Intrathoracic Injection for the Study of Adult Zebrafish Heart

In the cardiac research on adult zebrafish, drug treatments are usually done in a systemic way. With intrathoracic injection, one can locally deliver solution to the heart without causing myocardial damage. This method allows the delivery of small volumes of drugs, proteins, or other molecules in solution precisely around the heart of an adult zebrafish.

To begin this procedure, use a needle puller to pull microinjection-adapted borosilicate glass capillaries. Store the pulled capillaries in a nine-centimeter Petri dish with rails of modeling clay or adhesive tape. Using common scissors, cut a piece of sponge, and carve a fish-like silhouette in its middle.

Next, prepare small aliquots of the injection solutions with the tested proteins or other compounds. Adjust their concentration depending on the assay by diluting the substance in 1x Hanks'balanced salt solution complemented with 10%phenol red. To obtain the working concentration of anesthetics, add two to three milliliters of tricaine stock solution to a beaker containing 100 milliliters of fish water.

First, turn on the stereo microscope with the light from the top, and adjust the magnification to 16x. Soak the sponge with fish water, and place it on a nine-centimeter Petri dish on the microscope. Adjust the focus, and under the stereo microscope, use iridectomy scissors to cut the end of a microcapillary approximately seven millimeters from the base, as detailed in Figure 1A of the text protocol.

Insert the cut microcapillary into the needle holder of the microinjector apparatus. Using the Microloader tips, load a control solution to set up the pressure of injection in order to obtain the appropriate flow, then empty the needle. After this, load the selected volume of the injection solution into the tip of the capillary, making sure that there are no air bubbles.

Use a net to catch an adult fish, and transfer it into the anesthetic solution. After one to two minutes, when the 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. Next, quickly and carefully transfer the fish into the groove of the wet sponge, with the ventral side up.

The head of the fish should point away from the operator's dominant hand. Under the stereo microscope, 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.

Insert the tip of the capillary at an angle between 30 and 45 degrees relative to the body axis. Gently penetrate the skin with the tip of the microcapillary into the pericardium. Once the needle is inside the pericardium, press the pedal of the microinjector device to complete the injection.

After injection, gently withdraw the capillary from the thorax, and immediately transfer the fish into a tank with system water for recovery. Monitor the fish until total recovery from anesthesia. To analyze the effects of injection, collect the heart at the desired time point, and prepare it for further analysis.

In this study, exogenous compounds and proteins are delivered into the pericardial cavity in order to study their effects on the heart in adult zebrafish. To validate this method, two test experiments are performed by injecting color and fluorescent dyes into euthanized fish. In both assays, whole-mount analysis reveals labeling of the entire heart including the ventricle, the atrium, and the bulbus arteriosus.

These results reveal efficient spreading of the injection solutions on the heart surface. To compare the suitability of intrathoracic injection versus intraperitoneal injection for heart studies, a similar amount of DAPI is injected using both methods. The hearts are then fixed after either five minutes or 120 minutes.

No DAPI-positive cells are observed in the hearts after intraperitoneal injection at either time point. In contrast, the intrathoracic injections result in the presence of DAPI-labeled nuclei in the myocardium. These results demonstrate that the intrathoracic injection improves the delivery of the compound to the heart, as compared to the intraperitoneal injection.

To test the suitability of this method for heart regeneration studies, ventricles are cryoinjured, and then injections are performed at three and seven days post-cryoinjury. Both the myocardium and the injured tissue contain numerous DAPI-positive cells, indicating an efficient penetration of this dye into the intact heart and fibrotic tissue. Furthermore, injected phalloidin AF649 is also incorporated by cardiomyocytes of the peri-injury zone and some recruited fibroblasts of the wounded area.

This experiment reveals that the drugs can cross the epicardium and penetrate into the underlying myocardium. After testing the efficiency of intrathoracic injections using dyes, the effects of proteins injected on the heart are analyzed. For this, the effects of the exogenous cytokine on various processes is investigated.

The biological aspects of cardiomyocyte proliferation, extracellular matrix deposition, immune cell recruitment, and cardioprotective gene expression are activated by intrathoracic injection of cytokine. These results demonstrate that intrathoracic injection provides a suitable strategy for targeted delivery of proteins to study their effects on distinct heart tissues in a variety of assays. The insertion of the needle into the pericardium is a key step for a successful injection.

The shape of the needle, the angle of penetration, and the puncture sites are all factors that influence the needle insertion. Following or preceding intrathoracic injection, one can perform cryoinjury in order to analyze the effect of injected molecules on heart regeneration. Intrathoracic injection was used in zebrafish to study cardiac preconditioning.

A protein called CNTF was injected and has been shown to enhance cytoprotective and pro-regenerative programs in the heart.