Using a Cell-Tracer Injection to Investigate the Origin of Neointima-Forming Cells in a Rat Saccular Side Wall Model

By using a direct intra-aortal cell-tracer application, we can analyze the role of endothelial cells originating from the parent artery in neointima formation at two different time points. A big advantage of this technique is a direct, one-point, interpretive in-vivo injection which makes this model a robust and reproducible technique. Before the surgery, incubate the arterial pouches from donor rats in 0.1%sodium dodecyl sulfate for 10 hours at 37 degrees Celsius to obtain decellularized aneurysms.

Collect these pouches from donor animals a few days before the surgery. To begin, analyze the animal’s behavior and inspect the mucus membranes and turgor as part of the preoperative clinical examination. Record the weight of each animal.

For anesthesia induction, place all rats in a clean box provided with oxygen until loss of consciousness after five to 10 minutes. Check the depth of anesthesia by the absence of the pedal withdrawal reflex. Place the rats in a supine position and shave the thoracoabdominal area with an electric shaver.

Using tape, fixate the rat’s paws on a board covered by a heating pad connected to an auto-regulating rectal probe. Insert the rectal probe into the rat’s anus to maintain the desired temperature of 37 degrees Celsius with the help of a heating pad. Then install a sensor on the right-hind leg connected to a computerized system for checking vital signs intraoperatively.

For perianesthetic care, apply a sterile ophthalmic lubricant to the eyes, and cover them with an opaque foil mask to prevent drying and damage from the surgical lamp. Disinfect the surgical field with povidone iodine, and drape the surgical field in a sterile fashion. Put a micro swab with purple padding under the proximal and distal parts of the aorta to better visualize the artery as soon as it is separated from the caval vein.

Then protect the abdomen with white gauze. On the day of operation, dissolve two microliters of the cell-tracer by pipetting in one milliliter of PBS. Transfer the mixture to a one-milliliter syringe fitted with a cannula.

Turn off the light in the operating room. Using a microscope, perform the one-point injection in the middle ventral part of the aorta with micro forceps, and carefully inject one milliliter of heparinized 0.9%saline solution. Inject the cell-tracer carefully, and immediately turn off the operating microscope as well.

Protect the abdomen with wet gauze again. Let the dye incubate for at least 15 minutes. Then turn on the microscope and operating room lights.

Use micro forceps and micro scissors to perform the longitudinal arteriotomy so that its length averages the diameter of the harvested aneurysm. Place the aneurysm beside the aorta before performing arteriotomy to ensure the correct length. Suture the aneurysm with eight to 10 single stitches using a non-absorbable 10-0 suture.

Before the final stitch, deliver the coil with a packing density of one centimeter. Either coil or stent treatment can be performed in this model. Then carefully remove the temporary clamps, starting distally, under continuous irrigation with heparinized saline.

Close the wound in a layered fashion. At the end of the surgery, reverse the anesthesia with a subcutaneous injection. Let each operated animal recover in a clean cage until fully awake and warm as needed with a heating lamp.

Pulled baseline aneurysm volumes for day seven and day 21 did not differ significantly for decellularized or vital aneurysms between the coil or stent treatment groups. Pulled FU volumes for decellularized aneurysms showed a non-significant aneurysm growth in coiled compared to the stented aneurysms. Pulled FU volume was significantly greater in the vital coiled than in the stented group.

Amounts of cell-tracer positive cells in the neointima of decellularized aneurysms did not significantly differ between the stent or coiled-treated groups at day seven FU but were significantly higher in stented rats at day 21 FU.No significant differences were noted in vital-aneurysm sutured rats at either seven days or 21 days FU.In decellularized aneurysms at seven days FU, significantly more cell-tracer positive cells remained in the thrombus of the stent-treated compared to the coil-treated group. This difference was not observed in vital aneurysms at seven days FU.Remember to always turn off the lights in the operating room prior to injecting the cell-tracer to prevent fading. Also, let incubate the dye for at least 15 minutes.