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 regeneration2,3,4. 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 organisms5,6. 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 infarction8,9,10. 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 previously11,12, 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 applications13,14. QD are readily taken up by lymphatic vessels but not by blood vasculature when injected interstitially15,16. 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
Representative Results
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 regeneration2,18. 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 infarction19,2…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Adedeji Afolalu, Chaim Shapiro, Soji Hosten, and Chelsea Quaies for fish care (Weill Cornell Medicine), Caroline Pearson (Weill Cornell Medicine) for critical reading of the manuscript. Jingli Cao (Weill Cornell Medicine) for use of dissection scope and camera to record the procedure in addition to critical reading of the manuscript. Nathan Lawson (University of Massachusetts Medical School), Brant Weinstein (NICHD), Elke Ober (University of Copenhagen), and Stephan Schulte-Merker (WWU Münster) for transgenic zebrafish lines. Daniel Castranova (NICHD) for advice on QD and imaging and Yu Xia (Weill Cornell Medicine) for guidance on dissecting scope video capture. This work was supported by a NYSTEM Fellowship to NM, American Heart Association Career Development Award (AHA941434), National Institutes of Health (NIH) grant (R01NS126209), and Weill Cornell Medicine Startup Fund to MH.
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 |
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