arterial pressures as well as heart rate from the arterial blood pressure signal. We defined cor- onary vascular resistance as mean arterial pres- sure divided by total myocardial blood flow (Shen et al. 1994), and rate–pressure product as the product of heart rate and systolic blood pressure (Rooke and Feigl 1982). Statistical analysis. We calculated descrip- tive statistics for exposure measures (CAPs mass concentration, particle number con- centration, black carbon concentration) and cardiovascular parameters (coronary artery resistance, arterial blood pressure, rate–pressure product) and used graphical methods to assess the distribution of each outcome. Coronary artery resistance was log-transformed in all analyses to improve the normality assumption. We assessed the effect of CAPs exposure on myocardial blood flow using multilevel linear mixed models (Fitzmaurice et al. 2004) containing exposure type (CAPs, filtered air, initial baseline, final baseline) as fixed effects and tissue piece and dog as random effects. This modeling approach appropriately accounts for the correlation between multiple measurements on the same piece of tissue over time as well as for the correlation between measurements made in different tissue pieces within the same animal. We evaluated dose– response relationships by replacing the CAPs binary term with the 5-hr mean CAPs mass concentration, particle number concentration, or black carbon concentration. To assess whether the CAPs effects on myocardial blood flow differed in ischemic ver- sus nonischemic tissue, we dichotomized each piece according to its location. A tissue piece was estimated to be in or near the ischemic zone if the average blood flow during ischemia after filtered air exposure was less than under nonischemic baseline conditions and outside of the ischemic zone otherwise. We assessed differences in CAPs response in the ischemic versus nonischemic zone by incorporating fixed effects terms for zone (ischemic vs. non- ischemic) and zone-by-exposure interactions. In sensitivity analyses, we considered alter- native models that treated dogs as fixed effects rather than random effects and included a linear trend of time to account for the pos- sible effects on myocardial blood flow of repeat ischemic events or accumulation of microspheres in myocardial capillary beds over time. The results from these sensitivity analyses were not materially different from those of the primary analyses. As noted above, no reference blood sample could be collected from one animal in which the aortic catheter failed. Without the refer- ence blood sample, it was still possible to evalu- ate the effects of exposure on total myocardial blood flow under the unverifiable assumption that a similar number of microspheres was used for each determination. To include the data from this additional animal, we repeated the primary analysis using the number of micro- spheres in each tissue piece as the outcome rather than absolute values of flow. Specifically, the number of microspheres in each tissue piece was modeled using a Poisson log-linear mixed model containing fixed effects for exposure and random effects for tissue piece and each dog. Linear mixed model analyses were per- formed using PROC MIXED (version 9; SAS Institute Inc., Cary, NC, USA). We imple- mented Poisson log-linear mixed models in SAS using the GLIMMIX macro. Descriptive statistics were computed in SAS, and graphical inspection of all data was performed using R statistical software (R Development Core Team 2005). All statistical tests are two tailed, and p < 0.05 is considered statistically significant. Results Figure 2 illustrates regional myocardial blood flow in one animal during different exper- imental conditions. In this example, myo- cardial blood flow during coronary artery occlusion appears to be reduced to a greater extent after 5 hr of CAPs exposure compared with filtered air exposure. Among four dogs that completed the exposure protocol, total myocardial blood flow during coronary artery occlusion was lower after CAPs exposure than after filtered air exposure (Figure 3). In mixed-effect mod- els controlling for tissue piece and animal, CAPs exposure was associated with a statisti- cally significant 0.12 mL/min/g (SE = 0.02; p < 0.001) reduction in total myocardial blood flow during coronary artery occlusion. In an analysis of the relative flow (rather than absolute flow; n = 5 dogs), CAPs exposure was associated with a 5.7% [95% confidence Table 1. Average hemodynamic measures during a 5-min coronary artery occlusion after a 5-hr exposure to either CAPs or filtered air. Measure Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Mean arterial pressure (mmHg) Pulse pressure (mmHg) Heart rate (bpm) Rate–pressure product (bpm × mmHg) Filtered air 114.2 ± 20.7 73.6 ± 9.1 88.3 ± 12.0 40.7 ± 14.7 109.4 ± 23.4 12,197 ± 1,984 CAPs 122.7 ± 15.0 79.5 ± 7.1 93.7 ± 7.0 43.0 ± 16.0 99.4 ± 20.2 12,031 ± 1,951 ap-Value is for test of mean response in CAPs-exposed versus filtered-air–exposed animals, which is estimated from a mixed-effects model with fixed effects for CAPs and random dog effects. Environmental Health Perspectives • volume 117 | number 3 | March 2009 CAPs alter myocardial blood flow interval (CI), 1.7–9.5%; p = 0.005] reduction in myocardial blood flow versus filtered air. We calculated coronary vascular resistance and rate–pressure product, a standard index of cardiac metabolic demand, during myocardial ischemia in three of the four animals from the primary analysis where data were avail- able (Table 1). CAPs exposure was associated with a 13% (95% CI, 7.4–19.4%; p < 0.001) increase in coronary vascular resistance during coronary artery occlusion, whereas rate–pres- sure product remained unchanged (Table 1). Average values of systolic and diastolic blood pressure during LAD occlusion were higher after CAPs exposure versus filtered air expo- sure, but the difference was not statistically significant. We assessed whether CAPs effects were similar in ischemic versus nonischemic myo- cardium (Figure 4). In tissue outside of the ischemic zone, CAPs exposure decreased myocardial blood flow during coronary artery occlusion by 0.05 mL/min/g (SE = 0.03; p = 0.09). In contrast, in tissue within or near the ischemic zone, CAPs exposure decreased myocardial blood flow during coronary artery occlusion by 0.22 mL/min/g (SE = 0.03; p < 0.001). The interaction term between CAPs exposure and zone was statistically significant (p < 0.001). The CAPs-induced increase in cor- onary vascular resistance was similarly greater within the ischemic zone as compared with tis- sue outside the ischemic zone (33.7 vs. 5.8%, respectively; p for interaction < 0.001). CAPs characteristics. Daily CAPs mass con- centration ranged from 94.1 to 1556.8 μ g/m3 (mean ± SD, 349.0 ± 282.6 μ g/m3), particle number concentrations ranged from 3,000 to 69,300 particles/cm3 (20,381 ± 13,075 par- ticles/cm3), and black carbon concentrations ranged from 1.3 to 32.0 μ g/m3 (7.5 ± 5.6 μ g/ m3). Increases in CAPs mass, particle number, and black carbon concentrations were signifi- cantly associated with decreases in myocardial blood flow and increases in coronary vascu- lar resistance during coronary artery occlusion p = 0.09 1.0 w lfo 0.8 lood /)ng0.6 b i ilad /L0.4 m ra m ( oc 0.2 y M 0 p < 0.001 335 p-Valuea 0.20 0.11 0.21 0.78 0.19 0.90 Filtered air + CAPs + occlusion occlusion Nonischemic myocardium Filtered air + CAPs + occlusion occlusion Ischemic myocardium Figure 4. Myocardial blood flow in tissue not in the ischemic zone compared with tissue within or near the ischemic zone during coronary artery occlusion immediately after 5 hr exposure 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.