DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

The overall goal of this procedure is to visualize the vasculature of the axolotl. This method can help answer key questions in the regenerative medicine field about the intricacies of the revascularization of regenerating tissues in the axolotl. The main advantage of this vascular visualization technique is that, all of the blood vessels in the animal can be easily imaged on a standard fluorescent microscope.

Though this method can provide insight into axolotl limb regeneration, it can also be applied to other tissues, such as the tail, heart, eyes, gills or other organs. Before opening axolotl chest, first confirm the appropriate level of sedation by toe pinch. Then, use surgical forceps to pinch the skin along the central axis of the chest, just below the line of the shoulders.

Using a scalpel, make a incision where the skin is pulled up and remove a square patch of skin over the chest to reveal two cartilage plates. Next, open a window over the thoracic cavity and carefully tear the connective tissue without cutting any major blood vessels. Use forceps to lift each cartilage plate individually, excising each plate with surgical scissors.

Carefully pinch the exposed pericardium with the forceps. Pulling the tissue up and puncturing it with the surgical scissors, just deeply enough to remove the pericardium without injuring the heart. Then, gently remove the pericardium to expose the heart and aorta, to clearly view the heart at approximately five millimeters of the aorta, branching off of the heart.

To perfuse the axolotl, place a clamp stand with a loaded butterfly needle next to a polystyrene foam frame, such that the arm of the clamp is easily manipulated for needle insertion. Turn on a peristaltic pump to flush 0.7x PBS through the pump tubing and point the needle tip toward the rostral aspect. Slide the forceps under the aortic arch and pull up slightly.

Then, maneuver the needle clamp combination, so that the needle runs along the length of the aorta toward the head of the animal, and insert the needle about five millimeters into the aorta, keeping the forceps behind the aorta for support. A successful insertion can be confirmed by a visible enlargement of the heart atria. Quickly lacerate one atrium and allow the blood to drain.

Then, perfuse the axolotl with about 20 to 30 milliliters of PBS. After a successful perfusion, the animal should change from light pink to white in color. When all of the PBS has been perfused, pause the peristaltic pump and move the free end of the tubing, into a 15 milliliter tube of Dil solution.

Restart the pump, taking care to avoid creating bubbles in the tubing and perfuse the animal with the entire working Dil stock. In a successful perfusion, the axolotl will change to a bright pink color that will be most noticeable in the gills. When all of the Dil has been perfused, pause the pump and place the free end of the tubing into 4%paraformaldehyde solution.

Then, restart the pump and perfuse at least 10 milliliters of the fixative. When all of the paraformaldehyde has been administered, stop the pump and remove the needle. Transfer the axolotl onto a plastic dish, and place the dish onto a fluorescent microscope stage.

Then, with the lights off, select a filter that permits excitation of Dil, which emits a red orange light. After a successful perfusion, the Dil stain is visible as a red admission signal in all of the axolotl tissues, including the tail, limbs, gills and eyes. Once mastered, this technique can be completed in one hour if it is performed properly.

While attempting this procedure, it's important to remember to take care not to lacerate the major blood vessels or the heart while opening the chest cavity and to insert the needle in line with the aorta to prevent through and through punctures. Following this procedure, other methods like, skeletal preparations can be performed to answer additional questions. After its development, this technique paved the way for researchers in the biomedical field to explore the vasculature of different species.

After watching this video, you should have a good understanding of how to perform a cardiac perfusion of the axolotl with the carbocyanine dye, Dil. Don't forget, that working with needles and paraformaldehyde can be extremely hazardous and that precautions, such as wearing the appropriate personal protective equipment, should always be taken while performing the procedure.