Jonas-Closs RA and Peshkin L (2023), Effective Rapid Blood Perfusion in Xenopus.

XB-ART-59606

bioRxiv 2023 Feb 03; doi: 10.1101/2023.02.01.526649.

Show Gene links Show Anatomy links

Effective Rapid Blood Perfusion in Xenopus.

Jonas-Closs RA , Peshkin L .

???displayArticle.abstract???
UNLABELLED: Xenopus has been a powerful model organism for understanding vertebrate development and disease for over a hundred years. Here we define a rapid blood perfusion protocol in Xenopus aimed at a consistent and drastic reduction of blood across tissues. Perfusion is done by inserting a needle directly into the ventricle and pumping heparin in PBS through the vascular system. The whole procedure should take about 10 minutes per frog. Blood is dominated by a few highly abundant proteins and cell types which create numerous issues by masking most other molecules and cell types of interest. Reproducible characterization of adult Xenopus tissues with quantitative proteomics and single cell transcriptomics will gain from applying this protocol prior to organ dissections defined in companion papers. The procedure is aimed at standardization of practice across the animals of different gender, age and Xenopus species, specifically X.laevis and X.tropicalis . SUMMARY: An effective rapid blood perfusion protocol to prepare tissue samples for transcriptomics and proteomics studies.

???displayArticle.pubmedLink??? 36778320
???displayArticle.pmcLink??? PMC9915651
???displayArticle.link??? bioRxiv

???attribute.lit??? ???displayArticles.show???

References [+] :

Briggs, The dynamics of gene expression in vertebrate embryogenesis at single-cell resolution. 2018, Pubmed, Xenbase

	Figure 2. Mature female X. tropicalis pinned through each limb. Use toothed dissecting forceps to pull the skin near the cloaca taught to perforate it with dissection scissors and create 2 flaps as shown.
	Figure 3. With the ventral skin open but the peritoneum intact the linea alba is visible. To reduce the likelihood of damaging the underlying tissues grasp the linea alba and pull it taught prior to cutting. The coracoid bones are visible through the peritoneum. Once the coelomic cavity has been opened these bones should be reduced to give better access to the heart.
	Figure 4. The coelomic cavity of a mature X. tropicalis male. The coracoid bones have been reduced providing access to the pericardium-enclosed heart. The stomach has been shifted in front of the left lobe of the liver and its vasculature is clearly visible. The left lung has been pulled out of the coelomic cavity by its tip and pinned to ensure that it does not retract during the rinsing process.
	Figure 5. The pericardium is a thin tough membrane enclosing the heart. Using tissue forceps gently grasp the pericardium and then use the tip of iridectomy or iris scissors to perforate it. Once perforated, peel it up, away from the heart.
	Figure 6. (left) Ventral diagram of X. laevis heart. (right) Heart diagram, with pericardium removed, showing the correct needle and clamp placement.
	Figure 7. With the pericardium removed the 3 chambers of the heart and truncus arteriosus are easily visible. Use forceps to gently grasp the ventricle by its apex and then insert the needle through the forceps. Be careful not to cause unnecessary damage to the ventricle or other chambers as this will compromise the perfusion efficiency.
	Figure 8. Perfusion is underway at high pressure, the right auricle has been lanced, and the ventricle, truncus arteriosus, and left auricle are visibly engorged. The stomach is blanching but both the media running from the animal and the lung tissue are heavily saturated with blood.
	Figure 9. The coelomic cavity following successful rapid perfusion and rinsing. The vasculature of the stomach and other organs is no longer visible. Unless the Xenopus is albino the liver will remain heavily pigmented. Note that the apex of the lung still has a red tint due to blood vessels being broken from handling and pinning.
	Figure 10. X. laevis fat body lobe sampled before (left) perfusion and a fat body lobe of the same individual, sampled after (right) perfusion.
	Figure 11. An albino X. laevis both before (top) and after (bottom) rapid perfusion. The albinism makes it easier to determine the proficiency of the perfusion than it would be in a pigmented animal. This is especially apparent in the lung and liver tissues.
	Figure 12. Troubleshooting diagram of X. laevis heart. (left) The ventricle has perforation (in red) this perforation is isolated by the forceps and will not affect perfusion efficiency. (right) A heart with a severely damaged ventricle. The needle can be guided into the truncus arteriosus and clamped in place. It is especially important to ensure that the needle is well-blunted when using this technique [2].