Saline Lavage for Sampling of the Canine Nasal Immune Microenvironment

Summary

Saline nasal lavage can be used to sample the canine nasal immune microenvironment. Because the approach is relatively non-invasive and does not disrupt the nasal tissues, it can be performed serially. Cells and proteins collected from the nasal lavage technique can be processed for various laboratory analyses.

Abstract

Evaluating the local immune microenvironment of the canine nasal cavity can be important for investigating normal tissue health and disease conditions, particularly those associated with local inflammation. We have optimized a technique to evaluate the local nasal immune microenvironment of dogs via serial nasal lavage. Briefly, with dogs under anesthesia and positioned in sternal recumbency, prewarmed sterile saline is flushed into the affected nostril using a flexible soft rubber catheter. The fluid backflow is collected into conical tubes, and this process is repeated. The fluids containing dislodged cells and proteins are pooled, and the pooled nasal lavage samples are filtered through a cell strainer to remove large debris and mucus. Samples are centrifuged and the cell pellets are isolated for analysis. Once the samples have been processed, analyses that may follow nasal lavage include flow cytometry, transcriptomic analysis of cells via bulk or single-cell RNA seq, and/or quantification of cytokines present in the lavage fluid.

Introduction

Dogs routinely develop inflammatory nasal conditions throughout their lives. The underlying cause of acute or chronic rhinitis in dogs can range from infectious (viral: e.g., influenza, parainfluenza, herpesviruses; bacterial [e.g., Bordetella, mycoplasmas], fungal [e.g., aspergillosis, cryptococcosis]; parasitic [e.g., nasal mites]) to neoplastic (e.g., sinonasal malignancies, most commonly carcinoma or sarcoma histotypes) to foreign material (e.g., foreign body, intranasal migration of displaced teeth) to periodontal disease, as well as canine idiopathic inflammatory rhinitis^{1,2,3,4,5,6,7}.

In addition to a physical examination, various approaches are used to evaluate the condition of the sinonasal cavity in dogs with nasal inflammation. Imaging procedures may include radiographs (dental, skull), computed tomography (CT), or magnetic resonance imaging (MRI). Another approach to imaging the nasal cavity is rhinoscopy. Tissue sampling can involve acquiring nasal swabs, brush samples, or tissue biopsies, from which cytologic and/or histopathologic evaluation can be performed, as well as sample submission for fungal or bacterial culture. These samples may be obtained in a variety of approaches, ranging from "blind" sampling, to image-guided with rhinoscopy or advanced imaging, and acquired through the nares, from the nasopharynx, or with a surgical approach of trephination, rhinotomy, or sinusotomy.

Nasal lavage, which involves administering sterile saline into the nasal cavity, has also been used for sampling the canine nasal cavity for diagnostic and therapeutic purposes. An alternative version of the nasal lavage technique that has been used for nasal tumors is termed nasal hydropulsion, described as forceful nasal flushing, which can dislodge large tumor samples for diagnostic evaluation as well as provide therapeutic relief for the improvement of clinical signs associated with the nasal cancer⁸.

We present here another version of the nasal lavage technique for the intended purpose of collecting and analyzing cells and proteins of the nasal immune microenvironment. Through a gentle, relatively non-invasive approach, we have optimized this nasal lavage technique for serial nasal immune microenvironment sampling. In trials involving dogs with uninflamed nasal cavities, active herpesvirus infection, and sinonasal tumors, we have demonstrated the utility of nasal lavage for the collection and processing of samples for downstream applications^9,10.

In this manuscript, we describe a technique of saline nasal lavage for serial sampling of the canine nasal immune microenvironment. We provide protocol details for acquiring the nasal lavage sample effectively with minimal disruption to the tissues and then, processing the samples for a variety of analyses.

Protocol

This nasal lavage procedure has been approved by the Colorado State University Institutional Animal Care and Use Committee and Clinical Review Board (IACUC #2425). A schematic of the nasal lavage method is presented in Figure 1.

1. Preparing for the nasal lavage

1. The day before the nasal lavage procedure, fill five 20 mL syringes with sterile physiological saline (0.9% sodium chloride solution) and close with a cap. Place the syringes in an incubator set to 37 ^oC so they can warm overnight.

2. Positioning the dog for the nasal lavage

1. Anesthetize the dog for the procedure (e.g., with intravenous (IV) injection of dexmedetomidine (1-4 μg/kg) and butorphanol (0.2-0.5 mg/kg) through an IV catheter), followed by administration of propofol IV (2-5 mg/kg) titrated to effect for intubation; maintain anesthesia with 1-2% inhalant isoflurane to effect.) Confirm the appropriate depth of anesthesia by checking for palpebral response and jaw tone.
2. Lubricate the eyes to prevent drying during the procedure.
3. Monitor cardiac telemetry, blood pressure, capnography, and pulse oximetry values throughout the procedure.
4. Position the anesthetized dog in sternal recumbency. Place the dog's head so that it is angled naturally and comfortably at a downward angle from the edge of the treatment table for optimal nasal lavage sample collection.
5. Inflate the cuff of the endotracheal tube to ensure a tight seal of the airway.

3. Performing the nasal lavage

1. Cut an 8 FR sterile, red rubber catheter at the base so that it fits snugly onto one of the prefilled 20 mL syringes containing the warm saline. Measure a sterile red rubber catheter so that the distal tip of the catheter will extend to approximately midway into the nasal cavity when introduced through the nostril. If sampling a nasal tumor, use imaging or rhinoscopic guidance to estimate the location of the rostral aspect of the nasal tumor for measuring the length of the red rubber catheter so that the tip will extend to the rostral aspect of the tumor.
2. Then, cut the catheter at the tip for the defined intranasal catheter length so that the tip will land in the appropriate location when inserted into the nasal cavity. However, allow additional length for the base of the catheter extending outside of the nostril (catheter length from syringe fixation point to nostril entrance, approximately 3-5 cm). Apply a mark with a permanent marker to the base end of the catheter to indicate the starting point, corresponding to the nostril entrance; the predetermined catheter length will span intranasally, with the catheter tip positioned in the desired location within the nasal cavity.
3. Performing the nasal lavage procedure will require the participation of two individuals. Make one person (Person A) responsible for feeding the catheter into the nasal cavity and administering the lavage and the other person (Person B) for collecting the sample as it exits the nose.
4. Have Person A position themselves in front and below the dog's head. With gloved hands, have Person A gently guide the tip of the red rubber catheter into the medial aspect of the nasal cavity and advance it until the mark on the catheter lines up with the outer aspect of the nostril. During this process, ensure the catheter is connected to the syringe containing the warmed sterile saline. Have Person B hold a 50 mL conical tube below the nostril with the catheter in place.
5. Person A gently occludes the contralateral nostril and begins to infuse the saline into the nasal cavity with slow, steady pressure or with a pulsed infusion. As the dog's head is positioned at a downward angle, Person B collects the fluid that drains out of the nasal cavity via gravity into the 50 mL conical tube.
6. Repeat this nasal lavage technique for up to a total of five 20 mL saline lavages, changing out conical tubes, as needed, to collect the pooled samples. Record the total volume of nasal lavage fluid collected relative to the amount of saline infused following completion of the procedure.
7. Once completed, if anesthetized with dexmedetomidine, intramuscularly administer the same volume of atipamezole as used for dexmedetomidine, and allow the dog to recover from anesthesia. To be cautious, keep the dog in sternal recumbency with the head positioned downward to facilitate additional drainage of any residual saline from the nasal lavage.

4. Processing the nasal lavage sample

1. Gently vortex and/or pipette the nasal lavage samples contained within the conical tubes to break up clumps of debris and cells. Pass the pooled nasal lavage samples through a 70 µm cell straining filter to remove large debris and mucus. Centrifuge the samples at 300 × g for 5-10 min to form a cell pellet.
2. Collect the supernatant via aspiration with a pipette and deposit it in a clean tube for analysis of proteins of interest.
3. Resuspend cell pellets in phosphate-buffered saline (PBS) or preferred solution for cell assays of interest.
4. To clear red blood cells from the samples, perform ammonium-chloride-potassium (ACK) lysis to collect nucleated cells from nasal lavage^11,12.

Results

With this nasal lavage method, the collected sample will appear slightly cloudy, possibly with visible pieces of cellular debris and mucus when the tube is swirled. A sample would be considered contaminated with peripheral blood if the lavage procedure inadvertently induces bleeding, and the sample is tinged red. While some of the infused saline will be lost during the procedure, a negative lavage would be considered if the infused saline does not flow back out of the nostril and into the tube; potential causes could be ...

Discussion

There are several critical steps in the nasal lavage protocol. With respect to the dog undergoing the nasal lavage procedure, the dog must be within a deep enough plane of anesthesia so that they do not react to the catheter placement or lavage administration. If they are reactive under anesthesia, this may compromise the quality and quantity of lavage sample collection, as well as potentially increase risks for acute nasal tissue injury due to local trauma from the intranasal catheter or for the dog aspirating the lavag...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 mL  Tubes	Eppendorf	05-402
1000 µL Pipette	VWR	89079-974
1x PBS	Corning	21-040-CV
20 mL Syringes	VWR	BD302830
50 mL Conical Tubes	VWR	89039-656
70 µm Cell Strainer	Fisherbrand	22-363-548
8FR Sterile Red Rubber Catheter	Med Vet International	50-252-2428
ACK Lysis Buffer	Gibco	A1049201
Centrifuge	Beckman Coulter	366816
Physiological Saline (0.9%)	Vetivex	17033-492-01
Vortex	VWR	10153-838