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.
1. Hydrochloric Acid Preparation
<ol>
	<li>Make 56.2 mM HClsterile by adding 281 &#181;L of 1 N HCl to 4.72 mL sterile normal saline (SNS). Titrate to pH 1.25 with 1 N HCl (&#8776; 0 - 50 &#181;L).</li>
	<li>Make this preparation fresh prior to injury. As low pH SNS is not buffered it ensures the hydrochloric acid injury insult is transient.</li>
</ol>
2. Anesthesia and Tracheal Exposure Technique
NOTE: Ensure that the injury procedure is conducted in a fume hood with charcoal filtering for anesthetic gas scavenging. Maintenance of sterile conditions is essential as this is a survival surgery. Use sterile gloves and sterilize all instruments in a glass bead surgical instrument sterilizer prior to surgery on each animal. The use of drapes to isolate the surgical site is recommended to maintain sterility. Use isoflurane as the anesthetic gas as it provides a relatively low blood gas solubility allowing rapid induction of anesthesia, as well as rapid recovery following the injury.
<ol>
	<li>Start by inducing anesthesia in a chamber with 2.75% isoflurane in oxygen with flow rate of 2 L/min.</li>
	<li>Once the mouse is adequately anesthetized (unresponsive to toe pinch strong enough to not break skin or cause deep tissue damage), suspend the mouse by the superior incisors with a 15 cm length of 1-0 braided silk suture in a supine position on a 60o incline dissection board.</li>
	<li>Ensure that anesthesia is maintained throughout surgery by delivering isoflurane through a nose cone. Apply veterinary ointment on mouse&#39;s eye to prevent dryness.</li>
	<li>Shave the ventral portion of the neck with an electric trimmer and apply topical antiseptic solution (i.e., povidone-iodine). Remove excess antiseptic with gauze and let dry (~15 - 30 s).</li>
	<li>Infiltrate the midline of the neck from the sternal notch to inferior part of jaw with 100 &#181;L 0.5% bupivacaine (to provide pre-, peri-, and post-operative analgesia).</li>
	<li>Make a 1 - 1.5 cm longitudinal midline incision in the neck with a scalpel or curved fine scissors. Penetrate the fascial membrane between the salivary glands by blunt dissection with 2 curved serrated forceps to expose the trachea (identified by the white cartilage rings).</li>
	<li>Still using the 2 serrated forceps, grasp one side of the paratracheal musculature and pull it to the side while dissecting between the muscle fibers longitudinally next to the trachea.</li>
	<li>Once dissection plane is established, continue retracting the paratracheal muscle as the second pair of forceps is worked under the trachea. Use these forceps to place a 15 cm strand of 1-0 braided silk suture into the tip of the forceps and pull through to place the suture behind the trachea.</li>
</ol>
3. Intra-tracheal Instillation Procedure
<ol>
	<li>Prepare the injury instillation syringe by filling a 0.5 mL syringe attached to a 22 G needle with 0.2 mL of air followed by 3.6 mL/kg of hydrochloric acid. The air will act to aid in dispersion of the vehicle, as well as an alveolar recruitment maneuver for those alveoli that may have undergone atelectasis during anesthesia administration. &#160; 
	NOTE: Follow local IACUC guidelines regarding post-operative analgesia and consult with your local veterinarian. Be&#160;careful about using any analgesics with anti-inflammatory properties before employing this model of sterile lung injury.</li>
	<li>To administer the injury, first discontinue isoflurane administration. Allow mouse&#39;s anesthetic depth to rise for about 10 - 15 s until it begins to react to a forceps toe pinch.</li>
	<li>Immediately begin instillation steps of injury. If anesthetic depth is too deep the animal will not breathe spontaneously after injury instillation, which is especially true for hydrochloric acid containing injuries.</li>
	<li>Have an assistant squeeze the ribcage to expel vital capacity.</li>
	<li>Use the 1-0 braided silk suture placed previously around the trachea to provide traction by pulling it superiorly and then insert syringe needle into trachea between the 1st and 2nd cartilaginous rings below the larynx. The needle bevel should face the surgeon and advance until the bevel is just past the trachea with the needle as parallel with the trachea as possible.</li>
	<li>Release the chest just before bolus administration of hydrochloric acid in order to assure dispersion and deposition into the distal airway and alveoli. Deposit the bolus in roughly 0.5 - 0.75 s. Keep the needle in the trachea until the first spontaneous breath is taken to ensure that the entire injury bolus volume is inhaled into the lungs.</li>
	<li>Close incision using either 1 - 2 skin staples or sutures.</li>
</ol>
4. Recovery
<ol>
	<li>Using a 5 mL syringe with a 26 G needle, inject 1 mL SNS subcutaneously into scruff of neck for fluid resuscitation. Although there is minimal fluid loss during procedure, following surgery the mouse tends to not drink and can potentially dehydrate. Alternatively, provide SNS via an intraperitoneal injection.</li>
	<li>Place mouse in heated chamber at 37 &#176;C perfused with 100% O2. Ensure that the mouse is adequately recovered before being placed back into housing. Monitor animals and do not leave unattended during this time until ambulatory and fully recovered.</li>
</ol>
5. Bronchoalveolar Lavage and Lung Processing
<ol>
	<li>Induce and maintain isoflurane anesthesia as described in Step 2.1 and Step 2.2. Confirm adequate plane of anesthesia using toe pinch method described in Step 2.2.</li>
	<li>Secure in a supine position on a dissecting board and use a loop of 1-0 braided silk suture hooked onto the superior incisors to extend the neck. Apply veterinary ointment on eyes to prevent dryness.</li>
	<li>After removing the neck skin staples, make a midline longitudinal incision through the skin from the lower abdominal wall through the previous neck incision to the mandible with scissors.</li>
	<li>Make a midline incision in the peritoneal musculature with scissors and retract the abdominal organs with sponges to expose the retroperitoneal space. If blood is needed, collect from the abdominal aorta or inferior vena cava at this point.</li>
	<li>Euthanize the animal via exsanguination by transecting the abdominal aorta and inferior vena cava. By utilizing a bilateral thoracotomy in the following step a secondary confirmatory method of euthanasia will be completed humanely as established by IACUC guidelines.</li>
	<li>Perforate the diaphragm to cause lung collapse. Then using bone cutting scissors, cut away the diaphragm and cut through the rib cage parallel to and on both sides of the sternum. Use a locking hemostat to retract the sternal flap to facilitate injecting 5 mL warm (37 &#176;C) Hank&#39;s Balanced Salt Solution with calcium and magnesium into the right ventricle to flush the lung vasculature.
	<ol>
		<li>As the blood from the aorta is obtained to evaluate peripheral cytokine levels prior to the procedure described in this paper, make a small incision in the left ventricle to provide necessary outflow for blood to leave the circulation to adequate flush the lung vasculature if not obtaining blood.</li>
	</ol>
	</li>
	<li>Prop the dissection board to a 60&#176; incline and use the procedure described in Step 2.2 to facilitate inserting a 20 G x &#189;&#34; stainless steel cannula in the trachea and secure with a 1-0 suture.</li>
	<li>Place the dissection board horizontally and perform a bronchoalveolar lavage (BAL).
	<ol>
		<li>To perform the BAL attach two 5 mL syringes to a 3-way stopcock. Fill one syringe with 5 mL of SNS and screw into port of stopcock. Attach an empty 5 mL syringe to a 2nd port and finally attach open port of stopcock to intra-tracheal cannula placed in Step 5.6. Instill 1 mL of NS into lungs and lavage with second syringe.</li>
		<li>Repeat for 5 total 1 mL NS instillations being careful that the tracheal cannula remains secured and parallel to the trachea so it does not tear the trachea.</li>
	</ol>
	</li>
	<li>For histological analysis, cannulate the trachea as in Step 5.7. Fix the lungs by insufflating with 10% neutral buffered formalin at 20 cm H2O for 24 h.</li>
	<li>Remove each lung lobe carefully using scissors and forceps by transecting bronchus near the hilum. Place each lung lobe (5 total) in a histology cassette prior to being embedded in paraffin. Cut 5 &#181;m tissue sections with a microtome, and stain with hematoxylin and eosin using standard histological techniques.6 Morphometrics can also be performed on the histological slides to quantify the severity inflammation.4</li>
	<li>Process the recovered BAL by centrifuging at 1,500 x g for 3 min at 4 &#176;C. Aliquot the cell-free BAL supernatants in equivalent amounts into 2.0 mL microcentrifuge tubes for future analysis. Store at -80 &#176;C.</li>
	<li>Analyze the BAL for albumin concentration by ELISA, or any other biological product of interest.11The pelleted cells from the BAL can be counted, cytoslide specimens generated, and stained with Wright-Giemsa-based stain for white blood cell differential count determination to assess the lungs&#39; inflammatory reaction.</li>
</ol>

<ol>
	<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. G., Hamm, C. R., Coker-Flowers, P., Stober, M., Parker, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neutralization+of+alveolar+macrophages+reduces+barotrauma-induced+lung+injury.">The neutralization of alveolar macrophages reduces barotrauma-induced lung injury.</a> FASEB J. 16 (4), 410-411 (2002).</li><li>Hermans, C., Bernard, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+epithelium-specific+proteins:+characteristics+and+potential+applications+as+markers.">Lung epithelium-specific proteins: characteristics and potential applications as markers.</a> Am J Respir Crit Care Med. 159 (2), 646-678 (1999).</li><li>Segal, B. H., 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=Acid+aspiration-induced+lung+inflammation+and+injury+are+exacerbated+in+NADPH+oxidase-deficient+mice.">Acid aspiration-induced lung inflammation and injury are exacerbated in NADPH oxidase-deficient mice.</a> Am J Physiol Lung Cell Mol Physiol. 292 (3), 760-768 (2007).</li><li>Matthay, M. A., Robriquet, L., Fang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alveolar+epithelium:+role+in+lung+fluid+balance+and+acute+lung+injury.">Alveolar epithelium: role in lung fluid balance and acute lung injury.</a> Proc Am Thorac Soc. 2 (3), 206-213 (2005).</li><li>Richard, J. C., Factor, P., Welch, L. C., Schuster, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+spatial+distribution+of+transgene+expression+in+the+lungs+with+positron+emission+tomography.">Imaging the spatial distribution of transgene expression in the lungs with positron emission tomography.</a> Gene Ther. 10 (25), 2074-2080 (2003).</li></ol>

The authors have no competing financial interests to disclose.

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 following gastric acid aspiration in humans caustically damages the alveolar epithelium and vascular endothelial barrier function producing a leakage of serum proteins. BAL albumin has been shown to be a sensitive marker of pulmonary permeability as its size is smaller than other serum proteins. In addition, albumin can be assessed in a variety of ways from colorimetric assays to specific ELISA assays.7-9
Bronchoalveolar lavage is used in clinical settings to detect lung injury as plasma proteins and markers of inflammation are present in minimal amounts within the alveolar spaces of healthy lungs. Elevated BAL plasma proteins indicates a loss of endothelial barrier function.10
To elucidate the pathophysiology of gastric acid aspiration, it is important to develop and characterize an animal model within the scope of its limitations. Defining conditions associated with maximal acute nonlethal lung injury that mimic an aspiration event (i.e., pH, and volume of aspirate administered), one should reduce the variability inherent in such gastric aspiration/ALI models. Maximal nonlethal lung injury in mice occurs after aspiration of 3.6 mL/kg of pH 1.25 HCl, which simulates gastric acid.11 Gastric acid aspiration alters alveolar fluid clearance independent of pulmonary blood flow or vascular filtration.12 Histopathological characteristics of the gastric acid aspiration/ALI injury are an acute inflammatory response with patchy areas of neutrophilic inflammation, alveolar hemorrhage, intra-alveolar and interstitial edema. To improve the dispersion of the hydrochloric acid causing injury to the maximal number of alveoli, one should initiate a forced exhalation prior to instillation. This facilitates the dispersion to distal airspaces and produces maximal lung injury to the alveoli. This exhalation maneuver is produced by circumferential compression of the thorax and release post instillation. This is a modified version of a validated technique in a rat model delivering adenovirus to rat lungs that demonstrated the dispersion of a green fluorescent protein (Ad-CMV-mNLS-HSV1sr39tk-egfp) to the distal airspaces, thus delivering injury vehicle to the intended sites (e.g., alveoli).13
There are several advantages to utilizing the open tracheostomy method for hydrochloric acid aspiration instillation versus direct hydrochloric acid aspiration application into the oropharynx. Non-invasive instillation through the oropharynx is the easiest and least invasive method for delivery of hydrochloric acid aspiration to the lungs of small animals. However, due to the very low pH required of the aspirate to produce maximal nonlethal lung injury, any reflux following instillation will lead to post procedure obstructive conditions e.g., laryngeal edema, laryngospasm, sloughing of upper airway epithelium. In using the open tracheostomy method, hydrochloric acid is consistently able to reach the targeted area (e.g., distal airways and alveolar sacs) thus replicating the ALI mechanism. One can enhance ALI by combining hydrochloric acid aspiration with an alveoli recruitment maneuver using a bolus air &#34;chaser&#34; during hydrochloric acid administration. This maneuver opens up atelectatic alveoli following initiation of volatile anesthesia. Atelectasis is a well-known sequelae of volatile anesthetic inhalation caused by decreased smooth muscle tone which impedes the function of surfactant and causes the animal to have lower tidal volumes leading to V/Q mismatch. The air &#34;chaser&#34; equates to an alveolar recruitment maneuver regularly used in the operating room on patients following a surgical procedure. The recruitment maneuver provides a larger area for directed injury and aids in dispersion of the hydrochloric acid to the distal airway and alveoli. One can evaluate a successful intratracheal instillation by evaluating respiratory rate following return of spontaneous breathing as this is known to increase following delivery of the hydrochloric acid.
We use 5 mL NS total to lavage the lungs in 1 mL increments. Although the normal TLC of a mouse is 1 mL the large volume used ensures recovery of the maximal amount of albumin in order to quantify the injury. It is important to take into account that this will dilute the proteins and more so the pro-inflammatory mediators as they are usually in the order of pg/mL under normal circumstances. The variability can be mitigated by consistent processing of the BAL fluid. When utilizing this method, control animals utilizing a sham surgery should be performed in order to make appropriate comparisons as the tracheostomy itself produces inflammation.
An alternative approach to providing adequate anesthesia to mice would be via intraperitoneal injection of ketamine and xylazine compared to isoflurane inhalation as this paper describes. This option is best left to the researcher based on their comfort level in performing this alternative method. Advantages included minimized interactions with immune response as volatile anesthetics are known to alter normal immune system physiology.
There have been many advances in intubation methods used in mice, which require further investigation to reliably reproduce the histopathology of human ALI. The concerns that exist with intubation techniques include: proper endotracheal tube placement beyond the cords and above the carina; laryngospasm and bronchospasm; and laryngeal trauma caused by the technique itself. These are eliminated using the open tracheostomy method that this method employs. This method is not without limitations however as the tracheotomy itself produces injury and acid instillation performed with an injection needle may further increase the risk of severe injury. Mice awake from this procedure with pain, which subsequently worsens their perioperative hypovolemia secondary to lack of drinking. As such, this method provides an alternative to intubation techniques, which can decrease the amount of injury if performed correctly.
In conclusion, this animal model employs an open tracheostomy hydrochloric acid instillation simulating gastric acid aspiration to further the understanding of human ALI pathophysiology, and the subsequent development of ARDS. As human physiological mechanisms are unique, no single animal model reproduces all the characteristics of ALI. However, if the characteristics of this ALI murine model or any animal model are interpreted within the limits and scope of the model, the data generated can provide focused tests of key elements of the lung injury response in humans. The investigator must overcome the limitations common to all models of ALI and use one best suited to test their hypothesis and be cognizant about the differences between the model used and humans.9

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.

<table><tbody><tr><td>syringe, 1 cc</td><td>Becton Dickinson</td><td>309628</td><td></td></tr><tr><td>syringe, 5 cc</td><td>Becton Dickinson</td><td>309646</td><td></td></tr><tr><td>needle, 22 G x 1 1/2&quot;</td><td>Becton Dickinson</td><td>305159</td><td></td></tr><tr><td>needle, 26 G x 1 1/2&quot;</td><td>Becton Dickinson</td><td>305111</td><td></td></tr><tr><td>1-O Braided Silk Suture</td><td>Harvard Apparatus</td><td>517730</td><td></td></tr><tr><td>3&quot; Curved tissue serrated forceps</td><td>Fine Science Tools</td><td>11065-07</td><td></td></tr><tr><td>3&quot; Curved tissue &quot;toothed&quot; forceps, 1 x 2 teeth</td><td>Fine Science Tools</td><td>11067-07</td><td></td></tr><tr><td>4&quot; curved micro dissecting scissors</td><td>Fine Science Tools</td><td>14061-10</td><td></td></tr><tr><td>bone cutting spring scissors</td><td>Fine Science Tools</td><td>16144-13</td><td></td></tr><tr><td>3 1/2&quot; curved locking hemostat</td><td>Fine Science Tools</td><td>13021-12</td><td></td></tr><tr><td>Disposable Skin Stapler</td><td>3M</td><td>DS-25</td><td></td></tr><tr><td>tracheal cannula (20 gauge x 1/2&quot; stainless steal tubing adapter)</td><td>Becton Dickinson</td><td>408210</td><td></td></tr><tr><td>60-degree Incline Dissection Board</td><td></td><td></td><td></td></tr><tr><td>0.5% Bupivacaine</td><td></td><td></td><td></td></tr><tr><td>Isoflurane</td><td></td><td></td><td></td></tr><tr><td>Betadine and &quot;Q-tip&quot; cotton applicator</td><td></td><td></td><td></td></tr></tbody></table>

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 injury of the distal lung air spaces resulting in areas of patchy PMN infiltration and fibrinous protein deposition. This is very similar to the pattern observed in human ARDS patients histologically.
Albumin Leakage into Airways
A hallmark of acid aspiration is leakage of serum proteins into the airways. The severity of this pathogenic response to a low pH alveolar insult was determined by measuring BAL albumin concentrations. Albumin concentration in the BAL was increased following acid aspiration compared to normal saline (NS) controls at 2 h and 5 h (Figure 2). This is consistent with the pathophysiology observed in patients following acid aspiration. Time points were chosen as neutrophil infiltration occurs 2 h post acid aspiration and maximal non-lethal lung injury occurs at 5 h post acid aspiration.
<img alt="Figure 1" src="/files/ftp_upload/54700/54700fig1.jpg" /> 
Figure 1: Pulmonary Histopathology of CD-1 mice 24 h following Hydrochloric Acid Aspiration. H&#38;E-stained, 5 &#181;m sections from an uninjured mouse (A and B) and a mouse injured with hydrochloric acid (C-H). Necrotic cells (arrowhead), loss of parenchymal architecture, cells and debris within airspaces (arrows) and significant neutrophil infiltration (*) are seen throughout the lungs from the injured mice. A heterogeneous distribution the distal lung is observed with areas of patchy neutrophil infiltration and fibrinous protein deposition. This is very similar to the histopathology of ARDS in patients. Scale bars = A and C = 2 mm; B, D, F = 200 &#181;m; E = 100 &#181;m; and G and H = 60 &#181;m. Boxes in A and C indicate areas that are magnified in images B and D, respectively. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54700/54700fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54700/54700fig2.jpg" /> 
Figure 2: Albumin Leakage into Airways following Acid Aspiration. Albumin concentration of cell-free BAL fluid from C57BL/6 mice at 2 h and 5 h post acid aspiration. Acid aspiration was compared to 5 h NS aspiration control (n = 9) using an unpaired t test. Error bars indicate SEM. * = p &#60;0.05. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54700/54700fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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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Nanyang Technological University

Research

JoVE Journal

Medicine

9.7K Views.  University at Buffalo, State University of New York. 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.

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

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged mechanical ventilation, contributes to morbidity and mortality of critical illness, and studies on novel genetic or pharmacological targets are areas of intense investigation (1-3, 5, 8, 26, 30, 33-36). ALI is defined by the acute onset of the disease, which leads to non-cardiac pulmonary edema and subsequent impairment of pulmonary gas exchange (36). We have developed a murine model of ALI by using a pressure-controlled ventilation to induce ventilator-induced lung injury (2). For this purpose, C57BL/6 mice are anesthetized and a tracheotomy is performed followed by induction of ALI via mechanical ventilation. Mice are ventilated in a pressure-controlled setting with an inspiratory peak pressure of 45 mbar over 1 - 3 hours. As outcome parameters, pulmonary edema (wet-to-dry ratio), bronchoalveolar fluid albumin content, bronchoalveolar fluid and pulmonary tissue myeloperoxidase content and pulmonary gas exchange are assessed (2). Using this technique we could show that it sufficiently induces acute lung inflammation and can distinguish between different treatment groups or genotypes (1-3, 5). Therefore this technique may be helpful for researchers who pursue molecular mechanisms involved in ALI using a genetic approach in mice with gene-targeted deletion.

A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged ...

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

A Model of Self-limited Acute Lung Injury by Unilateral Intra-bronchial Acid Instillation

Selective intra-bronchial instillation of hydrochloric acid (HCl) to the murine left mainstem bronchus causes acute tissue injury with histopathologic findings similar to human acute respiratory distress syndrome (ARDS). The resulting alveolar edema, alveolar-capillary barrier damage, and leukocyte infiltration predominantly affect the left lung, preserving the right lung as an uninjured control and allowing animals to survive. This model of self-limited acute lung injury enables investigation of tissue resolution mechanisms, such as macrophage efferocytosis of apoptotic neutrophils and restitution of alveolar-capillary barrier integrity. This model has helped identify important roles for resolution agonists, including specialized pro-resolving mediators (SPMs), providing a foundation for the development of new therapeutic approaches for patients with ARDS.

Selective intra-bronchial acid instillation to the left lung in mice results in unilateral and self-limited acute lung injury that models human acute respiratory distress syndrome (ARDS) induced by gastric acid aspiration.

Selective intra-bronchial instillation of hydrochloric acid (HCl) to the murine left mainstem bronchus causes acute tissue injury with histopathologic ...

Immunology and Infection

Murine Oropharyngeal Aspiration Model of Ventilator-associated and Hospital-acquired Bacterial Pneumonia

Murine infection models are critical for understanding disease pathogenesis and testing the efficacy of novel therapeutics designed to combat causative pathogens. Infectious pneumonia is among the most common infections presented by patients in the clinic and thus warrants an appropriate in vivo model. Typical pneumonia models use intranasal inoculation, which deposits excessive organisms outside the lung, causing off-target complications and symptoms, such as sinusitis, gastritis, enteritis, physical trauma, or microparticle misting to mimic aerosol spread more typical of viral, tuberculous, or fungal pneumonia. These models do not accurately reflect the pathogenesis of typical community- or healthcare-acquired bacterial pneumonia. In contrast, this murine model of oropharyngeal aspiration pneumonia mimics the droplet route in healthcare-acquired pneumonia. Inoculating 50 &#181;L of the bacteria suspension into the oropharynx of anesthetized mice causes reflexive aspiration, which results in pneumonia. With this model, one can examine the pathogenesis of pneumonia-causing pathogens and new treatments to combat these diseases.

Infectious pneumonia is among the most common infections in human. An appropriate in vivo model is critical for understanding disease pathogenesis and testing the efficacy of novel therapeutics. With this murine oropharyngeal aspiration pneumonia model, one can examine the pathogenesis and new treatments against these deadly infections.

Murine infection models are critical for understanding disease pathogenesis and testing the efficacy of novel therapeutics designed to combat causative ...

Mouse Model of Oleic Acid-Induced Acute Respiratory Distress Syndrome

Acute respiratory distress syndrome (ARDS) is a significant threat to critically ill patients with a high fatality rate. Pollutant exposure, cigarette smoke, infectious agents, and fatty acids can induce ARDS. Animal models can mimic the complex pathomechanism of the ARDS. However, each of them has limitations. Notably, oleic acid (OA) is increased in critically ill patients with harmful effects on the lung. OA can induce lung injury by emboli, disrupting tissue, altering pH, and impairing edema clearance. OA-induced lung injury model resembles various features of ARDS with endothelial injury, increased alveolar permeability, inflammation, membrane hyaline formation, and cell death. Herein, induction of lung injury is described by injecting OA (in salt form) directly into the lung and intravenously in a mouse since it is the physiological form of OA at pH 7. Thus, the injection of OA in the salt form is a helpful animal model to study lung injury/ARDS without causing emboli or altering the pH, thereby getting close to what is happening in critically ill patients.

The present protocol describes a lung injury model in mice using oleic acid to mimic acute respiratory distress syndrome (ARDS). This model increases the inflammatory mediators on edema and decreases lung compliance. Oleic acid is used in the salt form (oleate) since this physiological form avoids the risk of embolism.

Acute respiratory distress syndrome (ARDS) is a significant threat to critically ill patients with a high fatality rate. Pollutant exposure, cigarette ...

A Mouse Model of Orotracheal Intubation and Ventilated Lung Ischemia Reperfusion Surgery

Ischemia reperfusion (IR) injury frequently results from processes that involve a transient period of interrupted blood flow. In the lung, isolated IR permits the experimental study of this specific process with continued alveolar ventilation, thereby avoiding the compounding injurious processes of hypoxia and atelectasis. In the clinical context, lung ischemia reperfusion injury (also known as lung IRI or LIRI) is caused by numerous processes, including but not limited to pulmonary embolism, resuscitated hemorrhagic trauma, and lung transplantation. There are currently limited effective treatment options for LIRI. Here, we present a reversible surgical model of lung IR involving first orotracheal intubation followed by unilateral left lung ischemia and reperfusion with preserved alveolar ventilation or gas exchange. Mice undergo a left thoracotomy, through which the left pulmonary artery is exposed, visualized, isolated, and compressed using a reversible slipknot. The surgical incision is then closed during the ischemic period, and the animal is awakened and extubated. With the mouse spontaneously breathing, reperfusion is established by releasing the slipknot around the pulmonary artery. This clinically relevant survival model permits the evaluation of lung IR injury, the resolution phase, downstream effects on lung function, as well as two-hit models involving experimental pneumonia. While technically challenging, this model can be mastered over the course of a few weeks to months with an eventual survival or success rate of 80%-90%.

A mouse surgical model to create left lung ischemia reperfusion (IR) injury while maintaining ventilation and avoiding hypoxia.

Ischemia reperfusion (IR) injury frequently results from processes that involve a transient period of interrupted blood flow. In the lung, isolated IR ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...

Experimental Model to Evaluate Resolution of Pneumonia

Acute respiratory distress syndrome (ARDS) causes acute lung injury, characterized by rapid alveolar damage and severe hypoxemia. This, in turn, leads to high morbidity and mortality. Currently, there are no pre-clinical models that recapitulate the complexity of human ARDS. However, infectious models of pneumonia (PNA) can replicate the main pathophysiological features of ARDS. Here, we describe a model of PNA induced by the intratracheal instillation of live Streptococcus pneumoniae and Klebsiella pneumoniae in C57BL6 mice. In order to evaluate and characterize the model, after inducing injury, we carried out serial measurements of body weight and bronchoalveolar lavage (BAL) for measuring markers of lung injury. Additionally, we harvested lungs for cell count and differentials, BAL protein quantification, cytospin, bacterial colony-forming unit counts, and histology. Lastly, high dimensional flow cytometry was performed. We propose this model as a tool to understand the immune landscape during the early and late resolution phases of lung injury.

This manuscript describes the establishment of an infectious model of pneumonia in mice and the respective characterization of injury resolution along with methods for growing bacteria and intratracheal instillation. A novel approach using high-dimensional flow cytometry to evaluate the immune landscape is also described.

Acute respiratory distress syndrome (ARDS) causes acute lung injury, characterized by rapid alveolar damage and severe hypoxemia. This, in turn, leads to ...

Halogenated Agent Delivery in Porcine Model of Acute Respiratory Distress Syndrome via an Intensive Care Unit Type Device

Acute respiratory distress syndrome (ARDS) is a common cause of hypoxemic respiratory failure and death in critically ill patients, and there is an urgent need to find effective therapies. Preclinical studies have shown that inhaled halogenated agents may have beneficial effects in animal models of ARDS. The development of new devices to administer halogenated agents using modern intensive care unit (ICU) ventilators has significantly simplified the dispensing of halogenated agents to ICU patients. Because previous experimental and clinical research suggested potential benefits of halogenated volatiles, such as sevoflurane or isoflurane, for lung alveolar epithelial injury and inflammation, two pathophysiologic landmarks of diffuse alveolar damage during ARDS, we designed an animal model to understand the mechanisms of the effects of halogenated agents on lung injury and repair. After general anesthesia, tracheal intubation, and the initiation of mechanical ventilation, ARDS was induced in piglets via the intratracheal instillation of hydrochloric acid. Then, the piglets were sedated with inhaled sevoflurane or isoflurane using an ICU-type device, and the animals were ventilated with lung-protective mechanical ventilation during a 4 h period. During the study period, blood and alveolar samples were collected to evaluate arterial oxygenation, the permeability of the alveolar-capillary membrane, alveolar fluid clearance, and lung inflammation. Mechanical ventilation parameters were also collected throughout the experiment. Although this model induced a marked decrease in arterial oxygenation with altered alveolar-capillary permeability, it is reproducible and is characterized by a rapid onset, good stability over time, and no fatal complications.
We have developed a piglet model of acid aspiration that reproduces most of the physiological, biological, and pathological features of clinical ARDS, and it will be helpful to further our understanding of the potential lung-protective effects of halogenated agents delivered through devices used for inhaled ICU sedation.

We describe a model of hydrochloric acid-induced acute respiratory distress syndrome (ARDS) in piglets receiving sedation with halogenated agents, isoflurane and sevoflurane, through a device used for inhaled intensive care sedation. This model can be used to investigate the biological mechanisms of halogenated agents on lung injury and repair.

Acute respiratory distress syndrome (ARDS) is a common cause of hypoxemic respiratory failure and death in critically ill patients, and there is an urgent ...

Mouse Model for Studying Lung Ischemia-Reperfusion Injury

Murine Left Pulmonary Hilar Clamp Model of Lung Ischemia Reperfusion Injury

Ischemia reperfusion injury (IRI) during lung transplantation is a major risk factor for post-transplant complications, including primary graft dysfunction, acute and chronic rejection, and mortality. Efforts to study the underpinnings of IRI led to the development of a reliable and reproducible mouse model of left lung hilar clamping. This model involves a surgical procedure performed in an anesthetized and intubated mouse. A left thoracotomy is performed, followed by careful lung mobilization and dissection of the left pulmonary hilum. The hilar clamp involves reversible suture ligation of the pulmonary hilum with a slipknot, which stops the arterial inflow, venous outflow, and airflow through the left mainstem bronchus. Reperfusion is initiated by careful removal of the suture. Our laboratory uses 30 min of ischemia and 1 h of reperfusion for the experimental model in the current investigations. However, these time periods can be modified depending on the specific experimental question. Immediately prior to sacrifice, arterial blood gas can be obtained from the left ventricle after a 4 min period of right hilar clamping to ensure that the PaO2 values obtained are attributed to the injured left lung alone. We also describe a method to measure cell extravasation with flow cytometry, which involves intravenous injection of a fluorochrome-labeled antibody specific for the cell(s) to be studied prior to sacrifice. The left lung can then be harvested for flow cytometry, frozen or fixed, paraffin-embedded immunohistochemistry, and quantitative polymerase chain reaction. This hilar clamp technique allows for&#160;detailed study of the cellular and molecular mechanisms underlying IRI. Representative results reveal decreased left lung oxygenation and histologic evidence of lung injury following hilar clamping. This technique can be readily learned and reproduced by personnel with and without microsurgical experience, leading to reliable and consistent results and serving as a widely adoptable model for studying lung IRI.

Author Spotlight: Insights into the Effect of Ischemia Reperfusion on Lung Transplantation

The protocol outlines a feasible, reliable, and reproducible method of left pulmonary hilar clamping that can be used to study lung ischemia-reperfusion injury in mouse models.

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

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