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

Blodperfusjon i afrikanske klofrosker for transkriptomikk og proteomikk

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 rask blodperfusjon i Xenopus  

Effektiv rask blodperfusjon 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 Disseksjon og perfusjon

Etter å ha fullført primær eutanasi, sjekk tap av smerterespons ved å klemme foten. Plasser frosken på ryggen. Fest ned hvert lem.Skjær gjennom midtlinjen av huden, og kutt deretter sideveis for å lage to hudklaffer. Ta tak i linea alba med tang, og trekk den bort fra coelomic cavity. Skjær gjennom muskulaturen.Klipp eller fest alle klaffene ut av veien. Skjær gjennom coracoid bein ved hjelp av disseksjon saks og fjerne overflødig vev for å få bedre tilgang til hjertet. Deretter skifter du forsiktig magen til den dissekerte Xenopus til toppen av venstre leverlapp.Ta forsiktig tak i en lungespiss ved hjelp av vevtang og trekk den utenfor coelomic cavity, og fest deretter lungen gjennom spissen. Trekk det tynne perikardiet stramt med vevtang. Bruk spissen av en irisektomi saks, forsiktig perforere perikardiet uten å kutte noe underliggende vev.Skrell perikardiet vekk fra hjertekamrene. Ta forsiktig tak i ventrikkelens apex med tang. Uten å perforere ventrikkelen, sett nålen gjennom lukkingen av tangen inn i ventrikkelkammeret.Klem vevtangen på plass ved hjelp av en nålholder eller hemostat. Start pumpens strømning på omtrent fem milliliter per minutt. Dette vil engorge hjertekamrene og arterielle stammen.Bruk saks til å lage et nick i høyre auricle. Fortsett pumpestrømmen til vaskulaturen i magen blancherer. Lance venstre hjerte auricle.Øk pumpens strømningshastighet til 10 milliliter per minutt. Bruk en overføringspipette til å skylle coelomic cavity og perfusjonsmedier. Hvis organene skifter under skylling, skift dem forsiktig tilbake for å opprettholde synligheten av mage og lunger.For å bidra til å visualisere perfusatet som strømmer ut av hjertet, kan auriklene ytterligere reduseres. Hold nålen på plass til magevaskulaturen og lungene har blanchert og perfusatet som strømmer fra hjertet virker klart. Vellykket perfusjon ble observert i alle vev unntatt leveren, noe som gjorde dem tydelig lettere og mindre mettet med blod.Dette er mest synlig hos 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 ...