Murine Nasal Lavage Fluid Collection without Blood Contamination

Summary

The present protocol describes a novel method for avoiding blood contamination in murine nasal lavage fluid (NLF). Since this method ensures the NLF collection without blood contamination, it allows for more accurate detections of immunological components and various respiratory pathogens.

Abstract

Upper airway samples, including nasal lavage fluid (NLF) and nasal swabs, are useful in detecting pathogens, including viruses and bacteria, which can cause respiratory diseases. NLF has been used to examine cellular and humoral components in the respiratory system, for example, in evaluating the induction of immunoglobulin (Ig) A following mucosal vaccinations. In experimental rodents, the NLF samples can be collected by either the trans-pharyngeal or trans-tracheal route. Although the trans-pharyngeal route has been reported to be more efficient in collecting the NLF samples than the trans-tracheal route, the NLF samples collected by the trans-pharyngeal route were often contaminated with blood, affecting the levels of cellular and humoral contents in the original NLF samples. Thus, this study aimed to establish a novel NLF collection method in experimental mice to minimize blood contamination. Briefly, before the lower jaw of the mouse was separated, cotton balls were placed in the mouth to absorb blood. When the bleeding was stopped, the NLF samples were collected by inserting a micropipette into the choana and flushing with 200 µL of phosphate-buffered saline twice (final volume: 400 µL/mouse). To detect blood contamination, a simple and sensitive forensic luminol test that detects hemoglobin was used. The luminol test demonstrated that blood contamination was detectable in the NLF samples harvested by the conventional method (without cotton balls), but not those harvested by the novel method (with cotton balls). Due to the contamination of blood, the total IgA and IgG concentrations in the NLF samples were higher in the conventional method than in the novel method; blood has been known to contain much higher levels of IgA and IgG than NLF. Therefore, this unique method can be used as a simple and sensitive method to collect NLF samples from experimental mice to prevent blood contamination.

Introduction

In both clinical and basic research, appropriate sample collection methods are most crucial to evaluating the results. Nasal lavage is an irrigation technique used to analyze the cellular and humoral components in the respiratory tract^1,2,3. Clinically, nasal lavage fluid (NLF) has been used as an effective noninvasive sample for biomarker discovery, with several studies supporting its use across various respiratory diseases, including sinusitis, cystic fibrosis, and general respiratory disorders^4,5,6,7. In experimental animals, NLF has also been used to study respiratory disease models, including influenza, coronavirus disease-2019 (COVID-19), and allergic disease^3,8,9. This method is widely used to determine key inflammatory biomarkers, such as polymorphonuclear cells and cytokines, which are often examined as indicators of responses to allergens, asthma, and respiratory infections^10,11,12. More recently, to investigate the efficacy and safety of potential intranasal vaccine candidates, NLF samples have often been collected from experimental animals, and the levels of pathogen and immunoglobulin (Ig) in NLF samples have been quantified as indicators¹³.

In experimental mice, NLF can be collected using the trans-pharyngeal or trans-tracheal method. In the trans-pharyngeal method, the murine head, including the palatopharyngeal region, was separated from the larynx, and then a catheter was inserted through the pharyngeal opening into the choana to inject phosphate-buffered saline (PBS) and collect NLF from the nostril¹⁴. The trans-tracheal method gained access to the respiratory tract through the trachea without severing the head, and NLF was collected from the nostril, minimizing the risk of blood contamination. Although these two primary methods have been employed for NLF collection in experimental mice, both conventional methods have their own limitations. The trans-tracheal method has been demonstrated to yield lower amounts of NLF than the trans-pharyngeal method due to the leakage of trans-tracheal lavage fluid into the oral cavity. The previous study showed that, following injection of PBS, the recovered fluid volumes in NLF were 70%-80% and 90%-100% in the trans-tracheal and trans-pharyngeal methods, respectively¹⁴. Although the trans-pharyngeal method can be considered better with larger amounts of NLF than the trans-tracheal method, blood contamination during the procedure was inevitable to some extent^14,15. Similarly, several studies have reported that NLF samples were often contaminated with high levels of blood serum proteins^16,17,18. This can interfere with the detection and quantification of NLF-specific biomarkers for respiratory diseases¹⁹. This may also affect the concentrations of Ig, particularly IgA and IgG, in NLF samples since blood contains larger amounts of IgA and IgG than NLF.

To address these challenges, this study aimed to establish a unique, simple, and reliable trans-pharyngeal method for collecting NLF samples from experimental mice, maximizing sample yield and minimizing blood contamination. During the experimental processes, cotton balls were placed into the mouth of the mouse to inhibit blood contamination, and then PBS was injected to collect NLF samples from the nostril. The levels of blood contamination in NLF were determined using a forensic technique, luminol-based chemiluminescence, that enables semi-quantification of hemoglobin (Hb) levels in NLF samples; the luminol reaction in NLF collected by the novel method was negative. Thus, the current novel method can improve the NLF sample quality for various biological experiments, such as antibody enzyme-linked immunosorbent assays (ELISAs), cytokine profiles, immunological and medical biomarker determinations, and murine respiratory disease models.

Protocol

All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Kindai University Faculty of Medicine and performed according to the criteria outlined by the National Institutes of Health (NIH)²⁰. 4-month-old male C57BL/6 mice were used in this study. Information on reagents and equipment used in this study are listed in the Table of Materials.

1. NLF collection

1. Euthanize a mouse with 5% isoflurane²¹ following institutionally approved protocols.
2. Place the mouse on its back on a surgical plate and fix it by pinning down all four limbs.
3. Take blood from the heart using a 1 mL syringe.
4. Open the abdominal cavity using scissors to expose the visceral organs and diaphragm.
5. Incise the diaphragm and ribs using scissors to make the right atrium visible.
6. Puncture the right atrium using scissors to remove blood further.
7. Place three cotton balls into the mouth of the animal while pulling the tongue.
8. Separate the lower jaw using scissors while raising the head (nose-up position) to inhibit blood contamination in NLF.
9. Replace the cotton balls with new ones to avoid blood contamination.
10. After bleeding has stopped, inject 200 µL of sterile PBS into the choana and collect NLF ejected from the nostrils into a 1.5 mL tube.
11. Repeat the step 1.10 once more.

2. Detection of blood contamination by luminol chemiluminescence

1. Dissolve 1 mg of luminol and 5 mg of sodium peroxide (Na₂O₂) with 1 mL of sterile water using a 1.5 mL tube to make the stock solution.
   NOTE: The final concentration is 1 mg/mL luminol and 5 mg/mL Na₂O₂.
2. Cover the tube with aluminum foil and store it at 4 °C until use.
3. Dilute the stock solution 10-fold with deionized water to make a working solution.
   NOTE: The final concentration is 0.1 mg/mL luminol and 0.5 mg/mL Na₂O₂.
4. Pipette 2 µL of the NLF samples into wells of a 96-well plate.
5. Add 100 µL of the diluted luminol solution to each well.
6. Briefly shake the 96-well plate to mix the samples with the luminol solution.
7. Directly observe luminol chemiluminescence in the dark.
8. Semi-quantify the Hb concentration using a luminometer, if necessary.

3. Quantification of IgA and IgG by ELISAs

1. Coat wells of 96-well plates with 100 µL/well of a total anti-mouse IgA (20 ng/well; 200 ng/mL) or IgG (10 ng/well; 100 ng/mL) antibody (see Table of Materials).
2. Seal the plates and incubate them at 4 °C overnight.
3. Wash the plates three times with 300 μL/well of a washing buffer containing 0.05% Tween 20 in PBS each time, and then blot the plates on paper towels.
4. Add 200 µL of an assay diluent containing 10% fetal bovine serum (FBS) and 0.2% Tween 20 in PBS to each well.
5. Incubate the plates at room temperature (RT) for 60 min.
6. Aspirate the assay diluent and then blot the plates on paper towels.
7. Prepare 100 ng/mL of a mouse IgA or IgG standard with the assay diluent and make two-fold serial dilutions: 50 ng/mL, 25 ng/mL, 12.5 ng/mL, 6.25 ng/mL, 3.13 ng/mL, 1.56 ng/mL, and 0.78 ng/mL.
   NOTE: The required volume of each standard is 100 µL per well.
8. Dilute the samples with the assay diluent at the appropriate dilutions: serum (10⁴ to 10⁶), NLF (10, 50, 200, and 500), and BALF (10¹ to 10⁴).
   NOTE: The required volume of each sample is 100 µL per well.
9. Add 100 µL of IgA or IgG standards and samples to the appropriate wells.
10. Incubate the plates at RT for 75 min.
11. Wash the plates three times with 300 µL/well of the washing buffer each time, and then blot the plates on paper towels.
12. Dilute a peroxidase-conjugated anti-mouse F(ab')₂ antibody (see Table of Materials) with the assay diluent at the appropriate dilution: 10⁴-fold dilution.
    NOTE: The required volume is 100 µL per well.
13. Add 100 µL of the diluted peroxidase-conjugated anti-mouse F(ab')₂ antibody to each well.
14. Incubate the plates at RT for 75 min.
15. Wash the plates five times with 300 µL/well of the washing buffer each time, and then blot the plates on paper towers.
16. Add 100 µL of a substrate solution containing tetramethylbenzidine (TMB) and hydrogen peroxide to each well.
17. Incubate the plates at RT for 5 min.
18. Add 50 µL of a stop solution, 2 N sulfuric acid (2N H₂SO₄), to each well.
19. Measure the absorbance at 450 nm using a microplate reader.
20. Determine the concentrations of IgA and IgG in each sample by utilizing the standard curves.

Results

Using the conventional or novel method, NLF samples were collected from C57BL/6 mice through the trans-pharyngeal route. As shown in Figure 1, the NLF samples collected by the conventional method were slightly pink-tinged (Figure 1A); the NLF samples collected by the novel method were clear (Figure 1B). Since the difference seemed to result from blood contamination in the NLF samples by the conventional method, blood contamination i...

Discussion

Nasal lavage is a valuable technique for acquiring biochemical, cytological, and biological information in respiratory diseases, such as influenza and COVID-19²³. Besides identifying various infiltrating cells, the non-cellular composition of the respiratory tract can be examined from the NLF samples using various methods, such as ELISAs, immunoblot, and quantitative polymerase chain reaction^1,19,24. Here...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Animal fixation needle	Natsume Seisakusho Co., Ltd., Tokyo, Japan	KN-358-A	We fixed a mouse with animal fixation needles. Stainless steel; total length, 23 mm; and needle tip, 15 mm.
BD OptEIATM TMB Substrate Reagent Set	BD Biosciences, San Diego, CA, USA	555214	We added 100 μL of TMB substrate solution per well.
Bell Jar	NA	NA	We used a bell jar for anesthesia.
Clear Flat-Bottom Immuno Nonsterile 96-Well Plates	ThermoFisher Scientific, Rochester, NY, USA	442404	We used Clear Flat-Bottom Immuno Nonsterile 96-Well Plates with MaxiSorp surface treatment for enzyme-linked immunosorbent assays.
Cotton ball	Sansho Co., Ltd., Tokyo, Japan	94-1951	We used cottons balls to avoid blood contamination. The diameter of cotton balls was 7 mm.
Fetal bovine serum	Sigma-Aldrich Co., St. Louis, MO, USA	172012-500 ML	We added 10% of fetal bovine serum to the assay diluent.
Forceps (Micro tweezers for research experiments, curved tip)	Sansho Co., Ltd., Tokyo, Japan	91-1506	We used forceps for disection. Model, HSC 551-10; and overll all length, 105 mm.
Forceps (Precision tweezers)	Sansho Co., Ltd., Tokyo, Japan	91-2772	We used forceps for disection. Model, HSC 607-12; overll all length, 120 mm; and tip diameter, 0.3 mm.
Goat Anti-Mouse IgA-UNLB	SouthernBiotech, Birmingham, AL, USA	1040-01	We coated Clear Flat-Bottom Immuno Nonsterile 96-Well Plates with 100 μL/well (20 ng/well) of total immunoglobulin A.
Isoflurane	Viatris Inc., Canonsburg, PA, USA	NA	We used isoflurane for anesthesia.
Luminol Rection Reagent Set	FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan	124-06511	We used a luminol reaction reagent set to detect hemoglobin in NLF samples.
Microtube	Watson Co., Ltd., Tokyo, Japan	131-7155C	We used  microtubes for collecting NLF and BALF (1.5-mL microtube with lockining-cap, round bottom, soft touch-cap).
Milli-Q water	Merck KGaA, Darmstadt, Germany	Milli-Q Direct 8	We used MilliQ water to make a luminol rection solution.
Mini-Shaker PSU-2T	Biosan Ltd., Riga, Latvia	PSU-2T	We shaked 96-well plates using the Mini-Shaler PSU-2T.
Mouse strain	CLEA Japan, Inc., Tokyo, Japan	NA	we used 4-month-old C57BL/6 male mice.
Nunc MicroWell 96-Well, Nunclon Delta-Treated, Flat-Bottom Microplate	Thermo Fisher Scientific Nunc A/S . Kamstrupvej,Roskilde, Denmark	136101	We used the NuncTM MicroWellTM 96-Well, Nunclon Delta-Treated, Flat-Bottom Microplate for the luminol test.
Peroxidase-conjugated AffiniPure F(ab')2 Fragment Goat Anti-Mouse IgG, F(ab')2 Fragment Specific (minimal cross-reaction to Human, Bovine, and Horse Serum Proteins)	Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA	115-036-072	We added 100 μL of peroxidase-conjugated AffiniPure F(ab')2 fragment goat anti-mouse IgG, F(ab')2 fragment specific, as the detection antibody (10,000-fold dilution) to each well.
Phosphate-buffered saline	Sigma-Aldrich Co., St. Louis, MO, USA	D8537-500ML	We used Dulbecco's Phosphate-Buffered Saline (modified without calcium chloride and magnesium chloride, liquid, sterile-filtered, suitable for cell cullture) for NLF and BALF collection.
Pipette	Watson Co., Ltd., Tokyo, Japan	NA	We used a 200-μL pipette for nasal lavage.
Pipette tip	Watson Co., Ltd., Tokyo, Japan	NA	We used a 200-μL multifit pipette tip for nasal lavage.
Polyoxyethylene(20) Sorbitan Monolaurate (Tween 20)	FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan	167-11515	We added 0.05% of Tween 20 to the washing buffer and 0.2% of Tween 20 to the assay diluent.
Purified Goat anti-mouse IgG (minimal x-reactivity) Antibody	BioLegend, San Diego, CA, USA	405301	We coated Clear Flat-Bottom Immuno Nonsterile 96-Well Plates with 100 μL/well (10 ng/well) of total immunoglobulin G.
Small scissors, double-pointed	Sansho Co., Ltd., Tokyo, Japan	91-7005	We used scissors for disection. Model, S-4A; and overll all length, 115 mm.
Sulfuric acid	Wako Pure Chemical Industries, Ltd, Tokyo, Japan	192-04755	We added 50 μL of 2 N sulfuric acid to each well to stop the reaction.
Surflo intravenous catheter	Terumo Corporation, Tokyo, Japan	SR-FS2032	We used a surflo intravenous catheter and injected PBS into the trachea for bronchoalveolar lavage. Catheter, 20G; catheter length, 32 mm; catheter inner diameter, 0.80 mm; catheter outer diameter, 1.1 mm; and inner needel, 22G.
Surgical straight scissors	Sansho Co., Ltd., Tokyo, Japan	91-7004	We used scissors for disection. Model, S-3B; and overll all length, 120 mm.
Surgilcal plate	Natsume Seisakusho Co., Ltd., Tokyo, Japan	KN-321-B	We used a cork board for dissection. Depth, 20 cm; width, 15 cm; and height 3 cm.
Synergy H1 Hybrid Multi-Mode Microplate Reader	Agilent Technologies, Inc., Santa Clara, CA, USA	NA	We measured the absorbance of samples at 450 nm, using the Synergy H1 Hybrid Multi-Mode Microplate Reader.
Syringe	Terumo Corporation, Tokyo, Japan	SS-01T	We used a 1-mL syringe for blood collection from the heart.
Thread	Niccho Kogyo Co., Ltd. Tokyo Japan	NA	We tied a catheter with thread to ensure proper positioning during the procedure.