The study was supported by a grant from National Institutes of Health, National Institute of Dental and Craniofacial Research (DE025030).

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
<ol>
	<li>To prepare a stock solution of pilocarpine hydrochloride, weigh 10 mg of the compound and dissolve it in 1.776 mL of sterile isotonic saline by vortexing, to give a stock solution of 5.63 mg/mL. There is no need to sterilize this solution. But if desired, filter it through a 0.2 &#181;M filter. Store this stock solution in multiple aliquots in a -80 oC freezer for up to 3 months. Each frozen stock is for single use. Once thawed, do not refreeze the pilocarpine solution.</li>
	<li>Take 0.6 mL microfuge tubes and carefully punch a small hole in the bottom of each tube with a heated 18-gauge needle. Set these tubes into 2 mL tubes. The tubes need not be sterile.</li>
	<li>Using a sharp, sterile razor blade, cut cylindrical absorbent swabs into pieces about 2 cm in length. Cut each 2 cm piece diagonally to give 2 conical shaped swabs. Place one swab in each of the 0.6 mL microfuge tubes.</li>
	<li>Weigh the 0.6 mL microfuge tube containing the dry swab.</li>
	<li>Transfer the mice to be studied into a new clean cage with water bottles. Keep the mice without food for at least 2 h prior to the start of saliva collection to prevent food particles from contaminating the saliva collected. 
	NOTE: It is not necessary to keep 1 mouse per cage for this procedure. However, the number of mice placed in each cage depends on the specific institution&#39;s rules and regulations for animal use.</li>
	<li>Just before the end of 2 h, prepare the working pilocarpine solution (0.0563 mg/mL) by diluting the stock solution 100x in sterile saline. It is not necessary to further filter sterilize this solution. Always keep the working pilocarpine solution on ice. 
	NOTE: Table 1 gives the amount of pilocarpine to be injected for a range of mouse body weights to give a final dose of 0.375 mg/kg body weight.</li>
</ol>
2. Procedure
<ol>
	<li>Weigh each mouse and determine the dose of the anesthetic mix for each mouse using Table 1. Inject the first mouse with the appropriate volume of the anesthetic mix by the intraperitoneal route. Set the timer for 2 min (most mouse strains tested in this protocol go to sleep by 2 min). Ensure that the mouse is properly anesthetized by lack of walking when placed on a flat surface. If the mouse is still walking, wait for additional 2 min before proceeding to the next step. Apply a drop of lubricant ophthalmic ointment to both eyes to prevent drying. 
	NOTE: In Table 1, the dose of the anesthetic mix is 0.007 mL/g body weight. The optimal range for this procedure is 0.006 to 0.008 mL/g body weight. However, after 4 min, if the mouse is still walking or exhibiting jerky movements, consult with the institutional veterinarian for appropriately increasing the dose of the anesthetic.</li>
	<li>Determine the dose of pilocarpine for the mouse using Table 1 and inject the appropriate amount by intraperitoneal route. Set the timer for 2 min and insert the mouse into the 50 mL restrainer tube until the head and ears stick out of the cut end. Place the tube at a 45 degree angle, with the head down, and the ventral surface facing upward. Tape the tube to the procedure board to keep it from moving. 
	NOTE: Table 1 provides dosage for mice more than 17 g, as this procedure has not been attempted on mice weighing below 17 g.</li>
	<li>At the end of 2 min, gently insert a closed pair of micro dissecting forceps into the mouth and lift the lower jaw upwards to open the mouth. Push the forceps 1-2 mm further into the mouth, making sure that the tongue is seen resting on the top arm of the forceps. With another pair of fine forceps, hold the pre-weighed dry swab close to its conical tip.</li>
	<li>Gently slide the conical tip of the swab into the mouth. Withdraw the forceps while leaving the tip of the swab in the oral cavity.</li>
	<li>Grasp the wider end of the swab extending outside of the mouth and rotate it to allow the maximum area of contact with the mouth. Keep the swab in this position for 15 min. 
	NOTE: This ensures that the swab remains in the mouth for the duration of the collection. Note that the conical tip of the swab acts as a wick and the wider portion of the swab remains outside the mouth.</li>
	<li>At the end of 15 min, gently rotate the swab to collect any saliva that has not been absorbed and place the wet swab in the 0.6 mL microfuge tube. Close the tube and set it in the 2 mL tube placed on ice. 
	NOTE: At this time, the oral mucosa will appear completely dry. The mouse can now be moved back into its cage for recovery from anesthesia.</li>
	<li>Transfer the mouse in the cage. Place moistened food pellets in the cage after saliva collection to help faster rehydration. 
	NOTE: The mice wake up within 10 min after the end of the collection. Subcutaneous injection of 0.2 mL pre-warmed isotonic saline may also be given to accelerate recovery.</li>
	<li>Proceed to the next mouse.&#160;All the mice should be monitored until they have completely recovered and are ambulatory.</li>
</ol>
3. Measurements
<ol>
	<li>At the end of all collections, weigh the 0.6 mL tubes with the wet swab. Calculate the difference between the wet weight and dry weight to get the weight of saliva produced. Place the 0.6 mL tube with saliva back into the 2-mL tube.</li>
	<li>Cut off the caps of the 2 ml tubes. Then, centrifuge the 2 mL tubes for 2 min at 7500 x g, at 4oC in a micro centrifuge to recover the saliva. Measure the volume of saliva obtained using a micropipette.</li>
	<li>Express the results as saliva weight (mg)10 over 15 min or as a ratio of saliva weight (mg)/mouse body weight (g). 
	NOTE: If the volume of saliva extracted out of the swab has been measured, express the results as the volume of saliva recovered (mL) or the ratio of saliva volume (mL)/mouse body weight (g).</li>
</ol>

<ol>
	<li>Proctor, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physiology+of+salivary+secretion.">The physiology of salivary secretion.</a> Periodontol 2000. 70 (1), 11-25 (2016).</li><li>Fox, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sj&#246;gren's+syndrome.">Sj&#246;gren's syndrome.</a> Lancet. 366 (9482), 321-331 (2005).</li><li>Eisbruch, A., Kim, H. M., Terrell, J. E., Marsh, L. H., Dawson, L. A., Ship, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xerostomia+and+its+predictors+following+parotid-sparing+irradiation+of+head-and-neck+cancer.">Xerostomia and its predictors following parotid-sparing irradiation of head-and-neck cancer.</a> Int J Radiat Oncol Biol Phys. 50 (3), 695-704 (2001).</li><li>Marmary, Y., Fox, P. C., Baum, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+secretion+rates+from+mouse+and+rat+parotid+glands+are+markedly+different+following+pilocarpine+stimulation.">Fluid secretion rates from mouse and rat parotid glands are markedly different following pilocarpine stimulation.</a> Comp Biochem Physiol A Comp Physiol. 88 (2), 307-310 (1987).</li><li>Lin, A. L., Johnson, D. A., Wu, Y., Wong, G., Ebersole, J. L., Yeh, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+short-term+gamma-irradiation+effects+on+mouse+salivary+gland+function+using+a+new+saliva+collection+device.">Measuring short-term gamma-irradiation effects on mouse salivary gland function using a new saliva collection device.</a> Arch Oral Biol. 46 (11), 1085-1089 (2001).</li><li>Scofield, R. H., Asfa, S., Obeso, D., Jonsson, R., Kurien, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunization+with+short+peptides+from+the+60-kDa+Ro+antigen+recapitulates+the+serological+and+pathological+findings+as+well+as+the+salivary+gland+dysfunction+of+Sjogren's+syndrome.">Immunization with short peptides from the 60-kDa Ro antigen recapitulates the serological and pathological findings as well as the salivary gland dysfunction of Sjogren's syndrome.</a> J Immunol. 175 (12), 8409-8414 (2005).</li><li>Deshmukh, U. S., Nandula, S. R., Thimmalapura, P. R., Scindia, Y. M., Bagavant, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+innate+immune+responses+through+Toll-like+receptor+3+causes+a+rapid+loss+of+salivary+gland+function.">Activation of innate immune responses through Toll-like receptor 3 causes a rapid loss of salivary gland function.</a> J Oral Pathol Med. 38 (1), 42-47 (2009).</li><li>Deshmukh, U. S., Ohyama, Y., Bagavant, H., Guo, X., Gaskin, F., Fu, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+stimuli+accelerate+Sj&#246;gren's+syndrome-like+disease+in+(NZB+x+NZW)F1+mice.">Inflammatory stimuli accelerate Sj&#246;gren's syndrome-like disease in (NZB x NZW)F1 mice.</a> Arthritis Rheum. 58 (5), 1318-1323 (2008).</li><li>Szczerba, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+innate+immunity+and+Ro52-induced+antibody+causes+Sj&#246;gren's+syndrome-like+disorder+in+mice.">Interaction between innate immunity and Ro52-induced antibody causes Sj&#246;gren's syndrome-like disorder in mice.</a> Ann Rheum Dis. 75 (3), 617-622 (2016).</li><li>Takakura, A., Moreira, T., Laitano, S., De Luca J&#250;nior, L., Renzi, A., Menani, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+muscarinic+receptors+signal+pilocarpine-induced+salivation.">Central muscarinic receptors signal pilocarpine-induced salivation.</a> J Dent Res. 82 (12), 993-997 (2003).</li><li>Yao, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharide-induced+elevation+and+secretion+of+interleukin-1beta+in+the+submandibular+gland+of+male+mice.">Lipopolysaccharide-induced elevation and secretion of interleukin-1beta in the submandibular gland of male mice.</a> Immunology. 116 (2), 213-222 (2005).</li><li>Knudsen, J., Nauntofte, B., Josipovic, M., Engelholm, S. A., Hyldegaard, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+isoflurane+anesthesia+and+pilocarpine+on+rat+parotid+saliva+flow.">Effects of isoflurane anesthesia and pilocarpine on rat parotid saliva flow.</a> Radiat Res. 176 (1), 84-88 (2011).</li><li>Kohrs, R., Durieux, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ketamine:+teaching+an+old+drug+new+tricks.">Ketamine: teaching an old drug new tricks.</a> Anesth Analg. 87 (5), 1186-1193 (1998).</li><li>Kozaki, T., Hashiguchi, N., Kaji, Y., Yasukouchi, A., Tochihara, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+saliva+collection+using+cotton+swab+on+cortisol+enzyme+immunoassay.">Effects of saliva collection using cotton swab on cortisol enzyme immunoassay.</a> Eur J Appl Physiol. 107 (6), 743-746 (2009).</li></ol>

The authors have no conflicts of interest to disclose.

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...

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 measure of salivary gland function.
Accurate measurement of saliva production is critical in the study of salivary gland diseases including Sj&#246;gren&#39;s syndrome2 and radiation injury following head and neck cancer treatment3. Several different methods have been developed to measure saliva production in rodents. These include direct cannulation of the excretory salivary duct4, the collection of saliva from the oral cavity under vacuum5, and collection using glass capillaries6 or a micropipette7,8,9. Direct cannulation of the salivary duct provides the most accurate and pure saliva. However, this is a technically challenging procedure, and the potential for causing ductal injury precludes repetitive saliva collections from the same animal. Collecting saliva under vacuum can lead to variable results due to drying of the saliva from the tube. This loss is further exaggerated in mice with reduced salivation. Glass capillaries allow the collection of saliva for subsequent analyses, however, changes in viscosity of the saliva secreted in the diseased state prevents efficient filling of the capillary. Further, attempts to sweep the oral cavity to collect residual saliva can lead to injury. The pipet method allows for complete collection, and for the same operator, it is remarkably consistent between experiments. However, for unknown reasons, this method shows significant variation between different operators. Therefore, to allow appropriate comparisons, it becomes imperative that the same operator performs all the experiments related to a specific project. Clearly, this represents a major disadvantage for a laboratory.
To overcome these issues, a procedure was developed that combines saliva collection methods used for humans and rodents. The swab method, described below for measuring pilocarpine-induced saliva volume is simple, reproducible, not influenced by the operator, and can be performed repeatedly in the same animal. In addition, it allows collecting the saliva for subsequent analyses of proteins, immunoglobulins, or other biomolecules.

<table>
	<tbody>
		<tr>
			<td>Chemicals for saliva collection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pilocarpine hydrochloride</td>
			<td>Alfa Aesar, Tewksbury, MA, USA</td>
			<td>B21410</td>
			<td>Dilute in sterile isotonic saline. Store single use aliquots of 100X stock at -80oC</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Anesthesia Mix solution</td>
			<td></td>
			<td></td>
			<td>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.</td>
		</tr>
		<tr>
			<td>Zetamine (Ketamine hydrochloride)</td>
			<td>Vet One, Boise, Idaho, USA</td>
			<td>C3N VT1</td>
			<td>Stock is 100 mg/mL</td>
		</tr>
		<tr>
			<td>Anased (Xylazine)</td>
			<td>Med-Vet International, Mettawa, IL, USA</td>
			<td>RXANASED-20</td>
			<td>Stock is 20 mg/mL</td>
		</tr>
		<tr>
			<td>Isotonic sterile saline</td>
			<td>Vet One, Boise, Idaho, USA</td>
			<td>501032</td>
			<td>Used as diluent</td>
		</tr>
		<tr>
			<td>Artificial tears (lubricant ophthalmic ointment)</td>
			<td>Henry Schien, Dublin, OH, USA</td>
			<td>48272</td>
			<td>Used to prevent eyes from drying during the procedure</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Materials for saliva collection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SalivaBio Children&#39;s swab</td>
			<td>Salimetrics, LLC Carlsbad, CA, USA</td>
			<td>N/A</td>
			<td>Individually wrapped swabs</td>
		</tr>
		<tr>
			<td>50 mL polypropylene tubes</td>
			<td>VWR, Radnor, PA, USA</td>
			<td>89004-364</td>
			<td>Cut off the bottom 1 cm of the tube. Make sure that the cut edge is smooth.</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes 0.6 mL</td>
			<td>VWR, Radnor, PA, USA</td>
			<td>87003-290</td>
			<td>Make holes in the bottom of tube with heated 18 gauge needles</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes 2 mL</td>
			<td>VWR, Radnor, PA, USA</td>
			<td>87003-298</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin Syringes with permanently attached needles</td>
			<td>Becton Dickinson and Company, Franklin Lakes, NJ, USA</td>
			<td>324702</td>
			<td></td>
		</tr>
		<tr>
			<td>18 gauge regular bevel needles</td>
			<td>Becton Dickinson and Company, Franklin Lakes, NJ, USA</td>
			<td>305195</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile scalpel blade #11</td>
			<td>Integra York Inc PA, USA</td>
			<td>4-311</td>
			<td></td>
		</tr>
		<tr>
			<td>Microdissecting forceps</td>
			<td>Roboz, Gaithersburg, MD, USA</td>
			<td>RS-5139</td>
			<td>Serrated angular 0.8 mm tip, 4&#34; length</td>
		</tr>
		<tr>
			<td>Label tape</td>
			<td>Santa Cruz Biotechnology, Dallas, TX, USA</td>
			<td>sc-224487</td>
			<td></td>
		</tr>
		<tr>
			<td>3 channel timer</td>
			<td>Amazon.com, Seattle, WA, USA</td>
			<td>B06W2KCYVN</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical Balance</td>
			<td>Mettler Toledo, Columbus OH, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Chemicals/ reagents for inducing salivary dysfunction</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LPS</td>
			<td>Invivogen, San Deigo, CA, USA</td>
			<td>tlrl-b5lps</td>
			<td>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</td>
		</tr>
		<tr>
			<td>Imject Alum adjuvant</td>
			<td>Thermo Scientific</td>
			<td>77161</td>
			<td>Dilute 1:1 in sterile saline. Inject intraperitoneally 0.1 mL/mouse</td>
		</tr>
		<tr>
			<td>Rabbit anti-Ro52 antiserum</td>
			<td>Generated in lab</td>
			<td></td>
			<td>Immunization of rabbits with recombinant mouse Ro52</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Chemicals/ Kits for saliva analyses</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Salivary lysozyme estimation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EnzChek Lysozyme Assay Kit</td>
			<td>Molecular Probes, Eugene, OR, USA</td>
			<td>E-22013</td>
			<td>Used as per manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Salivary IgA estimation by sandwich ELISA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgA</td>
			<td>Southern Biotech, Birmingham, AL, USA</td>
			<td>0106-01</td>
			<td>Standards for sandwich ELISA - range 30 ng/mL to 0.5 ng/mL</td>
		</tr>
		<tr>
			<td>Goat anti- mouse IgA unlabeled</td>
			<td>Southern Biotech, Birmingham, AL, USA</td>
			<td>1040-01</td>
			<td>Coat at 1 ug/mL in bicarbonate buffer</td>
		</tr>
		<tr>
			<td>Goat anti- mouse IgA HRP</td>
			<td>Southern Biotech, Birmingham, AL, USA</td>
			<td>1040-05</td>
			<td>Detection antibody used at 1:4000 dilution</td>
		</tr>
		<tr>
			<td>TMB substrate</td>
			<td>Becton Dickinson and Company, Franklin Lakes, NJ, USA</td>
			<td>555214</td>
			<td>Used as per manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>Immulon 4HBX Microtiter 96 well plates</td>
			<td>Thermo Scientific, Rochester, NY, USA</td>
			<td>3855</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Salivary protein estimation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Rad Protein Assay Dye Reagent Concentrate</td>
			<td>BioRad, Hercules, CA, USA</td>
			<td>5000006</td>
			<td>For protein estimation as per manufacturer&#39;s instructions</td>
		</tr>
	</tbody>
</table>

a method for the measurement of salivary gland function in mice

Patients with Sj&#246;gren&#39;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.

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...

Watch this Scientific Journal Video about A Method for the Measurement of Salivary Gland Function in Mice at JoVE.com

A Method for the Measurement of Salivary Gland Function in Mice

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.

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...

a-method-for-the-measurement-of-salivary-gland-function-in-mice

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Immunology and Infection

18.3K Views.  Oklahoma Medical Research Foundation. 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.

Video: A Method for the Measurement of Salivary Gland Function in Mice

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

A Method for Labeling Vasculature in Embryonic Mice

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in the thymus proper vasculature formation and patterning is essential for thymocyte entry into the organ and mature T-cell exit to the periphery. The spatial arrangement of blood vessels in the thymus is dependent upon signals from the local microenvironment, namely thymic epithelial cells (TEC). Several recent reports suggest that disruption of these signals results in thymus blood vessel defects 1,2. Previous studies have described techniques used to label the neonatal and adult thymus vasculature 1,2. We demonstrate here a technique for labeling blood vessels in the embryonic thymus. This method combines the use of FITC-dextran or Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL 1 - isolectin B4) facial vein injections and CD31 antibody staining to identify thymus vascular structures and PDGFR-&beta; to label thymic perivascular mesenchyme 3-5. The option of using cryosections or vibratome sections is also provided. This protocol can be used to identify thymus vascular defects, which is critical for defining the roles of TEC-derived molecules in thymus blood vessel formation. As the method labels the entire vasculature, it can also be used to analyze the vascular networks in multiple organs and tissues throughout the embryo including skin and heart 6-10.

This article describes a method for labeling embryonic skin and thymus blood vessels.

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

Measurement of &#947;HV68 Infection in Mice

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human &gamma;-HVs, such as EBV and KSHV, has severely hindered detailed pathogenic studies. Murine &gamma;-herpesvirus 68 (&gamma;HV68) shares extensive genetic and biological similarities with human &gamma;-HVs and is a natural pathogen of murid rodents2. As such, evaluation of &gamma;HV68 infection of mice inbred strains at different stages of viral infection provides an important model for understanding viral lifecycle and pathogenesis during &gamma;-HVs infection.
Upon intranasal inoculation, &gamma;HV68 infection results in acute viremia in the lung that is later resolved into a latent infection of splenocytes and other cells, which may be reactivated throughout the life of the host3,4. In this protocol, we will describe how to use the plaque assay to assess infectious virus titer in the lung homogenates on Vero cell monolayers at the early stage (5 - 7 days) of post-intranasal infection (dpi). While acute infection is largely cleared 2 - 3 weeks postinfection, a latent infection of &gamma;HV68 is established around 14 dpi and maintained later on in the spleen of the mice. Latent infection usually affects a very small population of cells in the infected tissues, whereby the virus stays dormant and shuts off most of its gene expression. Latently-infected splenocytes spontaneously reactivate virus upon explanting into tissue culture, which can be recapitulated by an infectious center (IC) assay to determine the viral latent load. To further estimate the amount of viral genome copies in the acutely and/or latently infected tissues, quantitative real-time PCR (qPCR) is used for its maximal sensitivity and accuracy. The combined analyses of the results of qPCR and plaque assay, and/or IC assay will reveal the spatiotemporal profiles of viral replication and infectivity in vivo.

&#947;-Herpesviruses (&#947;-HVs) establish life-long persistency in their host. Infection of mice with &#947;-HV68 provides a genetically tractable in vivo model for the characterization of the lifecycle/pathogenesis of &#947;HVs. This protocol describes the detection and quantitation of &#947;HV68 infection at acute and latent stages following infection by plaque-forming, infectious center, and qPCR assays.

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human ...

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes. Investigation of transcripts and gene regulators that are expressed in tissues in which the mosquito host and pathogen interact, and in organs involved in reproduction are of great interest for strategies to reduce mosquito-borne disease transmission and disrupt egg development. A number of tools have been employed to study and validate the temporal and tissue-specific regulation of gene expression. Here, we describe protocols that have been developed to obtain spatial information, which enhances our understanding of where specific genes are expressed and their products accumulate. The protocol described has been used to validate expression and determine accumulation patterns of transcripts in tissues related to mosquito-borne pathogen transmission, such as female salivary glands, as well as subcellular compartments of ovaries and embryos, which relate to mosquito reproduction and development. 
 The following procedures represent an optimized methodology that improves the efficiency of various steps in the protocol without loss of target-specific hybridization signals. Guidelines for RNA probe preparation, dissection of soft tissues and the general procedure for fixation and hybridization are described in Part A, while steps specific for the collection, fixation, pre-hybridization and hybridization of mosquito embryos are detailed in Part B.

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Temporal and spatial gene expression analyses have a crucial role in functional genomics. Whole-mount hybridization in situ is useful for determining the localization of transcripts within tissues and subcellular compartments. Here we outline a hybridization in situ protocol with modifications for specific target tissues in mosquitoes.

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes.  Investigation of transcripts and gene ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, including cell biology, oncology, dentistry, and immunology. The SMG is an exocrine gland comprised of secretory epithelial cells, myofibroblasts, endothelial cells, nerves, and extracellular matrix. Dynamic cellular processes in the rat and mouse SMG have previously been imaged, mostly using inverted multi-photon microscope systems. Here, we describe a straightforward protocol for the surgical preparation and stabilization of the murine SMG in anesthetized mice for in vivo imaging with upright multi-photon microscope systems. We present representative intravital image sets of endogenous and adoptively transferred fluorescent cells, including the labeling of blood vessels or salivary ducts and second harmonic generation to visualize fibrillar collagen. In sum, our protocol allows for surgical preparation of mouse salivary glands in upright microscopy systems, which are commonly used for intravital imaging in the field of immunology.

We describe a protocol to surgically expose and stabilize the murine submandibular salivary gland for intravital imaging using upright intravital microscopy. This protocol is easily adaptable to other exocrine glands of the head and neck region of mice and other small rodents.

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, ...

A Silver Nanoparticle Method for Ameliorating Biliary Atresia Syndrome in Mice

Biliary atresia (BA) is a severe type of cholangitis with high mortality in children of which the etiology is still not fully understood. Viral infections may be one possible cause. The typical animal model used for studying BA is established by inoculating a neonatal mouse with a rhesus rotavirus. Silver nanoparticles have been shown to exert antibacterial and antiviral effects; their function in the BA mouse model is evaluated in this study. Currently, in BA animal experiments, the methods used to improve the symptoms of BA mice are generally symptomatic treatments given via food or other drugs. The aim of this study is to demonstrate a new method for ameliorating BA syndrome in mice by the intraperitoneal injection of silver nanoparticles and to provide detailed methods for preparing the silver nanoparticle gel formulation. This method is simple and widely applicable and can be used to research the mechanism of BA, as well as in clinical treatments. Based on the BA mouse model, when the mice exhibit jaundice, the prepared silver nanoparticle gel is injected intraperitoneally to the surface of the lower liver. The survival status is observed, and biochemical indicators and liver histopathology are examined. This method allows a more intuitive understanding of both the establishment of the BA model and novel BA treatments.

This article describes in detail a method based on silver nanoparticles for ameliorating biliary atresia syndrome in an experimental biliary atresia mouse model. A solid understanding of the reagent preparation process and the neonatal mouse injection technique will help familiarize researchers with the method used in neonatal mouse model studies.

Biliary atresia (BA) is a severe type of cholangitis with high mortality in children of which the etiology is still not fully understood. Viral infections ...

Measurement of Specific Mycobacterial Mistranslation Rates with Gain-of-function Reporter Systems

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 per codon, the actual error rates vary widely, depending on the species, environment, and codons being studied. We have previously shown that mycobacteria use a two-step pathway for the generation of aminoacylated glutamine and asparagine tRNAs and that this is specifically associated with relatively high error rates due to the modulation of mistranslation rates by an essential component of the pathway, the amidotransferase GatCAB. We modified a previously employed Renilla-Firefly dual-luciferase system that had been used to measure mistranslation rates in Escherichia coli for use in mycobacteria to measure specific mistranslation rates of glutamate at glutamine codons and aspartate for asparagine codons. Although this reporter system was suitable for the accurate estimation of specific error rates, lack of sensitivity and requirements for excessive manipulation steps made it unsuitable for high-throughput applications. Therefore, we developed a second gain-of-function reporter system, using Nluc luciferase and green fluorescent protein (GFP), which is more amenable to medium/high-throughput settings. We used this system to identify kasugamycin as a small molecule that can decrease mycobacterial mistranslation. Although the reporters that we describe here have been used to measure specific types of mycobacterial mistranslation, they may be modified to measure other types of mistranslation in a number of model systems.

In this article, we present two complementary methods to measure specific rates of translational error and mistranslation in the model mycobacterium, Mycobacterium smegmatis, using gain-of-function reporter systems. The methods can be employed to measure accurate error rates in low throughput or relative error rates in a more high-throughput setting.

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 ...

A Method for the Measurement of Salivary Gland Function in Mice

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Chapters in this video

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A Method for Labeling Vasculature in Embryonic Mice

Measurement of γHV68 Infection in Mice

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Quantitative Measurement of the Immune Response and Sleep in Drosophila

Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy

A Silver Nanoparticle Method for Ameliorating Biliary Atresia Syndrome in Mice

Measurement of Specific Mycobacterial Mistranslation Rates with Gain-of-function Reporter Systems