Intrathoracic Injection: Compound Delivery to the Adult Zebrafish Heart

Overview

This video describes the technique of delivering compounds into the heart of adult zebrafish by using intrathoracic injection.

Protocol

1. Preparation of the Fish for Intrathoracic Injection

1. Catch an adult zebrafish (Danio rerio) with a net and transfer it into the anesthetic solution.
2. After 1−2 min, when fish stops swimming and the movement of operculum is reduced, touch the fish with a plastic spoon to make sure it does not react to any contact.
3. Quickly and carefully transfer the fish with the spoon into the groove of the wet sponge, with ventral side up. The head of the fish should point away from the operator’s dominant-hand.

2. Microinjection into the Pericardium

1. Under the stereomicroscope, carefully observe the movement of the beating heart under the skin of the fish. Visually determine the injection point above the beating heart and in the middle of the triangle defined by the ventral cartilaginous plates (Figure 1D). Insert the tip of the capillary at 30−45° degree angle relative to the body axis (Figure 1E). Gently penetrate the skin with the tip of the microcapillary into the pericardium (Figure 1C). An optimal entry point is closer to the abdomen than to the head.
   NOTE: Do not insert the capillary too deeply into the body and the heart, as this will cause injury to the organ. In case of heart puncture, the needle generally fills with blood. If this happens, remove the capillary and exclude the fish from the experiment.
2. Once the needle is inside the pericardium, complete injection by pressing the pedal of the microinjector device.
   NOTE: Be careful not to inject air into the thoracic cavity.
3. After injection, gently withdraw the capillary from the thorax and immediately transfer the fish into a tank with system water for recovery.
4. Monitor the fish until total recovery from anesthesia.
5. Collect heart at the desired time point and prepare it for further analysis.
   NOTE: In case the fish does not resume movement of the operculum within 30 s, reanimate the fish by squeezing water into the gills with a plastic pipette.

Representative Results

Figure 1: Intrathoracic (IT) injection in adult zebrafish. (A) Photograph of a pulled microinjection capillary with filament (6”, 1.0 mm in diameter) and values of the needle puller program used. (B) Photograph of a pulled microinjection capillary with filament (6”, 1.0 mm in diameter) filled up with 2.5 µL of solution containing 10% phenol red. The pulled tip of the needle is maximally 7 mm long. (C) Schematic representation of the IT injection procedure. (D) Photographs of the IT injection procedure. This figure has been modified from Bise et al. Numbers in panels C and D correspond to the same steps of the procedure: (1) fish is placed ventral side up on a humidified sponge. The puncture site (red dot in the triangle) is located in the center of the chest near the gills. (2) Penetration of the needle into the pericardium. Red dot indicates puncture site. (3) Injection is monitored by observing the spread of the red solution in the pericardial cavity. (E) Scheme of IT injection. Angle between the injection capillary and the body axis should be between 30° and 45° to avoid heart puncture. (F) Photographs of fish thorax at 1 hour after IT injection of indicated volumes. White arrows are pointing at the redish tissue, which might indicate internal bleeding

Materials

Name	Company	Catalog Number	Comments
Macroscope (binocular)	M400		with Apozoom
Micro-injector femtojet	Eppendorf	5247 0034 77
Microloaders femtotips	Eppendorf	5242 956.003
Micropipette glass needles type C	WPI	TW100F-6	thin-wall capillary
Micropipette puller model P-87	Flaming/Brown	20081016	filament box 2.5 mm x 4.5 mm
Sponge	any	any	dim. carved sponge 7cm x 3 cm x 1 cm
Tricaine (Anestethic)	Sigma	E10521