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.
