Six to eight slices per vessel were evaluated. A calibration bar was also digitized with each individual sample to determine the magnification of the system and to convert

the pixel values into millimeters. The measured parameters (IMT, average wall thickness) were expressed in millimeters. Mean values of the measured in vivo IMT, in vitro IMT and average wall thickness were calculated. Mean differences between in vivo and in vitro IMT were expressed in millimeters and percents according to the following formulas: IMT difference (mm)=in vitro IMT−in vivo IMT;IMT difference(%)=(in vitro IMT−in vivo IMT)/in vitro IMT×100, respectively Vessel circumference and lumen circumference on the digitized 3 mm thick arterial sections were measured with free available image analyzer software of the National Institute of Health and mean values were calculated. Subsequently, average wall thickness check details was determined based on the following formula: average wall thickness (mm) = (vessel circumference − lumen circumference)/2π. CCA specimens were processed for histology. Three millimeter thick frozen arterial slices prepared as described above and marked by the thread SB203580 at the level of in vitro IMT measurements were used. Afterwards, transverse sections (20 μm) of the marked slices were cut by cryomicrotome (Leica, CM 1850, Stockholm, Sweden) and were stained with hematoxylin & eosin (H&E) and Verhoeff–Van Gieson [33] and [34].

Sections with artificially damaged intima and/or media at the site of the measurement were excluded. Concordance analysis was performed between in vivo IMT and in vitro IMT measurements. Furthermore, Bland–Altman plots were applied to illustrate the agreement between in vitro and

in vivo IMT measurements Methane monooxygenase [20]. Linear regression analysis was preformed to correlate in vivo IMT, in vitro IMT and average wall thickness. In the present study we have compared postmortem IMT determination with in vivo IMT and average wall thickness. Furthermore, histological processing of selected snap frozen arterial specimens was performed. In vivo and in vitro IMT measurements were compared in n = 34 CCA specimens. Fig. 2 presents in vivo and in vitro IMT measurements as well as histological image of H&E stained snap frozen arterial section. Results are summarized in Table 2. According to our results the mean IMT was 0.93 ± 0.12 mm by in vivo US and 0.97 ± 0.18 mm by in vitro ultrasound. The concordance between the two groups was significant: concordance coefficient RC = 0.545, p < 0.0001, 95% confidence interval 0.336–0.755. Concordance analysis and Bland–Altman plots for both parameters are shown in Fig. 3. Average wall thicknesses were calculated in case of n = 34 CCA specimens. Both in vitro and in vivo IMT values correlated well with average wall thicknesses measured at the corresponding postmortem samples (r = 0.76, R2 = 0.571; r = 0.57, R2 = 0.328, respectively). Fig.