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>Using an analytical balance, weigh and identify all collection tubes and glass capillaries. The number of collection tubes and glass capillaries should match&#160;the number of mice.</li>
</ol>
2. Tracheostomy
<ol>
	<li>Weigh C57/BL6 mice using an analytical balance.</li>
	<li>Using a 0.5 mL syringe with 29G x &#189;&#34; needle, anesthetize mice with an intraperitoneally injected sterile saline solution of 100 mg/kg ketamine and 10 mg/kg xylazine. Proceed to the following step when the mouse no longer responds to stimuli (i.e.&#160;absence of&#160;the pedal withdrawal reflex), which generally occurs within 5 to 10 min following injection.</li>
	<li>After dispensing lubricant on a cotton-tipped applicator, gently apply to eyes and place the mouse in a supine position on the stage.&#160;The anesthetized animal must be kept on warm surface. 
	NOTE: All steps are performed at room temperature.</li>
	<li>Secure the nose, limbs, and tail to the stage using surgical tape.</li>
	<li>Clean and wet the neck with an alcohol wipe. 
	NOTE: This will help prevent fur from entering subsequent incisions.</li>
	<li>Raising the ventral midline neck skin with forceps, make a small, superficial cut using dissecting scissors. Guide the scissors into the opening and slowly open the blades subcutaneously to separate tissue planes.</li>
	<li>Keeping the skin raised, carefully cut to 1 cm below the mouth.</li>
	<li>Make two lateral incisions at the inferior and superior aspects of the first cut. Using forceps, gently reflect away the skin to enable access to head and neck structures.</li>
	<li>Using the dissecting microscope at 8X magnification, visualize the submandibular glands (midline: Figure 1).</li>
	<li>Using fine forceps, gently lift the glands to expose the four infrahyoid (strap) muscles overlying the trachea. Avoid tearing or disrupting the surrounding vasculature or excretory ducts.</li>
	<li>If collecting saliva from more than one mouse, repeat steps 2.1 - 2.10 with each mouse before continuing. 
	NOTE: This procedure can be safely performed on up to 5 mice concurrently.</li>
	<li>With small dissecting scissors, remove the medial portion of strap muscles while staying as close to midline as possible. Cut only as much as necessary to visualize trachea and keep strap muscles out of the way during the procedure. Avoid nicking any nearby vessels because if there is volume depletion secondary to significant hemorrhage, pilocarpine will not be effective at inducing secretion.</li>
	<li>Reflect the strap muscles away from the trachea.</li>
	<li>Visualize the larynx, trachea, and thyroid gland. Ensure that the trachea is clear of overlying tissue.</li>
	<li>Make a horizontal incision in the trachea inferior/posterior to the thyroid using small dissecting scissors. Ensure that the airway is patent and clear of fluid.</li>
</ol>
3. Saliva Collection
<ol>
	<li>Remove the tape near mouth and angle dissection stage downward (45&#176;, cranially) to assist with saliva flow.</li>
	<li>Using a 0.5 mL syringe with 29G x &#189;&#34; needle, perform intraperitoneal injection of 10 &#181;L/g body weight pilocarpine for a total dose of 100 mg/kg.</li>
	<li>Start a timer. If dosing multiple mice, reduce lead time by moving quickly and by having an assistant simultaneously inject the remaining mice.</li>
	<li>Using standard forceps, open the mouth for capillary tube access.</li>
	<li>Once a bead of saliva is observed in the mouth, place the proximal end of the capillary tube into the fluid, with the distal end placed into a collecting tube positioned below the dissecting stage (both tubes pre-weighed). 
	NOTE: In C57/BL6 female mice 6 - 10 weeks of age, gross salivation occurs within 2 min of pilocarpine injection.</li>
	<li>If saliva is building in the mouth, but not flowing through the tube, reattempt previous step. Ensure that the tube is not placed directly against a mucosal surface, which can obstruct the inlet.</li>
	<li>Collect saliva for a total of 12 min following pilocarpine stimulation. Ensure that the stoma remains clear. 
	NOTE: Increased tidal volume and secretions (salivary, urinary, fecal) are normal during this time.</li>
	<li>If mice have depressed saliva function (e.g., due to injury or intervention), transfer the remaining fluid from the mouth to the collection tube using a P200 micropipette. If collecting from multiple mice, ensure that tips are changed.</li>
	<li>Record the weight of collected saliva plus tubes 12 min after pilocarpine injection.</li>
</ol>
4. Gland Dissection
<ol>
	<li>Euthanize mice by CO2 euthanasia&#160;(as described in the AVMA Guidelines for Euthanasia of Animals: 2013 Edition) followed by thoracotomy, taking care to preserve the structures of the head and neck. For this reason, avoid cervical dislocation as a method of euthanasia.</li>
	<li>Place the mouse in a supine position on the stage. Reposition glands in the original site over the trachea (Figure 1).</li>
	<li>Using a dissecting microscope under 8X magnification, visualize the major glands: the PG, SMG, and SLG. To distinguish the glands during the dissection, identify that the PG is diffuse, lying lateral to the SMG and SLG. 
	NOTE: If dissecting the PG, this should be done first because it is the least defined and most likely to be torn.</li>
	<li>Grasp the tail of the PG with forceps, pulling it away from underlying structures, and gently apply tension before cutting the head of the PG with dissecting scissors.</li>
	<li>Breach the capsule surrounding the SMGs using forceps. Gently separate the left and right SMG.</li>
	<li>Free the dorsal aspect of the SMG from adhered nonglandular tissue using forceps.</li>
	<li>Remove the SMG from the neck by drawing Wharton&#39;s duct taut with forceps and cutting the duct with dissecting scissors.</li>
	<li>Gently separate the SLG from the SMG using forceps. 
	NOTE: The SLG is in the superolateral aspect of the SMG.</li>
	<li>Using an analytic balance, record SMG weights.</li>
</ol>

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The authors have nothing to disclose.

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><tbody><tr><td>Pilocarpine hydrochloride</td><td>Sigma Aldrich</td><td>P6503</td><td>Pilocarpine</td></tr><tr><td>Student Vannas Spring Scissors</td><td>Fine Science Tools</td><td>91500-9</td><td>Spring Scissors for Tracheostomy</td></tr><tr><td>Sterile Saline Solution</td><td>Medline</td><td>RDI30296H</td><td>Saline</td></tr><tr><td>Dumont #7 Forceps</td><td>Fine Science Tools</td><td>11274-20</td><td>Curved Forceps</td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11251-10</td><td>Straight Forceps</td></tr><tr><td>Standard Pattern Forceps</td><td>Fine Science Tools</td><td>11000-12</td><td>Blunt Forceps</td></tr><tr><td>Fine Scissors- Tungsten Carbide</td><td>Fine Science Tools</td><td>14568-09</td><td>Dissection Scissors</td></tr><tr><td>Microhematocrit Heparinized Capillary Tubes</td><td>Fisher Scientific</td><td>22362566</td><td>Capillary tubes</td></tr><tr><td>Lubricant Eye Ointment</td><td>Refresh</td><td>N/A</td><td>Refresh Lacri-Lube</td></tr><tr><td>Goat polyclonal anti-Nkcc1</td><td>Santa Cruz Biotech</td><td>SC-21545</td><td>Nkcc1 Antibody</td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>Thermo Fisher Scientific</td><td>D1306</td><td>DAPI</td></tr><tr><td>GraphPad Prism</td><td>GraphPad</td><td>ver6.0</td><td>Statistical Software</td></tr><tr><td>Cotton tipped applicator</td><td>Medline</td><td>MDS202000</td><td>Applicator for eye ointment</td></tr><tr><td>0.5cc Insulin Syringe, 29G x 1/2&quot;</td><td>BD</td><td>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 ...

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Research

JoVE Journal

Medicine

11.1K Views.  University of Rochester. We present a detailed approach to performing saliva collection, including murine tracheostomy and the isolation of three major salivary glands.

Video: Murine Salivary Functional Assessment via Pilocarpine Stimulation Following Fractionated Radiation

Cannulation of the Mouse Submandibular Salivary Gland via the Wharton's Duct

Severe salivary gland hypofunction is frequently found in patients with Sj&ouml;gren's syndrome and those who receiving therapeutic 
irradiation in their head and neck regions for cancer treatment. Both groups of patients experience symptoms such as xerostomia (dry mouth), dysphagia 
(impaired chewing and swallowing), severe dental caries, altered taste, oro-pharyngeal infections (candidiasis), mucositis, pain and discomfort.
One innovative approach of regenerative medicine for the treatment of salivary gland hypo-function is speculated in RS Redman, E Mezey 
et al. 2009: stem cells can be directly deposited by cannulation into the gland as a potent method in reviving the functions of the impaired organ. Presumably, 
the migrated foreign stem cells will differentiate into glandular cells to function as part of the host salivary gland. Also, this cannulation technique is an 
expedient and effective delivery method for clinical gene transfer application.
Here we illustrate the steps involved in performing the cannulation procedure on the mouse submandibular salivary gland via the Wharton's duct 
(Fig 1). C3H mice (Charles River, Montreal, QC, Canada) are used for this experiment, which have been kept under clean conventional conditions at 
the McGill University animal resource center. All experiments have been approved by the University Animal Care Committee and were in accordance with the 
guidelines of the Canadian Council on Animal Care.
For this experiment, a trypan blue solution is infused into the gland through the opening of the Wharton's duct using a 
insulin syringe with a 29-gauge needle encased inside a polyethylene tube. Subsequently, the mouse is dissected to show that the infusions migrated 
into the gland successfully.

A protocol for the cannulation of the mouse submandibular salivary gland via the Wharton's duct is described. For this experiment, the trypan blue solution is used as a dyer to demonstrate how this technique effectively delivers infusions into the targeted gland, and to suggest the reliability of this new approach as a potential clinical drug/cell therapy for the regeneration of salivary glands.

Severe salivary gland hypofunction is frequently found in patients with Sj&ouml;gren's syndrome and those who receiving therapeutic 
irradiation in their ...

Quantifying Cognitive Decrements Caused by Cranial Radiotherapy

With the exception of survival, cognitive impairment stemming from the clinical management of cancer is a major factor dictating therapeutic outcome. For many patients afflicted with CNS and non-CNS malignancies, radiotherapy and chemotherapy offer the best options for disease control. These treatments however come at a cost, and nearly all cancer survivors (~11 million in the US alone as of 2006) incur some risk for developing cognitive dysfunction, with the most severe cases found in patients subjected to cranial radiotherapy (~200,000/yr) for the control of primary and metastatic brain tumors1. Particularly problematic are pediatric cases, whose long-term survival plagued with marked cognitive decrements results in significant socioeconomic burdens2. To date, there are still no satisfactory solutions to this significant clinical problem.
We have addressed this serious health concern using transplanted stem cells to combat radiation-induced cognitive decline in athymic rats subjected to cranial irradiation3. Details of the stereotaxic irradiation and the in vitro culturing and transplantation of human neural stem cells (hNSCs) can be found in our companion paper (Acharya et al., JoVE reference). Following irradiation and transplantation surgery, rats are then assessed for changes in cognition, grafted cell survival and expression of differentiation-specific markers 1 and 4-months after irradiation. To critically evaluate the success or failure of any potential intervention designed to ameliorate radiation-induced cognitive sequelae, a rigorous series of quantitative cognitive tasks must be performed. To accomplish this, we subject our animals to a suite of cognitive testing paradigms including novel place recognition, water maze, elevated plus maze and fear conditioning, in order to quantify hippocampal and non-hippocampal learning and memory. We have demonstrated the utility of these tests for quantifying specific types of cognitive decrements in irradiated animals, and used them to show that animals engrafted with hNSCs exhibit significant improvements in cognitive function3.
The cognitive benefits derived from engrafted human stem cells suggest that similar strategies may one day provide much needed clinical recourse to cancer survivors suffering from impaired cognition. Accordingly, we have provided written and visual documentation of the critical steps used in our cognitive testing paradigms to facilitate the translation of our promising results into the clinic.

Cognitive impairment resulting from the radiotherapeutic management of brain tumors represents a clinically intractable condition that adversely impacts quality of life. The capability to critically evaluate potential interventions for ameliorating radiation-induced cognitive decrements ultimately depends on the capability to undertake rigorous quantitative assessments of cognition.

With the exception of survival, cognitive impairment stemming from the clinical management of cancer is a major factor dictating therapeutic outcome. For ...

Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral delivery or transgenic animals have potential confounds that make interpretation of results from these studies difficult. New radiological tools are emerging, however, that allow one to noninvasively investigate the function of select groups of adult-born neurons through accurate and precise anatomical targeting in small animals. Focal ionizing radiation inhibits the birth and differentiation of new neurons, and allows targeting of specific neural progenitor regions. In order to illuminate the potential functional role that adult hypothalamic neurogenesis plays in the regulation of physiological processes, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of adult-born neurons in the hypothalamic median eminence. We describe a method for Computer tomography-guided focal irradiation (CFIR) delivery to enable precise and accurate anatomical targeting in small animals. CFIR uses three-dimensional volumetric image guidance for localization and targeting of the radiation dose, minimizes radiation exposure to nontargeted brain regions, and allows for conformal dose distribution with sharp beam boundaries. This protocol allows one to ask questions regarding the function of adult-born neurons, but also opens areas to questions in areas of radiobiology, tumor biology, and immunology. These radiological tools will facilitate the translation of discoveries at the bench to the bedside.

The function of adult-born mammalian neurons remains an active area of investigation. Ionizing radiation inhibits the birth of new neurons. Using computer tomography-guided focal irradiation (CFIR), three-dimensional anatomical targeting of specific neural progenitor populations can now be used to assess the functional role of adult neurogenesis.

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral ...

Neuroscience

Assessment of Memory Function in Pilocarpine-induced Epileptic Mice

Cognitive impairment is one of the most common comorbidities in temporal lobe epilepsy. To recapitulate epilepsy-associated cognitive decline in an animal model of epilepsy, we generated pilocarpine-treated chronic epileptic mice. We present a protocol for three different behavioral tests using these epileptic mice: novel object location (NL), novel object recognition (NO), and pattern separation (PS) tests to evaluate learning and memory for places, objects, and contexts, respectively. We explain how to set the behavioral apparatus and provide experimental procedures for the NL, NO, and PS tests following an open field test that measures the animals&#8217; basal locomotor activities. We also describe the technical advantages of the NL, NO, and PS tests with respect to other behavioral tests for assessing memory function in epileptic mice. Finally, we discuss possible causes and solutions for epileptic mice failing to make 30 s of good contact with the objects during the familiarization sessions, which is a critical step for successful memory tests. Thus, this protocol provides detailed information about how to assess epilepsy-associated memory impairments using mice. The NL, NO, and PS tests are simple, efficient assays that are appropriate for the evaluation of different kinds of memory in epileptic mice.

This article presents experimental procedures for assessing memory impairments in pilocarpine-induced epileptic mice. This protocol can be used to study the pathophysiologic mechanisms of epilepsy-associated cognitive decline, which is one of the most common comorbidities in epilepsy.

Cognitive impairment is one of the most common comorbidities in temporal lobe epilepsy. To recapitulate epilepsy-associated cognitive decline in an animal ...

Behavior

Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research. Successful outcomes are dependent upon three critical components: test chambers that permit self-feeding while standing unrestrained in a confined space, recipes that mask the aversive taste/odor of commercially-available oral contrast agents, and a step-by-step test protocol that permits quantification of swallow physiology. Elimination of one or more of these components will have a detrimental impact on the study results. Moreover, the energy level capability of the fluoroscopy system will determine which swallow parameters can be investigated. Most research centers have high energy fluoroscopes designed for use with people and larger animals, which results in exceptionally poor image quality when testing mice and other small rodents. Despite this limitation, we have identified seven VFSS parameters that are consistently quantifiable in mice when using a high energy fluoroscope in combination with the new murine VFSS protocol. We recently obtained a low energy fluoroscopy system with exceptionally high imaging resolution and magnification capabilities that was designed for use with mice and other small rodents. Preliminary work using this new system, in combination with the new murine VFSS protocol, has identified 13 swallow parameters that are consistently quantifiable in mice, which is nearly double the number obtained using conventional (i.e., high energy) fluoroscopes. Identification of additional swallow parameters is expected as we optimize the capabilities of this new system. Results thus far demonstrate the utility of using a low energy fluoroscopy system to detect and quantify subtle changes in swallow physiology that may otherwise be overlooked when using high energy fluoroscopes to investigate murine disease models.

This study successfully adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research.

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Biology

Retroductal Nanoparticle Injection to the Murine Submandibular Gland

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally delivering bioactive compounds to the salivary glands, greater tissue concentrations can be safely achieved versus systemic administration. Furthermore, off target tissue effects from extra-glandular accumulation of material can be dramatically reduced. In this regard, retroductal injection is a widely used method for investigating both salivary gland biology and pathophysiology. Retroductal administration of growth factors, primary cells, adenoviral vectors, and small molecule drugs has been shown to support gland function in the setting of injury. We have previously shown the efficacy of a retroductally injected nanoparticle-siRNA strategy to maintain gland function following irradiation. Here, a highly effective and reproducible method to administer nanomaterials to the murine submandibular gland through Wharton's duct is detailed (Figure 1). We describe accessing the oral cavity and outline the steps necessary to cannulate Wharton's duct, with further observations serving as quality checks throughout the procedure.

Local drug delivery to the submandibular glands is of interest in understanding salivary gland biology and for the development of novel therapeutics. We present an updated and detailed retroductal injection protocol, designed to improve delivery accuracy and experimental reproducibility. The application presented herein is the delivery of polymeric nanoparticles.

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally ...

Bioengineering

Retroductal Submandibular Gland Instillation and Localized Fractionated Irradiation in a Rat Model of Salivary Hypofunction

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. Irreparable cell damage results in a chronic loss of salivary function that impairs basic oral activities, and increases the risk of oral infections and dental caries. Salivary hypofunction and its complications gravely impact a patient's comfort. Current symptomatic management of the condition is ineffective, and newer therapies to assuage the condition are needed. 
Salivary glands are exocrine glands, which expel their secretions into the mouth via excretory ducts. Cannulation of these ducts provides direct access to the glands. Retroductal delivery of a contrast agent to major salivary glands is a routine out-patient procedure for diagnostic imaging. Using a similar procedure, localized treatment of the glands is feasible. However, performing this technique in preclinical studies with small animals poses unique challenges. In this study we describe the technique of retroductal administration in rat submandibular glands, a procedure that was refined in Dr. Bruce Baum's laboratory (NIH)1, and lay out a procedure for local gland irradiation.

Salivary gland hypofunction, a major adverse effect of head and neck radiotherapy diminishes a patient's quality of life. The demonstration of efficacy of new therapies in animal models is a prerequisite before clinical transition. This protocol describes retroductal administration and local irradiation of rat submandibular glands.

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. ...

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.

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 ...

Immunology and Infection

Radiation Treatment of Organotypic Cultures from Submandibular and Parotid Salivary Glands Models Key In Vivo Characteristics

Hyposalivation and xerostomia create chronic oral complications that decrease the quality of life in head and neck cancer patients who are treated with radiotherapy. Experimental approaches to understanding mechanisms of salivary gland dysfunction and restoration have focused on in vivo models, which are handicapped by an inability to systematically screen therapeutic candidates and efficiencies in transfection capability to manipulate specific genes. The purpose of this salivary gland organotypic culture protocol is to evaluate maximal time of culture viability and characterize cellular changes following ex vivo radiation treatment. We utilized immunofluorescent staining and confocal microscopy to determine when specific cell populations and markers are present during a 30-day culture period. In addition, cellular markers previously reported in in vivo radiation models are evaluated in cultures that are irradiated ex vivo. Moving forward, this method is an attractive platform for rapid ex vivo assessment of murine and human salivary gland tissue responses to therapeutic agents that improve salivary function.

Using three-dimensional organotypic cultures to visualize morphology and functional markers of salivary glands may provide novel insights into the mechanisms of tissue damage following radiation. Described here is a protocol to section, culture, irradiate, stain, and image 50&#8211;90 &#956;m thick salivary gland sections prior to and following exposure to ionizing radiation.

Hyposalivation and xerostomia create chronic oral complications that decrease the quality of life in head and neck cancer patients who are treated with ...