The Focal Induction of Reactive Oxygen Species in Rats as a Trigger of Aortic Valve Degeneration

Figures

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  Figure 1. For the ROS, laser, and sham groups, a laser fiber was inserted under echocardiographic control. (a) The label “1” in (b) shows the fiber; the circle in (b) indicates the aortic valve. The label “2” in (b) indicates the left ventricle. (c) A typical specimen of the aortic valve in the aortic root with the adjacent parts of the myocardium and the aorta.

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  Figure 2. Indirect ROS detection by 8-OHdG fluorescence. After the initial procedure, the content of generated ROS was assessed indirectly via 8-OHdG fluorescence at t = 0 and t = 8 d ((a,d) = ROS; (b,e) = laser; (c,f) = sham at t = 0). Quantitative analysis of 8-OHdG (mean gray value normalized to the positive control of the staining run) displayed an increase in the laser and ROS groups at t = 0, with a significant difference between the ROS and the sham group (g). On the contrary, no differences between the groups were found at t = 8 d (h). 8-OHdG (red) with DAPI counterstain (blue); scale bars = 50 µm. ** p < 0.01.

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  Figure 3. Analyses of the cleaved-caspase-3 activity. Neither the cross-section images of the ROS groups at t = 0 (a) and t = 8 d (b) nor those of the corresponding laser (c,d) and sham groups (e,f) displayed distinctive cleaved-caspase-3 activity. Immunohistochemistry: cleaved-caspase-3 (DAB, brown) with hemalum counterstain (blue); scale bars = 1000 µm.

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  Figure 4. Concentration of monocytes was quantitatively assessed via CD 68 expression. For this purpose, the percentage of CD 68-specific signals in the total tissue area was calculated. Representative cross-section images at t = 8 d (ROS group: (a,c) = annulus, (b,d) = cusp; laser group: (e,g) = annulus; (f,h) = cusp; sham group: (i,k) = annulus; (j,l) = cusp). Quantitative analysis (m–p). CD 68 (red) with DAPI counterstain (blue); scale bars = 50 µm.

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  Figure 5. Analysis of the VCAM-1 expression. Representative cross-section images of the ROS groups at t = 0 (a,c) and at t = 8 d (b,d) displayed the increased expression of VCAM-1 compared to their respective control groups (laser group at t = 0 (e,g) and t = 8 d (f,h); sham group at t = 0 (i,k) and t = 8 d (j,l)). Semiquantitative analysis of VCAM-1 expression ((m–p); 0 = no expression; 5 = maximum). Immunohistological staining of VCAM-1 (DAB, brown) with hemalum counterstain (blue); scale bars = 1000 µm. * p < 0.05.

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  Figure 6. Analysis of the endothelial layer. While the cusps (a–l) displayed hardly any remaining endothelial layer (red = vWF) at any timepoint, neither in the ROS (a–d) nor in the laser (e–h) or the sham groups (i–l), the annulus area displayed a continuous, lumen-sided endothelial layer regardless of the timepoint or group (m–x). vWF (red), vimentin (green), with DAPI counterstain (blue); scale bars = 100 µm.

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  Figure 7. Analysis of degenerative calcification. Representative pairs of cross-section images and their details for all groups at t = 0 (ROS (a,c), laser (e,g), sham (i,k)) and at t = 168 d ((ROS (b,d), laser (f,h), sham (j,l)). Von Kossa staining: calcification (brownish/black) measured with nuclear fast red counterstain (red); scale bars = 1000 µm. This analysis showed hardly any hint of calcification in the cusps at t = 0 (m) but a slight increase over time, especially in the ROS group, which achieved significant differences at t = 168 d (n). * p < 0.05. On the contrary, the results of the semiquantitative analysis (von Kossa score: 0–5) displayed slight hints of calcification in the annulus areas in some of the animals at t = 0 and t = 168 d, regardless of the groups, with hardly any aggravation over time (o,p).

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  Figure 8. Analysis of the mean transvalvular pressure gradient. The measurement of the mean transvalvular pressure gradient just before termination of each experiment (a) displayed no significant differences at t = 0 (b), but an increase in all the groups over time up to t = 168 d (c) with a significant difference between the ROS and laser group as well as the ROS and sham group. * p < 0.05; ** p < 0.01.