Animal bioassay experiments

Name	Type	Comments
Intravital microscopy studies	Acute (<24 hr)	Male Sprague–Dawley rats (6–11 weeks old) were purchased from Hilltop Labs and housed at the WV University Health Sciences Center. Animal vital statistics are shown in Table 1. An N of 17 sham and 13 PMMTM-exposed animals were used for the intravital preparation, and an N of 11 sham and 8 PMMTM-exposed animals were used for the isolated arteriole preparation (Table 1). All animal procedures were approved by the WVU Institutional Animal Care and Use Committee. Air was sampled at two sites within 1 mile of an active MTM site (Sundial, WV, USA). PM was collected on 35 mm 5 lm pore size PTFE fiber-backed filters (Whatman, Springfield Mill, UK, Figure 1A) for 2–4 weeks. Air flow rate across the filters averaged 12 L/min. Following collection, the filters were stored at room temperature (20–25°C) and ambient humidity (10–30%) in the dark for 0.5–1 year prior to extraction. PM (Figure 1B) was removed from the filters by gentle agitation in 15 mL of ultrapure water (Cayman Chemical, Ann Arbor, MI, USA) in a glass jar for 96 hours. Storage and extraction of the particles from the filters are consistent with previously reported methods [14]. Aliquots of the particle suspension were dried down in 2 mL cryovials for 18 hours in a Speedvac (Savant, Midland, MI, USA). Total particle weight was determined by a microbalance (Metler-Toledo, Columbus, OH, USA). Intravital microscopy was performed as previously described [24]. Briefly, rats were anesthetized by an i.p. injection of Inactin (100 mg/kg) and maintained at 37ºC. The trachea was intubated to ensure a patent airway, and the right carotid artery was cannulated to measure arterial pressure. The right spinotrapezius muscle was exteriorized for microscopic observation over a clear pedestal, leaving all feed arteries and innervations intact. The tissue bath was continuously superfused with an electrolyte solution ([in mM] 119 NaCl, 25 NaHCO3, 6 KCl, and 3.6 CaCl2, pH 7.4, 290 mOsm), warmed to 35ºC, and equilibrated with 95% N2, 5% CO2 with a superfusion flow rate of 4–6 mL/min. The preparation was then transferred to the stage of an Olympus intravital microscope coupled to a CCD camera and was observed under a 209 water immersion objective (final image magnification was 7439). Greater than three images were digitally captured via DP controller (Olympus, Center Valley, PA, USA) during a baseline period and immediately following each experimental period. Arteriolar diameters from each digital image were measured with Microsuite analysis software (Olympus). Steady-state arteriolar diameters were averaged per experimental period to reduce sampling variability [24].
Isolated arteriole studies	Acute (<24 hr)	Male Sprague–Dawley rats (6–11 weeks old) were purchased from Hilltop Labs and housed at the WV University Health Sciences Center. Animal vital statistics are shown in Table 1. An N of 17 sham and 13 PMMTM-exposed animals were used for the intravital preparation, and an N of 11 sham and 8 PMMTM-exposed animals were used for the isolated arteriole preparation (Table 1). All animal procedures were approved by the WVU Institutional Animal Care and Use Committee. Air was sampled at two sites within 1 mile of an active MTM site (Sundial, WV, USA). PM was collected on 35 mm 5 lm pore size PTFE fiber-backed filters (Whatman, Springfield Mill, UK, Figure 1A) for 2–4 weeks. Air flow rate across the filters averaged 12 L/min. Following collection, the filters were stored at room temperature (20–25°C) and ambient humidity (10–30%) in the dark for 0.5–1 year prior to extraction. PM (Figure 1B) was removed from the filters by gentle agitation in 15 mL of ultrapure water (Cayman Chemical, Ann Arbor, MI, USA) in a glass jar for 96 hours. Storage and extraction of the particles from the filters are consistent with previously reported methods [14]. Aliquots of the particle suspension were dried down in 2 mL cryovials for 18 hours in a Speedvac (Savant, Midland, MI, USA). Total particle weight was determined by a microbalance (Metler-Toledo, Columbus, OH, USA). Coronary arterioles were isolated as previously described [26,27]. Arterioles from the mesentery were also removed in a similar manner. Briefly, the heart or the mesentery was removed from isoflurane anesthetized animals and placed into a silastic-coated dish containing chilled (4°C) PSS (in mM; 129.8 NaCl, 5.4 KCl, 1.1 NaH2PO4, 1.7 MgCl2, 19.0 NaHCO3, 1.8 CaCl2, and 5.5 glucose, pH 7.4, 290 mOsm). The heart was flushed of excess blood and the LAD artery was located. Arterioles 170 lm, which corresponded to third to fourth order arterioles in the heart or fourth and fifth order arterioles in the mesentery, were isolated and transferred to a vessel chamber containing fresh PSS oxygenated with normoxic gas (21% O2–5% CO2–74% N2), cannulated with glass micropipettes, and secured with nylon suture (10–0 ophthalmic; Alcon, Hemel Hempstead, UK). Arterioles were pressurized to 45 mmHg (coronary) or 80 mmHg (mesentery) with PSS using a servo controlled peristaltic pump (Living Systems Instrumentation, Burlington, VT, USA) without flow and superfused with oxygenated 37°C PSS at a rate of 10 mL/min. Vessel diameter was measured with a video caliper (Colorado Video, Boulder, CO, USA). Vessels without leaks were allowed to develop spontaneous tone ( 17% less initial diameter). Ca++-free PSS was superfused at the end of all experiments to determine passive arteriolar diameters.