Murine Salivary Functional Assessment via Pilocarpine Stimulation Following Fractionated Radiation

The overall goal of this procedure is to evaluate murine salivary function by pilocarpine stimulation. This method can be applied to both understand the natural history of salivary gland disfunction, and to test the efficacy of any potential interventions. Our protocol provides a modular platform of techniques, including murine tracheotomy, saliva collection, and gland dissection.

Clinically, this technique has implications for studies assessing either dry mouth, or over-production of saliva. And it can also be applied to other intra-oral procedures requiring airway management. Those first learning this protocol may find the tracheostomy and gland dissection challenging.

We hope this video will help you to learn proper gland isolation technique, as well as the relevant anatomy. Using an analytical balance, weigh out 20 milligrams of pilocarpine, then dissolve it in 2 milliliters of sterile saline. Pilocarpine solutions must be made fresh on their day of use.

Next, weigh all of the collection vessels for the procedure, and weigh the mouse to accurately dose it. Use the C57 black 6 strain. After anesthetizing the mouse, and confirming anesthesia with the toe pinch, apply ophthalmic ointment using a cotton-tipped applicator, and position the mouse supine.

Then, secure its limbs, nose, and tail with surgical tape. Next, clean and wet the neck using an alcohol wipe. Now, use forceps to pinch the ventral midline neck, and with scissors, make a small, superficial cut.

Then, guide the scissors into the opening and slowly open the blades subcutaneously to separate the tissue planes. While keeping the skin raised, carefully make another cut one centimeter below the mouth. Then, make two lateral incisions, inferior and superior to the first cut.

Now, using forceps, gently pull apart the skin to access head and neck anatomy. Proceed using a dissecting microscope at eight times magnification. Using fine forceps, gently lift the submandibular gland to expose the four infrahyoid muscles overlying the trachea.

Avoid tearing or disrupting the surrounding vasculature or excretory ducts. If collecting saliva from more than one mouse, prepare all the mice to this point before proceeding. One person can handle up to five mice concurrently.

Now, to continue the surgery, use small dissecting scissors to remove the medial portion of the strap muscles while staying close to the midline. Remove just enough muscle to visualize the trachea and prevent the muscles from interfering with the procedure. When dissecting the strap muscles, it is especially important to avoid nicking nearby vessels.

A nick can lead to volume depletion secondary to hemorrhage, and thus make pilocarpine ineffective for induction of salivary secretion. To proceed, reflect the strap muscles away from the trachea to visualize the larynx, trachea, and thyroid gland. Ensure that the trachea is clear of overlying tissue.

Finally, make a horizontal incision in the trachea, inferior to the thyroid, using small dissecting scissors, not a scalpel. Make certain that this airway is patent, and clear of fluid. For the saliva collection, first remove the tape near the mouth, and then tilt the dissection stage at 45 degrees to direct the saliva flow cranially.

Next, use a 0.5 milliliter syringe with a 29-gauge half-inch needle to administer an intraperitoneal pilocarpine injection. Then, start a timer. If multiple mice are being injected, work quickly with an assistant's help.

Next, use standard forceps to open the mouth and bring a capillary tube into position. A bead of saliva should form within a few minutes. Place one end of the capillary tube into the saliva, and put the other end into a collecting tube positioned below the dissecting stage.

As needed, reposition the tube to draw up the saliva. Avoid making contact with mucosal tissues, as mucus could obstruct the inlet. Collect saliva for 12 minutes from time of injection.

The stoma should remain clear throughout. If the mouse has depressed saliva function, transfer fluid from its mouth to the collection tube using a P200 micropipette. Always use a different tube or tip for each mouse.

After completing all of the collections, reweigh the collection vessels. After euthanizing the mouse by carbon dioxide asphyxiation, make a careful thoracotomy, and place the mouse supine on the dissection microscope stage. Then, reposition the glands at the original surgical site.

Under eight times magnification, visualize the major glands, the PG, SMG and SLG. The PG is diffuse, and is positioned lateral to the SMG and the SLG. Dissect the PG first.

Grasp the tail of the PG with forceps, pulling it away from the underlying structures. Build to a gentle tension, and then cut the head of the PG with dissecting scissors. Next, breach the capsule surrounding the SMGs with forceps.

Then, gently separate the left and right SMG. Next, free the dorsal aspect of the SMG from the surrounding non-glandular tissue. To remove the SMG, tighten the Wharton's duct with forceps, and cut the duct with dissecting scissors.

The SLG is in the superolateral aspect of the SMG. To separate the two glands, grasp each with separate forceps, and position one tine in the tissue plane between the glands. Then, slowly build tension, and the two glands will gradually come apart.

After isolating the glandular tissue, immediately reweigh the collection vessels. As a positive control for the analysis, mouse SMGs were irradiated and compared to controls. Two weeks after four fractions of radiation at 6.85 gray, saliva function significantly decreased.

Both total saliva secretion, and the secretion normalized to the gland wet weight showed salivary functional capacity was reduced, demonstrating the sensitivity of the assay. Next, SMGs were dissected. They could be fixed, sectioned, and stained.

Hematoxylin and eosin staining was used to compare the gross architecture with or without pilocarpine stimulation. Additionally, the tissues were immunostained for a membrane sodium-potassium chloride transporter, NKCC1. In either stain, the tissues showed similar cell morphology, irrespective of pilocarpine stimulation.

After watching this video, you should have a good understanding of how to evaluate murine salivary function by pilocarpine stimulation. Once mastered, this full technique can be done in under an hour. This method allows salivary researchers to collect whole saliva to quantify gland function, and to cleanly and reproducibly isolate the three major salivary glands.

While attempting this procedure, especially with more than one mouse, it's important to move quickly, and to terminate saliva collection at the correct time point. Following this procedure, composition and enzyme activity can be determined from whole saliva, while glandular tissues can be processed for histology or immunostaining.