We thank Dr. John Chang (Radiation Oncology, LSU Health Science Center) for assistance with radiation dose measurements. The study was supported by the American Cancer Society (Grant number: 116945-RSG-09-038-01-CCE), National Institute of Health (Grant number: R21CA173162) and the Feist-Weiller Cancer Center.

All procedures were approved by the LSU Health, Shreveport, Animal Care and Use Committee and were in accordance with the NIH guidelines for the care and use of laboratory animals.
1. Cannulation of Rat Submandibular Salivary Glands
<ol>
	<li>Preparation of Syringe-Tubing Assembly
	<ol>
		<li>Cut a 10 cm length of PE10 polyethylene tubing with a scalpel. Hold both ends of the tubing between the index finger and thumb. Heat the middle section of the tubing above a gentle flame, and gently stretch the softened tubing to double its length by pulling both sides.</li>
		<li>Cut the tubing in the middle with a scalpel at an angle of 45o to get 2 cannulae - each with a tapered end. Expand the non-tapered end of the cannula by fitting it over a 29 G needle of 0.5 cc Insulin syringe.</li>
		<li>Remove the cannula, and draw 220 &#956;l of saline solution into the syringe. Tap to dislodge air bubbles. Fit the cannula over the needle, and push the syringe plunger to expel air and ensure a free flow of solution through the cannula. Adjust the volume to 200 &#956;l.</li>
	</ol>
	</li>
	<li>Intra-Oral Cannulation of Submandibular Gland Duct 
	NOTE: Autoclave instruments prior to the procedure, and sterilize in-between procedures in a hot bead sterilizer.
	<ol>
		<li>Weigh the Sprague Dawley rat, and dispense the calculated volume of ketamine (42 mg/kg)/ xylazine (8 mg/kg)/ acepromazine (1.4 mg/kg) mix in a hypodermic syringe.</li>
		<li>Restrain the animal by grasping the base of the tail between the index finger and the thumb of one hand and sliding the other hand over the body to grasp it. Rest the index and middle fingers along the sides of the head while hold the torso with the thumb and remaining fingers.</li>
		<li>Inject the anesthetic in the hind limb musculature. Confirm depth of anesthesia by toe pinch and palpebral reflex. Apply eye lubricant to prevent dryness while animal is under anesthesia.</li>
		<li>Place the animal on a specially-designed platform (Figure 1), and engage the upper incisor teeth on the transverse bar. Pull the lower jaw down by looping a rubber band around the lower incisors and anchoring it to the platform.</li>
		<li>Pass a sterile suture through the tongue and lift it up to raise the floor of the mouth. Clamp the sutures with a hemostat, and pass it over the transverse bar.</li>
		<li>Expand the cheeks with a custom-built cheek spreader (Figure 1), and under a dissecting microscope locate the sublingual papillae on the floor of the mouth.</li>
		<li>Grasp the tapered end of the preformed PE10 tubing using a delicate forceps. Gently manipulate the tip of the cannula into the ductal orifice on the sublingual papillae. Confirm placement of the cannula by threading it 3 - 5 mm into the duct; ensure that it passes without any obstruction.</li>
	</ol>
	</li>
	<li>Submandibular Gland Instillation
	<ol>
		<li>Inject atropine (0.5 mg/kg) subcutaneously in the scruff of the neck, and wait 10 min for a reduction in salivary secretions.</li>
		<li>Secure the cannula to the duct orifice with a drop of cyanoacrylate (glue), and allow to dry. Instill the solution in the gland (200 &#956;l/gland) by slowly pressing the syringe plunger at a rate of ~ 50 &#956;l/min.</li>
		<li>Crush the tubing with a hemostat, and carefully remove the syringe. Retain the tubing in the duct for 30 - 60 min until the animal regains consciousness. Remove the suture that holds the tongue.</li>
		<li>Transfer the animal to a separate cage, and use a heat lamp to keep it warm during recovery. Do not leave the animal unattended until it has regained consciousness to maintain sternal recumbence.</li>
		<li>After the animal is fully ambulatory, house it at the vivarium with unrestricted access to food and water.</li>
	</ol>
	</li>
</ol>
2. Localized Fractionated Irradiation of Submandibular Glands
<ol>
	<li>Restrain the animal as described before, and anesthetize with intramuscular administration of ketamine (33 mg/kg) /xylazine (6 mg/kg)/ acepromazine (1 mg/kg) mix in the hind limb. Confirm depth of anesthesia, and apply lubricant to the eyes.</li>
	<li>Place the animal supine on the linear accelerator tabletop, and extend its neck by tilting the head. Collimate the radiation field (3 cm slit width) to encompass the area from the lower border of the mandible to the top of the sternum.</li>
	<li>Place a 1 cm tissue-equivalent bolus over the region, and adjust the distance between radiation source and top of the bolus to 100 cm.</li>
	<li>Irradiate the animal (2.5 Gy) using a 6 MV photon beam of a linear accelerator. Dose rate, field size, and distance from radiation source to bolus surface will dictate the exposure time. In the current set up, animals were irradiated at a dose rate of ~ 1 Gy/min.</li>
	<li>Repeat exposure; 2.5 Gy/day for a total of 8 days; 4 days/ week with a 2-day interval in between. Keep the animal warm during recovery. Transfer the animal to the vivarium after it is fully ambulatory.</li>
	<li>Collect stimulated submandibular gland saliva 8 weeks after radiation to measure gland function14. Euthanize animals under anesthesia by cardiac perfusion of cold 4% paraformaldehyde/ phosphate buffered saline pH 7.2. Extirpate submandibular glands for histologic and immunohistochemical analyses14.</li>
</ol>

<ol>
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J., Goldenberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+complications+of+radiotherapy.">Oral complications of radiotherapy.</a> Lancet Oncol. 7 (2), 175-183 (2006).</li><li>Rodrigues, N. A., 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=A+prospective+study+of+salivary+gland+function+in+lymphoma+patients+receiving+head+and+neck+irradiation.">A prospective study of salivary gland function in lymphoma patients receiving head and neck irradiation.</a> Int J Radiat Oncol Biol Phys. 75 (4), 1079-1083 (2009).</li><li>Coracin, F. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+irradiation-induced+salivary+hypofunction+by+microvessel+protection+in+mouse+salivary+glands.">Prevention of irradiation-induced salivary hypofunction by microvessel protection in mouse salivary glands.</a> Mol Ther. 15 (12), 2101-2106 (2007).</li><li>Zheng, 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=Prevention+of+radiation-induced+salivary+hypofunction+following+hKGF+gene+delivery+to+murine+submandibular+glands.">Prevention of radiation-induced salivary hypofunction following hKGF gene delivery to murine submandibular glands.</a> Clin Cancer Res. 17 (9), 2842-2851 (2011).</li><li>Palaniyandi, S., 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=Adenoviral+delivery+of+Tousled+kinase+for+the+protection+salivary+glands+against+ionizing+radiation+damage.">Adenoviral delivery of Tousled kinase for the protection salivary glands against ionizing radiation damage.</a> Gene Ther. 18 (3), 275-282 (2011).</li><li>Baum, B. J., Voutetakis, A., Wang, J. <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:+novel+target+sites+for+gene+therapeutics.">Salivary glands: novel target sites for gene therapeutics.</a> Trends Mol Med. 10 (12), 585-590 (2004).</li><li>Limesand, K. H., 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=Insulin-like+growth+factor-1+preserves+salivary+gland+function+after+fractionated+radiation.">Insulin-like growth factor-1 preserves salivary gland function after fractionated radiation.</a> Int J Radiat Oncol Biol Phys. 78 (2), 579-586 (2010).</li><li>Timiri Shanmugam, P. S., 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=Recombinant+AAV9-TLK1B+administration+ameliorates+fractionated+radiation-induced+xerostomia.">Recombinant AAV9-TLK1B administration ameliorates fractionated radiation-induced xerostomia.</a> Hum Gene Ther. 24 (6), 604-612 (2013).</li><li>Coppes, R. P., Vissink, A., Konings, A. W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+radiosensitivity+of+rat+parotid+and+submandibular+glands+after+different+radiation+schedules.">Comparison of radiosensitivity of rat parotid and submandibular glands after different radiation schedules.</a> Radiother Oncol. 63 (3), 321-328 (2002).</li><li>Sunavala-Dossabhoy, G., Palaniyandi, S., Richardson, C., De Benedetti, A., Schrott, L., Caldito, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TAT-mediated+delivery+of+Tousled+protein+to+salivary+glands+protects+against+radiation-induced+hypofunction.">TAT-mediated delivery of Tousled protein to salivary glands protects against radiation-induced hypofunction.</a> Int J Radiat Oncol Biol Phys. 84 (1), 257-265 (2012).</li><li>Baum, B. J., 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=Transfer+of+the+AQP1+cDNA+for+the+correction+of+radiation-induced+salivary+hypofunction.">Transfer of the AQP1 cDNA for the correction of radiation-induced salivary hypofunction.</a> Biochim Biophys Acta. 1758 (8), 1071-1077 (2006).</li><li>Tran, S. D., 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=Paracrine+effects+of+bone+marrow+soup+restore+organ+function,+regeneration,+and+repair+in+salivary+glands+damaged+by+irradiation.">Paracrine effects of bone marrow soup restore organ function, regeneration, and repair in salivary glands damaged by irradiation.</a> PLoS One. 8 (4), e61632 (2013).</li><li>Nanduri, L. S., 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=Salisphere+derived+c-Kit++cell+transplantation+restores+tissue+homeostasis+in+irradiated+salivary+gland.">Salisphere derived c-Kit+ cell transplantation restores tissue homeostasis in irradiated salivary gland.</a> Radiother Oncol. 108 (3), 458-463 (2013).</li><li>Arany, S., Benoit, D. S., Dewhurst, 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=Nanoparticle-mediated+gene+silencing+confers+radioprotection+to+salivary+glands+in+vivo.">Nanoparticle-mediated gene silencing confers radioprotection to salivary glands in vivo.</a> Mol Ther. 21 (6), 1182-1194 (2013).</li><li>Voutetakis, A., 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=Reengineered+salivary+glands+are+stable+endogenous+bioreactors+for+systemic+gene+therapeutics.">Reengineered salivary glands are stable endogenous bioreactors for systemic gene therapeutics.</a> Proc Natl Acad Sci U S A. 101 (9), 3053-3058 (2004).</li></ol>

The authors have nothing to disclose.

Salivary glands often receive radiation doses beyond the threshold of tissue recovery in patients undergoing radiotherapy for head neck cancer, elective ablation of neck nodes, or regional hematologic malignancies. Although the fluid-secreting acinar cells of the gland are terminally differentiated, they are paradoxically sensitive to radiation. The secretory function drops within the first weeks of radiation, and irreversible gland damage results in a chronic low saliva output. To combat poor gland function and oral dryness that ensues, the preservation or restoration of secretory function is crucial. Novel therapies to protect against or repair radiation damage are being investigated1, 8-11, 16-20.
Systemic administration of a therapeutic carries the inherent risk of unwanted effects in non-targeted tissues. Aimed delivery to the salivary glands diminishes this risk. Retroductal gene therapy is effective at preventing or remedying radiation-associated salivary gland affliction1, 9-, 11, 16,17,20. However, using the same technique, salivary glands can also be reengineered to express and secrete proteins, ectopically. Exploiting the basolateral endocrine secretory pathway in salivary cells, transgene expression and hormone secretion into the blood can be achieved21. Gene transfer to salivary glands can, therefore, be effectively extended to the correction of certain endocrine disorders.
Although cannulation of salivary gland ducts in small animals can be tricky, practice is central to honing the technique. The tapered end of the cannulae can kink easily during insertion, and make infusion difficult. Increased back-pressure while expelling the solution should forewarn of a bend in the tubing, and the necessity to re-cannulate the duct. Anatomical variations such as aberrant duct orifice or increased duct tortuosity, along with pathologies that limit mouth opening like trismus or submucous fibrosis can make submandibular gland delivery a challenge. Infusion of an impermeant dye when practicing the technique can be a useful indicator of precise delivery. Proper placement of the cannulae and gentle, resistance-free infusion are important factors for the success of the procedure.
Cancer patients are often treated with megavoltage photons generated by a linear accelerator. Radiation is delivered daily in small doses over a course of weeks. Dose fractionation provides periods of recovery between radiations to reduce toxicity on healthy tissues and sensitize tumors. Highly penetrating energy beams of a linear accelerator are useful for the treatment of deep-seated tumors. And, for treatment of tumors closer to the skin, a tissue-equivalent bolus is used to reduce the depth of photon penetration. Similar to clinical practice, a linear accelerator delivered fractionated radiation can be used to locally irradiate submandibular glands of small animals. However, the position of the glands immediately beneath the skin necessitates the placement of a bolus to achieve &#62; 90% radiation dose within the glands. The thickness of the bolus is calculated depending on the energy of photons and the desired depth of tissue penetration.
In this article, we provide experimental guidelines for performing retroductal submandibular gland instillation and local fractionated irradiation of rat submandibular glands. It is our hope that the highlighted methods will aid experimental setup for investigators venturing into the field of salivary gland radiotoxicity.

Collateral destruction of healthy tissues accounts for a number of deleterious side-effects of cancer treatments. A part or whole of the major salivary glands that lie with the radiation fields are inevitably destroyed. Therefore, most patients undergoing radiotherapy for head and neck cancer, cervical lymphoma, or full-body radiation before bone marrow transplantation suffer one of the most common and persistent adverse effects of radiation, salivary gland hypofunction2-6.
The fluid-producing acinar cells of the salivary glands are acutely sensitive to radiation. Damage to the salivary glands causes a drastic diminishing of salivary flow, a condition referred to as salivary hypofunction. The chronic reduction in salivary flow impairs key oral activities such as mastication, swallowing, speech, and taste, but the morbid sequelae of intense pain, mucosal tears, dysphagia, opportunistic infections, and dental caries worsens a patient&#39;s well-being and function2,3.
Since radiotherapy-associated salivary cell loss is irreversible, there is no corrective treatment of xerostomia. Current treatment that focuses on assuaging symptoms with artificial salivary substitutes and prosecretory drugs is ineffective for long-term relief6. Although improved radiation delivery techniques have helped diminish the severity of the condition, normal tissue toxicity and its complications remain a limiting factor in cancer treatment6,7. Pre-emptive measures to prevent radiotherapy-associated complications are, therefore, becoming the norm. Radio-protective agents that scavenge free radical oxygen species, foster cell repopulation, or enhance DNA repair are being explored to avert salivary hypofunction 8-11.
Secretions of exocrine salivary glands drain into the mouth through the main excretory ducts. Intra-oral cannulation of the excretory ducts for injection of contrast agents is done routinely as an outpatient procedure. Utilizing a similar approach, salivary glands can be directly targeted for localized treatment12. Apart from reducing the risk of systemic side-effects, retroductal gland instillation has added benefits. The monolayer arrangement of salivary cells around the ductal tree allows targeting of all salivary epithelial cells, and the fibrous encapsulation of the gland acts as a barrier to reduce unwanted therapeutic spread. In essence, salivary glands are optimally suited for targeted treatment of gland afflictions such as radiation-induced salivary hypofunction.
Conventional radiation for cancer treatment is delivered in small doses (1.8 - 2.5 Gy/fraction/day, five days a week) for a period of weeks. Therefore, a radio-protective therapeutic that shows efficacy against a protracted radiation scheme in experimental models has greater clinical bearing. Compromised salivary function after fractionated radiation has been recorded in small animals, but radiation source, dose fraction, and protocols used are varied9,10,13.
This report establishes methods for retroductal delivery to and localized radiation of rat submandibular glands using patient-relevant radiation source and dose fraction.

<table><tbody><tr><td>Intramedic Polyethylene tubing (PE10)</td><td>Becton Dickson</td><td>427401</td><td></td></tr><tr><td>1/2 cc Insulin Syringe U-100</td><td>Becton Dickson</td><td>309306</td><td></td></tr><tr><td>Artificial Tears</td><td>Miller Vet Supply&amp;nbsp;</td><td>5098-9840-64</td><td></td></tr><tr><td>Hot Bead Sterilizer</td><td>Fine Science Tools</td><td>18000-45</td><td></td></tr><tr><td>Perma-Hand silk suture</td><td>Ethicon</td><td>K833H</td><td></td></tr><tr><td>Graefe forcep</td><td>Fine Science Tools</td><td>11051-10</td><td></td></tr><tr><td>Olympus SZX16 Stereo Microscope</td><td>Hunt Optics and Imaging</td><td></td><td></td></tr><tr><td>6 MV Linear Accelerator</td><td>Elekta</td><td></td><td></td></tr><tr><td>Bolus - Skinless&amp;nbsp;</td><td>Civco</td><td>MTCB410</td><td></td></tr><tr><td>Heat Lamp</td><td>Braintree Scientific</td><td>HL-1&amp;nbsp;110V</td><td></td></tr></tbody></table>

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.

Adapting a minimally invasive sialography technique, local treatment of major salivary glands is feasible. Retroductal administration in rat submandibular salivary glands was attempted by intra-oral cannulation of Wharton&#39;s ducts (Figure 2). The salivary ducts of Wharton open on the sub-lingual papillae located on the floor of the mouth, but the orifices are not readily visible. Insertion of the cannula was, therefore, done by gentle probing. To avoid untoward bleeding or duct perforation, no force was used while placing the cannula. Smooth resistance-free passage of the cannula in the duct was confirmed by gentle back and forth movement of the tubing.
Cannulation during initial experiments was confirmed by infusing the gland with hematoxylin or trypan blue solution, and evaluating gland staining in euthanized animals (Figure 3). Gland fill volume was ascertained by visualizing staining of the ductal tree in extirpated glands. For animals weighing 150 - 250 g, complete filling of the gland was achieved with 200 - 250 &#956;l of solution. Rapid infusion can increase gland pressure and heighten the risk of injury and biospread. To avoid tissue destruction, the solution was administered gradually at a rate of ~ 50 &#956;l/min
The rat submandibular glands are comparable to parotid glands in their sensitivity to radiation15. Submandibular glands were studied because they can be selectively irradiated without incurring severe off-target effects (Figure 4). Sparing of the oral cavity and a large volume of the parotid glands minimized the influence of oral mucositis and acute xerostomia on animal health and experimental outcome. Our earlier studies in rats were performed with single, large doses of radiation11,16, but fractionated dosing is more pertinent to clinical practice. The neck region of the animal was, therefore, exposed daily to small doses of radiation. To achieve a uniform buildup of radiation dose within the submandibular glands located near the skin surface, a soft tissue-equivalent bolus made from a homogenous gel with a density of 1.03 g/cc was placed over the neck during radiation.
The effect of fractionated radiation scheme on salivary flow as determined at 8 weeks post-radiation show that daily exposure to increments of 2.5 Gy reduced salivary flow precipitously. A near 10-fold reduction in saliva output was recorded in irradiated animals in contrast to non-irradiated animals (2-sided Student&#39;s t-test, p &#60; 0.01; Figure 5)14. The results confirmed the establishment of radiation-induced salivary hypofunction in animals.
<img alt="Figure 1" src="/files/ftp_upload/53785/53785fig1.jpg" /> 
Figure 1. Construction of a Customized Platform and Cheek-Spreader. A stiff 4 mm wire was bent in a 3-sided rectangle (11 cm height X 13 cm width) as shown, and the transverse bar was bowed at the center. The ends of the wire were bent into loops, and screws were passed through them to secure the assembly to a plastic board (25 cm length x 15 cm width x 0.8 cm depth). A central screw was inserted 5 cm posterior to the wire assembly. It served to anchor the rubber band that pulled open the lower jaw. Inset: A custom-built cheek-spreader made of a disposable 1 mm thick wire bent in a &#34;U shape&#34; with flared ends. Plastic tubings were passed over the wire ends to prevent damage to the buccal mucosa. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53785/53785fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53785/53785fig2.jpg" /> 
Figure 2. Cannulation of Rat Submandibular Gland Excretory Ducts. The tongue is raised to elevate the floor of the mouth, and polyethylene cannulae were inserted into the submandibular duct orifices located on the floor of the mouth. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53785/53785fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53785/53785fig3.jpg" /> 
Figure 3. Infusion of Hematoxylin Solution in the Submandibular Gland to Confirm the Technique. Gland staining attests to the successful administration of the solution in the submandibular gland. Note that the sublingual gland in the upper left corner is not stained. A non-infused control gland is shown on the left. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53785/53785fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53785/53785fig4.jpg" /> 
Figure 4. Placement of the Animal for Irradiation. The animal is laid supine on the linear accelerator table, and the head titled back to extend the neck. The collimated slit width was 3 cm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53785/53785fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53785/53785fig5.jpg" /> 
Figure 5. Effect of Fractionated Radiation on Submandibular Gland Function. Saline was instilled in the submandibular glands before radiation. The neck of the animal was irradiated in fractions of 2.5 Gy/day for 8 days. The data shown are mean &#177; SEM of stimulated submandibular salivary flow rate 8 weeks after completion of radiation scheme. The figure is reproduced with copyright permission from Human Gene Therapy, Mary Ann Liebert, Inc. publisher15. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53785/53785fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Retroductal Submandibular Gland Instillation and Localized Fractionated Irradiation in a Rat Model of Salivary Hypofunction at JoVE.com

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

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. ...

retroductal-submandibular-gland-instillation-localized-fractionated

Nanyang Technological University

Research

JoVE Journal

Medicine

11.3K Views.  Louisiana State University Health Sciences Center. 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.

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

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 ...

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

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

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, ...

Ultrasound-guided Botulinum Toxin-A Injections: A Method of Treating Sialorrhea

Neurological diseases can be complicated by sialorrhea, an excessive flow of saliva. Patients suffering from moderate to severe sialorrhea have an impaired quality of life, often worsened by correlated complications such as aspiration pneumonia, oral infections, dental caries, and maceration of the skin. Diverse therapeutic approaches have been proposed for the treatment of sialorrhea, including surgery and the use of anticholinergic agents, with limited results and the possible occurrence of serious adverse events. Recently, botulinum toxin (BoNT) injection within the major salivary glands has been proposed in patients refractory to anticholinergic therapy, with the aim of inhibiting local acetylcholine release and gland activity. 
In order to obtain a better outcome in terms of reduction of saliva production, efficacy, duration, and avoidance of major adverse events, we developed an ultrasound-guided BoNT-type A injection technique accurately described in the text. Here we present a method of treating sialorrhea with bilateral parotid and submandibular gland BoNT-type A injections under ultrasound guidance. Four quadrants of the parotid gland and two quadrants of the submandibular gland are visualized and injected using two accesses and one access, respectively. 
The ultrasound-guided procedure provides a simple, non-invasive, real-time visualization of the muscular and glandular tissues and their surrounding structures, optimizing treatment efficacy and safety.

Botulinum toxin is injected into the salivary glands for the treatment of sialorrhea. The ultrasound-guided botulinum toxin injection in both the parotid and submandibular glands demonstrates a sustained efficacy and the absence of serious side effects.

Neurological diseases can be complicated by sialorrhea, an excessive flow of saliva. Patients suffering from moderate to severe sialorrhea have an ...

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).

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 ...

Retrograde Parotid Gland Infusion through Stensen's Duct in a Non-Human Primate for Vectored Gene Delivery

Salivary glands are an attractive tissue target for gene therapy with promising results already leading to human trials. They are inherently capable of secreting proteins into the bloodstream and are easily accessible, making them potentially superior tissue sites for replacement hormone production or vaccination by gene transfer. Suggested methods for gene delivery include transcutaneous injection and retrograde infusion through salivary ducts. We demonstrate how to perform Retrograde Salivary Gland Infusion (RSGI) in non-human primates. We describe the important anatomic landmarks including identification of the parotid papilla, an atraumatic method of cannulating and sealing Stensen&#39;s Duct utilizing basic dental tools, polyethylene tubing, and cyanoacrylate, and the appropriate rate of infusion. While this is the least traumatic method of delivery, the method is still limited by the volume able to be delivered (&#60;0.5 mL) and the potential for trauma to the duct and gland. We demonstrate using fluoroscopy that an infusate can be fully delivered into the gland, and further demonstrate by immunohistochemistry the transduction of a typical vector and expression of the delivered gene.

Salivary glands have been proposed as a tissue target site for gene therapy, especially in the area of vaccination by gene transfer. We demonstrate gene delivery in a non-human primate model utilizing retrograde parotid infusion.

Salivary glands are an attractive tissue target for gene therapy with promising results already leading to human trials. They are inherently capable of ...

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 ...

Real-Time Live Imaging of Mouse Submandibular Gland Regeneration

Interrogating Cell-Cell Interactions in the Salivary Gland via Ex Vivo Live Cell Imaging

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal role played by macrophages in driving the regenerative response. However, our understanding of this critical role has primarily relied on static views obtained from fixed tissue biopsies. To overcome this limitation and gain insights into these interactions in real time, this study outlines a comprehensive protocol for culturing salivary gland tissue ex vivo and capturing live images of cell migration.
The protocol involves several key steps: First, mouse submandibular salivary gland tissue is carefully sliced using a vibratome and then cultured at an air-liquid interface. These slices can be intentionally injured, for instance, through exposure to radiation, to induce cellular damage and trigger the regenerative response. To track specific cells of interest, they can be endogenously labeled, such as by utilizing salivary gland tissue collected from genetically modified mice where a particular protein is marked with green fluorescent protein (GFP). Alternatively, fluorescently-conjugated antibodies can be employed to stain cells expressing specific cell surface markers of interest. Once prepared, the salivary gland slices are subjected to live imaging using a high-content confocal imaging system over a duration of 12 h, with images captured at 15 min intervals. The resulting images are then compiled to create a movie, which can subsequently be analyzed to extract valuable cell behavior parameters. This innovative method provides researchers with a powerful tool to investigate and better understand macrophage interactions within the salivary gland following injury, thereby advancing our knowledge of the regenerative processes at play in this dynamic biological context.

Author Spotlight: Studying Macrophage-Epithelial Cell Interactions in Salivary Gland Regeneration After Injury 

Immunofluorescent imaging is constrained by the ability to observe complex, time-dependent biological processes in just a single snapshot in time. This study outlines a live-imaging approach conducted on precision-cut mouse submandibular gland slices. This approach allows for the real-time observation of cell-cell interactions during homeostasis and the processes of regeneration and repair.

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal ...

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

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