Labeling of Blood Vessels in the Teleost Brain and Pituitary Using Cardiac Perfusion with a DiI-fixative

The overall goal of this procedure is to label blood vessels in the brain and pituitary of teleost fish, and we use cardiac perfusion with a fixative containing DiI in the model species medaka. So this technique which is simple and efficient will help answer important questions regarding the vascular anatomy in the brain and pituitary of teleost fish as well as the relationship between neurons or endocrine cells and blood vessels. Some advantages of this technique include a fast and easy simple preparation, and its adaptability to the teleost fish species.

To prepare a fish holder, cut a piece of polystyrene to a five centimeter length, a three centimeter width, and a two centimeter thickness, and glue the polystyrene to a nine centimeter diameter plastic dish. Then use a scalpel to make a three centimeter boat shaped hole in the polystyrene. To prepare the perfusion system, add a 30 to 50 centimeter long plastic cannula at the extremity of the perfusion system needle.

To prepare five centimeter glass pipettes, use a pipette puller to stretch individual glass capillaries according to the manufacturer's instructions. Before beginning the dissection, load a 10 milliliters syringe with the prepared DiI-fixative solution and place a needle with a cannula at the extremity of the syringe. Then use several pieces of tape placed in different orientations to fix the syringe to the bench, and adjust the position of the microscope and the seat to obtain a good position for dissection and for pressing the syringe piston with the elbow.

Next, place a euthanized fish in the fish holder abdomen side up and secure the head and tail with pins. Use scissors to horizontally cut the superficial layer of skin to open the anterior abdomen, and use forceps to remove the skin above the heart until a clear access to the ventricle and the bulbus arteriosus is achieved. Add a glass pipe at the extremity of the capillary and break the tip of the pipette.

Bring the glass pipe close to the ventricle and add elbow pressure to the syringe piston to force the liquid out while pinning the heart ventricle with the pipette. Then immediately perforate the sinus venosus with forceps to release the blood from circulation. From the ventricle, adjust the angle of the glass pipette to locate the entrance of the bulbus arteriosus, and move the pipette opening inside the bulbus arteriosus.

Then add pressure to the syringe for an additional 30 to 60 seconds while keeping the pipette inside the bulbus arteriosus. For optimal labeling of all the blood vessels it is critical to perfuse into the bulbus arteriosus long enough to replace all the blood with the DiI-fixative solution. After about 60 seconds of perfusion, when no more blood leakage is observed, remove the glass pipette and needles from the fish, and harvest the brain and pituitary into fresh 4%paraformaldehyde for two hours at room temperature in the dark.

Then rinse the tissues two times in fresh PBS for 10 minutes per wash before preparing the samples for imaging. After cardiac injection of a fixative solution containing DiI as demonstrated, the labeled blood vessels can be observed on tissue slices by fluorescent stereo microscopy or on whole tissue by confocal microscopy. Note that if the solution is injected in the heart ventricle instead of the bulbus arteriosus or if the solution is injected with too low of a pressure and/or for too short a period, an inadequate labeling of the vessels will be observed.

In addition, when imaging the same tissue with the same imaging parameters, a decrease in the labeling intensity can be observed after several days, with the signal appearing more spread-out within the tissue sample. Tissue slices from transgenic lines within which the cells of interest express fluorescent reporter proteins can be further labeled for specific proteins of interest using standard immunofluorescence protocols allowing the investigation of interactions between blood vessels and other cell types. It is important to reach the bulbus arteriosus.

When this is achieved, adding pressure to the syringe piston should induce an expansion of the bulbus confirming a successful procedure. This technique can be used to investigate the interaction between endocrine cells and blood vessels in the teleost pituitary in general, but maybe more specifically to investigate possible differences or similarities between the portal systems in teleost and mammals. Because this political use toxic and volatile compounds precautions such as using a gas mask and a ventilated room should always be taken when performing these procedures.