The authors would like to thank Dr. Joseph Mizgerd for his contributions to the development of the selective intra-bronchial method and for his helpful comments and review of the manuscript. This work was supported by National Institutes of Health grants P01GM095467 (B.D.L.) and K08HL130540 (R.E.A.).

All animal procedures below have been reviewed and approved by the Institutional Animal Care And Use Committee at Brigham and Women&#8217;s Hospital (Protocol #2016N000356).
NOTE: Sterile technique was followed for all survival procedures. A sterile field was established for each surgery using a sterile drape towel, while surgeons wore sterile surgical gloves, caps, masks, and clean laboratory coats. All surgical instruments were sterilized using an autoclave, and sterility was maintained using a bead sterilizer.
1. Preparation of 0.1 N HCl
<ol>
	<li>Add 11 mL of ddH2O to an amber glass bottle. Slowly add 1 mL of 37% HCl (12 N) to create a 1 N HCl working stock. 
	CAUTION: Make sure HCl is added into the water. This is a safety concern because adding water directly to the acid can cause acid to boil and splash out of the bottle. When handling concentrated HCl, make sure the acid is kept in a vented chemical hood and the appropriate personal protective equipment is worn, including lab coat, gloves, and safety glasses.</li>
	<li>Slowly add 4 mL of the previously diluted HCl working stock into 35 mL of ddH2O in a 50 mL conical tube to create a 0.1 N HCl experimental stock.</li>
	<li>Measure the pH of the experimental stock using an electronic pH probe after two-point calibration using low pH solutions. Titrate to pH 1.1 using NaOH or HCl stock solutions as needed so that the final volume is 40 mL. 
	NOTE: Measuring the low pH values can be difficult. To ensure accurate measurement, make sure the pH probe is properly calibrated using low pH standards to avoid over-extrapolation of the measurement.</li>
	<li>Immediately before the experiment, filter 1&#8211;2 mL of the experimental HCl stock through a 0.22 &#181;m sterile filter into a sterile microcentrifuge tube.</li>
</ol>
2. Selective Intra-bronchial Instillation of HCl
<ol>
	<li>Preparing the surgical area
	<ol>
		<li>Induce general anesthesia by delivering a ketamine (100 mg/kg) and xylazine (10 mg/kg) mixture by intraperitoneal injection. Ensure the mouse is completely anesthetized by gently squeezing the tip of the tail or hind foot.&#160;Lack of withdrawal response is required prior to making a skin incision.&#160; Administer additional anesthesia boluses, if needed.</li>
		<li>Deliver 0.1 mg/kg of buprenorphine subcutaneously under the scruff of the neck. Pre-operative analgesic will strengthen the effect of the anesthesia and will ameliorate pre- and post-operative pain resulting from the procedure.</li>
		<li>Use electric clippers to gently shave the surgical area on the ventral surface of the mouse, below the chin in the cervical region of the throat, using slow downward strokes. Remove loose fur to fully expose the underlying skin.</li>
		<li>Prepare the surgical area by swabbing the shaved site with 10% povidone-iodine solution. After applying the aseptic solution, clean the site using a 70% isopropyl alcohol swab. Repeat this step 3x.</li>
	</ol>
	</li>
	<li>Isolating the trachea
	<ol>
		<li>Place the mouse in a supine position on a clean surgical board and cover the mouse in a sterile surgical drape while maintaining exposure of the surgical area. Secure the drape in place.</li>
		<li>Make a 0.5 cm longitudinal incision in the skin above the trachea and salivary glands. Use slightly curved serrated forceps to carefully pull back the skin and gently separate the salivary glands to expose the tracheal muscles.</li>
		<li>Using serrated forceps for blunt dissection, gently push apart the paratracheal muscles and tease away the fascia that is surrounding the trachea until the cartilaginous rings of the trachea are completely exposed.</li>
		<li>Use fully curved serrated forceps to lift the trachea and separate the connective tissue between the retro-trachea and the retro-fascia. Once the connective tissue is detached, the tip of the forceps should slide completely behind the trachea.</li>
		<li>Keep the curved forceps behind the trachea and grasp a 10&#8211;15 cm piece of 4-0 braided silk suture with the tips of the forceps. Pull the suture behind the trachea so that there is an even length on either side.</li>
		<li>Once the suture is in place, gently pull the sides of the suture towards the posterior of the mouse and hold the sides in place.</li>
	</ol>
	</li>
	<li>Selectively cannulating the left mainstem bronchus and instilling HCl
	<ol>
		<li>Take a 24 G x &#190;&#8221; angiocatheter and insert the needle, bevel up, into the anterior region of the trachea between the first and second tracheal rings. Once proper insertion is confirmed by direct visualization of the needle tip in the tracheal lumen, release the suture and advance the cannula over the needle and into the trachea until resistance is reached, then withdraw the needle. Angle the direction of insertion towards the left main stem bronchus for selective instillation into the left lung.</li>
		<li>Once the cannula is in place, firmly grasp the injection port to prevent the catheter from shifting.</li>
		<li>Using a P200 pipette and sterile P200 pipette tips, instill 2.5 mL/kg (50 &#181;L for a 20 g mouse) of sterile filtered 0.1 N HCl into the catheter, followed by an identical volume of air.</li>
		<li>Rapidly withdraw the catheter and lift the surgical board to a 60&#176; angle for 30 s.</li>
	</ol>
	</li>
	<li>Closing the surgical area
	<ol>
		<li>Lay the surgical board flat and remove the suture from behind the trachea.</li>
		<li>Use 4-0 wax coated braided silk suture to close the skin incision using 2&#8211;3 stitches.</li>
	</ol>
	</li>
</ol>
3. Post-operative Care
<ol>
	<li>Once the incision is closed, place the mouse on its left side on a warm heating pad until the mouse recovers from anesthesia. Begin monitoring the mouse for pain and activity level before returning it to normal housing. 
	NOTE: Buprenorphine should be administered at 0.1 mg/kg subcutaneously every 6-12 h for the first 24 h. If persistent breakthrough pain is present, extend the analgesic regimen until the pain subsides.</li>
</ol>
4. Whole Lung Bronchoalveolar Lavage (BAL) and Leukocyte Immunophenotyping
<ol>
	<li>Euthanize the mouse by administering 3x the dose of ketamine/xylazine used in step 2.1.1.
	<ol>
		<li>To differentiate interstitial and intravascular neutrophils, intravenously inject a selected fluorophore-labeled Ly6G antibody 5 min prior to euthanasia. This label should be suitable for detection by flow cytometry to distinguish intravascular neutrophils from lung interstitial and alveolar neutrophils, which will be labeled during the tissue preparation with a different fluorophore (see below).</li>
	</ol>
	</li>
	<li>Place the mouse on a surgical board and hook the anterior incisors around a loop of 2-0 braided silk suture.</li>
	<li>Follow steps 2.2.2&#8211;2.2.6. to prepare the trachea for cannulation. 
	NOTE: Make sure the diaphragm is not punctured to maximize trans-alveolar inflating pressure during lung lavage; left lung compliance decreases following injury, which may require a higher trans-alveolar pressure requirement for lung lavage.</li>
	<li>Cannulate the trachea following step 2.3.1, but do not advance the catheter below the carina; insert the catheter parallel to the trachea.</li>
	<li>With the catheter inserted, tie the suture around the trachea to hold the catheter in place.</li>
	<li>Instill and withdraw two consecutive 1 mL aliquots of ice-cold PBS -/- (without magnesium or calcium) with 0.6 mM EDTA using a 1 cc syringe. For immunophenotyping by flow cytometry, remove each aliquot and return to a 5 mL polystyrene FACS tube on ice.</li>
	<li>To ensure euthanasia, perform a thoracotomy using surgical scissors followed by cardiac puncture. The lungs may be harvested for further processing.</li>
	<li>Centrifuge the BAL for 10 min at 800 g at 4 &#176;C to pellet the cells.</li>
	<li>Decant the supernatant into a 2 mL microcentrifuge tube and aliquot into 1.5 mL microcentrifuge tubes. Store at -80 &#176;C for subsequent analysis.</li>
	<li>Resuspend the cell pellet in PBS -/- with 2% FBS for leukocyte differential analysis by flow cytometry.</li>
	<li>To differentiate interstitial and intravascular neutrophils, remove the left and right lung separately and process the lungs for flow cytometry as in Abdulnour et al. 20146.</li>
	<li>Stain the resulting cell suspension using selected FACS antibodies, making sure to stain for Ly6G that is conjugated with a different fluorophore than the Ly6G antibody from step 4.1.1.</li>
</ol>
5. Assessment of Alveolar Barrier Permeability Using Evan&#8217;s Blue Dye (EBD)
<ol>
	<li>Intravenously inject Evan&#8217;s Blue Dye (40 mg/kg) 30 min prior to euthanasia.</li>
	<li>Euthanize the mouse using ketamine/xylazine overdose (step 4.1).</li>
	<li>To measure alveolar barrier integrity, follow steps 4.2&#8211;4.9 for BAL collection.</li>
	<li>Transfer 100 &#181;L of BALF to a clear bottom 96-well microplate, along with 100 &#181;L of duplicate EBD standards. Use PBS -/- as a blank.</li>
	<li>Use a microplate reader to measure the absorbance of the BALF at 620 nm and 740 nm. Use the absorbance at 740 nm to correct for heme contamination in the samples7.</li>
	<li>To measure vascular barrier integrity, perfuse the lungs by slowly injecting 5 mL of ice-cold PBS -/- through the right ventricle of the heart. Remove the left lung.</li>
	<li>Dry the left lung for 72 h at 58 &#176;C to remove excess water.</li>
	<li>Process the dried lung tissue as in Radu and Chernoff 20138, and measure the absorbances at 620 nm and 740 nm.</li>
</ol>
6. Lung Histology
<ol>
	<li>Cannulate the trachea by following steps 4.1&#8211;4.5.</li>
	<li>To pressure fix the lungs at 20 cm H2O, use a ring stand and clamp to elevate a 60 mL syringe fitted with valve-controlled tubing and filled with a select fixative solution (e.g., zinc fixative) so that the meniscus of the fixative solution is 20 cm above the lungs.</li>
	<li>Attach the tubing to the catheter and open the value. Slowly fill the lungs with fixative until they stop inflating.</li>
	<li>Remove the catheter &#190; of the way out of the trachea. Tie off the trachea with suture before fully removing the catheter to minimize loss of fixative.</li>
	<li>Remove the lungs and heart en bloc. 
	NOTE: Make sure the lungs are not punctured during the removal to retain the pressure infused fixative.</li>
	<li>Fix the lungs for 24 h in 25 mL of fixative at room temperature.</li>
	<li>Wash the fixed lungs for sequential 20 min intervals in PBS -/-, 30% ethanol, and 50% ethanol.</li>
	<li>After the last wash, store the lungs in 70% ethanol for histology processing as in Eickmeier et al. 20139.</li>
</ol>

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The authors have nothing to disclose.

The intra-bronchial instillation method described here uses selective cannulation of the left mainstem bronchus to instill HCl into the left lung, resulting in unilateral and self-limited murine acute lung injury. This murine acid lung injury model closely represents the inflammatory response, histopathology, and physiological dysfunction seen in human ARDS, where gastric acid aspiration is a common precipitant or contributing factor4. Exposure of the murine airway to low pH HCl results in increased permeability of the alveolar-capillary barrier, alveolar edema, and profound neutrophil infiltration at the site of injury. These events are not observed in the uninjured right lung. In addition, this model produces rapid inflammatory responses that peak within 24 h following acid instillation, and shares changes in gene expression with human ARDS, such as the differential expression of phospholipase D isoforms10.
Although this murine preclinical model reproduces many of the features of ARDS at the molecular, cellular, and tissue levels, it does not fully recapitulate human ARDS. The definition of ARDS includes bilateral lung involvement3, whereas the instillation method described here results by design in unilateral lung disease. Moreover, the animals do not require continuous mechanical ventilation, immobility, or parenteral sedation. Results presented here (vide supra) and elsewhere6,9,11,12,13 demonstrate that unilateral acid-induced lung injury reproduces most of the pathological hallmarks of ARDS while providing the unique opportunity to use the right lung as internal control and to study the resolution phase of this disease. As such, the model discussed here models ARDS pathobiology, but also enables mechanistic investigation of fundamental lung tissue responses to injury and resolution mechanisms that may be relevant for addressing this important disease.
Instillation of HCl represents direct acute lung injury, so it is modeling aspects of the pathophysiology associated with aspiration pneumonitis. In addition, the initial left lung insult in this model is generated using sterile HCl rather than bacteria-laden gastric contents seen in some human aspiration events that can also lead to pneumonia14. In humans, aspiration of pathogenic bacteria can result in secondary bacterial pneumonia that exacerbates the acute inflammatory response, prolonging the initial lung injury and increasing patient susceptibility to develop ARDS14. This potential limitation has been addressed by investigators purposely instilling pathogenic bacteria Escherichia coli (E. coli)15 after sterile HCl. Additionally, this method has been used to investigate pathogen-mediated inflammation; unilateral bacterial pneumonia can be induced by selective left lung instillation of bacteria, such as E. coli16,17, Pseudomonas aeruginosa16, and Streptococcus pneumoniae18. The self-limited acute lung injury model described here may also be used to study ventilator-induced lung injury (VILI), an important cause of increased mortality in human ARDS19. Experimental animal models of VILI usually involve mechanical ventilation in na&#239;ve mice with tidal volumes that are much higher than what is clinically used to cause lung injury (&#62;15 mL/kg; see previous work20,21). Towards a more clinically relevant model of VILI, intra-bronchial acid instillation as described here may be used first to induce non-lethal lung injury followed by mechanical ventilation at tidal volumes within clinical range (6-12 mL/kg). This hypothetical animal model may allow investigators to study VILI in a clinically relevant manner once developed and validated. Together, these murine models highlight the versatility of the selective intrabronchial instillation method to generate unilateral lung insults that closely resemble pathologies associated with human lung diseases.
In addition to allowing selective instillation of various noxious agents to the left lung, the technique of intra-bronchial instillation after tracheostomy does not require extended training, long procedure time, or complex equipment, and in experienced hands causes minimal distress to the animals. Despite this, several issues may occur during the selective HCl instillation procedure that can impact experimental results. Improper cannulation of the left main stem bronchus can result in bilateral lung injury that decreases survival of experimental mice and confounds the use of the right lung as an uninjured internal control. This can be avoided by angling the catheter sufficiently towards the left lung during cannulation until resistance is reached. After the injection of HCl, a bolus of air should be injected, the catheter rapidly removed, and the surgical board brought upright to a 60&#176; angle. These steps are crucial for ensuring that the acid reaches the distal airways of the left lung and prevents the reflux of acid into the right lung and trachea, which may cause proximal injury. Within 24 h following instillation, the injury in the left lung is diffuse with extensive pulmonary edema, affecting both the distal and proximal left lung.
During method development in adult 8-12 week old mice, 2.5 mL/kg of intra-bronchial HCl produced substantial yet sublethal acute lung injury; lower doses of HCl did not result in reproducible and homogenous lung injury. Although we have not performed this model in younger (e.g., 3-6 weeks old) or older mice (e.g., 10-14 months old), we anticipate that weight-based dosing of HCl will result in a lung injury phenotype similar to what is noted in 8-12 week old mice. We recommend that investigators titrate HCl doses to achieve the desired degree of lung injury prior to performing experiments with mice at extremes of weight.
This selective acid instillation procedure offers a non-lethal murine model of sterile tissue inflammation that reduces the need for supportive care, such as mechanical ventilation. With extended survival of injured mice, the acid-induced inflammation has enough time to self-resolve. The resolution phase of this model has been used to identify temporally regulated endogenous bioactive lipid mediators, termed specialized pro-resolving mediators (SPMs), such as lipoxin A4 (LXA4), maresin 1 (MaR1), and resolvins6,11,12,16. Administering exogenous SPMs to injured mice quickens the resolution of acid induced lung injury by dampening inflammatory mechanisms and promoting catabasis of the injured lung tissue. These SPMs promote the clearance of alveolar edema12, increase the efferocytosis of apoptotic neutrophils by recruited macrophages16, and accelerate the re-epithelialization of the airways and alveoli12 to reduce vascular leakage and tissue hypoxia. In a model of pathogen-induced lung injury, 15-epi-resolvin D1 also exhibited antimicrobial actions through increased bacterial phagocytosis by macrophages and enhanced bacterial clearance from the infected lung16. Investigating these endogenous resolution mechanisms provides insight into potential novel therapeutic strategies for patients with ARDS5.
To best study the spatiotemporal regulation of resolution mechanisms, in vivo experimental models are needed. Acute lung injury models must include relevant acute inflammatory responses and organ dysfunction with engagement of host resolution promoting molecular and cellular processes. These mechanisms can be quantified using established resolution indices22. The selective intra-bronchial instillation method to generate unilateral acute lung injury has proven useful in this regard to probe endogenous resolution mediators and pathways. Future studies that deepen our understanding of these active resolution processes have the promise of leading to therapeutic agonists that mimic the bioactions of endogenous lipid mediators to enhance the resolution of inflammation and mitigating the morbidity and mortality of ARDS and other important lung diseases.

Acute respiratory distress syndrome (ARDS) is an important cause of acute respiratory failure1. It is a common and lethal or disabling disease that occurs in 10% of all patients admitted to intensive care units worldwide2. According to the Berlin definition3, ARDS is defined by the acute onset of hypoxemic respiratory failure (&#60;1 week) and bilateral pulmonary infiltrates on chest radiographs that are not explained by cardiac failure4. The underlying pathobiology is characterized by an excessive inflammatory response. The lung can be injured directly, such as in pneumonia or with gastric acid aspiration, or indirectly, such as in sepsis or after multiple blood transfusions4. Following the initial insult, ARDS pathogenesis progresses in three phases: exudative, proliferative, and fibrotic phases1. These phases are characterized by distinct molecular and cellular immune and repair mechanisms that determine the prognosis for ARDS patients. Supportive care remains the mainstay for ARDS patients; currently, there are no effective pharmacological treatments for ARDS, so there is an urgent need for new research on this devastating condition4.
Dysregulation of the innate immune response during the exudative phase contributes to the acute onset of ARDS and associated respiratory failure1. Potent pro-inflammatory mediator signaling orchestrates the initial immune responses, leading to disruption of the alveolar-capillary barrier, diffuse alveolar edema, and neutrophil infiltration to the site of lung tissue injury4. In ARDS, ineffective braking signals for acute inflammation predispose to lung failure and can delay timely catabasis of the injured lung tissue5. To that end, preclinical investigation into the endogenous initiating and pro-resolution mechanisms of ARDS may uncover novel therapeutic strategies. Such investigation requires self-limited experimental in vivo models of acute lung injury that closely resemble features of human ARDS, permitting interrogation of mechanisms underlying the initiation and resolution phases of tissue injury.
The murine model presented here produces direct acute lung injury that demonstrates the cardinal pathobiological processes of exudative ARDS, namely alveolar-capillary barrier disruption and neutrophil infiltration. The method relies on selective intra-bronchial instillation of HCl through cannulation of the left mainstem bronchus, localizing the injury and inflammatory response to the left lung; the uninjured right lung can be used as an internal control for select determinations of tissue injury and inflammation. In addition, unilateral lung injury is non-lethal and unveils a resolution program. This offers a distinct window into resolution of lung inflammation that can be leveraged for identification of endogenous pro-resolving mediators and cellular mechanisms and to open new therapeutic avenues for ARDS that emphasizes resolution physiology and pharmacology.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>10x Zinc Fixative</td>
			<td>BD Biosciences</td>
			<td>552658</td>
			<td></td>
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			<td>2-0 Braided Silk Suture</td>
			<td>Surgical Specialties</td>
			<td>SP118</td>
			<td></td>
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			<td>24G x 3/4&#34; Disposable Safelet I.V. Catheter</td>
			<td>Excel</td>
			<td>26751</td>
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			<td>33 mm, 0.22 &#181;m syringe filter unit</td>
			<td>Millipore-Sigma</td>
			<td>SLGP033RS</td>
			<td></td>
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			<td>4&#34; Long Serrated Slight Curve Graefe Forceps</td>
			<td>Roboz</td>
			<td>RS-5135</td>
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			<td>4&#34; Long Tip Serrated Full Curve Graefe Forceps</td>
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			<td>4.5 &#34; Micro Dissecting Scissors</td>
			<td>Roboz</td>
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			<td>RS-7860</td>
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			<td>60 mL syringe</td>
			<td>BD Biosciences</td>
			<td>309653</td>
			<td></td>
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			<td>Anti-mouse FITC-Ly6G antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>11-9668-82</td>
			<td>Preferred fluorophore can be used</td>
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		<tr>
			<td>Anti-mouse PE-Ly6G antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>12-9668-82</td>
			<td>Preferred fluorophore can be used</td>
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			<td>Bead sterilizer</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>Betadine Solution Swabstick</td>
			<td>Betadine</td>
			<td>67618-153-01</td>
			<td></td>
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			<td>Buprenex</td>
			<td>Reckitt Benckiser</td>
			<td></td>
			<td>NDC: 12496-0757-1, 12496-0757-5</td>
		</tr>
		<tr>
			<td>Clear flat-bottomed 96-well microplate</td>
			<td>Thermo Fisher Scientific</td>
			<td>12565501</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbeccos&#39;s Phosphate Buffered Saline (PBS) without Ca2+ or Mg+</td>
			<td>life technologies</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Electric clippers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Millipore-Sigma</td>
			<td>E6758</td>
			<td></td>
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		<tr>
			<td>Evans Blue Dye</td>
			<td>Millipore-Sigma</td>
			<td>E-2129</td>
			<td></td>
		</tr>
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			<td>Heating pad</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>Hydrochloric acid, 37%</td>
			<td>Millipore-Sigma</td>
			<td>258148</td>
			<td></td>
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			<td>Ketamine</td>
			<td>Henry-Schein</td>
			<td>56344</td>
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			<td>Microplate reader (640, 720 nm)</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>P200 Pipette</td>
			<td></td>
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			<td>P200 Pipette Tips</td>
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			<td>pH probe</td>
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			<td>Ring stand with extension clamp</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>Sterile Alcohol Prep Pads</td>
			<td>Thermo Fisher Scientific</td>
			<td>22-363-750</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Mouse Drape 8&#34; x 8&#34; with Oval Adhesive Fenestration</td>
			<td>Steris</td>
			<td>88VCSTF</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Nitrile Gloves</td>
			<td>Kimberly-Clark</td>
			<td>56890</td>
			<td></td>
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		<tr>
			<td>Sterile Towl Drape</td>
			<td>Dynarex</td>
			<td>4410</td>
			<td></td>
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		<tr>
			<td>Wax Coated 4-0 Braided Silk Suture</td>
			<td>Covidien</td>
			<td>SS733</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>AKORN</td>
			<td></td>
			<td>NDC: 59399-111-50</td>
		</tr>
	</tbody>
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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 HCl instillation results in unilateral acute lung injury
The method of selective intra-bronchial instillation of HCl into the left mainstem bronchus is illustrated in Figure 1A. The consequent acute lung injury involves the entire left lung, and following intravenous administration of EBD and lung perfusion, the EBD remained only in the left lung (Figure 1B). EBD extravasation into the left lung was quantified and found to be significantly increased relative to sham selective instillation (Figure 1C; adapted from Abdulnour et al. 20146). In response to lung injury, circulating leukocytes diapedese into the inflamed tissue. In this model, vascular neutrophils undergo trans-endothelial migration into the injured lung interstitium. Interstitial neutrophils accumulated in the left lung 24 h after HCl instillation, in contrast to the right lung where few interstitial neutrophils are observed (Figure 1D). These results indicate that the selective left mainstem intra-bronchial instillation method resulted in murine acute lung injury that was largely localized to the left lung and produced pathological changes that are also seen with human ARDS, including increased alveolar-capillary barrier breach and neutrophil infiltration.
Unilateral acute lung injury enables investigation of resolution mechanisms
To study the resolution phase of acid-induced acute lung injury mice must be able to survive the initial insult. Distinct from intratracheal HCl, instillation into only the left mainstem bronchus leads to a self-limited injury with uniform survival in otherwise healthy mice. Lungs can be obtained from mice at either early or later timepoints as in Figure 2A. Lung histology shows tissue injury and inflammation at the organ and cellular level with exudative inflammation 24 h after injury characterized by marked alveolar edema and neutrophil infiltration in the left lung. Note that there is no significant injury or leukocyte influx into the uninjured control right lung (Figure 2A). 72 h after injury, edema and cellular infiltrates are substantially decreased, representing a resolving exudative phase. Alveolar neutrophils can be monitored by flow cytometry (CD45+/CD68-/F4/80-/Ly6G+/CD11b+) obtained by whole lung lavage. Neutrophils increase in the left lung 24 h following the initial injury and decrease substantially at 48 and 72 h (Figure 2B). If later time points are investigated, the neutrophil numbers will return to baseline and mechanisms in later phases of catabasis, such as fibroproliferative responses, can be studied.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60024/60024fig1v2.jpg" /> 
Figure 1: Selective intra-bronchial HCl instillation produces unilateral lung injury defined by alveolar barrier breach and neutrophil infiltration. (A) Representation of the cannulation of the murine left mainstem bronchus for selective instillation of HCl into the left lung. (B) Resected right (RL) and left (LL) lungs exposed to selective acid instillation and perfused following intravenous Evan&#39;s blue dye. (C) Quantification of interstitial Evan&#39;s blue dye from homogenized, perfused lung 24 h after acid injury or sham control; figure adapted from Abdulnour et al. 20146. Values represent mean &#177; SEM, where n &#8805; 5. *p &#60; 0.05, Mann-Whitney U Test. (D) Representative flow cytometry of intravascular (I.V.; fluorophore 1) and interstitial (I.S.; fluorophore 2) neutrophils as percent of total CD45+ cells in processed lung 24 h after acid injury. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60024/60024fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60024/60024fig2.jpg" /> 
Figure 2: Unilateral acute lung injury is self-resolving. (A) Representative H&#38;E histology (10x) of left lungs obtained from na&#239;ve mice (0 h) or mice 24, 48, 72 h after injury, along with the associated right lung from the same mouse (Scale bar = 250 &#181;m). (B) Representative flow cytometry of alveolar neutrophils (Ly6G+ CD11b+) obtained from whole lung lavage as percent of total CD45+ cells in na&#239;ve (0 h) mice or mice 24, 48, and 72 h after acid injury. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60024/60024fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Model of Self-limited Acute Lung Injury by Unilateral Intra-bronchial Acid Instillation at JoVE.com

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

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 ...

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Nanyang Technological University

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JoVE Journal

Immunology and Infection

8.9K Views.  Brigham and Women's Hospital, Harvard Medical School. 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.

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

Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, in particular, are known to migrate to the lung draining lymph nodes (LDLNs) where they present inhaled antigens to T cells initiating an 
appropriate immune response to a variety of immunogens1,2. To model interactions between the lungs and airborne antigens in mice, antigens can be 
administered intranasally1,3,4, intratracheally5 or as aerosols6. Delivery by each route involves distinct technical skills and limitations that need to be 
considered before designing an experiment. For example, intranasal and aerosolized exposure delivers antigens to both the lungs and the upper respiratory 
tract. Hence antigens can access the nasal associated lymphoid tissue (NALT)7, potentially complicating interpretation of the results. In addition, swallowing, 
sneezing and the breathing rate of the mouse may also lead to inconsistencies in the doses delivered. Although the involvement of the upper respiratory tract may 
be preferred for some studies, it can complicate experiments focusing on events specifically initiated in the lungs. In this setting, the intratracheal (i.t) 
route is preferable as it delivers test materials directly into the lungs and bypasses the NALT. Many i.t injection protocols involve either blind intubation of the 
trachea through the oral cavity or surgical exposure of the trachea to access the lungs. Herein, we describe a simple, consistent, non-surgical method for i.t 
instillation. The opening of the trachea is visualized using a laryngoscope and a bent gavage needle is then inserted directly into the trachea to deliver the 
innoculum. We also describe procedures for harvesting and processing of LDLNs and lungs for analysis of antigen trafficking by flow cytometry.

We illustrate non-surgical delivery of test materials into the lungs of anesthetized mice via the trachea. This method permits lung exposure to bacterial and viral pathogens, cytokines, antibodies, beads, chemicals, or dyes. We further describe harvesting and processing of lungs and lung draining lymph nodes (LDLNs) for flow cytometry.

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, ...

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their mucus-plugged bronchioles create a favorable habitat for microbial colonization. Various pathogens thrive in this environment interacting with each other and driving many of the symptoms associated with CF disease. Like any microbial community, the chemical conditions of their habitat have a significant impact on the community structure and dynamics. For example, different microorganisms thrive in differing levels of oxygen or other solute concentrations. This is also true in the CF lung, where oxygen concentrations are believed to drive community physiology and structure. The methods described here are designed to mimic the lung environment and grow pathogens in a manner more similar to that from which they cause disease. Manipulation of the chemical surroundings of these microbes is then used to study how the chemistry of lung infections governs its microbial ecology. The method, called the WinCF system, is based on artificial sputum medium and narrow capillary tubes meant to provide an oxygen gradient similar to that which exists in mucus-plugged bronchioles. Manipulating chemical conditions, such as the media pH of the sputum or antibiotics pressure, allows for visualization of the microbiological differences in those samples using colored indicators, watching for gas or biofilm production, or extracting and sequencing the nucleic acid contents of each sample.

The mucus plugged airways of cystic fibrosis (CF) patients are an ideal environment for microbial pathogens to thrive. The manuscript describes a novel method for studying the CF lung microbiome in an environment that mimics where they cause disease and how alterations of chemical conditions can drive microbial dynamics.

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

Transurethral Instillation Procedure in Adult Male Mouse

Transurethral instillation can be used to deliver different solutions with active ingredients (e.g., drugs, chemicals, bacteria, and viruses) locally into the urinary bladder to either induce animal models of bladder pathologies or evaluate the effectiveness of intravesical treatments. Most rodent models of lower urinary tract (LUT) pathologies are induced in female mice due to ease of intravesical instillation of the substances via the female urethra. However, due to anatomical differences between the female and male LUT, transurethral instillation in a male mouse has been deemed a very challenging procedure, and it has not been previously described. In this manuscript, we provide a detailed description of how to prepare polyethylene (PE) tubing for subsequent insertion into the urethra of a male mouse. In addition, we discuss the ideal types of PE tubing to be used depending on the desired site of inoculation. Furthermore, we describe point by point how to prepare an animal for a successful transurethral instillation to avoid injury to the urethra and ensure the delivery of the solution to the desired location. The procedure is started by retracting the prepuce and the glans to expose the opening of the urethral meatus. Next, the glans are grasped by blunt non-crushing forceps to stabilize the penis and the PE tubing. The PE tubing is first inserted into the urethral meatus parallel to the animal body, then its angle is adjusted by tilting the catheter to maneuver it to follow the natural curvature of the urethra. This technique can be used to induced murine models of bladder pathologies and/or evaluate the effectiveness of intravesical treatments in male mice.

Transurethral instillation is a challenging procedure and has not been well described in the literature. The aim of this manuscript is to describe a technique for transurethral insertion of a catheter for intravesical delivery of liquids with active substances into the urinary bladder and/or prostate in adult male mice.

Transurethral instillation can be used to deliver different solutions with active ingredients (e.g., drugs, chemicals, bacteria, and viruses) locally into ...

Measuring Deformability and Red Cell Heterogeneity in Blood by Ektacytometry

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of disease or occurrence of serious complications. Ektacytometry uses laser diffraction viscometry to measure the deformability of red blood cells subject to either increasing shear stress or an osmotic gradient at a constant value of applied shear stress. However, direct deformability measurements are difficult to interpret when measuring heterogenous blood that is characterized by the presence of both rigid and deformable red cells. This is due to the inability of rigid cells to properly align in response to shear stress and results in a distorted diffraction pattern marked by an exaggerated decrease in apparent deformability. Measurement of the degree of distortion provides an indicator of the heterogeneity of the erythrocytes in blood. In sickle cell anemia, this is correlated with the percentage of rigid cells, which reflects the hemoglobin concentration and hemoglobin composition of the erythrocytes. In addition to measuring deformability, osmotic gradient ektacytometry provides information about the osmotic fragility and hydration status of erythrocytes. These parameters also reflect the hemoglobin composition of red blood cells from sickle cell patients. Ektacytometry measures deformability in populations of red cells and does not, therefore, provide information on the deformability or mechanical properties of individual erythrocytes. Regardless, the goal of the techniques described herein is to provide a convenient and reliable method for measuring the deformability and cellular heterogeneity of blood. These techniques may be useful for monitoring temporal changes, as well as disease progression and response to therapeutic intervention in several disorders. Sickle cell anemia is one well-characterized example. Other potential disorders where measurements of red cell deformability and/or heterogeneity are of interest include blood storage, diabetes, Plasmodium infection, iron deficiency, and the hemolytic anemias due to membrane defects.

Here we present techniques to measure red cell deformability and cellular heterogeneity by ektacytometry. These techniques are applicable to general investigations of red cell deformability and specific investigations of blood diseases characterized by the presence of both rigid and deformable red cells in circulation, such as sickle cell anemia.

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of ...

Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models continue to be an essential tool for developing new antifibrotic strategies. Here we provide an effective method to investigate in vivo antifibrotic activity of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC) in attenuating bleomycin-induced lung injury. C57BL/6 mice receive a single endotracheal injection of bleomycin (1.5 U/kg body weight) followed by a double infusion of hUC-MSC (2.5 x 105) into the tail vein, 24 h and 7 days after the bleomycin administration. Upon sacrifice at days 8, 14, or 21, inflammatory and fibrotic changes, collagen content, and hUC-MSC presence in explanted lung tissue are analyzed. The injection of bleomycin into the mouse&#160;trachea allows the direct targeting of the lungs, leading to extensive pulmonary inflammation and fibrosis. The systemic administration of a double dose of hUC-MSC results in the early blunting of the bleomycin-induced lung injury. Intravenously infused hUC-MSC are transiently engrafted into the mouse lungs, where they exert their anti-inflammatory and antifibrotic activity. In conclusion, this protocol has been successfully applied for the preclinical testing of hUC-MSC in an experimental mouse model of human pulmonary fibrosis. However, this technique can be easily extended both to study the effect of different endotracheally administered substances on the pathophysiology of the lungs and to validate new anti-inflammatory and antifibrotic systemic therapies.

Here we present an effective method to investigate the antifibrotic activity of intravenously infused human mesenchymal stromal cells obtained from the whole umbilical cord following the induction of lung injury by an endotracheal injection of bleomycin in C57BL/6 mice. This protocol can be easily extended to the preclinical testing of other therapeutics.

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models ...

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Here, we present a protocol for creating an immunocompetent ICR (Institute of Cancer Research) murine model of central nervous system infection to display the development of neuropathy. Monitoring acute viral encephalitic disorders by identical disease scores could be performed for showing dengue virus-induced neuropathy in vivo.

Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is ...

Design of Cecal Ligation and Puncture and Intranasal Infection Dual Model of Sepsis-Induced Immunosuppression

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple organs. With the development of therapies, most patients survive the hyperinflammatory phase but progress to an immunosuppressive phase, which increases the emergence of secondary infections. Therefore, improved understanding of the pathogenesis underlying secondary hospital-acquired infections in the immunosuppressive phase during sepsis is of tremendous importance. Reported here is a model to test infectious outcomes by creating double-hit infections in mice. A standard surgical procedure is used to induce polymicrobial peritonitis by cecal ligation and puncture (CLP) and followed by intranasal infection of Staphylococcus aureus to simulate pneumonia occurring in immune suppression that is frequently seen in septic patients. This dual model can reflect the immunosuppressive state occurring in patients with protracted sepsis and susceptibility to secondary infection from nosocomial pneumonia. Hence, this model provides a simple experimental approach to investigate the pathophysiology of sepsis-induced secondary bacterial pneumonia, which may be used for discovering novel treatments for sepsis and its complications.

This protocol describes techniques to measure infectious outcomes underlying secondary hospital-acquired infections in the immunosuppressive condition, first by establishing cecal ligation/puncture mice then challenging them with intranasal infection to create a clinically relevant model of immunosuppression sepsis.

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple ...

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

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Design of Cecal Ligation and Puncture and Intranasal Infection Dual Model of Sepsis-Induced Immunosuppression