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 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.
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 (<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.
All animal procedures below have been reviewed and approved by the Institutional Animal Care And Use Committee at Brigham and Women’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
2. Selective Intra-bronchial Instillation of HCl
3. Post-operative Care
4. Whole Lung Bronchoalveolar Lavage (BAL) and Leukocyte Immunophenotyping
5. Assessment of Alveolar Barrier Permeability Using Evan’s Blue Dye (EBD)
6. Lung Histology
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.
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's blue dye. (C) Quantification of interstitial Evan'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 ± SEM, where n ≥ 5. *p < 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. Please click here to view a larger version of this figure.
Figure 2: Unilateral acute lung injury is self-resolving. (A) Representative H&E histology (10x) of left lungs obtained from naïve mice (0 h) or mice 24, 48, 72 h after injury, along with the associated right lung from the same mouse (Scale bar = 250 µm). (B) Representative flow cytometry of alveolar neutrophils (Ly6G+ CD11b+) obtained from whole lung lavage as percent of total CD45+ cells in naïve (0 h) mice or mice 24, 48, and 72 h after acid injury. Please click here to view a larger version of this figure.
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 naïve mice with tidal volumes that are much higher than what is clinically used to cause lung injury (>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° 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.
The authors have nothing to disclose.
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.).
10x Zinc Fixative | BD Biosciences | 552658 | |
2-0 Braided Silk Suture | Surgical Specialties | SP118 | |
24G x 3/4" Disposable Safelet I.V. Catheter | Excel | 26751 | |
33 mm, 0.22 µm syringe filter unit | Millipore-Sigma | SLGP033RS | |
4" Long Serrated Slight Curve Graefe Forceps | Roboz | RS-5135 | |
4" Long Tip Serrated Full Curve Graefe Forceps | Roboz | RS-5137 | |
4.5 " Micro Dissecting Scissors | Roboz | RS-5912 | |
6" Crile Wood Needle Holder | Roboz | RS-7860 | |
60 mL syringe | BD Biosciences | 309653 | |
Anti-mouse FITC-Ly6G antibody | Thermo Fisher Scientific | 11-9668-82 | Preferred fluorophore can be used |
Anti-mouse PE-Ly6G antibody | Thermo Fisher Scientific | 12-9668-82 | Preferred fluorophore can be used |
Bead sterilizer | |||
Betadine Solution Swabstick | Betadine | 67618-153-01 | |
Buprenex | Reckitt Benckiser | NDC: 12496-0757-1, 12496-0757-5 | |
Clear flat-bottomed 96-well microplate | Thermo Fisher Scientific | 12565501 | |
Dulbeccos's Phosphate Buffered Saline (PBS) without Ca2+ or Mg+ | life technologies | 14190-144 | |
Electric clippers | |||
Ethylenediaminetetraacetic acid (EDTA) | Millipore-Sigma | E6758 | |
Evans Blue Dye | Millipore-Sigma | E-2129 | |
Heating pad | |||
Hydrochloric acid, 37% | Millipore-Sigma | 258148 | |
Ketamine | Henry-Schein | 56344 | |
Microplate reader (640, 720 nm) | |||
P200 Pipette | |||
P200 Pipette Tips | |||
pH probe | |||
Ring stand with extension clamp | |||
Sterile Alcohol Prep Pads | Thermo Fisher Scientific | 22-363-750 | |
Sterile Mouse Drape 8" x 8" with Oval Adhesive Fenestration | Steris | 88VCSTF | |
Sterile Nitrile Gloves | Kimberly-Clark | 56890 | |
Sterile Towl Drape | Dynarex | 4410 | |
Wax Coated 4-0 Braided Silk Suture | Covidien | SS733 | |
Xylazine | AKORN | NDC: 59399-111-50 |