Name: Xenopus Dissection and Perfusion
Uploaded: 2023-05-16T12:50:00
Description: Watch this Scientific Journal Video about Xenopus Dissection and Perfusion at JoVE.com

The video provides a detailed guide on the dissection and perfusion process of a frog (Xenopus), outlining steps such as euthanasia, incision techniques, and heart perfusion to visualize blood flow. It emphasizes monitoring tissue responses and ensuring successful perfusion while noting the visual effects on various organs, especially in albino specimens.

xenopus dissection and perfusion

Blodperfusion i afrikanske klofrøer til transkriptomik og proteomik

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.

Forfatter Spotlight: Effektiv hurtig blodperfusion i Xenopus  

Effektiv hurtig blodperfusion i 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 Xenopus Dissection and Perfusion at JoVE.com

Xenopus Dissection and Perfusion  

Xenopus Dissektion og perfusion

Efter at have afsluttet den primære eutanasi, skal du kontrollere tabet af smerterespons ved at klemme foden. Placer frøen på ryggen. Fastgør hvert lem.Skær gennem hudens midterlinje, og skær derefter sideværts for at skabe to hudflapper. Grib linea alba med tang, og træk den væk fra det coelomiske hulrum. Skær gennem muskulaturen.Klip eller fastgør alle flapperne af vejen. Skær gennem coracoid knogler ved hjælp af dissektion saks og fjern overskydende væv for at få bedre adgang til hjertet. Skift derefter forsigtigt maven på den dissekerede Xenopus til toppen af venstre leverlap.Tag forsigtigt fat i en lungespids ved hjælp af vævspincet og træk den uden for det coelomiske hulrum, og fastgør derefter lungen gennem spidsen. Træk det tynde perikardium tæt med vævspincet. Brug spidsen af en iridectomy saks til forsigtigt at perforere perikardiet uden at skære noget underliggende væv.Skræl perikardiet væk fra hjertekamrene. Tag forsigtigt fat i ventrikelspidsen med tang. Uden perforering af ventriklen skal nålen indsættes gennem lukningen af tangen i ventrikelkammeret.Klem vævspincetten på plads ved hjælp af en nåleholder eller hæmostat. Start pumpens flow med cirka fem ml pr. Minut. Dette vil oversvømme hjertekamrene og arteriestammen.Brug en saks til at lave et hak i den rigtige auricle. Fortsæt pumpestrømmen, indtil vaskulaturen i maven blancherer. Lance venstre hjerte auricle.Forøg pumpens strømningshastighed til 10 ml pr. Minut. Brug en overførselspipette til at skylle det coelomiske hulrum og perfusionsmediet. Hvis organerne skifter under skylning, skal du forsigtigt flytte dem tilbage for at opretholde synligheden af maven og lungerne.For at hjælpe med at visualisere perfusat, der strømmer ud af hjertet, kan auriklerne reduceres yderligere. Hold nålen på plads, indtil mavevaskulaturen og lungerne er blancheret, og perfusatet, der strømmer fra hjertet, ser klart ud. Vellykket perfusion blev observeret i alle væv undtagen leveren, hvilket gjorde dem tydeligt lettere og mindre mættet med blod.Dette er mest synligt i albinoer.

Research

JoVE Journal

Biology

Xenopus Dissection and Perfusion

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 ...