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,....

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 i.......

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

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