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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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

Abstract

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

Introduction

Ashbaugh et al.1, in 1967, first described the acute respiratory distress syndrome (ARDS) and since then has been through multiple revisions. According to the Berlin definition, ARDS is a pulmonary inflammation that leads to an acute respiratory failure and hypoxemia (PaO2/FiO2 > 300 mm Hg) due to imbalance in the ventilation to perfusion ratio, diffuse bilateral alveolar damage (DAD) and infiltrate, increased lung weight, and edema2,3. The pulmonary parenchyma is a complex cellular environment compounded by epithelial, endothelial, and other cells. These cells form barriers and structures responsible for gas exchange and homeostasis in the alveoli3. The most abundant cells within the epithelial barrier are alveolar type I cells (AT1) with a larger surface area for gas exchange and fluid management through Na/K-ATPase. Also, the alveolar type II cells (AT2) produce surfactant, reducing surface tension in the alveoli4. Underneath, endothelial cells form a semipermeable barrier separating the pulmonary circulation from the interstitium. Its functions include detecting stimuli, coordinating inflammatory responses, and cellular transmigration5. The endothelial cells also regulate gas exchange, vascular tonus, and coagulation5. Therefore, endothelial and epithelial function disturbances may exacerbate a proinflammatory phenotype, causing lung damage leading to ARDS5.

ARDS development is risk-associated with bacterial and viral pneumonia or indirect factors such as non-pulmonary sepsis, trauma, blood transfusions, and pancreatitis6. These conditions cause the release of pathogens-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), inducing proinflammatory cytokines and chemokines such as TNF-α, IL-1β, IL-6, and IL-85. TNF-α is linked to vascular-endothelial cadherin (VE-cadherin) degradation in endothelial barrier disruption and leucocyte infiltration into the lung parenchyma. Neutrophils are the first cells to migrate, attracted by IL-8 and LTB45,7,8. Neutrophils further increase proinflammatory cytokines, reactive oxygen species (ROS)9, and neutrophil extracellular traps (NETs) formation generating extra endothelial and epithelial damage10. Epithelial damage prompts inflammation and activation of Toll-like receptors in AT2 cells and resident macrophages, inducing the release of chemokines attracting inflammatory cells to the lungs4. Also, the production of cytokines like interferon-β (INFβ) causes TNF-related apoptosis-inducing receptors (TRAIL), leading ATII cells to apoptosis, impairing fluid and ion clarence4. The disruption of endothelial and epithelial barrier structure allows the influx of fluid, proteins, red blood cells, and leukocytes into the alveolar space, causing edema. With edema established, the pulmonary effort to maintain breathing and gas exchange is altered11. Hypercapnia and hypoxemia induce cell death and sodium transport disturbance, aggravating alveolar edema due to poor clearance capacity10. ARDS also has elevated levels of IL-17A, associated with organ dysfunction, increased percentage of alveolar neutrophils, and alveolar permeability9.

There have been ongoing advances in research on the pathophysiology, epidemiology, and treatment of ARDS in recent years12,13. However, ARDS is a heterogeneous syndrome despite the progress in therapeutic research resulting in mechanical ventilation and fluid therapy optimization. Thus, a more effective direct pharmacological treatment is still needed10, and animal studies may help unveil ARDS mechanisms and targets for intervention.

Current ARDS models are not able to fully replicate the pathology. Thus, researchers often choose the model that could better fit their interests. For instance, the lipopolysaccharide (LPS) induction model induces ARDS by endotoxic shock triggered mainly by TLR414. HCl induction mimics acid aspiration, and the damage is neutrophilic-dependent14. On the other hand, the current sodium oleate model induces endothelial damage that increases vascular permeability and edema. Furthermore, using sodium oleate instead of oleic acid in liquid form avoids embolism risks and alteration in the blood pH15.

Animals models for ARDS
Preclinical studies in animal models help understand the pathology and are essential for new ARDS treatments research. The ideal animal model needs to have characteristics resembling the clinical situation and good reproducibility of disease mechanisms with relevant pathophysiological features of each disease stage, evolution, and repair14. Several animal models are used to assess acute lung injury in ARDS pre-clinically. However, as all models have limitations, they do not fully reproduce the human pathology6,14,16. The oleic acid-induced ARDS is used in different animal species17. Pigs18, sheeps19, and dogs20 submitted to OA injection present numerous clinical features of the disease with alveolar-capillary membrane dysfunction and increased permeability with protein and cell infiltration.

For instance, OA at 1.25 µM intravenously injected blocked transepithelial transport leading to alveolar edema15. Alternatively, in the in vitro model using A549 cells, OA at a concentration of 10 µM did not change the epithelial sodium channel (eNAC) or the expression of Na/K-ATPase. However, OA seems to associate with both channels, directly inhibiting their activity21. OA intravenous injection at 0.1 mL/kg caused lung tissue congestion and swelling, reduced alveolar spaces with thickened alveolar septa, and increased inflammatory and red blood cell counts22. Also, OA induced apoptosis and necrosis in endothelial and epithelial cells in the lung15. The injection of a tris-oleate solution, intratracheally in mice, enhanced neutrophil infiltration and edema as early as 6 h after stimulation23. OA injection at 24 h increased proinflammatory cytokine levels (i.e., TNF-α, IL-6, and IL-1β)23. In addition, intravenous (orbital plexus) injection of 10 µM of a tris-oleate inhibits pulmonary Na/K-ATPase activity, similar to ouabain at 10-3 µM, a selective enzyme inhibitor. Also, OA induces inflammation with cell infiltration, formation of lipid bodies, and production of leukotriene B4 (LTB4) and prostaglandin E2 (PGE2)22,24. Therefore, oleic acid-induced ARDS generates edema, hemorrhage, neutrophil infiltration, increased myeloperoxidase (MPO) activity, and ROS24. Hence, OA administration is a well-established model for lung injury22,25. All the results presented in this article that has OA represents the salt form, sodium oleate.

Protocol

The procedures used in this study were approved by the Ethics Committee on the Use of Animals of the Oswaldo Cruz Foundation (CEUA licenses n°002-08, 36/10 and 054/2015). Male Swiss Webster mice weighing between 20-30 g, provided by the Institute of Science and Technology in Biomodels (ICTB) of the Oswaldo Cruz Foundation (FIOCRUZ), were used for the experiments. The animals were kept in ventilated isolators in the Pavilhão Ozório de Almeida's vivarium, and water and food were available ad libitum. They were exposed to a 12 h/12 h light and dark cycle. 

1. Preparation of sodium oleate solution

  1. Use oleic acid to prepare a 100 mmol/L of sodium oleate stock solution in any sterile tube or glass flask.
    NOTE: A 50 mL (final volume) solution was prepared for the present work, but the volume must be adjusted as per the experimental need. The solution must be always prepared in sterile tubes or glass containers.
    1. First, add NaOH tablets or solution in ultrapure water to elevate the pH. A pH value of 12-13 is recommended for a 25 mL volume.
      NOTE: Alternatively, Tris base can be used to prepare the Tris-oleate solution.
    2. Add the oleic acid (see Table of Materials) very slowly, drop by drop, under constant agitation in an ultrasonic bath at 37 °C.
      NOTE: If oleic acid precipitation occurs, start all over from the beginning.
    3. Once oleic acid is completely dissolved, carefully adjust the pH to 7.4, drop by drop under stirring, with ultra-pure diluted HCl and then adjust to the final volume of 50 mL.
      ​NOTE: Freshly prepare the working oleate solutions. Alternatively, the solution may be aliquoted, stocked, and maintained at -20 °C in a nitrogen-enriched environment to avoid oxidation for no longer than a month. Avoid frozen-refrozen cycles.

2. Induction of lung injury by oleic acid

  1. Perform the intratracheal administration of oleic acid.
    1. Anesthetize the mice using 5% isoflurane with 2 L/min of O2 employing a veterinary anesthetic vaporizer (Figure 1A). Remove the fur at the incision area with depilatory cream and disinfect the area with three alternating rounds of betadine scrub and alcohol using sterile gauze. Confirm the depth of anesthesia by toe pinch.
      NOTE: Use sterile gloves and instruments during the procedure. Use a drape to cover the animal and expose only the incision site. Perform the experiment in a biological safety cabinet to avoid isofluorane escape to the environment. Analgesics are not administered as they may inhibit the inflammatory response.
    2. After anesthesia, lay the animal in a dorsal decubitus position and make an incision (0.5-1 cm) in V-shape at the thyroid level. Gently displace the thyroid to expose the trachea (Figure 1B) and inject 50 µL of the prepared oleate solution (step 1).
      NOTE: The mice were divided into two groups, with eight animals in each group. The lung injury group receives sodium-oleate solution at 25 mM (1.25 µmol), and the control group receives 50 µL of sterile saline by instillation into the trachea of each mouse with an insulin syringe (volume 300 µL, 30 G) (Figure 1C).
    3. Suture the mice's incision site with a synthetic non-absorbable monofilament suture, return it to their cage, and monitor it until complete recovery from the surgery. During all procedures, maintain the animals on a heating pad at 37 °C.
      NOTE: Mice usually take up to 15 min to recover from surgery.
  2. Perform intravenous administration of oleic acid.
    1. After anesthesia (step 2.1.1, Figure 2A), inject intravenously into the orbital plexus by inserting the ultrafine needle (see Table of Materials) in the medial canthus of the eye socket (Figure 2B).
      ​NOTE: The mice were divided into two groups, with eight animals in each group. Each group receives 100 µL of the sodium-oleate solution at 10 µmol of OA per animal, while the control group receives 100 µL of sterile saline.
  3. After the surgery, monitor the animals daily for adverse reactions. Humane endpoints for euthanasia include adverse reactions, convulsions, and coma.

3. Bronchoalveolar lavage fluid collection (BALF)

  1. Euthanize the mice with an intraperitoneal lethal dose of ketamine (300 mg/Kg) and xylazine (30 mg/Kg) (see Table of Materials).
  2. Lay the animal in the dorsal decubitus position, make an incision of approximately 1 cm with surgical scissors in the animals' anterior region, expose the trachea and make a small cut to introduce an intravenous catheter (20 G).
  3. Connect the catheter to a 1 mL sterile syringe, slowly and gradually inject 0.5 mL of sterile saline into the lungs, and then aspirate the fluid from the BALF with the same syringe. Repeat it 3-5 times, and transfer it to a sterile microtube, placing them in ice.
    ​NOTE: The samples can be stored at -20 °C for up to 6 months.

4. Total and differential cell analysis in BALF

  1. For total cell count, dilute 20 µL of BALF in 180 µL (10x dilution) of Turk's solution (see Table of Materials). Perform the counting using a Neubauer chamber under an optical microscope with a 40x objective.
  2. For differential count, put 100 µL of BALF in the cellular funnel containing slides and centrifuge it at 22.86 x g for 5 min at 4 °C in a cytocentrifuge, and stain with May-Grunwald (15%, pH 7.2)-Giemsa (1:10) (see Table of Materials). Proceed with cell count in a light microscope with immersion objective.

5. Determination of total protein in BALF

  1. Determine the total BALF supernatant protein by a commercial protein quantification kit and read the absorbance at 562 nm using a spectrophotometer following the manufacturer's instructions (see Table of Materials).

6. Enzyme immunosorbent assays

  1. Centrifuge BALF at 1,200 x g for 10 min at 4 °C. Then collect the supernatant with a pipette and store it at -80 °C for assays of TNF-α, IL-1β, IL-6, and PGE215,23,25.
    NOTE: The centrifugation in step 6.1 makes the BALF cell-free.
    1. Perform the cytokines assays on cell-free BALF using a commercial ELISA kit according to the manufacturer's instructions. Carry out the PGE2 assay using an enzyme immunoassay (EIA) kit following the manufacturer's instructions (see Table of Materials).

7. Lipid body staining and counting

  1. Fix the leukocytes on cytospin slides using 3.7% formaldehyde in Ca2+, Mg2+ free Hank's buffered salt solution (HBSS, pH 7.4) and stain with 1.5% OsO4 while still moist3 (see Table of Materials). Then, count the lipid bodies per cell in 50 consecutive leukocytes from each slide using the oil-immersion objective lens of the microscope.

8. Statistical analysis

  1. Perform statistical analysis using graphing and statistics software (see Table of Materials). Express the results as mean ± SEM and analyze by one-Way Anova followed by a post-test Newman-Keuls-Student26. Consider the differences significant when P < 0.05.

Results

In an uninjured lung, alveolar fluid clearance occurs by the transport of ions through the intact alveolar epithelial layer. The osmotic gradient carries fluid from the alveoli into the pulmonary interstitium, where it is drained by lymphatic vessels or reabsorbed. Na/K-ATPase drives this transport11. OA is an inhibitor of Na/K-ATPase27 and sodium channel21, which may contribute to edema formation, as we have already suggested23

Discussion

Selecting the correct ARDS model is essential to carry out the preclinical studies, and the evaluator must consider all the possible variables, such as age, sex, administration methods, and others6. The chosen model must reproduce the disease based on risk factors such as sepsis, lipid embolism, ischemia-reperfusion of the pulmonary vasculature, and other clinical risks14. However, no animal model used for ARDS can recreate all the human syndrome's features. Multiple in...

Disclosures

The authors declare no conflict of interest.

Acknowledgements

This research was funded by the Instituto Oswaldo Cruz, Fundação Oswaldo Cruz (FIOCRUZ), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Grant 001, Programa de Biotecnologia da Universidade Federal Fluminense (UFF), Universidade Federal do Estado do Rio de Janeiro (UNIRIO), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Figure 1 and Figure 2 are created with BioRender.com.

Materials

NameCompanyCatalog NumberComments
Anesthetic vaporizerSurgiVetmodel 100
Braided slik thread with needle number 5Shalon medicalN/A
Cabinet vivariumInsight Model EB273
CentrifugeEppendorf5430/5430R
CytofunnelThermoFisher11-025-48
Drontal puppyBayerN/A
Hank's balanced SaltsSigma-AldrichH4981
HeatpadtkreproduçãoTK-500
Hydrocloric AcidSigma-Aldrich30721
Insulin syringe UltrafineBD328322
Isoforine 1mL/mLCristáliaN/A
KetamineSyntecN/A
May-Grunwald-GiemsaSigma-Aldrich205435
Micro BCA Protein Assay KitThermoFisher23235
Microscope  PrimoStarCarl Zeiss
Mouse IL-1 beta duoSet ELISAR&D systemDY401
Mouse IL-6 duoSet ELISAR&D systemDY406
Mouse TNF-alpha duoSet ELISAR&D systemDY410
Neubauer chamber improved bright-lineGlobal optics
Oleic Acid (99%)Sigma-AldrichO1008
Osmium tetroxide solution (4%)Sigma-Aldrich75632
Peripheral Intravenous Catherter 20 GBD Angiocath388333
Prism 8 (graphic and statistic software)GraphpadN/A
Prostaglandin E2 ELISA Kit -MonoclonalCayman Chemical514010
Shandon Cytospin 3ThermoFisherN/A
Sodium hydroxideMerck1,06,49,81,000
SpectrophotometerMolecular DevicesSpectraMax ABS plus
Swiss webster miceICTB/FIOCRUZN/A
Syringe 1 mLBD990189
Tris-baseBio Rad161-0719Electrophoresis purity reagent
Türk's solutionSigma-Aldrich93770
XilazineSyntecN/A

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