The video outlines a protocol for euthanizing and preparing Xenopus frogs, detailing the necessary anesthetic procedures and injections with heparin. It emphasizes proper techniques for blood drainage and equipment setup to ensure a humane and efficient process for handling these amphibians in a laboratory setting.

preparation for perfusion in xenopus

Перфузия крови у африканских когтистых лягушек для транскриптомики и протеомики

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.

В центре внимания автора: Эффективная быстрая перфузия крови при Xenopus  

Эффективная быстрая перфузия крови у Xenopus

This procedure centers around the humane utilization of amphibian models in a manner that maximizes efficiency. Standardization practices can be applied to samples taken and preserved at the originating colony, mitigating biosecurity risks and eliminating animal transport stress. Profusion can be completed quickly, maximizing the time available to sample tissues while they are still fresh.This freshness is especially important for proteomic and transcriptomic profiling. This protocol is detailed and explicit, making it usable for individuals with a wide variety of backgrounds. This facilitates the sharing of high-quality samples between Xenopus labs.Along with this profusion technique, a similarly comprehensive protocol is being worked on for the standardized sampling of all major organs.

Watch this Scientific Journal Video about Preparation for Perfusion in Xenopus at JoVE.com

Preparation for Perfusion in Xenopus   

Подготовка к перфузии при ксенопусе

После 15 минут погружения в раствор для эвтаназии убедитесь, что Xenopus обезболивается, ущипнув лапку лягушки. Запишите вес и любые другие параметры, необходимые в это время. Используя иглу 31 калибра, введите Xenopus laevis в каждую мышцу передней конечности 250 микролитров гепарина в PBS.ввести Xenopus tropicalis 100 микролитров гепарина в PBS. Обрежьте кончик перфузионной иглы кусачками. Присоедините обрезанную иглу к насосу и циркулируйте 54 единицы на миллилитр гепаринизированного перфузата PBS.Очистите трубку от всех пузырьков воздуха. Поместите диссекционный лоток под наклоном во вторичный контейнер, чтобы облегчить дренаж крови.

Research

JoVE Journal

Biology

Preparation for Perfusion in Xenopus

187 Views.  Harvard Medical School. После 15 минут погружения в раствор для эвтаназии убедитесь, что Xenopus обезболивается, ущипнув лапку лягушки. Запишите вес и любые другие параметры, необходимые в это время. Используя иглу 31 калибра, введите Xenopus laevis в каждую мышцу передней конечности 250 микролитров гепарин?...

Video: Preparation for Perfusion in Xenopus   

Effective Rapid Blood Perfusion in Xenopus

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.

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

Following 15 minutes of submersion in the euthanasia solution, ensure the Xenopus is anesthetized by pinching the foot of the frog. Record the weight and any other parameters needed at this time. Using a 31 gauge needle, inject Xenopus laevis in each forelimb muscle with 250 microliters of heparin in PBS.inject Xenopus tropicalis with 100 microliters of heparin in PBS. Trim the tip off a perfusion needle with wire cutters. Attach the trimmed needle to the pump and circulate 54 units per milliliter of heparinized PBS perfusate.Purge all air bubbles from the tubing. Place the dissection tray at an incline within a secondary container to facilitate blood drainage.

Xenopus Dissection and Perfusion

Xenopus Dissection and Perfusion  

After completing the primary euthanasia, check the loss of pain response by pinching the foot. Place the frog on its back. Pin down each limb.Cut through the midline of the skin, then cut laterally to create two skin flaps. Grab the linea alba with forceps, and pull it away from the coelomic cavity. Cut through the musculature.Cut or pin all the flaps out of the way. Cut through the coracoid bones with the help of dissection scissors and remove any excess tissue to gain better access to the heart. Next, gently shift the stomach of the dissected Xenopus to the top of the left liver lobe.Gently grasp a lung tip using tissue forceps and pull it outside the coelomic cavity, then pin the lung through the tip. Pull the thin pericardium tight with tissue forceps. Using the tip of a pair of iridectomy scissors, gently perforate the pericardium without cutting any underlying tissue.Peel the pericardium away from the heart chambers. Gently grasp the ventricle apex with forceps. Without perforating the ventricle, insert the needle through the closure of the forceps into the ventricle chamber.Clamp the tissue forceps in place using a needle holder or hemostat. Start the flow of the pump at approximately five milliliters per minute. This will engorge the heart chambers and the arterial trunk.Use scissors to make a nick in the right auricle. Continue the pump flow until the vasculature of the stomach blanches. Lance the left heart auricle.Increase the flow rate of the pump to 10 milliliters per minute. Using a transfer pipette, rinse the coelomic cavity and perfusion media. If the organs shift during rinsing, gently shift them back to maintain visibility of the stomach and lungs.To help visualize the perfusate flowing out of the heart, the auricles may further be reduced. Keep the needle in place until the stomach vasculature and lungs have blanched and the perfusate flowing from the heart appears clear. Successful perfusion was observed in all tissues except the liver, making them distinctly lighter and less saturated with blood.This is most visible in albinos.