Intramyocardial Injection for the Study of Cardiac Lymphatic Function in Zebrafish

Summary

The present protocol allows efficient and stable delivery of fluorescent microspheres (MS) and quantum dots (QD) into the myocardium of live fish that can be tracked (traced) over time.

Abstract

Zebrafish have proved to be an important model for studying cardiovascular formation and function during postembryonic development and regeneration. The present protocol describes a method for injecting fluorescent tracers into the zebrafish myocardium to study interstitial fluid and debris uptake into cardiac lymphatic vessels. To do so, microspheres (200 nm diameter) and quantum dots (<10 nm diameter) are introduced into the myocardium of live zebrafish, which can be tracked using ex vivo confocal microscopy. These tracers are then tracked intermittently over several hours to follow clearance from the myocardium into cardiac lymphatic vessels. Quantum dots are transported through cardiac lymphatic vessels away from the heart, while larger microspheres remain at the injection site for over three weeks. This method of intramyocardial injection can be extended to other uses, including the injection of encapsulated MS or hydrogels to locally release cells, proteins, or compounds of interest to a targeted region of the heart.

Introduction

The lymphatic system is essential for maintaining tissue-fluid balance, modulation of the immune response following injury, and absorption of lipids in the gut¹. Accumulating evidence supports the broad roles of the lymphatic system in various disease and developmental contexts. However, mechanistic studies are hampered because lymphatic vessels can be hard to visualize, and their functionality can be uncertain. Early imaging techniques relied on the natural ability of the lymphatic system to interstitially absorb injected tracers, and then transport them through the lymphatic vessel network, allowing detection and visualization

Protocol

All animal procedures were approved by the Institutional Animal Care and Use Committee at Weill Cornell Medicine (protocol 2020-0027) and followed proper guidelines. The following experiments were performed with male and female AB wild-type zebrafish aged 14-to-20-months post fertilization for adults, and 35-days post fertilization for juveniles.

1. Needle pulling and reagent preparation

1. Pull a 1.2 mm OD (outer diameter) standard borosilicate glass capillary using a needle puller (Figure 1A,B) (see Table of Materials), into two needles using optimal....

Results

Immediately after injection, a small white region of the myocardial wall must be visible (Figure 3F). This region will show bright fluorescent labeling of the injected MS and QD (Figure 4B,E). In addition, there may be weak and sporadic fluorescence puncta on the heart's outer surface from any QD and MS in the pericardial space following the procedure (Figure 4B,E). The injected tracers can be t.......

Discussion

The present article has described a method to introduce exogenous material into the myocardium of zebrafish. This technique was developed to introduce QD and MS into the myocardium to study lymphatic function in homeostasis and regeneration^2,18. A similar approach has also been used to introduce QD into the myocardium of mice to investigate the presence and function of lymphatics after myocardial infarction^19,2.......

Materials

Name	Company	Catalog Number	Comments
Crystallization dish	VWR	89000-288
Dissection Scope	Zeiss	495010-0007-000
Fish facility water	N/A	N/A	RO water with sea salt and sodium bicarbonate added to a conductivity of 226uS and pH of 7.35
Forceps	Dumont	11252-20
Glass Capillaries	WPI	1B120-3	no filament
ImageJ			https://imagej.nih.gov/ij/download.html
Iridectomy scissors	Fine Scientific Tools	15000-00
Microinjector	Warner Instruments	64-1735
Microloader femtotips	Eppendorf	5242 956.003
Micropipette puller	Sutter Instrument	P-97	Gated pedal input
Microspheres	Thermo Fisher Scientific	B200	Blue
PBS	Corning	46-013-CM
Quantum dots (QD)	Thermo Fisher Scientific	Q21061MP	Qtracker705 vascular label
Sponge	any	any	(1.5 × 5 × 3 cm) with groove (0.5 × 2.5 cm)
Syringe filter	Corning	431220
Tricaine	Sigma-Aldrich	A5040	concentration: 4 mg/mL