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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Effective Rapid Blood Perfusion in Xenopus

Published: May 16th, 2023

DOI:

10.3791/65287

1Department of Systems Biology, Harvard Medical School

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

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.

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. lae....

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All experiments were performed in accordance with the rules and regulations of the Harvard Medical School IACUC (Institutional Animal Care and Use Committee) (IS 00001365_3).

NOTE: Though the primary method of euthanasia described is deemed an acceptable technique for euthanasia by the American Veterinary Medical Association12, it has not been found to lead to the cessation of a heartbeat13. Even the frequently used secondary method of double pit.......

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Following successful perfusion, all tissues (excluding the liver in pigmented Xenopus) will be distinctly lighter and less saturated with blood. Major blood vessels will become less noticeable (Figure 10), and tissues (excluding the liver) will rinse cleanly in the buffer after being sampled. While the successful execution of the protocol can ultimately only be confirmed by the quality of the data from exsanguinated tissue samples, several typical problems, their possible causes, an.......

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This protocol describes traditional dissection techniques for accessing the coelomic cavity. Other techniques are also acceptable, provided they cause minimal damage to the tissues, the heart is accessible, and the lung and stomach are visible. Similarly, most dissection tools listed can be easily substituted with comparable items.

While attempts have been made to optimize the efficacy of this procedure, results may vary depending on one's experience and variability between individual frogs. O.......

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This work was supported by NIH's OD R24 grant OD031956 and NICHD R01 grant HD073104. We thank Darcy Kelly for helpful discussions and initial input on this protocol. We would also like to thank Samantha Jalbert, Jill Ralston, and Wil Ratzan for their assistance and support as well as our three anonymous peer reviewers for their feedback.

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Name Company Catalog Number Comments
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

  1. 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).
  2. 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).
  3. 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).
  4. 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).
  5. 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).
  6. 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).
  7. Nenni, M. J., et al. Xenbase: Facilitating the use of Xenopus to model human disease. Frontiers in Physiology. 10, 154 (2019).
  8. Tandon, P., Conlon, F., Furlow, J. D., Horb, M. E. Expanding the genetic toolkit in Xenopus: Approaches and opportunities for human disease modeling. Developmental Biology. 426 (2), 325-335 (2017).
  9. Peshkin, L., et al. The protein repertoire in early vertebrate embryogenesis. bioRxiv. , (2019).
  10. Briggs, J. A., et al. The dynamics of gene expression in vertebrate embryogenesis at single-cell resolution. Science. 360 (6392), (2018).
  11. Liao, Y., et al. Cell landscape of larval and adult Xenopus laevis at single-cell resolution. Nature Communications. 13 (1), 4306 (2022).
  12. AVMA (American Veterinary Medical Association). AVMA guidelines for the euthanasia of animals, 2020 edition. AVMA. , 37 (2020).
  13. 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).
  14. Wu, J., et al. Transcardiac perfusion of the mouse for brain tissue dissection and fixation. Bio-Protocol. 11 (5), e3988 (2021).
  15. 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).
  16. Stephenson, A., Adams, J. W., Vaccarezza, M. The vertebrate heart: an evolutionary perspective. Journal of Anatomy. 231 (6), 787-797 (2017).
  17. Hoops, D. A perfusion protocol for lizards, including a method for brain removal. MethodsX. 2, 165-173 (2015).

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