The overall goal of this procedure is to accurately and reproducibly collect and measure mouse saliva following Pilocarpine stimulation. Measuring saliva production in rodents is a challenging procedure, and is prone to high variability. This method is simple and it can be repeated on the same animal in the multiple time points of the whole study.
This method can not only measure the volume of the saliva produced, it can also be used to collect the saliva to further analysis of bioactive molecules that may be secreted in saliva. Ultimately, this will be of a great use when investigating Sjogren Syndrome, salivary gland fibrosis, or salivary gland regeneration in rodent model systems. For this procedure, have Pilocarpine hydrochloride aliquots prepared and frozen, not more than three months old.
Do not re-freeze any thawed aliquots. Begin with preparing 0.6 mL microfuge tubes. Carefully punch a small hole in the bottom of the tubes with a heated 18 gauge needle.
Make one tube per mouse. Then, set these tubes into 2 mL tubes. Sterility is not a concern.
Next, using aseptic methods, cut cylindrical absorbent swabs into two centimeter lengths. Then, cut each two centimeter piece diagonally to make two conically shaped swabs. Place one conical swab into each of the prepared microfuge tubes.
Then, weigh each 0.6 mL tube with its dry swab. Now, transfer the mice for the study into a new clean cage. They may be housed together.
For the next two hours, give them ice water, but no food. Just before the two hour period ends, prepare the working Pilocarpine solution by diluting an aliquot of 100x stock in sterile saline down to 1x. Vortex the tube, and keep the solution on ice.
To begin, weigh each mouse to calculate doses. Then, anesthetize the mice with intraperitoneal injections of ketamine plus xylazine. If after four minutes a mouse is not anesthetized, then adjust the dosage.
Now, apply a thalmic ointment to both eyes to prevent drying. Next, deliver the calculated dose of pilocarpine to the mouse using an intraperitoneal route, and set the timer for two minutes. During this two minute interval, insert the mouse into a 50 mL restrainer tube, so only the head and ears stick out.
Position the tube at a 45 degree angle, with the mouse's head down, and the ventral surface facing upwards. Then, secure the tube with tape. Just after the two minutes, lift the lower jaw to open the mouth, and gently insert a closed dissecting forceps, one to two millimeters into the mouth.
Ensure that the tongue is resting on the top arm of the forceps. With the other hand, use a pair of fine forceps to hold the pre-weighed dry swab close to its conical tip. Then, gently slide the conical tip of the swab into the mouth, and withdraw the forceps while leaving the tip of the swab in the oral cavity.
Now, grip and rotate the swab until it is making maximum contact with the mouth, and so that it will not fall out. The wide end of the swab must remain outside the mouth. Keep the swab in this position for fifteen minutes.
After fifteen minutes, gently rotate the swab to collect any saliva that has not been absorbed. Then, transfer the saliva soaked swab in the 0.6 mL microfuge tube. Cap the tube and set it into the 2 mL tube, placed on ice.
Now, transfer the mouse back to a recovery cage. Provide diet gel or moistened food pellets in the cage to help it re-hydrate. A subcutaneous injection of 0.2 mL isotonic saline may also be given to accelerate recovery.
After collecting all the saliva samples, weigh them to determine the mass of collected saliva. Cut off the caps from the 2 mL tubes containing the smaller tube of saliva sample, and then load them into a refrigerated centrifuge, and spin them down for two minutes at 7, 500 g, and at four degrees Celsius. The saliva will pool into the two mL tube.
Then, measure the volumes of the samples using a micropipette. Saliva production was measured in eleven week old C57 black six female mice using the described method. At the dose of 1 microgram of pilocarpine per gram of body weight, some of the mice started exhibiting signs of distress.
There was a good dose response relationship between pilocarpine and saliva production. There was also a significant concordance between saliva weight and volume. Using the described technique, strain to strain differences in saliva were found.
The 129 S6 strain of mice produced the least amount of saliva. In an experiment to detect salivary gland hypofunction, lipopolysaccharides were injected to activate an innate immune response. During the response, mice produced less saliva.
Another tactic to induce salivary gland hypofunction was to inject the mice with an adjuvant Alum plus anti-Ro52 antibodies. The vehicle control was treated only with Alum. This was also detectable with the described technique.
After watching this video, you should have a good understanding of how to accurately and reproducibly measure saliva production in mice. Once mastered, ten mice can be assessed in about two and a half hours. While attempting this procedure, it's important to remember to be extremely consistent at every step, right from intraperitoneal injections, to placing the swab, and managing time intervals for each step.
Don't forget that pilocarpine hydrochloride acts on the cardiovascular system, causing bradycardia and hypotension. Therefore, injecting pilocarpine in the mice should be done with care.
