Research reported in this publication was supported by the National Institute of Dental and Craniofacial Research (NIDCR) and the National Cancer Institute (NCI) of the National Institutes of Health under Award Number R56 DE025098, UG3 DE027695, and F30 CA206296. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This work was also supported by the NSF DMR 1206219 and the IADR Innovation in Oral Care Award (2016). 
We would like to thank Dr. Eri Maruyama and Andrew Hollomon for their assistance with saliva collection. We would like to thank Pei-Lun Weng for his assistance with gland dissection. We would like to thank Matthew Ingalls for his assistance in figure preparation. We would like to thank Dr. Elaine Smolock and Emily Wu for critical reading of this manuscript.

All in vivo procedures outlined below were approved by the University Committee on Animal Resources at the University of Rochester, Rochester. NY.
1. Preparation
<ol>
	<li>Using an analytical balance, weigh 20 mg of pilocarpine. Dissolve it in 2 mL of sterile saline in a microcentrifuge tube. 
	NOTE: Because pilocarpine is light sensitive and loses activity over time, this solution should be prepared the day of injection, and protected from light until administered.</li>
	<li>U...

<ol>
	<li>Bradley, P., O'Hara, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diseases+of+the+salivary+glands.">Diseases of the salivary glands.</a> Surgery (Oxford). 33 (12), 614-619 (2015).</li><li>Fox, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salivary+enhancement+therapies.">Salivary enhancement therapies.</a> Caries Research. 38 (3), 241-246 (2004).</li><li>Konings, A. W. T., Coppes, R. P., Vissink, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+mechanism+of+salivary+gland+radiosensitivity.">On the mechanism of salivary gland radiosensitivity.</a> International Journal of Radiation Oncology Biology Physics. 62 (4), 1187-1194 (2005).</li><li>Burlage, F. R., Coppes, R. P., Meertens, H., Stokman, M. A., Vissink, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parotid+and+submandibular/sublingual+salivary+flow+during+high+dose+radiotherapy.">Parotid and submandibular/sublingual salivary flow during high dose radiotherapy.</a> Radiotherapy and Oncology. 61 (3), 271-274 (2001).</li><li>Aure, M. H., Konieczny, S. F., Ovitt, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salivary+gland+homeostasis+is+maintained+through+acinar+cell+self-duplication.">Salivary gland homeostasis is maintained through acinar cell self-duplication.</a> Developmental Cell. 33 (2), 231-237 (2015).</li><li>Aure, M. H., Arany, S., Ovitt, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salivary+Glands:+Stem+Cells,+Self-duplication,+or+Both?.">Salivary Glands: Stem Cells, Self-duplication, or Both?.</a> Journal of Dental Research. 94 (11), 1502-1507 (2015).</li><li>Ono, K., 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=Distinct+effects+of+cevimeline+and+pilocarpine+on+salivary+mechanisms,+cardiovascular+response+and+thirst+sensation+in+rats.">Distinct effects of cevimeline and pilocarpine on salivary mechanisms, cardiovascular response and thirst sensation in rats.</a> Archives of Oral Biology. 57 (4), 421-428 (2012).</li><li>Proctor, G. B., Carpenter, G. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiation-induced+xerostomia:+pathophysiology,+clinical+course+and+supportive+treatment.">Radiation-induced xerostomia: pathophysiology, clinical course and supportive treatment.</a> Support Care Cancer. 5 (4), 281-288 (1997).</li><li>Lin, A. L., 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=Measuring+short-term+&#947;-irradiation+effects+on+mouse+salivary+gland+function+using+a+new+saliva+collection+device.">Measuring short-term &#947;-irradiation effects on mouse salivary gland function using a new saliva collection device.</a> Archives of Oral Biology. 46 (11), 1085-1089 (2001).</li><li>Montenegro, M. F., 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=Profound+differences+between+humans+and+rodents+in+the+ability+to+concentrate+salivary+nitrate:+Implications+for+translational+research.">Profound differences between humans and rodents in the ability to concentrate salivary nitrate: Implications for translational research.</a> Redox biology. 10, 206-210 (2016).</li><li>Choi, J. S., Park, I. S., Kim, S. K., Lim, J. Y., Kim, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphometric+and+Functional+Changes+of+Salivary+Gland+Dysfunction+After+Radioactive+Iodine+Ablation+in+a+Murine+Model.">Morphometric and Functional Changes of Salivary Gland Dysfunction After Radioactive Iodine Ablation in a Murine Model.</a> Thyroid. 23 (11), 1445-1451 (2013).</li><li>Imamura, T. K., 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=Inhibition+of+pilocarpine-induced+saliva+secretion+by+adrenergic+agonists+in+ICR+mice.">Inhibition of pilocarpine-induced saliva secretion by adrenergic agonists in ICR mice.</a> Clinical and Experimental Pharmacology and Physiology. 39 (12), 1038-1043 (2012).</li><li>Ma, T., 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=Defective+Secretion+of+Saliva+in+Transgenic+Mice+Lacking+Aquaporin-5+Water+Channels.">Defective Secretion of Saliva in Transgenic Mice Lacking Aquaporin-5 Water Channels.</a> Journal of Biological Chemistry. 274 (29), 20071-20074 (1999).</li><li>Parkes, M. W., Parks, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supersensitivity+of+salivation+in+response+to+pilocarpine+after+withdrawal+of+chronically+administered+hyoscine+in+the+mouse.">Supersensitivity of salivation in response to pilocarpine after withdrawal of chronically administered hyoscine in the mouse.</a> British Journal of Pharmacology. 46 (2), 315-323 (1972).</li><li>Nishiyama, T., 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=Up-Regulated+PAR-2-Mediated+Salivary+Secretion+in+Mice+Deficient+in+Muscarinic+Acetylcholine+Receptor+Subtypes.">Up-Regulated PAR-2-Mediated Salivary Secretion in Mice Deficient in Muscarinic Acetylcholine Receptor Subtypes.</a> Journal of Pharmacology and Experimental Therapeutics. 320 (2), 516 (2007).</li><li>Yang, B., Song, Y., Zhao, D., Verkman, A. 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We present a multistep method to assess salivary gland function, which can be applied to study gland injury and therapeutics. Our procedure involves tracheostomy, saliva collection, and gland dissection, each of which has experimental applications that can support an integrated study of salivary gland biology. For example, murine tracheostomy has been used for general airway management during procedures obstructing the oral cavity.
Proper dissection and tracheal incision are required for piloc...

Salivary gland disorders include syndromes of dysregulated or impaired secretion leading to either overproduction (sialorrhea) or underproduction (xerostomia and hyposalivation) of saliva1. In both cases, there is an interest in improving our understanding of salivary gland biology towards the end goal of therapeutic development2.
The salivary glands are highly radiosensitive organs, and are often damaged during head and neck cancer radiotherapy, leading to permanent dry mouth (xerostomia)3,4. Unlike other radiosensitive tissues, however, salivary gland turnover rate is relatively low, and the mechanism of secretory loss is poorly understood5,6. In this unique injury setting, tissue regeneration and radioprotection strategies require salivary functional assessment. Experimentally, murine saliva collection is a particularly valuable tool in evaluating the gland response to both radiation and therapeutic agents.
Here, we present a method for performing and quantifying stimulated saliva secretion using pilocarpine, a potent muscarinic agonist7. Pilocarpine stimulates the autonomic nervous system to induce gland secretion8,9. To complete this test appropriately, a tracheostomy is required to ensure that the mouse maintains a patent airway throughout the procedure, and to reduce the risks of choking and aspiration from pooled secretions in the oral cavity10.
This is a terminal procedure, culminating in the removal of the three major salivary glands: the parotid (PG), the submandibular (SMG), and the sublingual (SLG). For functional studies, gland weights are recorded and are often used to normalize saliva measurement11,12,13. This data is particularly important in radiation studies, wherein gland atrophy is an expected outcome14,15
There is variability in the literature with regards to how stimulated saliva secretion is performed and reported16. For example, pilocarpine doses within the literature span at least three orders of magnitude17,18,19,20,21,22,23. Here, we present an optimized high dose pilocarpine protocol with the intent of improved reproducibility in method execution, as well as providing a modular platform of techniques (tracheostomy, saliva collection, and gland dissection) that can be adapted as needed.
In addition to protocol demonstration, we include representative functional data of saliva flow at 2 weeks following fractionated radiation (4 doses of 6.85 Gy) to the SMG region.

<table border="0" cellpadding="0" cellspacing="0" width="690">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Pilocarpine hydrochloride</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:119px;">P6503</td>
			<td style="width:225px;">Pilocarpine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Student Vannas Spring Scissors</td>
			<td style="width:128px;">Fine Science Tools</td>
			<td style="width:119px;">91500-9</td>
			<td>Spring Scissors for Tracheostomy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Sterile Saline Solution</td>
			<td style="width:128px;">Medline</td>
			<td style="width:119px;">RDI30296H</td>
			<td>Saline</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:219px;">Dumont #7 Forceps</td>
			<td style="width:128px;">Fine Science Tools</td>
			<td style="width:119px;">11274-20</td>
			<td>Curved Forceps</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:219px;">Dumont #5 Forceps</td>
			<td style="width:128px;">Fine Science Tools</td>
			<td style="width:119px;">11251-10</td>
			<td>Straight Forceps</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:219px;">Standard Pattern Forceps</td>
			<td style="width:128px;">Fine Science Tools</td>
			<td style="width:119px;">11000-12</td>
			<td>Blunt Forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Fine Scissors- Tungsten Carbide</td>
			<td style="width:128px;">Fine Science Tools</td>
			<td style="width:119px;">14568-09</td>
			<td>Dissection Scissors</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Microhematocrit Heparinized Capillary Tubes</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:119px;">22362566</td>
			<td>Capillary tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Lubricant Eye Ointment</td>
			<td style="width:128px;">Refresh</td>
			<td style="width:119px;">N/A</td>
			<td>Refresh Lacri-Lube</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Goat polyclonal anti-Nkcc1</td>
			<td style="width:128px;">Santa Cruz Biotech</td>
			<td style="width:119px;">SC-21545</td>
			<td>Nkcc1 Antibody</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">D1306</td>
			<td>DAPI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">GraphPad Prism</td>
			<td style="width:128px;">GraphPad</td>
			<td style="width:119px;">ver6.0</td>
			<td>Statistical Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Cotton tipped applicator</td>
			<td style="width:128px;">Medline</td>
			<td style="width:119px;">MDS202000</td>
			<td>Applicator for eye ointment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">0.5cc Insulin Syringe, 29G x 1/2&#34;</td>
			<td style="width:128px;">BD</td>
			<td style="width:119px;">7629</td>
			<td>Syringe for intraperitoneal injection</td>
		</tr>
	</tbody>
</table>

murine salivary functional assessment via pilocarpine stimulation following fractionated radiation

Hyposalivation is commonly observed in the autoimmune reaction of Sj&#246;gren's syndrome or following radiation injury to the major salivary glands. In these cases, questions remain regarding disease pathogenesis and effective interventions. An optimized technique that allows functional assessment of the salivary glands is invaluable for investigating exocrine gland biology, dysfunction, and therapeutics. Here, we present a step by step approach to performing pilocarpine stimulated saliva secretion, including tracheostomy and the dissection of the three major murine salivary glands. We also detail the appropriate murine head and neck anatomy accessed during these techniques. This approach is scalable, allowing for multiple mice to be processed simultaneously, thus improving the efficiency of the work flow. We aim to improve the reproducibility of these methods, each of which has further applications within the field. In addition to saliva collection, we discuss metrics for quantifying and normalizing functional capacity of these tissues. Representative data are included from submandibular glands with depressed salivary gland function 2 weeks following fractionated radiation (4 doses of 6.85 Gy).

When performing high dose pilocarpine stimulated saliva collection, it is important to maintain the airway to prevent aspiration or choking from secretions in the oral cavity. A schematic of the tracheostomy is provided (Figure 1). Following tracheal incision, the stoma must remain clear of tissue and fluids.
To enhance capillary action during saliva collection, mice should be positioned with their ...

Watch this Scientific Journal Video about Murine Salivary Functional Assessment via Pilocarpine Stimulation Following Fractionated Radiation at JoVE.com

Murine Salivary Functional Assessment via Pilocarpine Stimulation Following Fractionated Radiation

We present a detailed approach to performing saliva collection, including murine tracheostomy and the isolation of three major salivary glands.

Hyposalivation is commonly observed in the autoimmune reaction of Sj&#246;gren's syndrome or following radiation injury to the major salivary glands. In ...

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.

murine-salivary-functional-assessment-via-pilocarpine-stimulation

Research

JoVE Journal

Medicine

10.8K Views.  University of Rochester. 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 s...