How to Measure Alterations in Alveolar Barrier Function as a Marker of Lung Injury

Raquel Herrero1, Gustavo Matute‐Bello2

1 Hospital Universitario de Getafe, Servicio de Cuidados Intensivos, CIBER de Enfermedades Respiratorias, Getafe, Madrid, 2 Medical Research Service of the Veterans Affairs Puget Sound Health Care Center and Center for Lung Biology, Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Washington, Seattle, Washington
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 24.3
DOI:  10.1002/0471140856.tx2403s63
Online Posting Date:  February, 2015
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The alveolar capillary membrane maintains the proper water and solute content of the epithelial lining fluid at the alveolar air‐liquid interface, which is critical for adequate gas exchange in the lung. This is possible due to the alveolar fluid clearance (AFC) capacity of this membrane that assists in the removal of salt and water from the alveolar air spaces. The alveolar capillary membrane also provides a barrier that restricts the passage of proteins and water from the interstitial and vascular compartments into the alveolar air spaces. This restricted passage is due to the presence of tight junctions between adjacent alveolar epithelial cells. Severe injury to the alveolar epithelial/endothelial membrane results in increased protein permeability and impairment of AFC, which leads to the formation of protein‐rich edema with the consequent deterioration of gas exchange. Many animal models of lung injury, focused on damage of the alveolar‐capillary membrane, assess the AFC capacity and the barrier function. We describe a simple method to assess the AFC rate in normal and pathological conditions in mice. We also describe two complementary methods to assess the alveolar‐capillary barrier function, which require measuring the concentration of endogenous plasma proteins in bronchoalveolar lavage fluid and detection of tight‐junction proteins in lung tissue by immunofluorescence. © 2015 by John Wiley & Sons, Inc.

Keywords: lung injury; alveolar fluid clearance; barrier function; protein permeability; tight‐junction proteins; mice

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Measurement of Alveolar Fluid Clearance (AFC) in Situ in Mouse Lungs
  • Basic Protocol 2: Protein Permeability of the Alveolar‐Capillary Membrane in Mice
  • Basic Protocol 3: Evaluation of Expression of Tight‐Junction Proteins in Mouse Lungs by Immunofluorescence
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Measurement of Alveolar Fluid Clearance (AFC) in Situ in Mouse Lungs

  Materials
  • 8 week‐old mice (body weight: 25 to 30 g)
  • Human serum albumin (stock 50 mg/ml; Baxter)
  • Ringer's lactate solution (Baxter)
  • Fluorescein isothiocyanate (FITC)‐tagged human serum albumin (Sigma‐Aldrich)
  • 7% sodium chloride solution (see recipe)
  • Pentobarbital (Nembutal sodium; Abbot Laboratories)
  • Alcohol
  • Osmometer
  • 18‐G Terumo Surflo ETFE i.v. catheter (Terumo Corporation)
  • Rectal temperature probe
  • Thermostatically controlled pad
  • Infrared lamp (Thermo Fisher Scientific)
  • Mouse dissection kit (including dissection board, gauze, tweezers, scissors, and curved sharp‐tip small scissors)
  • 2‐0 silk suture thread
  • Balance (to weigh mice)
  • 1‐ml syringes
  • 25‐G needle
  • P200 pipet
  • For continuous positive airway pressure and 100% oxygen flow:
    • Oxygen regulator
    • PVC tubing (Value Plastics)
    • T‐tube fittings with 200 series barbs (Value Plastics)
    • 3‐way stopcocks with luer connections and male lock (Value Plastics)
  • 96‐well plate (white/clear bottom, TC surface; BD Falcon)
  • Fluorescence spectrophotometer (Thermo Fisher Scientific)
  • Centrifuge
  • NOTE: Avoid lipopolysaccharide (LPS) and microbial contamination throughoutthe instillation procedure. Perform all instillations inside a biosafety hood.

Basic Protocol 2: Protein Permeability of the Alveolar‐Capillary Membrane in Mice

  Materials
  • 8 week‐old mice (body weight: 25 to 30 g)
  • Pentobarbital (Nembutal sodium; Abbot Laboratories)
  • Heparin
  • Bronchoalveolar lavage fluid buffer (see recipe)
  • ELISA kit (IgM, α 2‐macroglobulin; bicinchoninic acid method [BCA assay])
  • Mouse dissection kit (including dissection board, gauze, tweezers, scissors, and curved sharp‐tip small scissors)
  • 2‐0 silk suture thread
  • Balance (to weigh mice)
  • 1‐ml syringes equipped with 25‐G needles
  • 23‐G sterile needles (if sampling blood)
  • 18‐G Terumo Surflo ETFE i.v. catheter (Terumo Corporation)
  • 5‐ml polypropylene tubes
  • Centrifuge
  • Thermostatically controlled pad

Basic Protocol 3: Evaluation of Expression of Tight‐Junction Proteins in Mouse Lungs by Immunofluorescence

  Materials
  • Paraffin‐embedded murine lung tissue sections (4‐μm thick)
  • Xylene
  • Ethanol (100%, 95%, 70%)
  • 1× Phosphate‐buffered saline (PBS; see recipe)
  • Distilled H 2O
  • Citrate buffer, pH 6.0 (see recipe)
  • Proteinase K working solution (see recipe)
  • Phosphate‐buffered saline/Tween (PBST; see recipe)
  • Blocking buffer (5% bovine serum albumin [BSA] in PBST, freshly prepared)
  • Antibody diluent buffer (1% bovine serum albumin in PBS)
  • Primary antibody against the protein of interest (diluted at an appropriate concentration in antibody diluent buffer)
  • Secondary antibody against host species of primary antibody, conjugated to a fluorescent dye (e.g., fluorescein, Alexa Fluor, Texas Red; diluted at an appropriate concentration in 1× PBS)
  • DAPI nucleic acid stain (stored at 5 mg/ml; working concentration: 0.5 μg/ml; Invitrogen)
  • Fluorescent mounting medium (Dako)
  • Clear nail polish (with applicator)
  • Antifade reagent (optional)
  • Oven or slide warmer
  • Pressure cooker
  • Plastic Coplin jar, with slide racks
  • Liquid repellent slide marker pen
  • Cover slips 50 × 22 mm (No. 1.5; Fisherbrand cat. no. 12‐544‐D; Fisher Scientific)
  • Humidified chamber
  • Paper towels
  • Kimwipes (Fisher Scientific)
  • Fluorescence microscope
  • NOTE: Fixed tissue should be embedded in paraffin and sectioned onto slides. Do not allow slides to dry at any time during the whole procedure.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Basset, G., Crone, C., and Saumon, G. 1987. Fluid absorption by rat lung in situ: Pathways for sodium entry in the luminal membrane of alveolar epithelium. J. Physiol. 384:325‐345.
  Carter, E.P., Matthay, M.A., Farinas, J., and Verkman, A.S. 1996. Transalveolar osmotic and diffusional water permeability in intact mouse lung measured by a novel surface fluorescence method. J. Gen. Physiol. 108:133‐142.
  Effros, R.M., Mason, G.R., Sietsema, K., Silverman, P., and Hukkanen, J. 1987. Fluid reabsorption and glucose consumption in edematous rat lungs. Circ. Res. 60:708‐719.
  Farnand, A.W., Eastman, A.J., Herrero, R., Hanson, J.F., Mongovin, S., Altemeier, W.A., and Matute‐Bello, G. 2011. Fas activation in alveolar epithelial cells induces KC (CXCL1) release by a MyD88‐dependent mechanism. Am. J. Respir. Cell Mol. Biol. 45:650‐658.
  Garat, C., Carter, E.P., and Matthay, M.A. 1998. New in situ mouse model to quantify alveolar epithelial fluid clearance. J. Appl. Physiol. 84:1763‐1767.
  Garat, C., Rezaiguia, S., Meignan, M., D'Ortho, M.P., Harf, A., Matthay, M.A., and Jayr, C. 1995. Alveolar endotoxin increases alveolar liquid clearance in rats. J. Appl. Physiol. 79:2021‐2028.
  Garty, H. and Palmer, L.G. 1997. Epithelial sodium channels: Function, structure, and regulation. Physiol. Rev. 77:359‐396.
  Herrero, R., Tanino, M., Smith, L.S., Kajikawa, O., Wong, V.A., Mongovin, S., Matute‐Bello, G., and Martin, T.R. 2013. The Fas/FasL pathway impairs the alveolar fluid clearance in mouse lungs. Am. J. Physiol. Lung Cell Mol. Physiol. 305:L377‐L388.
  Herrero, R., Kajikawa, O., Matute‐Bello, G., Wang, Y., Hagimoto, N., Mongovin, S., Wong, V., Park, D.R., Brot, N., Heinecke, J.W., Rosen, H., Goodman, R.B., Fu, X., and Martin, T.R. 2011. The biological activity of FasL in human and mouse lungs is determined by the structure of its stalk region. J. Clin. Invest. 121:1174‐1190.
  Jayr, C., Garat, C., Meignan, M., Pittet, J.F., Zelter, M., and Matthay, M.A. 1994. Alveolar liquid and protein clearance in anesthetized ventilated rats. J. Appl. Physiol. 76:2636‐2642.
  Lipke, A.B., Matute‐Bello, G., Herrero, R., Kurahashi, K., Wong, V.A., Mongovin, S.M., and Martin, T.R. 2010. Febrile‐range hyperthermia augments lipopolysaccharide‐induced lung injury by a mechanism of enhanced alveolar epithelial apoptosis. J. Immunol. 184:3801‐3813.
  Matalon, S., Benos, D.J., and Jackson, R.M. 1996. Biophysical and molecular properties of amiloride‐inhibitable Na+ channels in alveolar epithelial cells. Am. J. Physiol. 271:L1‐L22.
  Matthay, M.A., Landolt, C.C., and Staub, N.C. 1982. Differential liquid and protein clearance from the alveoli of anesthetized sheep. J. Appl. Physiol. Respir. Environ. Exerc. Physiol. 53:96‐104.
  Matthay, M.A., Robriquet, L., and Fang, X. 2005. Alveolar epithelium: Role in lung fluid balance and acute lung injury. Proc. Am. Thorac. Soc. 2:206‐213.
  Mazzon, E. and Cuzzocrea, S. 2007. Role of TNF‐α in lung tight junction alteration in mouse model of acute lung inflammation. Respir. Res. 8:75.
  Modelska, K., Pittet, J.F., Folkesson, H.G., Courtney Broaddus, V., and Matthay, M.A. 1999. Acid‐induced lung injury. Protective effect of anti‐interleukin‐8 pretreatment on alveolar epithelial barrier function in rabbits. Am. J. Respir. Crit. Care Med. 160:1450‐1456.
  Mutlu, G.M. and Sznajder, J.I. 2005. Mechanisms of pulmonary edema clearance. Am. J. Physiol. Lung Cell Mol. Physiol. 289:L685‐L695.
  Pittet, J.F., Brenner, T.J., Modelska, K., and Matthay, M.A. 1996. Alveolar liquid clearance is increased by endogenous catecholamines in hemorrhagic shock in rats. J. Appl. Physiol. 81:830‐837.
  Rezaiguia, S., Garat, C., Delclaux, C., Meignan, M., Fleury, J., Legrand, P., Matthay, M.A., and Jayr, C. 1997. Acute bacterial pneumonia in rats increases alveolar epithelial fluid clearance by a tumor necrosis factor‐alpha‐dependent mechanism. J. Clin. Invest. 99:325‐335.
  Sakuma, T., Pittet, J.F., Jayr, C., and Matthay, M.A. 1993. Alveolar liquid and protein clearance in the absence of blood flow or ventilation in sheep. J. Appl. Physiol. 74:176‐185.
  Sakuma, T., Folkesson, H.G., Suzuki, S., Okaniwa, G., Fujimura, S., and Matthay, M.A. 1997. Beta‐adrenergic agonist stimulated alveolar fluid clearance in ex vivo human and rat lungs. Am. J. Respir. Crit. Care Med. 155:506‐512.
  Sartori, C. and Matthay, M.A. 2002. Alveolar epithelial fluid transport in acute lung injury: New insights. Eur. Respir. J. 20:1299‐1313.
  Smedira, N., Gates, L., Hastings, R., Jayr, C., Sakuma, T., Pittet, J.F., and Matthay, M.A. 1991. Alveolar and lung liquid clearance in anesthetized rabbits. J. Appl. Physiol. 70:1827‐1835.
  Suzuki, S., Zuege, D., and Berthiaume, Y. 1995. Sodium‐independent modulation of Na(+)‐K(+)‐ATPase activity by beta‐adrenergic agonist in alveolar type II cells. Am. J. Physiol. 268:L983‐L990.
  Ware, L.B. and Matthay, M.A. 2001. Alveolar fluid clearance is impaired in the majority of patients with acute lung injury and the acute respiratory distress syndrome. Am. J. Respir. Crit. Care Med. 163:1376‐1383.
  Xie, W., Wang, H., Wang, L., Yao, C., Yuan, R., and Wu, Q. 2013. Resolvin D1 reduces deterioration of tight junction proteins by upregulating HO‐1 in LPS‐induced mice. Lab. Invest. 93:991‐1000.
  Yue, G. and Matalon, S. 1997. Mechanisms and sequelae of increased alveolar fluid clearance in hyperoxic rats. Am. J. Physiol. 272:L407‐L412.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library