A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

Summary

We describe a method for inducing neutrophilic pulmonary inflammation by challenge to aerosolized lipopolysaccharide by nebulization, to model acute lung injury. In addition, basic surgical techniques for lung isolation, tracheal intubation and bronchoalveolar lavage are also described.

Abstract

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models of ALI are key in enhancing our understanding of disease pathogenesis. Lipopolysaccharide (LPS) derived from gram positive bacteria induces neutrophilic inflammation in the airways and lung parenchyma of mice. Efficient pulmonary delivery of compounds such as LPS is, however, difficult to achieve. In the approach described here, pulmonary delivery in mice is achieved by challenge to aerosolized Pseudomonas aeruginosa LPS. Dissolved LPS was aerosolized by a nebulizer connected to compressed air. Mice were exposed to a continuous flow of LPS aerosol in a Plexiglas box for 10 min, followed by 2 min conditioning after the aerosol was discontinued. Tracheal intubation and subsequent bronchoalveolar lavage, followed by formalin perfusion was next performed, which allows for characterization of the sterile pulmonary inflammation. Aerosolized LPS generates a pulmonary inflammation characterized by alveolar neutrophilia, detected in bronchoalveolar lavage and by histological assessment. This technique can be set up at a small cost with few appliances, and requires minimal training and expertise. The exposure system can thus be routinely performed at any laboratory, with the potential to enhance our understanding of lung pathology.

Introduction

Lipopolysaccharide (LPS) is a cell wall component of gram negative bacteria¹. Challenge to LPS is a well-documented model of acute lung injury, a syndrome characterized by acute neutrophilic inflammation and edema². In addition, pulmonary neutrophilia is also a hallmark of chronic obstructive pulmonary disease (COPD)³, and LPS challenge in humans has been used to model COPD exacerbations⁴. Thus, experimental models of LPS exposure are clinically relevant and valuable tools to understand human pathology.

The objective of the pulmonary delivery of aerosolized LPS described here is to generate a neut....

Protocol

The animal studies were approved by the Northern Stockholm animal welfare ethics committee. The experimental procedures were performed in compliance with Swedish law.

1. Generating an LPS Aerosol

1. Dissolve 0.5 g purified P. aeruginosa LPS in 50 ml sterile saline with gentle agitation and verify dissolution. Dilute 1 ml dissolved LPS in 9 ml sterile saline, to a final concentration of 1 mg/ml. Protect from light with aluminum foil and store at -20 °C.
2. Thaw solubilized LPS in the dark at room temperature and mix well immediately before use.
3. In a ventilated level II biohazard hood, insert a red inlet int....

Results

Challenge to aerosolized P. aeruginosa LPS usually yields a marked inflammatory response in the airway lumen and alveolar space, characterized by a predominance of neutrophils at both early and late time points.

Aerosolized LPS induces pulmonary neutrophilia

C57BL/6by and BALB/c mice were exposed to aerosolized P. aeruginosa LPS or vehicle alone and neutrophils were enumerated in BALF. The total cell number in BALF of C57BL/6by mice exposed to an aero.......

Discussion

Aerosolized LPS generates an inflammatory response in the airways, characterized by neutrophils in the epithelial submucosa, spaces surrounding the conducting airways, as well as the alveolar spaces. This is, together with the increased total protein content in BALF, indicative of plasma leakage, representative of the pathology of acute lung injury. As LPS induces a sterile inflammation, the reaction is independent of the adaptive immune response, and there are limitations to the relevance to bacterial infections. The te.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Purified Pseudomonas aeruginosa LPS	Sigma-Aldrich		Harmful. Recomended purification. LPS purified from other bactria may be used.
Pari LC sprint star nebulizer	PARI Respiratory Equipment Inc.	023G1250
TSI mass flowmeter 4040	TSI	4040	Alternative product from supplier may be used.
Saint-Gobain 15.9 mm Tygon tube	Sigma-Aldrich	Z685704	Recomended brand.
Plexiglas boxes with removable lids	Custom built	N/A	150 x 163 x 205 mm (a 2 mm hole on the side).
3M Half Facepiece Reusable Respirator	3M	7503	Recomended brand.
3M Advanced Particulate Filters (P100)	3M	2291	Recomended brand.
Scissors	VWR	233-1104	Preferred scissors may be used.
Forceps	VWR	232-1313	Preferred forceps may be used.
Intramedic PE50 polyethylene tube	BD	427411	Recomended brand.
Ethicon 2-0 Perma-hand silk tread	VWR	95056-992	Recomended brand.
26 ½ gage needle			Alternative suppliers exist.
1 ml BD slip-tip syringe, non-sterile	BD	301205	Alternative suppliers exist.
60 ml BD Luer-Lok syringe, non-sterile, polypropolene	BD	301035	Alternative suppliers exist.
Fluka Hematoxylin-Eosin	Sigma-Aldrich	3972	Alternative suppliers exist.
Türk's solution	Merck Millipore	109277
Table top centrifuge			Alternative manufacturers exist.
Cytospin 4 cytocentrifuge	Thermo Scientific	A78300003	Alternative centrifuge can be used.
HEMA-3 stat pack	Fisher Scientific	23-123-869	Alternative staining kits exists.
Formalin solution, neutral buffered, 10%	Sigma-Aldrich	HT501128	Alternative suppliers exist.