Establishment of a Simple and Effective Rat Model for Intraoperative Parathyroid Gland Imaging

To date, the development of parathyroid gland (PG) identification methods is limited by the lack of animal models in preclinical research. Here, we establish a simple and effective rat model for intraoperative PG imaging and evaluate its effectiveness by using iron oxide nanoparticles as a novel PG contrast agent.

Parathyroid gland is a small organ with very important functions. Our protocol can be used for developing new PG identification technologies which can help surgeons to protect it during the thyroidectomy for thyroid cancer. The main advantage of this model is that it can observe a clear difference between the thyroid and PGs through the negative imaging of IONP.

In addition, the cost and operational difficulty are considerably lower for this rat model than for large animal models. To begin, place a prewarmed heating pad on the surgical table in order to sustain the animal's body temperature during the surgery, and then transfer the anesthetized rat down to a surgical drape placed on the operation table. Using rubber bands, fix the rat's limbs to the operation table and place a cylindrical pillow made of drape under the rat's shoulder to lean its head back, completely exposing the neck area, then apply depilatory cream to the neck area, up to the submandibular space, down to the xiphoid process, and on both sides to the outside of the sternocleidomastoid muscle.

After three minutes, gently wipe the hair and depilatory cream with a tissue. Next, using an iodophor cotton ball, sterilize the middle of the neck and surrounding area. from which hair is removed.

Cover the animal with a surgical drape, keeping the hole aligned with disinfected area. Using the scalpel, make approximately a five-centimeter longitudinal incision in the anterior midline of the rat's neck. Lift the skin along both sides of the incision and using scissors, longitudinally cut along the linea alba cervicalis.

Use forceps to separate the sternohyoid and sternothyroid muscle. Clamp the separated muscles in front of the neck using vascular forceps and pull the clamped tissue outside. Using the needle, pass the suture through the clamped tissue.

Make a knot and fix the suture to the surgical drape of the operation table. Locate the thyroid cartilage based on its shield shape and cricoid cartilage based on its ring shape as the upper boundary in the operation area, then locate the trachea based on its tubular cartilage ring shape in the front and middle of the neck as the lower boundary in the operation area. Locate the thyroid gland, a red butterfly-shaped gland on the opposite side of the trachea between the upper and lower boundaries.

Next, locate two parathyroid glands in a fusiform shape that are reddish, but lighter than the surrounding thyroid gland with a certain boundary on the upper and outer sides of the thyroid gland. Take a frontal photograph of the parathyroid glands with the trachea, thyroid, and larynx. Dissect the back of the esophagus and using a retractor, expose the right side of the parathyroid glands.

Take a right-side photograph of the parathyroid glands with the thyroid gland and trachea, and similarly expose and capture the left side of the glands. Use an insulin syringe to locally inject 10 microliters of black iron oxide nanoparticles suspension into the center of the thyroid gland, and gently press the injection site with gauze for five seconds, then observe the rapid diffusion of the black iron oxide nanoparticles within the thyroid glands, but not the parathyroid glands, as it negatively stains the parathyroid glands and differentiates them from the surrounding thyroid. Capture the front, left, and right-side photographs of the negatively stained parathyroid glands together with the trachea, thyroid gland, and larynx.

After injection, the contrast agent, black iron oxide nanoparticles, readily diffuse within the thyroid gland and stain it black, but cannot infiltrate the parathyroid gland due to their high tissue density. Histological analysis of black iron oxide nanoparticles injected-thyroid gland revealed that the parathyroid glands are enriched with closely aligned chief cells, whereas the thyroid gland features many loose lumens indicating much lower tissue density. Two important things should be remembered in this procedure.

First, the posture of the rat. We recommended to lean ears and head back, completely exposing the neck area. Second, when we inject the IONPs, we should be use fine-ensuring needle because the thyroid surface is full of blood vessels.

This procedure can also be performed by autofluorescence imaging of the PG, which is non-invasive, provides real-time information, and does not use dye as a contrast agent, but the autofluorescence signal is weak and the imaging efficiency is also poor. Other researchers can use this simple and effective rat model to develop and evaluate new PG identification technologies and products instead of using more complicated and expensive large animals such as dogs and pigs.