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 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>syringe, 1cc</td>
			<td>Becton Dickinson</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr>
			<td>syringe, 5cc</td>
			<td>Becton Dickinson</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr>
			<td>needle, 22 ga x 1 1/2&#34;</td>
			<td>Becton Dickinson</td>
			<td>305159</td>
			<td></td>
		</tr>
		<tr>
			<td>needle, 26 ga x 1 1/2&#34;</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&#34; Curved tissue serrated forceps</td>
			<td>Fine Science Tools</td>
			<td>11065-07</td>
			<td></td>
		</tr>
		<tr>
			<td>3&#34; Curved tissue &#34;toothed&#34; forceps, 1x2 teeth</td>
			<td>Fine Science Tools</td>
			<td>11067-07</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#34; 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&#34; 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 ga x 1/2&#34; 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 &#34;Q-tip&#34; 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 ...

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 ...

open-tracheostomy-gastric-acid-aspiration-murine-model-acute-lung

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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

Expired CO2 Measurement in Intubated or Spontaneously Breathing Patients from the Emergency Department

Carbon dioxide (CO2) along with oxygen (O2) share the role of being the most important gases in the human body. The measuring of expired CO2 at the mouth has solicited growing clinical interest among physicians in the emergency department for various indications: (1) surveillance et monitoring of the intubated patient; (2) verification of the correct positioning of an endotracheal tube; (3) monitoring of a patient in cardiac arrest; (4) achieving normocapnia in intubated head trauma patients; (5) monitoring ventilation during procedural sedation. The video allows physicians to familiarize themselves with the use of capnography and the text offers a review of the theory and principals involved. In particular, the importance of CO2 for the organism, the relevance of measuring expired CO2, the differences between arterial and expired CO2, the material used in capnography with their artifacts and traps, will be reviewed. Since the main reluctance in the use of expired CO2 measurement is due to lack of correct knowledge concerning the physiopathology of CO2 by the physician, we hope that this explanation and the video sequences accompanying will help resolve this limitation.

Expired CO2 Measurement in Intubated or Spontaneously Breathing Patients from the Emergency Department

This video describes how to perform CO2 measurement in intubated as well as spontaneously breathing patients. The main clinical indications refer to emergency situations: (1) verifying adequate positioning of an endotracheal tube; (2) achieving normocapnia in trauma patients; (3) monitoring ventilation in the case of procedural sedation.

Carbon dioxide (CO2) along with oxygen (O2) share the role of being the most important gases in the human body. The measuring of expired CO2 at the mouth ...

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 ...

Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia3. Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI4-5. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically5. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery4-5. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.

We describe two tactile sensory testing methods for acute or chronic periods of spinal cord injury in rats. These validated procedures can detect the development and maintenance of allodynia-like sensations.

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Induction and Phenotyping of Acute Right Heart Failure in a Large Animal Model of Chronic Thromboembolic Pulmonary Hypertension

The development of acute right heart failure (ARHF) in the context of chronic pulmonary hypertension (PH) is associated with poor short-term outcomes. The morphological and functional phenotyping of the right ventricle is of particular importance in the context of hemodynamic compromise in patients with ARHF. Here, we describe a method to induce ARHF in a previously described large animal model of chronic PH, and to phenotype, dynamically, right ventricular function using the gold standard method (i.e., pressure-volume PV loops) and with a non-invasive clinically available method (i.e., echocardiography). Chronic PH is first induced in pigs by left pulmonary artery ligation and right lower lobe embolism with biological glue once a week for 5 weeks. After 16 weeks, ARHF is induced by successive volume loading using saline followed by iterative pulmonary embolism until the ratio of the systolic pulmonary pressure over systemic pressure reaches 0.9 or until the systolic systemic pressure decreases below 90 mmHg. Hemodynamics are restored with dobutamine infusion (from 2.5 &#181;g/kg/min to 7.5 &#181;g/kg/min). PV-loops and echocardiography are performed during each condition. Each condition requires around 40 minutes for induction, hemodynamic stabilization and data acquisition. Out of 9 animals, 2 died immediately after pulmonary embolism and 7 completed the protocol, which illustrates the learning curve of the model. The model induced a 3-fold increase in mean pulmonary artery pressure. The PV-loop analysis showed that ventriculo-arterial coupling was preserved after volume loading, decreased after acute pulmonary embolism and was restored with dobutamine. Echocardiographic acquisitions allowed to quantify right ventricular parameters of morphology and function with good quality. We identified right ventricular ischemic lesions in the model. The model can be used to compare different treatments or to validate non-invasive parameters of right ventricular morphology and function in the context of ARHF.

We present a protocol to induce and phenotype an acute right heart failure in a large animal model with chronic pulmonary hypertension. This model can be used to test therapeutic interventions, to develop right heart metrics&#160;or to improve the understanding of acute right heart failure pathophysiology.

The development of acute right heart failure (ARHF) in the context of chronic pulmonary hypertension (PH) is associated with poor short-term outcomes. The ...

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. ...

Inducing Acute Liver Injury in Rats via Carbon Tetrachloride (CCl4) Exposure Through an Orogastric Tube

Acute liver injury (ALI) plays a crucial role in the development of hepatic failure, which is characterized by severe liver dysfunction including complications such as hepatic encephalopathy and impaired protein synthesis. Appropriate animal models are vital to test the mechanism and pathophysiology of ALI and investigate different hepatoprotective strategies. Due to its ability to perform chemical transformations, carbon tetrachloride (CCl4) is widely used in the liver to induce ALI through the formation of reactive oxygen species. CCl4 exposure can be performed intraperitoneally, by inhalation, or through a nasogastric or orogastric tube. Here, we describe a rodent model, in which ALI is induced by CCl4 exposure through an orogastric tube. This method is inexpensive, easily performed, and has minimal hazard risk. The model is highly reproducible and can be widely used to determine the efficacy of potential hepatoprotective strategies and assess markers of liver injury.

Inducing Acute Liver Injury in Rats via Carbon Tetrachloride CCl4 Exposure Through an Orogastric Tube

This protocol describes a common and feasible method of inducing acute liver injury (ALI) via CCl4 exposure through an orogastric tube. CCl4 exposure induces ALI through the formation of reactive oxygen species during its biotransformation in the liver. This method is used to analyze the pathophysiology of ALI and examine different hepatoprotective strategies.

Acute liver injury (ALI) plays a crucial role in the development of hepatic failure, which is characterized by severe liver dysfunction including ...

Acute Kidney Injury Model Induced by Cisplatin in Adult Zebrafish

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney due to its low molecular weight. Such accumulation causes the death of tubular cells and can induce the development of Acute Kidney Injury (AKI), which is characterized by a quick decrease in kidney function, tissue damage, and immune cells infiltration. If administered in specific doses cisplatin can be a useful tool as an AKI inducer in animal models. The zebrafish has appeared as an interesting model to study renal function, kidney regeneration, and injury, as renal structures conserve functional similarities with mammals. Adult zebrafish injected with cisplatin shows decreased survival, kidney cell death, and increased inflammation markers after 24 h post-injection (hpi). In this model, immune cells infiltration and cell death can be assessed by flow cytometry and TUNEL assay. This protocol describes the procedures to induce AKI in adult zebrafish by intraperitoneal cisplatin injection and subsequently demonstrates how to collect the renal tissue for flow cytometry processing and cell death TUNEL assay. These techniques will be useful to understand the effects of cisplatin as a nephrotoxic agent and will contribute to the expansion of AKI models in adult zebrafish. This model can also be used to study kidney regeneration, in the search for compounds that treat or prevent kidney damage and to study inflammation in AKI. Moreover, the methods used in this protocol will improve the characterization of tissue damage and inflammation, which are therapeutic targets in kidney-associated comorbidities.

This protocol describes the procedures to induce Acute Kidney Injury (AKI) in adult zebrafish using cisplatin as a nephrotoxic agent. We detailed the steps to evaluate the reproducibility of the technique and two techniques to analyze inflammation and cell death in the renal tissue, flow cytometry and TUNEL, respectively.

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney ...

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

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Expired CO₂ Measurement in Intubated or Spontaneously Breathing Patients from the Emergency Department