Name: open tracheostomy gastric acid aspiration murine model of acute lung injury results in maximal acute nonlethal lung injury
Uploaded: 2017-02-26T14:00:00
Description: Watch this Scientific Journal Video about Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury at JoVE.com

Ravi Alluri and Hilliard L. Kutscher are supported by Ruth L. Kirschstein National Research Service Award (NRSA) Institutional Research Training Grant 1T32GM099607.

All materials and equipment need to be gathered and adequately organized prior to procedure. The procedure is to be conducted with seamless transitions from one step to the next in order to provide consistent, reproducible data. This protocol follows institutional policies set by the Institutional Animal Care and Use Committee at the Buffalo Veterans Affairs Medical Center. 
NOTE: Both male and female C57BL/6 mice were used for this protocol. There is no significant difference of albumin leakage between sexes.
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	<li>Knight, P. R., Rutter, T., Tait, A. R., Coleman, E., Johnson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+gastric+particulate+lung+injury:+a+comparison+and+interaction+with+acidic+pneumonitis.">Pathogenesis of gastric particulate lung injury: a comparison and interaction with acidic pneumonitis.</a> Anesth Analg. 77 (4), 754-760 (1993).</li><li>Raghavendran, K., Nemzek, J., Napolitano, L. M., Knight, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aspiration-induced+lung+injury.">Aspiration-induced lung injury.</a> Crit Care Med. 39 (4), 818-826 (2011).</li><li>Rubenfeld, G. D., 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=Incidence+and+outcomes+of+acute+lung+injury.">Incidence and outcomes of acute lung injury.</a> N Engl J Med. 353 (16), 1685-1693 (2005).</li><li>Kennedy, T. P., 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=Acute+acid+aspiration+lung+injury+in+the+rat:+biphasic+pathogenesis.">Acute acid aspiration lung injury in the rat: biphasic pathogenesis.</a> Anesthesia &amp; Analgesia. 69 (1), 87-92 (1989).</li><li>Kudoh, I., 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=The+effect+of+pentoxifylline+on+acid-induced+alveolar+epithelial+injury.">The effect of pentoxifylline on acid-induced alveolar epithelial injury.</a> Anesthesiology. 82 (2), 531-541 (1995).</li><li>Prophet, E. B., Mills, B., Arrington, J. B., Sobin, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory methods in histotechnology. , (1995).</li><li>Kyriakides, 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=Membrane+attack+complex+of+complement+and+neutrophils+mediate+the+injury+of+acid+aspiration.">Membrane attack complex of complement and neutrophils mediate the injury of acid aspiration.</a> J Appl Physiol. 87 (6), 2357-2361 (1999).</li><li>Yoshikawa, S., 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=Acute+ventilator-induced+vascular+permeability+and+cytokine+responses+in+isolated+and+in+situ+mouse+lungs.">Acute ventilator-induced vascular permeability and cytokine responses in isolated and in situ mouse lungs.</a> J Appl Physiol. 97 (6), 2190-2199 (2004).</li><li>Eyal, F. 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The objective was to develop an ALI animal model using gastric acid aspiration that closely resembles the pathophysiology that occurs in humans during development of acid pneumonitis and subsequent ARDS. In developing a model, we chose an animal species that offers high throughput data acquisition due to its low cost, short reproductive cycle, and a well understood immunological system with an abundance of investigative tools (i.e., monoclonal antibodies, transgenic strains).
ALI foll...

ARDS is characterized by widespread lung inflammation and is clinically seen as acute shortness of breath with hypoxemia. These symptoms often occur less than 24 h after an inciting event such as trauma, sepsis, blood transfusion related reactions or aspiration. It is characterized histopathologically by neutrophilic alveolitis (i.e., widespread inflammation) localized to alveolar epithelium and vascular endothelium leading to protein leakage and subsequently hyaline membrane formation. Aspiration is categorized as chemical pneumonitis or aspiration pneumonia.1 The acidic component of the gastric aspiration contributes to both the pneumonitis and predilection to develop a secondary bacterial pneumonia. Aspiration pneumonia is one of the leading risk factors for ALI and subsequent development of ARDS.2
Gastric aspiration is an acute event defined as inhalation of materials from the stomach with or without oropharyngeal flora into the airways beyond the vocal cords. The aspirate contents may contain low pH stomach fluid, bacteria, blood, or food particles. Gastric aspiration often occurs in patients in the Intensive Care Unit (ICU), whom are typically in a fasting state and thus placed on a proton pump inhibitor to limit aspiration of acidified gastric contents. The incidence of ALI in the ICU population in the US is 2.5 - 5 times higher compared to the general patient population.3 Unfortunately, these predisposing conditions often lead to a state of bacterial overgrowth in the stomach that may lead to more severe sequelae in the lung following an aspiration event, as gastric aspiration is an independent risk factor for the development of secondary bacterial pneumonia (SBP), ALI and ARDS.
Gastric aspiration has two major components: hydrochloric acid and gastric contents, which may or may not contain bacteria or food particulate. In the rodent model the acid component alone of gastric aspiration produces an initial inflammatory response as a result of the direct caustic injury of low pH on airway epithelium. This is followed by a neutrophilic infiltration and an inflammatory response at 4 - 6 h.4 These two factors ultimately lead to destruction of pulmonary microvascular integrity thus leading to extravasation of fluid and proteins into alveoli and airways. To understand this pathophysiology and to further investigate possible therapeutic interventions, it is important to develop and characterize an animal model that elucidates the underlying mechanisms involved. An acidic aspirate alone must be voluminous or with a low enough pH to bypass the buffering capacity of the respiratory tree and reach the alveoli. If this does not occur, only a transient upper, conducting airways injury occurs which is less likely to lead to the severe sequelae of ARDS.5
To emulate an aspiration event accurately with injured alveolar epithelium, it is important to bypass an animal&#39;s natural defenses. Using a mouse aspiration model to produce an ALI that mimics the gastric acid injury seen in humans, one must account for the differences in the trachea-bronchial tree. The open tracheostomy technique that this method utilizes bypasses the differences between murine and human respiratory trees and models the injury in a manner that reproduces ALI both physiologically and histologically. Historically, intratracheal intubation was used to generate ALI, however it is considered difficult to perform in mice without laryngeal injury. Therefore, this method offers a potential alternative that has yielded consistent results across multiple researchers and with minimal procedure attributed mortality.

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			<td>Fine Science Tools</td>
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			<td>tracheal cannula (20 ga x 1/2&#34; stainless steal tubing adapter)</td>
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open tracheostomy gastric acid aspiration murine model of acute lung injury results in maximal acute nonlethal lung injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

Pulmonary Histopathology of the Murine Model of Acid Aspiration Pneumonitis
Mice were injured as described above and lungs were removed 24 h post low pH insult, sectioned and H&#38;E stained (Figure 1). Necrotic cells, loss of lung parenchymal architecture, cells and debris within airspaces and significant PMN infiltration are clearly observed. Similar to aspiration clinically, our model results in a heterogeneous ...

Watch this Scientific Journal Video about Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury at JoVE.com

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

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