Bartoli et al. from analysis. The aortic catheter failed in one additional animal, which we excluded from the primary analysis (n = 4) but included in a sensitivity analysis (n = 5), as described below. Exposure technology and characterization. To evaluate the effects of ambient PM on myocardial blood flow, we repeatedly exposed pairs of animals via the permanent tracheos- tomy to 5 hr of either filtered air or CAPs. The characteristics of the Harvard Ambient Particle Concentrator (HAPC) have been pre- viously described (Godleski et al. 2000; Sioutas et al. 1995). Briefly, the HAPC concentrates ambient particles ≤ 2.5 μm in aero dynamic diameter to approximately 30 times ambient levels without altering its size distribution or chemical composition. Particles with diam- eters > 2.5 μm are removed upstream of the HAPC, whereas ultrafine particles (≤ 0.1 mμ in aerodynamic diameter) and ambient gases are neither enriched nor excluded. Exposures typically took place between 0800 and 1430 hours each day. We measured CAPs mass concentration using a tapered- element oscillating microbalance (TEOM series 1400a; Rupprecht and Patashnick Co. Inc., East Greenbush, NY, USA); particle number concentration using a condensation particle counter (CPC model 3022A; TSI, Inc., Shoreview, MN, USA), and black car- bon concentration by optical transmission (Aethalometer model AE-9; Magee Scientific, Berkeley, CA, USA), as previously described (Godleski et al. 2000). Experimental design. To evaluate the effects of CAPs on myocardial blood flow, we Sequence 1 Initial baseline Sequence 2 Initial baseline Filtered air + occlusion Filtered air + occlusion CAPS + occlusion Filtered air + occlusion Filtered air + occlusion CAPS + occlusion CAPS + occlusion Filtered air + occlusion Filtered air + occlusion CAPS + occlusion Final baseline Final baseline Figure 1. Exposure protocol for assessing the effects of CAPs on myocardial blood flow during coronary artery occlusion. After recovery from surgery, dogs were randomized to one of two exposure sequences. Myocardial blood flow was determined with fluorescent microspheres at baseline (no exposure, no coronary artery occlu- sion) and during a 5-min coronary artery occlusion immediately after 5 hr exposure to either CAPs or filtered air. Exposures in the same animal were separated by a minimum of 1 week. 334 repeatedly exposed pairs of animals to 5 hr of either filtered air or CAPs in a crossover pro- tocol (Figure 1). Exposures in the same ani- mal were separated by a minimum of 1 week. Immediately after each exposure, animals underwent two 5-min occlusions of the LAD coronary artery separated by a 20-min rest period. Acute myocardial ischemia during each occlusion was confirmed by observing real- time electrocardiographic changes. We evalu- ated myocardial blood flow with fluorescent microspheres during the second occlusion after 3 min of ischemia. This occlusion protocol is consistent with our prior work in this animal model showing that CAPs exacerbate myocar- dial ischemia during the second LAD occlusion (Godleski et al. 2000; Wellenius et al. 2003). To control for changes in myocardial blood flow due to repeated microsphere injection, each animal received a baseline injection of microspheres (initial baseline) without inter- vention 1 week before the first CAPs or filtered air exposure. One week after the last CAPs or filtered air exposure, animals received a second baseline injection of microspheres (final base- line) without exposure or intervention. Measurement of myocardial blood flow. We measured myocardial blood flow using standard fluorescent microsphere techniques as previously described (Bartoli et al. 2008b). The principle of this technique is as follows. Fluorescent microspheres (15 μ m diameter) were injected into the left atrium, where they were mixed with blood in the left ven- tricle, ejected into the aorta, and distributed throughout the body in a pattern similar to Baseline Filtered air + occlusion CAPs + occlusion Blood flow (mL/min/g) > 0.75 0.5–0.75 0.25–0.5 0–0.25 Figure 2. Transverse sections through the ventricles showing myocardial blood flow in one animal at baseline (left), during coronary artery occlusion immediately after 5 hr exposure to filtered air (cen- ter), and during coronary artery occlusion immedi- ately after 5 hr exposure to CAPs (right). At baseline, myocardial blood flow is uniform except for a minor flow deficit at the location of hydraulic balloon occluder implantation. During coronary artery occlusion after exposure to filtered air, myocardial blood flow was diminished in the anterolateral left ventricular myocardium. During coronary artery occlusion after exposure to CAPs, myocardial blood flow to the same region was further diminished. that of red blood cells. Microspheres of this diameter should lodge within the smallest precapillary arterioles based on regional tis- sue blood flow patterns. Postmortem analyses quantified relative differences in blood flow among organs or within regions of a single organ. We used blood sampled from the descending aorta at a known rate using a cali- brated withdrawal pump (Harvard Apparatus; Holliston, MA, USA) for 100 sec to deter- mine the absolute flow to tissue samples in milliliters per minute or milliliters per minute per gram of tissue. Microspheres with dif- ferent fluorescent properties permit multiple determinations of myocardial blood flow over time in the same animal. For each determination, microspheres were vortexed for 1 min and sonicated in an ultrasonic water bath for 15 min before injection into the left atrium via the vascular access port. The bolus of microspheres was injected over a period of 10 sec, followed by a flush of 5 mL warmed heparinized saline (38°C; 20 IU/mL). Blood was sampled from the descending aorta at a rate of 7.75 mL/ min using a calibrated Harvard Apparatus withdrawal pump for 100 sec starting 10 sec before the microsphere injection. After euthanasia, the heart and surround- ing structures were harvested. The ventricles were sectioned into 40–60 pieces of approxi- mately 1 g each and mapped on a three- dimensional grid (Figure 2). We sent tissue and reference blood samples to IMT/Stason Laboratories (Irvine, CA, USA) for automated digestion of tissue samples and counting of fluorescent microspheres using flow cytometry. Instantaneous flow values were calculated for each piece of myocardial tissue as milliliters per minute per gram of tissue. Hemodynamic measurements. Continuous arterial blood pressure was monitored and recorded throughout exposures in three ani- mals (DSI Dataquest ART 3.1; Data Sciences International). Data were exported and ana- lyzed using custom-designed Matlab software (Mathworks, Inc., Natick, MA, USA) to obtain 5-min averages for each outcome. We derived systolic, diastolic, mean, and pulse p < 0.001 p < 0.001 1.0 w lfo 0.8 lood /)ng0.6 b i ilad /L0.4 m ra m ( oc 0.2 y M 0 Initial baseline Filtered air + occlusion CAPs + occlusion Final baseline Figure 3. Total myocardial blood flow at baseline and during coronary artery occlusion after a 5 hr expo- sure to either filtered air or CAPs. Bars represent model-estimated mean ± SE from four dogs exposed to CAPs on 7 days and filtered air on 9 days. ovlume 117 | number 3 | March 2009 • Environmental Health Perspectives