Name: the utilization of oropharyngeal intratracheal pamp administration and bronchoalveolar lavage to evaluate the host immune response in mice
Uploaded: 2014-04-02T17:00:00
Description: Watch this Scientific Journal Video about The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice at JoVE.com

The authors thank the VA-MD Regional College of Veterinary medicine for providing core and technical support for this project. This work is supported by an NIH Career Development Award (K01DK092355).

All studies were conducted under the approval of the Institutional Care and Use Committee (IACUC) for Virginia Tech and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
1. Intratracheal (i.t.) Inoculation of LPS Using Oropharyngeal Administration
<ol>
	<li>Ensure that each animal is uniquely identified using either an ear punch, ear tag, or other institutionally approved method.</li>
	<li>Record the baseline body weight and body tem...

<ol>
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Biol. 44, 725-738 (2011).</li><li>Egger, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Administration+of+bleomycin+via+the+oropharyngeal+aspiration+route+leads+to+sustained+lung+fibrosis+in+mice+and+rats+as+quantified+by+UTE-MRI+and+histology.">Administration of bleomycin via the oropharyngeal aspiration route leads to sustained lung fibrosis in mice and rats as quantified by UTE-MRI and histology.</a> PloS one. 8, (2013).</li><li>Rayamajhi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonsurgical+intratracheal+instillation+of+mice+with+analysis+of+lungs+and+lung+draining+lymph+nodes+by+flow+cytometry.">Nonsurgical intratracheal instillation of mice with analysis of lungs and lung draining lymph nodes by flow cytometry.</a> J. Vis. Exp. , (2011).</li><li>Revelli, D. A., Boylan, J. A., Gherardini, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+non-invasive+intratracheal+inoculation+method+for+the+study+of+pulmonary+melioidosis.">A non-invasive intratracheal inoculation method for the study of pulmonary melioidosis.</a> Front. Cell. Infect. Microbiol. 2, 164 (2012).</li><li>Allen, I. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+NLRP3+in+the+development+of+allergic+airway+disease+in+mice.">Analysis of NLRP3 in the development of allergic airway disease in mice.</a> J. Immunol. 188, 2884-2893 (2012).</li><li>Allen, I. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+NLRP12+during+the+in+vivo+host+immune+response+to+Klebsiella+pneumoniae+and+Mycobacterium+tuberculosis.">Characterization of NLRP12 during the in vivo host immune response to Klebsiella pneumoniae and Mycobacterium tuberculosis.</a> PloS one. , (2013).</li><li>Allen, I. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+function+of+NPSR1/GPRA+in+the+lung+before+and+after+induction+of+asthma-like+disease.">Expression and function of NPSR1/GPRA in the lung before and after induction of asthma-like disease.</a> Am. J. Physiol. Lung Cell. Mol. 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The most critical steps for successfully evaluating the host immune response in mouse lungs is as follows: 1) choose the appropriate mouse strain and sex for the model being evaluated; 2) optimize PAMP delivery to the lungs; 3) correctly collect and process the BALF; and 4) properly fix and prepare of the lungs for histopathological assessments.
The choice of mouse strain is an important factor in evaluating the host immune response. C57Bl/6 mice are typically considered the optimal mouse back...

Pulmonary infections associated with pathogenic bacteria species are a common cause of global morbidity and mortality. Determining the mechanisms that drive the host immune response to these pathogens will promote the development of novel prevention strategies and therapeutic agents that will attenuate the impact of these infections. The overall goal of the protocol described here is to provide the user with a flexible method to evaluate the host innate immune response to pathogen infection using a pathogen associated molecular pattern (PAMP) as a surrogate for live bacteria. The majority of previous studies evaluating the host innate immune response to bacteria have focused on peritoneal models due to the relative ease of execution. While these models are highly useful and have resulted in significant advances in the field of host-pathogen interactions and systemic inflammation, the data generated from these models are not always appropriate for studies involving the respiratory system. Here, a pulmonary model of acute lung inflammation is proposed as a practical and clinically relevant expansion of the classical intraperitoneal (i.p.) injection models. The proposed technique allows for the local assessment of the innate immune response in an organ specific model system.
The methods described here are designed to provide a simple and robust technique to allow users to evaluate the host immune response to LPS, which is a common PAMP. The methods are based on intratracheal (i.t.) instillation of LPS, which induces a robust innate immune response in the lungs of mice and mimics many of the pathophysiological features observed in human patients suffering from respiratory infections and acute lung injury1. A primary advantage of this technique is that it allows the user to evaluate the host immune response without the confounding factors and safety concerns associated with conducting in vivo studies using live bacteria. Likewise, the oropharyngeal i.t. administration route of exposure described in this protocol has significant advantages over other commonly utilized techniques, including intranasal (i.n.) administration and surgical i.t. administration. For example, oropharyngeal i.t. administration allows relatively accurate dosaging and lung deposition compared to i.n. administration, which typically suffers from increased variability of lung deposition due to the loss of agents in the nasal cavity and sinuses2-4. The i.t. administration route circumvents these cavities and allows direct access to the trachea and airway. Likewise, the surgical i.t. approach is a significantly more morbid administration method and requires extensive training to master. The protocols described here also include a description the common techniques and surrogate markers used to evaluate inflammation progression and end with a protocol describing the proper techniques for preparing the lungs for histopathology assessments. These protocols are focused on minimizing the number of mice required for each study by maximizing the data generated from each individual animal.
The protocols described are highly flexible and can be readily modified to evaluate a diverse range PAMPs and damage associated molecular patterns (DAMPs). Furthermore, with a few additional modifications, these protocols can also be applied to studies evaluating allergic airway disease progression or host-pathogen interactions with live bacteria, viruses or fungi5-10.

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the utilization of oropharyngeal intratracheal pamp administration and bronchoalveolar lavage to evaluate the host immune response in mice

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen interactions take advantage of intraperitoneal injections of select bacteria or pathogen associated molecular patterns (PAMPs) in mice. While these techniques have yielded tremendous data associated with infectious disease pathobiology, intraperitoneal injection models are not always appropriate for host-pathogen interaction studies in the lung. Utilizing an acute lung inflammation model in mice, it is possible to conduct a high resolution analysis of the host innate immune response utilizing lipopolysaccharide (LPS). Here, we describe the methods to administer LPS using nonsurgical oropharyngeal intratracheal administration, monitor clinical parameters associated with disease pathogenesis, and utilize bronchoalveolar lavage fluid to evaluate the host immune response. The techniques that are described are widely applicable for studying the host innate immune response to a diverse range of PAMPs and pathogens. Likewise, with minor modifications, these techniques can also be applied in studies evaluating allergic airway inflammation and in pharmacological applications.

The cell walls of gram-negative bacteria are composed of LPS, which is highly abundant in the environment. Inhalation of LPS in sensitive human populations exacerbates airway reactivity and is capable of triggering a robust immune response11. LPS is also a common PAMP used in mouse models to elicit a robust innate immune response. In the protocol described here, the mice received an i.t. dose of LPS isolated from E. coli (serotype 0111:B4) using oropharyngeal i.t. administration. In models of LPS exposure, both ...

Watch this Scientific Journal Video about The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice at JoVE.com

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

The host immune response to pathogen infection is a tightly regulated process. Utilizing a lipopolysaccharide lung exposure model in mice, it is possible to conduct high resolution evaluations of the complex mechanisms associated with disease pathogenesis.

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen ...

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