Animal bioassay experiments

Name	Type	Comments
Intratracheal instillation	Acute (<24 hr)	The animal experiments in this study were approved by the West Virginia University Animal Care and Use Committee and conformed to the most current National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals manual. Male Sprague-Dawley rats were housed in the West Virginia University Health Sciences Center animal facility. Rats were given access to a rodent diet and water ad libitum. PMMTM preparation: PM was collected on 35-mm, 5-m pore size polytetrafluoroethylene fiber-backed filters (Whatman, Springfield Mill, UK) for 2– 4 wk at two sites within 1 mile of an active MTM site. Particle storage and extraction from the filters following collection are consistent with previous reports (24). Briefly, filters were stored at room temperature (20 –25°C) and ambient humidity (10 – 30%) before extraction. PM extraction was accomplished by gentle agitation in ultrapure water for 96 h. Particle suspension aliquots were then dried in a Speedvac (Savant, Midland, MI), and total particle weight was determined using a microbalance (Metler-Toledo, Columbus, OH). Intratracheal instillation: Intratracheal instillation was performed according to the method of Brain et al. (4) as previously described (36, 45, 46, 49, 50). Briefly, following anesthesia with isoflurane, a ball needle attached to a 1-ml tuberculin syringe was inserted under the glottis into the trachea, and 300 l of either vehicle (5% fetal bovine serum in phosphate-buffered saline) or vehicle with 300 g of PMMTM was instilled directly into the trachea. We have previously shown that this dose of PMMTM partially impaired endotheliumdependent microvascular dysfunction (36). The PM characterization and resuspension were carried out as described previously (36).