Optimized Intravenous Injection in Adult Zebrafish

Summary

Here, we present an optimized and effective protocol for the intravenous injection of cells or pharmacological agents into adult zebrafish, resulting in enhanced cell engraftment and increased survival rates of the treated zebrafish.

Abstract

Intravenous (IV) injection is widely recognized as the most effective and commonly utilized method for achieving systemic delivery of substances in mammalian research models. However, its application in adult zebrafish for drug delivery, stem cell transplantation, and regenerative and cancer studies has been limited due to the challenges posed by their small body size and intricate blood vessels. To overcome these limitations, alternative injection techniques such as intracardiac and retro-orbital (RO) injection have been explored in the past for stem cell transplantation in adult zebrafish. However, these techniques have their drawbacks, including the need for meticulous injection techniques or increased risk of mortality.

In this study, we have developed a refined and optimized IV injection procedure specifically tailored to adult zebrafish, addressing the challenges associated with their unique anatomy. To demonstrate the effectiveness of this technique, we performed successful IV injections of whole kidney marrow cells from Tg(mpo: EGFP) fish and FITC-dextran dye into adult Casper fish. The subsequent visualization of injected cells and dyes using a fluorescence microscope confirmed their successful delivery and engraftment within the zebrafish. Furthermore, we demonstrated that compared with the intracardiac and RO injections, the IV injection resulted in improved survival rates and engraftment efficiency in treated zebrafish. This approach enables precise delivery and localization of substances and holds great potential for large-scale drug and chemical screening using adult zebrafish. Additionally, the ability to visually track the injected cells and dyes provides invaluable insights into their engraftment, migration, and interactions with host tissues, enabling a more comprehensive evaluation of therapeutic effects and biological processes in zebrafish models.

Introduction

Zebrafish (Danio rerio) has emerged as a valuable model organism in biomedical research, primarily due to its genetic similarity to humans, with more than 70% of human genes having zebrafish counterparts^1,2. This genetic resemblance, combined with zebrafish's compact size, rapid developmental cycle, and capacity for extensive genetic manipulation, makes it a powerful tool for scientific exploration. These attributes are particularly advantageous for experiments involving cell transplantation, drug delivery, and cell tracing. Furthermore, the optical clarity of transparent larvae and specific pigme....

Protocol

All animal procedures were approved by the Committee of the Use of Laboratory and Research Animals (CULATR) at the University of Hong Kong (HKU).

1. Preparation of injection material

1. Prepare cell suspension medium: 0.9x PBS + 5% fetal bovine serum (FBS) + 1% penicillin/streptomycin (Pen/Strep).
2. Sterilize the Hamilton syringe and attached needle (34 G, 10 mm length, 10 µL capacity, see Table of Materials) under UV light with a 270-280.......

Discussion

In this study, we developed an IV injection protocol tailored for adult zebrafish to enhance the precision and consistency of delivering cells or drugs. A critical element of this method is the ability to localize and visualize the primary vein, which is crucial for the accurate administration of substances. While the translucent Casper zebrafish strain is recommended for optimal vessel visibility, our protocol is adaptable for use with various adult zebrafish strains, provided suitable adjustments are made for .......

Materials

Name	Company	Catalog Number	Comments
Autoclaves Steam Sterilizer	HIRAYAMA	HRG-140
Centrifuge 5424	Eppendorf
Countess 3 Automated Cell Counter	Thermofisher
Countess II FL	Invitrogen
Ethyl 3-aminobenzoate methanesulfonate salt (Tricaine)	Sigma-aldrich	MKCL9483
Falcon 40 µm Cell Strainer	CORNING		Blue, Sterile, Individually Packaged, 50/Case
FBS, Qualified	Gibco	26140079
FITC-Dextran (MW 10000)	MedChemExpress	60842-46-8
Injection needle	Hamilton	HAMI207434	34 G, 10 mm length
Micro syringe	Hamilton	7635-01	10 µL capacity, Model 701 RN
Nikon SMZ18
PBS pH 7.4 (1x)	Gibco	10010023
Penicillin-Streptomycin (5,000 U/mL)	Gibco™	15070063	100x
Pipette Tips	Eppendorf	epTIPS
Single Channel Pipette	Eppendorf	05-403-151
UltraPure Distilled Water	Invitrogen	10977015