Ex vivo fluorescence microscopy provides simple and accurate assessment of neutrophil-endothelial adhesion in the rat lung.
Neutrophil adhesion to the pulmonary endothelium is prerequisite to neutrophil transmigration and activation, both of which may lead to lung injury. A simple method to evaluate neutrophil adherence in the lung would be useful for developing new strategies for neutrophil-mediated lung injury. The purpose was to establish a simple method to evaluate neutrophil adhesion in the lung using ex vivo fluorescence microscopy. Rats were anesthetized, and the right jugular veins were catheterized. Neutrophils were isolated from another set of rats and labeled with 5,(6)-carboxyfluorescein diacetate. Animals were killed 120 s after a 1 x 10(6) labeled neutrophil injection. The pulmonary labeled neutrophil number was counted under a fluorescence microscope. In the first experiment, rats were given 0, 20, 200, or 2000 microg/kg lipopolysaccharide (LPS) i.p. At 4 h after challenge, the pulmonary labeled neutrophil number was determined. Kinetic studies were also performed at 0, 1, 4, and 8 h after 200 microg/kg LPS. Finally, anti-ICAM-1 Ab was injected i.v. before LPS 200 microg/kg, and the labeled neutrophil number in the lung was determined at 4 h. The number of pulmonary labeled neutrophils was higher after LPS 200 or 2000 microg/kg than after the other doses. The pulmonary labeled neutrophil number was increased at 4 h compared with the other time points. ICAM-1 blocking normalized the pulmonary labeled neutrophil number in the LPS group. In conclusion, our method seems to reflect ICAM-1-mediated neutrophil adherence to the endothelium in the present setting. This simple technique may be useful for evaluating neutrophil adhesion.
Han
I
I
Department of Surgery, University of Tokyo, Japan.
Saito
H
H
Fukatsu
K
K
Inoue
T
T
Yasuhara
H
H
Furukawa
S
S
Matsuda
T
T
Lin
M T
MT
Ikeda
S
S
D000818:Animals; D002448:Cell Adhesion / Q000502:physiology; D004730:Endothelium, Vascular / Q000166:cytology / Q000502:physiology*; D004926:Escherichia coli; D066298:In Vitro Techniques; D018799:Intercellular Adhesion Molecule-1 / Q000276:immunology; D007958:Leukocyte Count; D008070:Lipopolysaccharides / Q000633:toxicity; D008168:Lung / Q000502:physiology*; D008297:Male; D008856:Microscopy, Fluorescence / Q000379:methods; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D011652:Pulmonary Circulation / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012044:Regression Analysis
D008070:Lipopolysaccharides; D018799:Intercellular Adhesion Molecule-1