Pleural tissue hyaluronan produced by postmortem ventilation in rabbits

We developed a method that used Alcian blue bound to hyaluronan to measure pleural hyaluronan in rabbits postmortem. Rabbits were killed, then ventila ted with 21% O-2-5% CO2-74% N-2 for 3 h. The pleural liquid was removed by suction and 5 mi Alcian blue stock solution (0.33 mg/ml, 3.3 pH) was inject ed into each chest cavity. After 10 min, the Alcian blue solution was remov ed and the unbound Alcian blue solution (supernatant) separated by centrifu gation and filtration. The supernatant transmissibility (T) was measured sp ectrophotometrically at 613 nm. Supernatant Alcian blue concentration (Cab) was obtained from a calibration curve of T versus dilutions of stock solut ion Cab. Alcian blue bound to pleural tissue hyaluronan was obtained by sub tracting supernatant Cab from stock solution Cab. Pleural tissue hyaluronan was obtained from a calibration curve of hyaluronan versus Alcian blue bou nd to hyaluronan. Compared with control rabbits, pleural tissue hyaluronan (0.21 +/- 0.04 mg/kg) increased twofold, whereas pleural liquid volume decr eased by 30% after 3 h of ventilation. Pleural effusions present 3 h postmo rtem without ventilation did not change pleural tissue hyaluronan from cont rol values. Thus ventilation-induced pleural liquid shear stress, not incre ased filtration, was the stimulus for the increased hyaluronan produced fro m pleural mesothelial cells.