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An Intraperitoneal Injection Technique in Adult Zebrafish that Minimizes Body Damage and Associated Mortality

Published: March 29th, 2024



1Department of Biochemistry and Molecular Biology, Mayo Clinic, 2Department of Cardiovascular Medicine, Mayo Clinic, 3Cardiovascular Surgery Department, Guilin Hospital of 2nd Xiangya Hospital, Central South University, 4Cardiovascular Surgery Department, 2nd Xiangya Hospital, Central South University
* These authors contributed equally

A new intraperitoneal (IP) injection method in adult zebrafish is described. When handling toxic compounds such as doxorubicin, this procedure is more effective than the two previously reported IP methods. The technique is designed to be easily adopted by researchers with limited experience in the zebrafish model.

The adult zebrafish (Danio rerio), which is genetically accessible, is being employed as a valuable vertebrate model to study human disorders such as cardiomyopathy. Intraperitoneal (IP) injection is an important method that delivers compounds to the body for either testing therapeutic effects or generating disease models such as doxorubicin-induced cardiomyopathy (DIC). Currently, there are two methods of IP injection. Both methods have limitations when handling toxic compounds such as doxorubicin, which result in side effects manifesting as severe damage to the body shape and fish death. While these shortcomings could be overcome by extensive investigator training, a new IP injection method that has minimal side effects is desirable. Here, a unique IP injection method that is able to handle toxic compounds is reported. Consistently reduced cardiac function can result without incurring significant fish death. The technique can be easily mastered by researchers who have minimal experience with adult zebrafish.

Zebrafish (Danio rerio) has gained attention as an experimental model for studying human diseases because this animal encompasses high gene and organ homology to humans, external fertilization, ease of genetic manipulation, and body transparency into early maturity, which facilitates a myriad of imaging applications1. Unlike the straightforward process of delivering drugs directly to the water for zebrafish embryos and larvae, administering drugs to adult zebrafish presents a more intricate and challenging endeavor2.

In adult fish, compounds can be delivered through passive drug ....

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All procedures conducted were approved by the Mayo Clinic Institutional Animal Care and Use Committee, adhering to the standards outlined in the 'Guide for the Care and Use of Laboratory Animals' (National Academies Press, 2011). All zebrafish in the study belong to the Wild Indian Karyotype (WIK) strain. The details of the reagents and the equipment used for the study are listed in the Table of Materials.

1. Preparation and storage of doxorubicin stock solution<.......

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Previously, two intraperitoneal (IP) methods have been employed for the administration of doxorubicin in adult zebrafish4,10. In method I, also known as the Classic IP injection method as described by Kinkel et al.4, the needle was inserted at a 45° angle to the midline between the pelvic fins with the abdomen facing upward. In method II, or the Alternative IP injection method as described by Ma et al.10, the n.......

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Different from the two existing IP injection methods4,10, the new IP injection method is characterized by the following distinct features.Firstly, a unique needle penetration angle is used (close to zero); secondly, the needle penetrates the fish via a unique location, i.e., a natural hole on the ventral surface of a fish, which would facilitate the injection; and finally, the movement of the needle is from anterior to posterior. These adjustments effect.......

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This study was supported by the NIH (HL107304 and HL081753) and the Mayo Foundation (Center for Biomedical Discovery and Cardiovascular Research Center) to X.X. J.L. is funded by the Fundamental Research Funds for the Central Universities of Central South University, No. 56021702. Special thanks to Beninio Gore and Quentin Stevens for managing the zebrafish facility.


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NameCompanyCatalog NumberComments
10 μL NanoFil-syringeWorld Precision Instruments, IncNANOFILinjection tool
34 G needleWorld Precision Instruments, IncNI34BV-2injcetion tool
60 mm Petri dishfisher scientific/fisherbrandFB0875713Aplacing the sponge 
Dissecting microscopeNikonSMZ800Injceting the Dox
Doxorubicin hydrochlorideSigmaD1515-10MGdrug for creating DIC model 
EchocardiographyVISUAL SONICSVevo 3100measuring cardiac function
Foam SpongeJaece IndustriesL800-Dplacing the fish
Hank's balanced salt solution (HBBS)Thermo Fisher14025076Vehicle for Dox
Microcentrifuge southernlabwareMyFuge/C1012collect the Dox solution 
Precision Balance ScaleTorbalAD60Digital scales
TricaineArgentMS-222Anesthetizing fish
TubeEppendorf1.5 mLstorage 
vevo LAB  softwareFUJIFILM VISUAL SONICS 5.6.0quantification of the heart

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