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
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		<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 flam...

<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. 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=Major+salivary+gland+damage+in+allogeneic+hematopoietic+progenitor+cell+transplantation+assessed+by+scintigraphic+methods.">Major salivary gland damage in allogeneic hematopoietic progenitor cell transplantation assessed by scintigraphic methods.</a> Bone Marrow Transplant. 37 (10), 955-959 (2006).</li><li>Jensen, S. B., 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+systematic+review+of+salivary+gland+hypofunction+and+xerostomia+induced+by+cancer+therapies:+management+strategies+and+economic+impact.">A systematic review of salivary gland hypofunction and xerostomia induced by cancer therapies: management strategies and economic impact.</a> Support Care Cancer. 18 (8), 1061-1079 (2010).</li><li>de Castro, G., Federico, M. 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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>

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 dry...

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.

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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 bleedin...

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. ...

The overall goal of this procedure is to retroductally administer therapeutics to the submandibular glands to assess their efficacy in combating salivary disfunction. This method can help answer key questions in the field of salivary gland disorders such as what therapies can protect against or restore salivary gland function against radiation. The main advantage of this technique is that salivary glands can be treated locally thereby reducing the risk of nontarget tissue side effects.Generally people new to this method will struggle because the duct openings are not visible and the cannula has to be manipulated blindly into the duct. Before beginning the procedure cut a ten centimeter length of PE 10 polyethylene tubing with a scalpal, then grasp both ends of the tubing between the index finger and thumb of each hand and heat the middle section of the tubing above a gentle flame carefully pulling both sides to stretch the softened tubing to double it's length. Next use a scalpal to cut the tubing in the middle at a 45 degree angle to obtain two cannuli each with a tapered end.Stretch the non-tapered end of one of the cannuli over the 29 gauge needle of 0.5cc insulin syringe. Then remove the cannula and draw 220 microliters of saline solution into the syringe. Tap the syringe shaft to dislodge any air bubbles and reattach the cannula over the needle.Depress the plunger to expell the air from the cannula and to ensure a free flow of solution through the tubing and adjust the volume of saline in the syringe to 200 microliters. Now confirm the appropriate level of sedation of the rat by toe pinch and palpable reflex and apply eye lubricant over the exposed corneas. Place the amimal on a specially designed restraining platform and engage the upper incisor teeth on the transverse bar.Loop a rubber band around the lower incisors to pull down the jaw. Anchoring it to the platform. Next pass a sterile suture through the tongue and lift it up to raise the floor of the mouth.Then use a hemostat to clamp the sutures and pass the hemostat over the transverse bar. Next use a custom built cheek spreader to expand the cheeks and a disecting microscope to locate the sublingual papilli on the floor of the mouth. Using delicate forcebs grasp the tapered end of the preformed PE 10 tubing and gently manipulate the tip of the cannula into the ductal orifice on the sublingual papilla.Then thread the cannula three to five millimeters into the duct making sure that the cannula passes throught the duct without obstruction. Before instilling the saline inject atrophine subcutaniously into the scruff of the animal's neck. Ten minutes after the injection secure the cannula to the duct orifice with a drop of cyanoacrylate.After the glue has dried slowly depress the syringe plunger at 50 microliters per minute until 200 microliters of the saline solution has been instilled into the gland. If you encounter back preesure during instillation stop and then withdraw the tubing then recanulate with a new cannula. Then clamp the tubing with a hemostat and carefully remove the syringe.Leave the tubing in the duct for 30 to 60 minutes until the animal regains conciousness. Then remove the suture that holds the tongue and transfer the animal into a separate cage under a heat lamp until it is fully recovered. To locally apply fractionated irradiation to the submandibular gland.Columnate a three centimeter slit of radiation field that emcompasses the area from the lower border of the manible to the top of the sternum of the anesthetized rat in the supine position. After placing a one centimeter tissue equivilant bolus over the region. Adjust the distance between the radiation source and the top of the bolus to 100 centimetes using a six megavolt photon beam on the linear accelerator.Irradiate the animal with 2.5 grays for the appropriate length of exposure. At the end of the radiation exposure period warm the animal during it's recovery as just demonstated. During the initial experiments the proper placement of the cannula was confirmed by infusing the gland with hematoxylin or tryplan blue solution and evaluating the gland staining in euthanized animals.The submandibular glands were studied because they can be selectively irradiated with columnated radiation to the neck avoiding severe oral off target effects that can effect the animals health and experimental outcome. Analysis of the effects of this fractionated radiation scheme on the salivary flow at eight weeks post radiation demonstrates that daily exposure to 2.5 grey increments precipitously reduces the salivary flow. Within your 10 fold reduction in the saliva output in irradiated animals compared to non-irradiated animals.Once mastered this technique can be completed in a few minutes if it is performed properly. While attempting this procedure it is important to remember to ensure the unrestricted passage of cannula into the excratory duct of salivary gland. After it's development this technique paved the way for researchers in the field of salivary gland disorders to explore treatments for radiation and autoimmune salivary gland afflictions.After watching this video you should have a good understanding of how to perform retroductal installation in the rat's submandibular gland and to locally irradiate the glands to assess the the therapeutic efficacy of radiation toxicity amelioration. Don't forget that working with gene and protein therapeutics can be extremely dangerous and the precautions such as avoiding needle pricks and minimizing skin exposure to biological agents should alwasys be taken while performing this procedure.

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11.2K Aufrufe.  Louisiana State University Health Sciences Center. The overall goal of this procedure is to retroductally administer therapeutics to the submandibular glands to assess their efficacy in combating salivary disfunction. This method can help answer key questions in the field of salivary gland disorders such as what therapies can protect against or restore salivary gland function against radiation. The main advantage of this technique is that salivary glands can be treated locally thereby reducing the risk of nontarget tissue side effects....