A Method for the Measurement of Salivary Gland Function in Mice

Summary

Salivary gland hypofunction is a frequent consequence of autoimmune disease and radiation therapy. Reproducible evaluation of salivary gland function in mouse models of these diseases is a technical challenge. Here, a simple method for accurate and reproducible measurement of saliva production in mice is described.

Abstract

Patients with Sjögren's syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Introduction

The salivary glands produce saliva in response to a variety of neurological and mechanical stimuli¹. The stimuli are carried through the sympathetic and parasympathetic nervous system to the adrenergic and cholinergic receptors in the gland. Pilocarpine is a cholinergic, para-sympathomimetic agent that acts predominantly on muscarinic receptors. In the salivary gland, it induces the production of saliva by acting on the muscarinic acetylcholine receptor M3¹. Saliva production following pilocarpine administration is an indicator of the ability of the salivary glands to respond to stimulation and is commonly used as a meas....

Protocol

The protocol described below was approved by the Institutional Animal Care and Use Committee and it follows the ethical guidelines established by the National Institutes of Health. All data presented in this report were generated by using female mice that were 10-12 weeks of age. The following strains of mice were used: C57BL/6, BALB/c, DBA1, 129S, and (B6XA/J) F1. All mice were housed in barrier cages (5 animals per cage) in specific pathogen-free conditions and provided feed and water ad libitum.

1. Preparation

1. To prepare a stock solution of pilocarpine hydrochloride, weigh 10 mg of the compound and dissolve it in 1.776 mL of ....

Results

In the experimental mouse model systems, the ability to produce saliva in response to pilocarpine stimulation is used as an indicator of salivary gland function. Saliva production was measured in 11-week-old C57BL/6 female mice by the swab method. In Figure 1A, the results are expressed as the amount of saliva (mg) collected following increasing doses of pilocarpine. At the dose of 1 mg/kg body weight, some of the mice started exhibiting distress (involuntary.......

Discussion

Saliva production is a complex process and is influenced by many factors. Therefore, measuring salivary gland function in laboratory animals can be a challenge. An additional challenge is that repeated measurements of salivary gland function in the same animal are required to establish the onset of disease or to demonstrate recovery following treatment.

The swab method described in this report is technically simple to perform with multiple options to represent the data. The results obtained ar.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Chemicals for saliva collection
Pilocarpine hydrochloride	Alfa Aesar, Tewksbury, MA, USA	B21410	Dilute in sterile isotonic saline. Store single use aliquots of 100X stock at -80oC
Name	Company	Catalog Number	Comments
Anesthesia Mix solution			Mix 1 mL ketamine hydrochloride + 0.5 mL xylazine + 8.5 mL sterile isotonic saline in a sterile vial. Can be used for 3 months.
Zetamine (Ketamine hydrochloride)	Vet One, Boise, Idaho, USA	C3N VT1	Stock is 100 mg/mL
Anased (Xylazine)	Med-Vet International, Mettawa, IL, USA	RXANASED-20	Stock is 20 mg/mL
Isotonic sterile saline	Vet One, Boise, Idaho, USA	501032	Used as diluent
Artificial tears (lubricant ophthalmic ointment)	Henry Schien, Dublin, OH, USA	48272	Used to prevent eyes from drying during the procedure
Name	Company	Catalog Number	Comments
Materials for saliva collection
SalivaBio Children's swab	Salimetrics, LLC Carlsbad, CA, USA	N/A	Individually wrapped swabs
50 mL polypropylene tubes	VWR, Radnor, PA, USA	89004-364	Cut off the bottom 1 cm of the tube. Make sure that the cut edge is smooth.
Microcentrifuge tubes 0.6 mL	VWR, Radnor, PA, USA	87003-290	Make holes in the bottom of tube with heated 18 gauge needles
Microcentrifuge tubes 2 mL	VWR, Radnor, PA, USA	87003-298
Insulin Syringes with permanently attached needles	Becton Dickinson and Company, Franklin Lakes, NJ, USA	324702
18 gauge regular bevel needles	Becton Dickinson and Company, Franklin Lakes, NJ, USA	305195
Sterile scalpel blade #11	Integra York Inc PA, USA	4-311
Microdissecting forceps	Roboz, Gaithersburg, MD, USA	RS-5139	Serrated angular 0.8 mm tip, 4" length
Label tape	Santa Cruz Biotechnology, Dallas, TX, USA	sc-224487
3 channel timer	Amazon.com, Seattle, WA, USA	B06W2KCYVN
Analytical Balance	Mettler Toledo, Columbus OH, USA
Name	Company	Catalog Number	Comments
Chemicals/ reagents for inducing salivary dysfunction
LPS	Invivogen, San Deigo, CA, USA	tlrl-b5lps	Dissolve in endotoxin free water, and store stock solution at 5 mg/mL . dilute in sterile HBSS 100 ug/mL - inject 100 uL/ moue ip
Imject Alum adjuvant	Thermo Scientific	77161	Dilute 1:1 in sterile saline. Inject intraperitoneally 0.1 mL/mouse
Rabbit anti-Ro52 antiserum	Generated in lab		Immunization of rabbits with recombinant mouse Ro52
Name	Company	Catalog Number	Comments
Chemicals/ Kits for saliva analyses
Salivary lysozyme estimation
EnzChek Lysozyme Assay Kit	Molecular Probes, Eugene, OR, USA	E-22013	Used as per manufacturer's instructions
Name	Company	Catalog Number	Comments
Salivary IgA estimation by sandwich ELISA
Mouse IgA	Southern Biotech, Birmingham, AL, USA	0106-01	Standards for sandwich ELISA - range 30 ng/mL to 0.5 ng/mL
Goat anti- mouse IgA unlabeled	Southern Biotech, Birmingham, AL, USA	1040-01	Coat at 1 ug/mL in bicarbonate buffer
Goat anti- mouse IgA HRP	Southern Biotech, Birmingham, AL, USA	1040-05	Detection antibody used at 1:4000 dilution
TMB substrate	Becton Dickinson and Company, Franklin Lakes, NJ, USA	555214	Used as per manufacturer's instructions
Immulon 4HBX Microtiter 96 well plates	Thermo Scientific, Rochester, NY, USA	3855
Name	Company	Catalog Number	Comments
Salivary protein estimation
Bio-Rad Protein Assay Dye Reagent Concentrate	BioRad, Hercules, CA, USA	5000006	For protein estimation as per manufacturer's instructions