Standardization of a new non-invasive device for assessment of arterial stiffness in rats: Correlation with age-related arteries’ structure

Figures

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  Fig. 1. Image illustrating the correct positioning of rat and the two photodiodes: one on right forearm, close to elbow and another one on hindlimb, close to the knee on anesthetized rat, placed on heating plate. Photodiodes are connected to a computer and transit time (TT, ms) between probes (carotid-femoral path) can be detected by pOpet 1.0 software, which calculates pulse wave velocity (PWV, m/s). Distance between photodiodes is included in the software before each animal analysis, for calibration. See animal and room conditions in Methods details section.

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  Fig. 2. Test and re-test schedule for internal consistency, stability and objectivity analyses. Experiment 1, first day of measurement; Experiment 2, second day of measurement; rater A (evaluator A); rater B (evaluator B); test: first measurement on each day for each evaluator; re-test: second measurement (after reconnection of device) on each day for each evaluator. Sequence of tests: 1: test-retest of rater A on Experiment 1; 2: test-retest of rater B on Experiment 1; 3: test-retest of rater A on Experiment 2; 4: test-retest of rater B on Experiment 2; 5: Comparison of first measurement (test) between raters A and B on Experiment 1; 6: Comparison of second measurement (re-test) between raters A and B on Experiment 1; 7: Comparison of first measurement (test) between raters A and B on Experiment 2; 8: Comparison of second measurement (re-test) between raters A and B on Experiment 2; 9: comparison of test results of rater A between Experiment 1 and Experiment 2; 10: comparison of re-test results of rater A between Experiment 1 and Experiment 2; 11: comparison of test results of rater B between Experiment 1 and Experiment 2; 12: comparison of re-test results of rater B between Experiment…

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  Fig. 3. Estimated values of pulse wave velocity (PWV, m/s) in all analyzed groups: young (n = 16), adult (n = 16) and old (n = 10). Transit time was 17.65 ± 0.40 ms, 21.9 ± 0.66 ms and 18.93 ± 0.93 ms for young, adult and old rats, respectively. Distance between probes 6.38 ±0.13cm, 10.5 ± 0.11 cm and 11.5 ± 0.13 cm, for young, adult and old rats, respectively. Significance: One way ANOVA test with Tukey as post-hoc: * vs young, + vs adult, p <0.05

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  Fig. 4. Left panel: Histological cross-sectional photos (10 µm) stained with hematoxylin and eosin, obtained from fragments of aorta (upper), carotid (middle) and femoral (bottom) arteries from 1 rat of each group (young, adult and old). Bars represent 200µm (objective: 40x for aorta artery and 100x for carotid and femoral arteries). Right panel: Pearson correlation between vessel wall cross-sectional area (CSA, µm2) of aorta (r=0.508. p=0.0113), carotid (r=0.519, p=0.0158) and femoral (r=0.683, p=0.0002) arteries. Significance: * p<0.05

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  Fig. 5. Upper panel: Picrosirius red-stained cross-sections of thoracic aorta (left panel), carotid (center panel) and femoral (right panel) arteries of all groups: young (Y), adult (A) and old (O) viewed under light microscope to show collagen fibers. 400 x magnification, Bar= 200µm. Middle panel: Values of % of collagen deposition area on transverse sections of thoracic aorta (left panel), carotid (center panel) and femoral (right panel) arteries. Bottom panel: Pearson correlation between % of collagen deposition area and estimated pulse wave velocity (PWV, m/s) in picrosirius-stained aorta (left panel, r=0.817, p=0.0004), carotid (center panel, r=0.862, p=0.0002) and femoral (right panel, r=0.885, p<0.0001) arteries among all rats. Significance: One way ANOVA test with Tukey as post hoc: * vs young, + vs adult, p<0.001

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  Fig. 6. Elastic fiber networks in transverse sections of thoracic aorta (400x magnification) after Weigert-hematoxylin staining. Red arrow mean thin elastic fiber and black arrow means thicker elastic fibers. Both young and adult aorta show oriented thinner (red arrow) and thicker (black arrow) elastic fibers. Elastin disarray was obvious in aorta sections of older rats with presence of thinner elastic fibers probably from rupture of thicker fibers or remodeling.

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  Fig. 7. Results of estimated pulse wave velocity (PWV, m/s), systolic arterial pressure (SAP) and Pearson´s correlation test between PWV and SAP (r= 0.923, p< 0.0001) of normotensive (Wistar=6) and hypertensive rats (SHR=6). Significance: + vs Wistar, Student T-test, p<0.01; Pearson correlation test *** P<0.0001