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

Summary

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.

Abstract

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.

Introduction

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 delivery techniques, such as direct administration into the water, or through oral drug delivery methods like gavaging². Other approaches include coating fish food with the compounds and subsequently feeding the fish³, and direct administration of water-insoluble medications at a predetermined concentration, including retro-orbital or intraperitoneal injections^4,5. Intraperitoneal administration is preferred for in vivo studies of disease models due to its distinct pharmacokinetic advantages⁶. This method provides a high drug concentration and an extended half-life within the peritoneal cavity, offering an effective route for drug delivery^7,8. The approach is commonly utilized in research settings to ensure optimal drug absorption and distribution ⁹. While injection-based methods prove efficient for single delivery, prolonged and repeated injections often lead to body damage and chronic infection².

Currently, there are two methods of IP injection in adult zerbafish^4,10. However, both methods have limitations when delivering toxic compounds like doxorubicin, leading to severe damage to the body shape and fish mortality. The side effects can significantly complicate data interpretation. Although these challenges may be addressed with extensive training¹⁰, there is a clear need for a new IP injection method that minimizes side effects.

Here, our goal is to develop a new method of IP injection optimized for the effective delivery of doxorubicin into adult zebrafish, facilitating the generation of reliable doxorubicin-induced cardiomyopathy (DIC) models with minimized body damage and associated mortality.

Protocol

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<…

Representative Results

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…

Discussion

Different from the two existing IP injection methods^4,10, 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…

Materials

10 μL NanoFil-syringe	World Precision Instruments, Inc	NANOFIL	injection tool
34 G needle	World Precision Instruments, Inc	NI34BV-2	injcetion tool
60 mm Petri dish	fisher scientific/fisherbrand	FB0875713A	placing the sponge
Dissecting microscope	Nikon	SMZ800	Injceting the Dox
Doxorubicin hydrochloride	Sigma	D1515-10MG	drug for creating DIC model
Echocardiography	VISUAL SONICS	Vevo 3100	measuring cardiac function
Foam Sponge	Jaece Industries	L800-D	placing the fish
Hank's balanced salt solution (HBBS)	Thermo Fisher	14025076	Vehicle for Dox
Microcentrifuge	southernlabware	MyFuge/C1012	collect the Dox solution
Precision Balance Scale	Torbal	AD60	Digital scales
Tricaine	Argent	MS-222	Anesthetizing fish
Tube	Eppendorf	1.5 mL	storage
vevo LAB  software	FUJIFILM VISUAL SONICS	5.6.0	quantification of the heart