Ex vivo fluorescence microscopy provides simple and accurate assessment ofneutrophil-endothelial adhesion in the rat lung

Neutrophil adhesion to the pulmonary endothelium Is prerequisite to neutrop hil 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 pur pose was to establish a simple method to evaluate neutrophil adhesion in th e lung using ex vivo fluorescence microscopy. Rats were anesthetized, arid the right jugular veins were catheterized. Neutrophils were isolated from a nother set of rats and labeled with 5,(6)-carboxyfluorescein diacetate. Ani mals were killed 120 s after a 1 x 10(6) labeled neutrophil injection. The pulmonary labeled neutrophil number was counted under a fluorescence micros cope. In the first experiment, rats were given 0, 20, 200, or 2000 pg/kg li popolysaccharide (LPS) Lp. At 4 h after challenge, the pulmonary labeled ne utrophil number was determined. Kinetic studies were also performed at 0, 1 , 4, and 8 h after 200 pg/kg LPS. Finally, anti-ICAM-1 Ab was injected im. before LPS 200 pg/kg, and the labeled neutrophil number in the lung was det ermined at 4 h. The number of pulmonary labeled neutrophils. was higher aft er LPS 200 or 2000 pg/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 neutr ophil adherence to the endothelium In the present setting. This simple tech nique may be useful for evaluating neutrophil adhesion.