The animals were killed with a lethal injection of pentobarbital (USP Pharmacy). Immediately before the pentobarbital injection, a blood sample from each animal was collected for comparison with normal blood parameters from noninjected rabbits. After death, the animals were exsanguinated by aortic section through a middle abdominal incision, and a 21-gauge needle was inserted through the diaphragm to aspirate the fluid. After the fluid was aspirated, the thoracic cavity was opened and grossly inspected for the presence of TL. Animals injected with saline solution were used as benign noninflammatory controls. Blood samples were collected from a group of six noninjected rabbits as controls on the measurements in the blood of the injected animals. The study was approved by the Ethics Committee of the Heart Institute (InCor), University of Sao Paulo Medical School, which oversees research involving both animals and humans.
TL, a magnesium silicate asbestos-free particle with a mean length of 25.4 |j,m (length range, 6.4 to 50.5 |xm), was suspended under sterile conditions in endotoxin-free saline solution to a concentration of 400 mg/kg. SN was suspended in endotoxin-free H2O at a concentration of 0.5% for intrapleural injection.
Cytologic and Biochemical Analysis
Blood and pleural liquid were collected in ethylenediaminetet-raacetic acid for cytologic and cytokine analysis, and in dry tubes for LDH evaluation. Samples were placed in a hemocytometer for determination of the total number of leukocytes, and onto slides for Leishman preparation and determination of the percentage of neutrophils. Other samples were centrifuged at 1,000 revolutions per minute for 10 min at 4°C, and the supernatants were withdrawn immediately for LDH analysis and were stored at — 80°C for cytokine determination. LDH was measured by a standard biochemical kinetic UV method (range of normal serum values for the method, 120 to 240 IU/L).