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 visualization¹. Not only can this method be used to visualize the lymphatics, but it can also be used to quantify their ability to uptake fluid and macromolecules from the tissue.

The vast lymphatic network also encompasses the cardiac lymphatic system, which has been shown to play an integral role in zebrafish regeneration^2,3,4. Understanding the differences and similarities in lymphatic function across different species is crucial to utilizing this knowledge clinically. Therefore, there is a need to explore the technologies that can measure and visualize lymphatic function across different model organisms^5,6. Lymphatics are blunt-end vessels that transport fluid in one direction, away from the tissue⁷. Intramyocardial injection of fluorescent dyes is required to observe lymphatic drainage from cardiac tissue. Intramyocardial injections have also been used clinically and in pre-clinical mammalian models to transplant stem and progenitor cells or exogenous compounds such as hydrogel to test for the improvement of heart function after myocardial infarction^8,9,10. Zebrafish intramyocardial injection has not been described in detail, which has limited the use of such experimental approaches to the zebrafish heart.

Injections into the zebrafish's pericardial space and the systemic blood flow within the lumen of the heart have been described in detail previously^11,12, and successful intramyocardial injection of fluorescent tracers in adult zebrafish has been reported². The present article provides a detailed protocol for carrying out intramyocardial injections in adult zebrafish. Several transgenic zebrafish lines can identify lymphatic vessels; however, there is a need to explore approaches to understanding lymphatic drainage or to visualize lymphatics in the absence of transgenic markers. Fluorescent tracers, microspheres (MS), and quantum dots (QD) are used here to visualize the injection site and fluid flow into the cardiac lymphatics. QD are fluorescent nanocrystals of <10 nm in diameter whose optical properties can be tuned and adapted to serve many biomedical applications^13,14. QD are readily taken up by lymphatic vessels but not by blood vasculature when injected interstitially^15,16. MS are fluorescently coated polystyrene beads of approximately 200 nm in diameter¹⁵. As such, MS are considerably larger than QD and significantly more persistent when injected into the myocardium, allowing consistent identification of the injection site. This method is useful to study lymphatic function during cardiac regeneration but can be adapted to study various aspects of cardiac biology using the stable localized introduction of coated beads, hydrogels, or cell preparations.

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 Pull a 1.2 mm OD (outer diameter) standard borosilicate glass capillary using …

Representative 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

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