Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging

This protocol gives precise, step-by-step guidance for casting the pulmonary arterial vasculature in both adult and early postnatal mice. This technique enables users to fully cast the entire vascular network for visualization by a variety of methods, including micro-CT, while maintaining soft tissue integrity. This technique is particularly useful for looking at pulmonary-vascular morphology and architecture, but can easily be adapted for the targeting of other vascular beds.

Like many surgeries and dissections, there’s no substitute for actually visualizing a procedure, and viewers may benefit from seeing some of the more challenging maneuvers being performed. Once the xiphoid process is exposed, gently grasp and elevate the rib cage. Carefully make an incision in the now-exposed, semi-transparent diaphragm.

The lungs will visibly collapse, and retract away from the diaphragm. After exposing the lung and trachea, thread 15 centimeters of PE10 tubing onto the hub of a 30-gauge needle. And attach this unit to a one-milliliter syringe, containing 10 to the 4 molar of sodium nitroprusside, in PBS.

Advance the plunger to prime the tubing until all of the air has been purged. As an effective alternative to the text, penetrate the apex of the heart on one side, and pass the tips of curved forceps through the muscle, and out the other side of the tissue. Grasp one end of a 10-centimeter length of 7-0 silk, and pull approximately two centimeters through the tissue.

After tying off the suture, use the remaining eight centimeters of suture to pull the heart caudally. And tape the end of the suture to the surgical board. Hook the tips of curved forceps under both the ascending aorta and pulmonary artery trunk.

And pull a three-centimeter length of 7-0 silk back through the opening to create a single-throw loose suture. Use scissors to make a one-to-two-millimeter incision towards the apex of the heart, penetrating the thin-walled right ventricle. And introduce the primed tubing into the right ventricle, gently advancing the catheter into the semi-transparent, thin-walled pulmonary artery trunk.

Visually verify that the catheter has not advanced into either the left or right pulmonary branches, and does not abut the pulmonary artery branch point. Tape the distal portion of the tubing to the surgical board, and gently tighten the loose suture around both of the great vessels. Cut the eight-centimeter piece of suture to return the heart to a natural resting position.

And clip the left oracle of the heart to allow the perfusate to exit the vasculature. Then, use a syringe pump, to infuse the sodium nitroprusside at a 0.5 ml/min flow rate to both flush the blood, and to maximally dilate the vasculature, until the perfusate runs clear. To construct the lung inflation unit, cut the flexible plastic 24-gauge intravenous catheter from a tub and connect to the needle of a butterfly infusion set.

Attach this unit to the stopcock, and attach the stopcock to an open 50-milliliter syringe. After loading the syringe with formalin and priming the catheter, with the stopcock closed, position the syringe in a ring stand, to the point at which the meniscus is 20 centimeters above the trachea. Place two loose sutures, two to four millimeters apart, inferior to the cricoid cartilage, and use scissors to make a small incision in the cricothyroid ligament, superior to the sutures.

Insert the catheter into the opening, and advance the tip beyond the two loose sutures. Tighten the sutures around the trachea, and open the stopcock to allow the formalin to enter the lungs by gravity. Wait for five minutes for the lungs to fully inflate.

If the lungs adhere to the rib cage during inflation, use blunt-tipped forceps to grasp the outside of the rib cage, and move the ribs in all directions to assist in freeing the lobes without making direct contact with the lungs. After five minutes, retract the catheter behind the first suture and ligate. Then retract the catheter behind the second suture and ligate.

The lungs should now be inflated in a closed, pressurized state. To cast the vasculature, load one milliliter of an 8-1-1 solution of polymer diluent curing agent into a one-milliliter syringe. And carefully insert the plunger.

With the syringe inverted, depress the plunger to remove the air, and to allow the formation of a meniscus at the tip of the syringe. Remove the sodium nitroprusside syringe from the hub of the needle, and drip addition PBS into the hub, to create a meniscus. Join the polymer-compound syringe to the hub, and start the infusion at 0.02 millimeters per minute.

Monitor the compound as it moves freely through the tubing, noting the syringe volume as it enters the pulmonary artery trunk. Continue the profusion until all lobes are completely filled, down to the smallest vessel. Stop the pump, and once again, note the syringe volume.

Cover the lungs with a fiber-optic cleaning wipe, and liberally apply PBS. Allow the specimen to sit undisturbed for 30 to 40 minutes at room temperature. Once the polymer compound is cured and hardened, sever the limbs and lower half of the mouse.

And place the head and thorax into a 50-milliliter conical filled 10%buffered formalin, overnight. The next morning, grasp the trachea to gently separate the heart and lung unit from the remaining rib cage and thorax. Place the harvested tissue block in a formalin-filled scintillation vial.

A successful cast will result in a uniform filling of the entire pulmonary arterial network. If there is damage to the lung or lung airways, small leaks will prevent the lungs from holding pressure. Patchy or incomplete filling can arise from an airlock as a result of air being introduced into the vascular system via the catheter, blocking downstream flow of the compound.

Underfilling occurs when too little compound is introduced into the vasculature. Alternatively, overfilling, or introducing too much polymer compound too rapidly, can cause arterial rupture, or, more commonly, venous transit. Advancing the catheter too far down the pulmonary trunk can cause the tip to wedge into one pulmonary artery branch, creating an imbalance in flow, and causing one side to fill faster than the other.

Careful performance of the technique as demonstrated, using the appropriate direct monitoring of distal vasculature end points, and standard infusion rates can result in optimal filling of pulmonary vasculature. Furthermore, adapting this technique to the systemic vasculature can yield equally favorable results in other vascular beds. After casting, samples can be processed for micro-computed tomography scanning.

For post-processing, a commercial software package can be used to generate a 3D volume rendering of the pulmonary-vascular tree. When attempting this technique for the first time, be especially careful around the fragile lungs, and the pulmonary artery. Most importantly, avoid introducing air into the vascular circuit.

Following a successful cast, a sample can be scanned by micro-CT, and questions involving vascular morphometry and architecture can be explored.