Saline Lavage for Sampling of the Canine Nasal Immune Microenvironment

We study the immune responses associated with radiation therapy and dogs with nasal tumors. We're trying to understand how we can improve cancer patient outcomes to radiation therapy by modulating the immune system. Shifts in the immune microenvironment can be important in studying treatment effects. Therefore, serially analyzing the cells of the microenvironment is desired. However, it can be challenging to sample the tissues of the nasal cavity with repeated biopsies due to the complex anatomy and the propensity of the mucosa to bleed.

The saline nasal lavage technique allows serial sampling of the immune microenvironment of the nasal cavity with minimal tissue disruption. Cells and proteins collected with the nasal lavage technique can be analyzed with various assays to investigate the status of the nasal immune microenvironment over time.

One day before the procedure, fill 520 milliliter syringes with sterile physiological saline. Cap the syringes securely and place them in an incubator at 37 degrees Celsius to warm overnight. Then position the anesthetized dog external recumbent on the treatment table. Adjust the dog's head to be angled downward, naturally and comfortably off the table's edge to facilitate optimal nasal lavage sample collection. Inflate the cuff of the endotracheal tube to ensure a tight seal of the airway. For nasal lavage, cut an eight French sterile red rubber catheter at the base to fit snugly onto one of the prefilled 20 milliliter syringes containing warm saline. Using a permanent marker, mark the base-end of the catheter to indicate the starting point at the nostril entrance. Confirm that the predetermined length spans intranasally with the tip positioned in the desired location. Next, cut the catheter tip to match the defined intranasal catheter length, ensuring it will land in the appropriate location within the nasal cavity. To perform the nasal lavage procedure, designate one person to feed the catheter into the nasal cavity and administer it, and the other person to collect the sample as it exits the nose. Position one person in front and below the dog's head, wearing gloves. Gently guide the red rubber catheter into the medial aspect of the nasal cavity until the mark on the catheter aligns with the nostril entrance. Simultaneously, the other person holds a 50-milliliter conical tube below the nostril with the catheter in place. Using one hand, gently occlude the contralateral nostril. Begin infusing the saline into the nasal cavity with slow, steady pressure or with a pulse infusion while maintaining the dog's head at a downward angle. Collect the draining fluid in a 50-milliliter conical tube positioned below the nostril. After the procedure, allow the dog to recover from anesthesia while keeping it external recumbent with its head downward. Now record the total volume of nasal lavage fluid collected relative to the total amount of saline infused. To process the sample, gently vortex or pipette the nasal lavage samples within the conical tubes to break up clumps of debris and cells. Pass the pooled samples through a 70-micrometer cell filter to remove large debris in mucus. Centrifuge the filtered samples at 300 G for five to 10 minutes to form a cell pellet. Using a pipette, carefully aspirate the supernatant from the centrifuge samples. Deposit the supernatant into a clean tube for subsequent protein analysis. Finally, resuspend the cell pellets in PBS or the preferred solution for cell assays of interest.