The research conducted is funded by President&#39;s NSERC grant as well as start-up funds from the Sylvia Fedoruk Canadian Center for Nuclear Innovation. The Sylvia Fedoruk Canadian Center for Nuclear Innovation is funded by Innovation Saskatchewan. Fluorescence imaging was performed at the WCVM Imaging Centre, which is funded by NSERC. Jessica Brocos (MSc Student) and Manpreet Kaur (MSc Student) were funded by the start-up funds from the Sylvia Fedoruk Canadian Center for Nuclear Innovation.

<ol>
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The authors have no conflicts of interest or disclosures to make.

The methods presented in the current study highlight the usefulness of multiple compartment analysis to study multiple cellular events during lung inflammation. We have summarized the findings in Table 2. We and many labs have extensively studied the murine response to intranasal LPS instillation, which is marked by rapid recruitment of lung neutrophils, which peaks between 6-24 h following which, resolution kicks in. And recently, we have shown that sub-clinical O3 (at 0.05 ppm for 2 h) alone...

Acute lung injury (ALI) is a crucial pathologic response of lungs to infectious or other harmful stimuli which is marked by simultaneous activation of coagulative, fibrinolytic and innate immune systems1. Neutrophils promptly sense microbial as well as intracellular damage patterns through the Toll-like receptor (TLR) family2,3,4. Neutrophils release preformed cytokines and cytotoxic granule contents, which can then cause collateral tissue damage. The ensuing alveolar damage is marred with secondary cell death leading to release of molecules such as adenosine triphosphate (ATP)5, thus setting in a vicious cycle of immune-dysregulation.
An unsolved problem in the understanding of ALI relates to the question of how the injury is initiated within the alveolar membrane. The electron transport complex V, F1F0 ATP synthase, is a mitochondrial protein known to be expressed ubiquitously, on cell (including endothelial, leukocyte, epithelial) plasma membrane during inflammation. The cell cytoskeleton which is comprised of actin and tubulin, harbors many cell shape and function modulating as well as mitochondrial proteins, respectively. We have recently shown that blockade of the ATP synthase by an endogenous molecule, angiostatin, silences neutrophil recruitment, activation and lipopolysaccharide (LPS) induced lung inflammation6. Thus, both biochemical (ATP synthase) and immune (TLR4) mechanisms might regulate the alveolar barrier during lung inflammation.
Exposure to ozone (O3), an environmental pollutant, impairs lung function, increases susceptibility to pulmonary infections, and short low-levels of O3 exposures increase the risk of mortality in those with underlying cardiorespiratory conditions7,8,9,10,11,12,13,14. Thus, exposure to physiologically relevant concentrations of O3 provides a meaningful model of ALI to study fundamental mechanisms of inflammation7,8. Our lab has recently established a murine model of low-dose O3 induced ALI15. After performing a dose and time-response to low O3 concentrations, we observed that exposure to 0.05 ppm O3 for 2 h, induces acute lung injury that is marked by lung ATP synthase complex V subunit &#946; (ATP&#946;) and angiostatin expression, similar to the LPS model. Intravital lung imaging revealed disorganization of alveolar actin microfilaments indicating lung damage, and ablation of alveolar septal reactive oxygen species (ROS) levels (indicating abrogation of baseline cell signaling) and mitochondrial membrane potential (indicating acute cell death) after 2 h exposure to 0.05 ppm O315 which correlated with a heterogeneous lung 18FDG retention16, neutrophil recruitment and cytokine release, most notably IL-16 and SDF-1&#945;. The take-home message from our recent studies is that O3 produces exponentially high toxicity when exposed at concentrations below the allowed limits of 0.063 ppm over 8 h (per day) for human exposure. Importantly, no clear understanding exists on whether these sub-clinical O3 exposures can modulate TLR4-mediated mechanisms such as by bacterial endotoxin17. Thus, we studied a dual-hit O3 and LPS exposure model and observed the immune and non-immune cellular adaptations.
We describe a comprehensive fluorescent microscopic analysis of various lung and systemic body compartments, namely the broncho-alveolar lavage fluid (i.e., BAL) which samples the alveolar spaces, the lung vascular perfusate (i.e., LVP) that samples the pulmonary vasculature and the alveolar septal interstitium in the event of a compromised endothelial barrier, left lung cryosections, to look into resident parenchymal and adherent leukocytes left in the lavaged lung tissue, peripheral blood which represents the circulating leukocytes and the sternal and femur bone marrow perfusates that sample the proximal and distal sites of hematopoietic cell mobilization during inflammation, respectively.

<table><tbody><tr><td>33-plex Bioplex chemokine panel</td><td>Biorad</td><td>12002231</td><td></td></tr><tr><td>63X oil (NA 1.4-0.6) Microscope objectives</td><td>Leica</td><td>HCX PL APO CS (11506188)</td><td></td></tr><tr><td>Alexa 350 conjugated goat anti-mouse IgG (H+L)</td><td>Invitrogen</td><td>A11045</td><td></td></tr><tr><td>Alexa 488 conjugated goat anti-mouse IgG (H+L)</td><td>Invitrogen</td><td>A11002</td><td></td></tr><tr><td>Alexa 488 conjugated phalloidin</td><td>Invitrogen</td><td>A12370</td><td></td></tr><tr><td>Alexa 555 conjugated mouse anti-&amp;alpha; tubulin clone DM1A</td><td>Millipore</td><td>05-829X-555</td><td></td></tr><tr><td>Alexa 568 conjugated goat anti-hamster IgG (H+L)</td><td>Invitrogen</td><td>A21112</td><td></td></tr><tr><td>Alexa 568 conjugated goat anti-rat IgG (H+L)</td><td>Invitrogen</td><td>A11077</td><td></td></tr><tr><td>Alexa 633 conjugated goat anti-rabbit IgG (H+L)</td><td>Invitrogen</td><td>A21070</td><td></td></tr><tr><td>Armenian hamster anti-CD61 (clone 2C9.G2) IgG1 kappa</td><td>BD Pharmingen</td><td>553343</td><td></td></tr><tr><td>C57BL/6 J Mice</td><td>Jackson Laboratories</td><td>64</td><td></td></tr><tr><td>Confocal laser scanning microscope</td><td>Leica</td><td>Leica TCS SP5</td><td></td></tr><tr><td>DAPI (4&amp;prime;,6-diamidino-2-phenylindole)</td><td>Invitrogen</td><td>D1306</td><td>aliquot in 2 &amp;micro;l stocks and store at -20&amp;deg;C</td></tr><tr><td>Inverted fluorescent wide field microscope</td><td>Olympus</td><td>Olympus IX83</td><td></td></tr><tr><td>Ketamine (Narketan)</td><td>Vetoquinol</td><td>100 mg/ml</td><td>Dilute 10 times to make a 10 mg/ml stock</td></tr><tr><td>Live (calcein)/Dead (Ethidium homodimer-1) cytotoxicity kit</td><td>Invitrogen</td><td>L3224</td><td></td></tr><tr><td>Mouse anti-ATP5A1 IgG2b (clone 7H10BD4F9)</td><td>Invitrogen</td><td>459240</td><td></td></tr><tr><td>Mouse anti-ATP5&amp;beta; IgG2b (clone 3D5AB1)</td><td>Invitrogen</td><td>A-21351</td><td></td></tr><tr><td>Mouse anti-NK1.1 IgG2a kappa (clone PK136)</td><td>Invitrogen</td><td>16-5941-82</td><td></td></tr><tr><td>Pierce 660 nm protein assay</td><td>Thermoscientific</td><td>22660</td><td></td></tr><tr><td>Rabbit anti-angiostatin (mouse aa 98-116) IgG</td><td>Abcam</td><td>ab2904</td><td></td></tr><tr><td>Rabbit anti-CX3CR1 IgG (RRID 467880)</td><td>Invitrogen</td><td>14-6093-81</td><td></td></tr><tr><td>Rat anti-Ki-67 (clone SolA15) IgG2a kappa</td><td>Invitrogen</td><td>14-5698-82</td><td></td></tr><tr><td>Rat anti-Ly6G IgG2a kappa (clone 1A8)</td><td>Invitrogen</td><td>16-9668-82</td><td></td></tr><tr><td>Rat anti-Ly6G/Ly6C (Gr1) IgG2b kappa (clone RB6-8C5)</td><td>Invitrogen</td><td>53-5931-82</td><td></td></tr><tr><td>Rat anti-mouse CD16/CD32 Fc block (clone 2.4G2)</td><td>BD Pharmingen</td><td>553142</td><td></td></tr><tr><td>Reduced mitotracker orange</td><td>Invitrogen</td><td>M7511</td><td></td></tr><tr><td>Xylazine (Rompun)</td><td>Bayer</td><td>20 mg/ml</td><td>Dilute 2 times to make a 10 mg/ml stock</td></tr></tbody></table>

visualizing lung cellular adaptations during combined ozone and lps induced murine acute lung injury

Lungs are continually faced with direct and indirect insults in the form of sterile (particles or reactive toxins) and infectious (bacterial, viral or fungal) inflammatory conditions. An overwhelming host response may result in compromised respiration and acute lung injury, which is characterized by lung neutrophil recruitment as a result of the patho-logical host immune, coagulative and tissue remodeling response. Sensitive microscopic methods to visualize and quantify murine lung cellular adaptations, in response to low-dose (0.05 ppm) ozone, a potent environmental pollutant in combination with bacterial lipopolysaccharide, a TLR4 agonist, are crucial in order to understand the host inflammatory and repair mechanisms. We describe a comprehensive fluorescent microscopic analysis of various lung and systemic body compartments, namely the broncho-alveolar lavage fluid, lung vascular perfusate, left lung cryosections, and sternal bone marrow perfusate. We show damage of alveolar macrophages, neutrophils, lung parenchymal tissue, as well as bone marrow cells in correlation with a delayed (up to 36-72 h) immune response that is marked by discrete chemokine gradients in the analyzed compartments. In addition, we present lung extracellular matrix and cellular cytoskeletal interactions (actin, tubulin), mitochondrial and reactive oxygen species, anti-coagulative plasminogen, its anti-angiogenic peptide fragment angiostatin, the mitochondrial ATP synthase complex V subunits, &#945; and &#946;. These surrogate markers, when supplemented with adequate in vitro cell-based assays and in vivo animal imaging techniques such as intravital microscopy, can provide vital information towards understanding the lung response to novel immunomodulatory agents.

Combined O3 and LPS exposure leads to systemic inflammation and bone marrow mobilization at 72 h: Cell counts in different compartments revealed significant changes in peripheral blood and the femur bone marrow total cell counts upon combined O3 and LPS exposures. Although combined O3 and LPS exposures did not induce any changes in the total BAL (Figure 1A) or LVP (Figure 1B</str...

Watch this Scientific Journal Video about Visualizing Lung Cellular Adaptations during Combined Ozone and LPS Induced Murine Acute Lung Injury at JoVE.com

Visualizing Lung Cellular Adaptations during Combined Ozone and LPS Induced Murine Acute Lung Injury

Combined ozone and bacterial endotoxin exposed mice show wide-spread cell death, including that of neutrophils. We observed cellular adaptations such as disruption of cytoskeletal lamellipodia, increased cellular expression of complex V ATP synthase subunit &#946; and angiostatin in broncho-alveolar lavage, suppression of the lung immune response and delayed neutrophil recruitment.

Lungs are continually faced with direct and indirect insults in the form of sterile (particles or reactive toxins) and infectious (bacterial, viral or ...

visualizing-lung-cellular-adaptations-during-combined-ozone-lps

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

5.0K Views.  University of Saskatchewan. Combined ozone and bacterial endotoxin exposed mice show wide-spread cell death, including that of neutrophils. We observed cellular adaptations such as disruption of cytoskeletal lamellipodia, increased cellular expression of complex V ATP synthase subunit β and angiostatin in broncho-alveolar lavage, suppression of the lung immune response and delayed neutrophil recruitment.

Video: Visualizing Lung Cellular Adaptations during Combined Ozone and LPS Induced Murine Acute Lung Injury

Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) represent a heterogeneous group of lung diseases which continues to have a high morbidity and mortality. The molecular pathogenesis of ALI is being better defined; however, because of the complex nature of the disease molecular therapies have yet to be developed. Here we use a lipopolysaccharide (LPS) induced mouse model of acute septic lung injury to delineate the role of exosomes in the inflammatory response. Using this model, we were able to show that mice that are exposed to intraperitoneal LPS secrete exosomes in Broncho-alveolar lavage (BAL) fluid from the lungs that are packaged with miRNA and cytokines which regulate inflammatory response. Further using a co-culture model system, we show that exosomes released from macrophages disrupt expression of tight junction proteins in bronchial epithelial cells. These results suggest that 1) cross talk between innate immune and structural cells through the exosomal shuttling contribute to the inflammatory response and disruption of the structural barrier and 2) targeting these miRNAs may provide a novel platform to treat ALI and ARDS.

Mice exposed to intraperitoneal LPS secrete exosomes in Broncho-alveolar lavage (BAL) fluid that are packaged with miRNAs. Using a co-culture system, we show that exosomes released in the BAL fluid disrupt expression of tight junction proteins in bronchial epithelial cells and increase expression of pro-inflammatory cytokines that accentuate lung injury.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) represent a heterogeneous group of lung diseases which continues to have a high ...

Genetics

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH oxidase has critical signaling functions that modulate the inflammatory response 1. Thus, the development of a method to measure in "real-time" the kinetics of NADPH oxidase-derived ROS generation is expected to be a valuable research tool to understand mechanisms relevant to host defense, inflammation, and injury.
Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by severe infections and excessive inflammation. Activation of the phagocyte NADPH oxidase requires translocation of its cytosolic subunits (p47phox, p67phox, and p40phox) and Rac to a membrane-bound flavocytochrome (composed of a gp91phox and p22phox heterodimer). Loss of function mutations in any of these NADPH oxidase components result in CGD. Similar to patients with CGD, gp91phox -deficient mice and p47phox-deficient mice have defective phagocyte NADPH oxidase activity and impaired host defense 2, 13. In addition to phagocytes, which contain the NADPH oxidase components described above, a variety of other cell types express different isoforms of NADPH oxidase.
Here, we describe a method to quantify ROS production in living mice and to delineate the contribution of NADPH oxidase to ROS generation in models of inflammation and injury. This method is based on ROS reacting with L-012 (an analogue of luminol) to emit luminescence that is recorded by a charge-coupled device (CCD). In the original description of the L-012 probe, L-012-dependent chemiluminescence was completely abolished by superoxide dismutase, indicating that the main ROS detected in this reaction was superoxide anion 14. Subsequent studies have shown that L-012 can detect other free radicals, including reactive nitrogen species 15, 16. Kielland et al. 16 showed that topical application of phorbol myristate acetate, a potent activator of NADPH oxidase, led to NADPH oxidase-dependent ROS generation that could be detected in mice using the luminescent probe L-012. In this model, they showed that L-012-dependent luminescence was abolished in p47phox-deficient mice.
We compared ROS generation in wildtype mice and NADPH oxidase-deficient p47phox-/- mice 2 in the following three models: 1) intratracheal administration of zymosan, a pro-inflammatory fungal cell wall-derived product that can activate NADPH oxidase; 2) cecal ligation and puncture (CLP), a model of intra-abdominal sepsis with secondary acute lung inflammation and injury; and 3) oral carbon tetrachloride (CCl4), a model of ROS-dependent hepatic injury. These models were specifically selected to evaluate NADPH oxidase-dependent ROS generation in the context of non-infectious inflammation, polymicrobial sepsis, and toxin-induced organ injury, respectively. Comparing bioluminescence in wildtype mice to p47phox-/- mice enables us to delineate the specific contribution of ROS generated by p47phox-containing NADPH oxidase to the bioluminescent signal in these models.
Bioluminescence imaging results that demonstrated increased ROS levels in wildtype mice compared to p47phox-/- mice indicated that NADPH oxidase is the major source of ROS generation in response to inflammatory stimuli. This method provides a minimally invasive approach for "real-time" monitoring of ROS generation during inflammation in vivo.

NADPH oxidase is the major source of reactive oxygen species (ROS) in phagocytes. Because of the ephemeral nature of ROS, it is difficult to measure and monitor ROS levels in living animals. A minimally invasive method for serial quantification of ROS in living mice is described.

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH ...

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen interactions take advantage of intraperitoneal injections of select bacteria or pathogen associated molecular patterns (PAMPs) in mice. While these techniques have yielded tremendous data associated with infectious disease pathobiology, intraperitoneal injection models are not always appropriate for host-pathogen interaction studies in the lung. Utilizing an acute lung inflammation model in mice, it is possible to conduct a high resolution analysis of the host innate immune response utilizing lipopolysaccharide (LPS). Here, we describe the methods to administer LPS using nonsurgical oropharyngeal intratracheal administration, monitor clinical parameters associated with disease pathogenesis, and utilize bronchoalveolar lavage fluid to evaluate the host immune response. The techniques that are described are widely applicable for studying the host innate immune response to a diverse range of PAMPs and pathogens. Likewise, with minor modifications, these techniques can also be applied in studies evaluating allergic airway inflammation and in pharmacological applications.

The host immune response to pathogen infection is a tightly regulated process. Utilizing a lipopolysaccharide lung exposure model in mice, it is possible to conduct high resolution evaluations of the complex mechanisms associated with disease pathogenesis.

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen ...

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. ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high incidence in aging populations. The current conventional therapies for COPD focus mainly on symptom-modifying drugs; thus, the development of new therapies is urgently needed. Qualified animal models of COPD could help to characterize the underlying mechanisms and can be used for new drug screening. Current COPD models, such as lipopolysaccharide (LPS) or the porcine pancreatic elastase (PPE)-induced emphysema model, generate COPD-like lesions in the lungs and airways but do not otherwise resemble the pathogenesis of human COPD. A cigarette smoke (CS)-induced model remains one of the most popular because it not only simulates COPD-like lesions in the respiratory system, but it is also based on one of the main hazardous materials that causes COPD in humans. However, the time-consuming and labor-intensive aspects of the CS-induced model dramatically limit its application in new drug screening. In this study, we successfully generated a new COPD model by exposing mice to high levels of ozone. This model demonstrated the following: 1) decreased forced expiratory volume 25, 50, and 75/forced vital capacity (FEV25/FVC, FEV50/FVC, and FEV75/FVC), indicating the deterioration of lung function; 2) enlarged lung alveoli, with lung parenchymal destruction; 3) reduced fatigue time and distance; and 4) increased inflammation. Taken together, these data demonstrate that the ozone exposure (OE) model is a reliable animal model that is similar to humans because ozone overexposure is one of the etiological factors of COPD. Additionally, it only took 6 - 8 weeks, based on our previous work, to create an OE model, whereas it requires 3 - 12 months to induce the cigarette smoke model, indicating that the OE model might be a good choice for COPD research.

This study describes the successful generation of a new chronic obstructive pulmonary disease (COPD) animal model by repeatedly exposing mice to high concentrations of ozone.

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high ...

Medicine

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

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

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

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

In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce pulmonary inflammation, but little is known regarding how exposure alters processes important to the resolution of inflammation. Efferocytosis is a resolution process, whereby macrophages phagocytize apoptotic cells. The purpose of this protocol is to measure alveolar macrophage efferocytosis following O3-induced lung injury and inflammation. Several methods have been described for measuring efferocytosis; however, most require ex vivo manipulations. Described in detail here is a protocol to measure in vivo alveolar macrophage efferocytosis 24 h after O3 exposure, which avoids ex vivo manipulation of macrophages and serves as a simple technique that can be used to accurately represent perturbations in this resolution process. The protocol is a technically non-intensive and relatively inexpensive method that involves whole-body O3 inhalation followed by oropharyngeal aspiration of apoptotic cells (i.e., Jurkat T cells) while under general anesthesia. Alveolar macrophage efferocytosis is then measured by light microscopy evaluation of macrophages collected from bronchoalveolar (BAL) lavage. Efferocytosis is finally measured by calculating an efferocytic index. Collectively, the outlined methods quantify efferocytic activity in the lung in vivo while also serving to analyze the negative health effects of O3 or other inhaled insults.

This manuscript describes a protocol for determining whether exposure to ozone, a criteria air pollutant, impairs alveolar macrophage efferocytosis in vivo. This protocol utilizes commonly used reagents and techniques and can be adapted to multiple models of pulmonary injury to determine effects on alveolar macrophage efferocytosis.

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce ...

Intravital Widefield Fluorescence Microscopy of Pulmonary Microcirculation in Experimental Acute Lung Injury Using a Vacuum-Stabilized Imaging System

Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) and other respiratory pathologies in vivo is difficult due to the limited accessibility and inherent motion artifacts of the lungs. Nonetheless, various approaches have been developed to overcome these challenges. This protocol describes a method for intravital fluorescence microscopy to study real-time leukocyte-endothelial interactions in the pulmonary microcirculation in an experimental model of ALI. An in vivo lung imaging system and 3-D printed intravital microscopy platform are used to secure the anesthetized mouse and stabilize the lung while minimizing confounding lung injury. Following preparation, widefield fluorescence microscopy is used to study leukocyte adhesion, leukocyte rolling, and capillary function. While the protocol presented here focuses on imaging in an acute model of inflammatory lung disease, it may also be adapted to study other pathological and physiological processes in the lung.

Intravital fluorescence microscopy can be utilized to study leukocyte-endothelial interactions and capillary perfusion in real-time. This protocol describes methods to image and quantify these parameters in the pulmonary microcirculation using a vacuum-stabilized lung imaging system.

Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung ...

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

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

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

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

Visualizing Lung Cellular Adaptations during Combined Ozone and LPS Induced Murine Acute Lung Injury

Summary

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Chapters in this video

Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

Pseudomonas aeruginosa Induced Lung Injury Model

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure

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

In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure

Intravital Widefield Fluorescence Microscopy of Pulmonary Microcirculation in Experimental Acute Lung Injury Using a Vacuum-Stabilized Imaging System

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