Two Techniques to Create Hypoparathyroid Mice: Parathyroidectomy Using GFP Glands and Diphtheria-Toxin-Mediated Parathyroid Ablation

The overall goal of this procedure is to generate two mouse models of acquired hypoparathyroidism that are easy to use, produce a stable phenotype, and leave thyroid function intact. The main advantage of these two models is that they enable the precise removal of parathyroid glands in mice, either by fluorescence guided surgery, or by PTH specific expression of the diphtheria toxin receptor, allowing the cells to be selectively targeted and destroyed without interfering with the neighboring thyroid gland. We hope that our mouse models will be able to answer key questions in the field of hypoparathyroidism.

For example, can PTH analog therapies help maintain normal serum calcium levels without increasing urinary calcium excretion? These models can also be used to study other clinical questions in hypoparathyroidism. For example, is bone strength affected by hypoparathyroidism and does it change with treatment?

Questions like these are hard to answer in patients, but these mouse models can be used to address them. Begin by anesthetizing eight-to 10-week-old mice, expressing GFP specifically in parathyroids according to an approved protocol. Place the animal in supine position and ensure an appropriate depth of anesthesia by the absence of a toe pinch withdrawal reflex or other means.

Extend the ventral neck region and prepare for surgery by shaving the fur. Disinfect the shaved skin with a povidone iodine antiseptic pad, and then prepare the incision site using a surgical scrub and alcohol rinse per your approved protocol. Next, cover the animal with sterile surgical drapes to reduce contamination of the surgical site.

After cutting a 2 centimeter longitudinal incision with a surgical scalpel, use curved, serrated forceps to dissect the fascia, and push the salivary glands to the side. Then while viewing the surgical site through a dissection microscope, use the tips of sharp forceps to cut and separate the paratracheal muscles and expose the trachea. Identify the right and left lobes of the thyroid gland located next to the trachea.

Then switch the light source to fluorescent light and visualize the two green fluorescent parathyroid glands. Rodents typically only have two parathyroid glands. If you cannot identify the two parathyroid glands next to the thyroid on each side, ectopically located parathyroid glands should be suspected, and a careful search for them along the trachea should be performed.

Next, use surgical forceps and scissors to carefully remove the green parathyroid glands and carefully check along the trachea to ensure that all green tissue has been removed. Use sterile gauze for hemostasis. Close the paratracheal muscles with interrupted sutures.

Close the skin incision with Halstead sutures. Place the postsurgical animal into a separate cage on a warming pad for body temperature recovery. Once the mouse is awake, put pellet chow and water with some gelatin food on the cage floor.

After a postsurgical observation period of two hours, return the mouse to the animal facility. Three days after parathyroidectomy, analyze 10 microliters of tail blood for ionized calcium using an analyzer such as the blood gas system, calcium and pH analyzer. If parathyroidectomy is successful, blood ionized calcium will be equal to or less than minus two standard deviations of that of sham operated control mice.

Begin by preparing diphtheria toxin solution by diluting diphtheria toxin powder with sterile saline to a concentration of 0.5 micrograms per milliliter. Sterile filter the solution and alloquot. Inject five micrograms per kilogram body weight diphtheria toxin into eight-to 10-week-old mice, expressing the diphtheria toxin receptors specifically in the parathyroids.

Three days after a second injection of diphtheria toxin, analyze 10 microliters of tail blood for ionized calcium, as before. The following are representative images of the various locations of the parathyroid glands in mice expressing GFP specifically in the parathyroid. Mice had either two or three green parathyroid glands.

Most parathyroid glands were located near the superior border or near the inferior border of the thyroid gland. In rare cases, some parathyroid glands were located ectopically. One of the advantages of our mouse model is that the thyroid gland stays intact.

This figure shows TSH levels of GFP-PTX mice three months after parathyroidectomy. This figure shows T4 measurements. GFP-PTX mice show TSH concentrations and T4 concentrations that were not different from control mice.

This image shows blood calcium levels and PTH levels in GFP-PTX mice. This image shows blood calcium levels and PTH levels in diphtheria toxin injected PTHcre-iDTR mice. Both GFP-PTX mice and DT injected PTHcre-iDTR mice exhibited stable hypocalcemia and reduced PTH levels over a three month observation period.

Once mastered, the GFP guided parathyroidectomy can be done in about 20 minutes for the entire procedure. A single injection of diphtheria toxin takes no more than a minute if it is performed properly. Following this procedure, these mice develop hypoparathyroidism and can be used as a preclinical disease model.

Questions can be addressed such as the efficacy of different PTH hormone analogs in the treatment of hypoparathyroidism. Don't forget that diphtheria toxin is hazardous, and that workers should have the usual diphtheria toxin immunizations. Personal protective equipment such as masks and gloves should always be worn while performing these procedures.