Technical Reference #1664

1664.

Novel Critical Role of Toll-like Receptor 4 in Lung Ischemia-reperfusion Injury and Edema Giorgio Zanotti; Monica Casiraghi; John B. Abano; Jason R. Tatreau; Mayura Sevala; Hilary Berlin; Susan Smyth; William K. Funkhouser; Keith Burridge; Scott H. Randell; and Thomas M. Egan1, Cystic Fibrosis Pulmonary Research and Treatment Center, American Journal of Physiology: Cell and Molecular Physiology, 297(1664), (2009)
Link To Paper

Abstract:
Toll-like receptors (TLRs) of the innate immune system contribute to noninfectious inflammatory processes.

Keywords:
microvascular permeability; endothelial cell; pulmonary edema

Materials & Methods:
Cell culture model of warm lung IRI. Because mice were ventilated with 100% O2 before hilar clamping our in vivo model of IRI was likely not associated with lung hypoxia. Thus we developed an in vitro normothermic (37°C) model of IRI employing nutrient depletion in 100% oxygen to model ischemia with reperfusion modeled by supplying fresh medium to culture dishes in sealed Plexiglas containers. Human pulmonary microvascular endothelial cells (HMVECs; Cambrex Bio Science Walkersville MD) maintained in Clonetics EGM-2 MV BulletKits medium (Cambrex Bio Science) at 37°C in a humidified incubator in 5% CO2 were seeded at 2000 cells/cm2 on collagencoated 30-mm diameter glass bottom dish coverslips (MatTek Ashland MA) and grown until 100% confluent. Sealed Plexiglas containers housing culture dishes at 37°C were ventilated with 95% O2-5% CO2. To model warm ischemia (WI) cell medium was suddenly replaced with 2 ml of nutrient-depleted clinical grade Ringer lactate (RL) containing 100 U/ml penicillin and 100 g/ml streptomycin and the chamber was ventilated with 100% O2. Dishes were pretreated with 1 g/ml CRX-526 (GlaxoSmithKline Duluth MN) a competitive inhibitor of TLR4 (14) or vehicle (2% glycerin) 1 h before WI. CRX-526 or vehicle was added whenever medium was changed. After 1 h of simulated WI RL was replaced with EGM-2 MV medium to simulate reperfusion ventilating the chamber with 5% CO2 in room air. Dishes were removed in triplicate during WI and reperfusion and immediately fixed in 4% paraformaldehyde for phalloidin staining. Cells with inhibitor or vehicle maintained in EGM-2 MV medium at 37°C in humidified 5% CO2 incubator served as controls. Culture medium was changed at the same time that medium was changed in experimental dishes. Probes inserted through sealed ports continuously recorded temperature and pH in a representative dish in the Plexiglas box using voltmeters with data output recorded by Pico software (Pico St. Neots United Kingdom).

Microscopic Technique
Fluorescence Microscopy, differential interference contrast

Cell Type(s)
Pulmonary microvascular