非洲爪蟾的有效快速血液灌注
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 5x Magnifying glass with LED light and stand | amazon.com | B08QJ6J8P1 | light must not produce heat |
| Disposable transfer pipets | VWR | 414004-036 | |
| Dissecting fine-pointed forceps | Fisher Scinetific | 08-875 | |
| Dissecting scissors sharp piont, straight 6.5" | VWR | 76457-374 | |
| Dissection tray | Fisher Scinetific | 14-370-284 | styrofoam sheets are an acceptable alternative |
| Euthanasia container | US Plastic | Item 2860 | alternative opaque containers acceptable |
| Euthanasia container lid | US Plastic | Item 3047 | |
| Fine dissection pins | Living Systems Instrumentation | PIN-#3 | |
| General use hypodermic needles, 22 G | Fisher Scientific | 14-826-5A | for X. laevis |
| General use hypodermic needles, 25 G | Fisher Scientific | 14-826AA | for X. tropicalis |
| Heparin, porcine intestinal mucosa | MilliporeSigma | 37-505-410MG | |
| Iridectomy scissors 6" | vwr | 470018-938 | iris scissors are an acceptable alternative |
| Luer-to-barb adapter male Luer with lock ring | amazon.com | B09PTX6M2Z | size will be dependant on the hosing of the pump used |
| Mayo-Hegar needle holder | Fisher Scinetific | 08-966 | mosquito forceps are an acceptable alternative |
| MS-222: Syncaine (formerly tricaine) | Pentair AES | TRS1 | |
| PBS 1x | Corning | 21-040-CV | |
| Peristaltic liquid pump dosing pump 5–100 mL/min | amazon.com | B07PWY4SM6 | any peristaltic pump capable of pumping 5-10mL/min is acceptable |
| Sharpening stone | VWR | 470150-112 | optional; for dulling needles |
| Sodium bicarbonate, powder, USP | Fisher Scientific | 18-606-333 | |
| Specimen forceps, serrated | VWR | 82027-442 | |
| T-Pins for dissecting | Fisher Scinetific | S99385 | |
| Ultra-fine short insulin syringes, 31 G | VWR | BD328438 | |
| Wire flush cutters, 6-inch ultra sharp & powerful side cutter clippers | amazon.com | B087P191LP |
References
- Saltman, A. J., Barakat, M., Bryant, D. M., Brodovskaya, A., Whited, J. L. DiI perfusion as a method for vascular visualization in Ambystoma mexicanum. Journal of Visualized Experiments. (124), e55740 (2017).
- Lametschwandtner, A., Minnich, B. Microvascular anatomy of the brain of the adult pipid frog, Xenopus laevis (Daudin): A scanning electron microscopic study of vascular corrosion casts. Journal of Morphology. 279 (7), 950-969 (2018).
- Lametschwandtner, A., Minnich, B. Microvascular anatomy of the urinary bladder in the adult African clawed toad, Xenopus laevis: A scanning electron microscope study of vascular casts. Journal of Morphology. 282 (3), 368-377 (2021).
- Lametschwandtner, A., et al. Microvascular anatomy of the gallbladder of the adult South African clawed toad, Xenopus laevis Daudin: A scanning electron microscope study of vascular corrosion casts. Microscopy and Microanalysis. 13, 492-493 (2007).
- Lametschwandtner, A., Spornitz, U., Minnich, B. Microvascular anatomy of the non-lobulated liver of adult Xenopus laevis: A scanning electron microscopic study of vascular casts. Anatomical Record. 305 (2), 243-253 (2022).
- Miodoński, A. J., Bär, T. Arterial supply of the choriocapillaris of anuran amphibians (Rana temporaria, Rana esculenta). Scanning electron-microscopic (SEM) study of microcorrosion casts. Cell and Tissue Research. 249 (1), 101-109 (1987).
- Nenni, M. J., et al. Xenbase: Facilitating the use of Xenopus to model human disease. Frontiers in Physiology. 10, 154 (2019).
- Tandon, P., Conlon, F., Furlow, J. D., Horb, M. E. Expanding the genetic toolkit in Xenopus: Approaches and opportunities for human disease modeling. 발생학. 426 (2), 325-335 (2017).
- Peshkin, L., et al. The protein repertoire in early vertebrate embryogenesis. bioRxiv. , (2019).
- Briggs, J. A., et al. The dynamics of gene expression in vertebrate embryogenesis at single-cell resolution. Science. 360 (6392), (2018).
- Liao, Y., et al. Cell landscape of larval and adult Xenopus laevis at single-cell resolution. Nature Communications. 13 (1), 4306 (2022).
- AVMA (American Veterinary Medical Association). AVMA guidelines for the euthanasia of animals, 2020 edition. AVMA. , 37 (2020).
- Navarro, K., Jampachaisri, K., Chu, D., Pacharinsak, C. Bupivacaine as a euthanasia agent for African Clawed Frogs (Xenopus laevis). PLoS One. 17 (12), e0279331 (2022).
- Wu, J., et al. Transcardiac perfusion of the mouse for brain tissue dissection and fixation. Bio-Protocol. 11 (5), e3988 (2021).
- Heinz-Taheny, K. M. Cardiovascular physiology and diseases of amphibians. Veterinary clinics of North America. The Veterinary Clinics of North America. Exotic Animal Practice. 12 (1), 39-50 (2009).
- Stephenson, A., Adams, J. W., Vaccarezza, M. The vertebrate heart: an evolutionary perspective. Journal of Anatomy. 231 (6), 787-797 (2017).
- Hoops, D. A perfusion protocol for lizards, including a method for brain removal. MethodsX. 2, 165-173 (2015).
