Effective Rapid Blood Perfusion in Xenopus

Summary

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

Abstract

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.

Introduction

The whole body perfusion of amphibians is routinely completed for the purposes of preservation and fixation^1,2,3,4,5,6. 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....

Protocol

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 pithing does not prevent this, nor does removing the heart from the animal. Exsanguination of anesthetized animals is considered a humane and effective m....

Discussion

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.......

Materials

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