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Abstract

Introduction

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Representative Results

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Acknowledgements

Materials

References

Immunology and Infection

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Published: September 28th, 2016

DOI:

10.3791/54508

1Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under 'blind' and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Community-acquired pneumonia remains the leading cause of death from infection in the U.S., with little overall change in mortality rates over the past 40 years despite improvements in vaccination and antibiotic strategies1,2. Despite the lack of perceptible progress at the public health level, in recent years dramatic advances have been made in our understanding of the molecular and cellular pathogenesis of pneumonia, with many of these steps forward made possible by the use of mouse models of lung infection. The genetic tractability of the mouse, the similarity of the murine and human immune systems, and the vast array of murine-targeted immunologic reage....

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All experiments were performed in accordance with the Animal Welfare Act and the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals after review by the Animal Care and Use Committee of the NIEHS.

1. Preparation of K. pneumoniae Culture

Caution: Perform all steps in a biosafety level 2 (BSL2) hood or other BSL2 designated area and discard waste per institute BSL2 guidelines.

  1. For suspension growth of K. pneumoniae

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C57BL/6 mice were infected with 2000 CFU of K. pneumoniae 43816 (serotype 2) via oropharyngeal aspiration into the lungs. At this dose, mice typically begin to show clinical symptoms 12-24 hr post-infection including lethargy, ruffled fur, and weight loss of 5-10% (Figure 2A). Within 48-72 hr post-infection, many of the mice show symptoms of illness and morbidity that is typically preceded by an average of 20% weight loss and results in hunched postures with decr.......

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Murine models of bacterial pneumonia, partnered with gene targeting and in vivo biological and pharmacological interventions, have provided critical insights into the pulmonary host defense response. Great advances have been made in particular in our understanding of the chemokines and adhesion molecules that govern recruitment of neutrophils to the infected airspace10,11. In vivo models of pneumonia, unlike cell-based or alternative approaches, have also provided key insights into endocrine .......

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This work was supported in part by the Intramural Research Program of the National Institutes of Health, National Institute of Environmental Health Sciences (Z01 ES102005).

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Name Company Catalog Number Comments
Klebsiella pneumoniae, serotype 2 ATCC 43816
Tryptic soy broth Becton Dickenson 211825
Excel Safelet IV Catheters, 18G x 1 1/4" Claflin Medical Equipment MEDC-031122
Hema 3 Solution 1 Fisher 23-122-937
Hema 3 Solution 2 Fisher 23-122-952
Hema 3 Fixative Fisher 23-122-929
27½ gauge tuberculin syringes Fisher 14-826-87
Lithium heparin plasma collectors Fisher 2675187
L-shaped disposable spreaders Lab Scientific DSC
1x PBS, pH 7.4 prepared in-house n/a Distilled water (5 L), NaCl (40 g), KCl (1 g), Na2HPO4 (5.75 g), KH2PO4 (1 g)   
ACK lysis buffer prepared in-house n/a NH4Cl (4.145 g), KHCO3 (0.5 g), EDTA (18.6 mg), bring up to 500 ml with distilled water and pH to 7.4

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