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

Summary

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.

Abstract

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.

Introduction

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 hypofunction^2-6.

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's well-being and function^2,3.

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 treatment^6,7. 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 varied^9,10,13.

This report establishes methods for retroductal delivery to and localized radiation of rat submandibular glands using patient-relevant radiation source and dose fraction.

Protocol

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

1. Preparation of Syringe-Tubing Assembly
   1. 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.
   2. Cut the tubing in the middle with a scalpel at an angle of 45^o 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.
   3. Remove the cannula, and draw 220 μl 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 μl.
2. Intra-Oral Cannulation of Submandibular Gland Duct
   NOTE: Autoclave instruments prior to the procedure, and sterilize in-between procedures in a hot bead sterilizer.
   1. 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.
   2. 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.
   3. 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.
   4. 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.
   5. 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.
   6. 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.
   7. 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.
3. Submandibular Gland Instillation
   1. Inject atropine (0.5 mg/kg) subcutaneously in the scruff of the neck, and wait 10 min for a reduction in salivary secretions.
   2. Secure the cannula to the duct orifice with a drop of cyanoacrylate (glue), and allow to dry. Instill the solution in the gland (200 μl/gland) by slowly pressing the syringe plunger at a rate of ~ 50 μl/min.
   3. 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.
   4. 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.
   5. After the animal is fully ambulatory, house it at the vivarium with unrestricted access to food and water.

2. Localized Fractionated Irradiation of Submandibular Glands

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.

Results

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'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...

Discussion

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...

Materials

List of materials used in this article

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