animal for X. tropicalis and less than 15 min per X. laevis animal. The secondary priorities are the ease of replication and the use of easily acquired equipment so that high-quality samples can be shared widely between Xenopus labs.
Xenopus frogs are used widely in biomedical research to study fundamental biological and pathological processes conserved across species. This tetrapod has a closer evolutionary relationship with mammals than other aquatic models, having lungs, a three-chambered heart, and limbs with digits. The international community effectively uses Xenopus to gain a deeper understanding of human disease through in-depth disease modeling and molecular analysis of disease-related gene function. The numerous advantages of Xenopus as an animal model make them invaluable tools to study the molecular basis of human development and disease; these advantages include: large oocyte and embryo size, high fecundity, ease of housing, rapid external development, and ease of genomic manipulation. Xenopus have been estimated to share ~80% of the identified human disease genes7.
Compared to popular mammalian models, Xenopus is a rapid, cost-effective model, with the ease of morpholino knock-down and availability of efficient transgenics and targeted gene mutations using CRISPR8. Quantitative mass spectrometry and single-cell transcriptomics have been successfully applied to Xenopus embryos9,10,&#160;but a recent cell atlas from Xenopus laevis shows that the composition of most tissues is dominated by blood cell types11. By developing a technique that exsanguinates tissue at a rapid rate and using chilled media, the sample freshness is minimally affected by perfusion. This is particularly important for applications where the goal is to profile physiologically unperturbed mRNA or protein expression.

作者聚焦：非洲爪蟾中的有效快速血液灌注  

非洲爪蟾的有效快速血液灌注

成功灌注后，所有组织（色素沉着爪蟾中的肝脏除外）将明显变浅，血液饱和度较低。主要血管将变得不那么明显（图10），取样后组织（不包括肝脏）将在缓冲液中干净冲洗。虽然该方案的成功执行最终只能通过出血组织样本的数据质量来确认，但故障排除表中提供了几个典型问题、可能的原因和建议的补救措施（表1和图11<...

Watch this Scientific Journal Video about Effective Rapid Blood Perfusion in Xenopus at JoVE.com

这里介绍的是一种有效的快速血液灌注方案，用于制备非洲爪蛙的组织样本，用于转录组学和蛋白质组学研究。

Xenopus have been powerful model organisms for understanding vertebrate development and disease for over 100 years. Here, a rapid blood perfusion protocol ...

该程序以以最大化效率的方式人道地利用两栖动物模型为中心。标准化实践可以应用于在原产地采集和保存的样本，从而减轻生物安全风险并消除动物运输压力。浓缩可以快速完成，最大限度地延长了在组织新鲜时取样的时间。这种新鲜度对于蛋白质组学和转录组学分析尤为重要。该协议详细而明确，使其可用于具有各种背景的个人。这有助于非洲爪蟾实验室之间共享高质量样品。除了这种丰富的技术外，正在制定一项类似的综合方案，用于所有主要器官的标准化取样。

effective-rapid-blood-perfusion-in-xenopus

Research

JoVE Journal

Biology

Effective Rapid Blood Perfusion in Xenopus

3.7K 次观看.  Harvard Medical School. 该程序以以最大化效率的方式人道地利用两栖动物模型为中心。标准化实践可以应用于在原产地采集和保存的样本，从而减轻生物安全风险并消除动物运输压力。浓缩可以快速完成，最大限度地延长了在组织新鲜时取样的时间。这种新鲜度对于蛋白质组学和转录组学分析尤为重要。该协议详细而明确，使其可用于具有各种背景的个人。这有助于非洲爪蟾实验室之间共享?...

视频: 作者聚焦：非洲爪蟾中的有效快速血液灌注  

Xenopus have been powerful model organisms for understanding vertebrate development and disease for over 100 years. Here, a rapid blood perfusion protocol in Xenopus, aimed at a consistent and drastic reduction of blood within all tissues, is defined. Perfusion is carried out by inserting a needle directly into the ventricle of the heart and pumping heparinized phosphate-buffered saline (PBS) through the vascular system. The procedure can be completed in approximately 10 min per animal. The blood is dominated by a few highly abundant proteins and cell types, creating numerous issues as these proteins mask most other molecules and cell types of interest. The reproducible characterization of adult Xenopus tissues with quantitative proteomics and single-cell transcriptomics will benefit from applying this protocol prior to organ sampling. The protocols for tissue sampling are defined in companion papers. These procedures are aimed at the standardization of practices across Xenopus of different sex, age, and health status, specifically X. laevis and X. tropicalis.

Presented here is an effective rapid blood perfusion protocol to prepare tissue samples from African clawed frogs for transcriptomics and proteomics studies.