Collagenase treatment reduces the anisotropy of ultrasonic backscatter in rat myocardium by reducing collagen crosslinks

FIGURE 1. Effect of Collagenase Treatment on Ultrasonic Backscatter. (a) Mid‐myocardial short‐axis ultrasonic backscatter of one control (top) and one collagenase treated (bottom) heart at three time points. Horizontal center represents the generalized backscatter at each of the four cardinal positions analyzed. The LV positions as shown on the inset for (b) are: ●‐anterior myocardium; ►‐lateral myocardium; ■‐ posterior myocardium, ◄‐septal myocardium. Scale bar shows 50 μm square for full short‐axis views (top/bottom); center images are each 25 μm square. (b) Statistical analysis. Treatment duration and myocardial position show a significant interaction, with 30 min data suggesting an increase in backscatter intensity in myocardial sections that align parallel to the angle of insonification, and indicate minimal alteration in backscatter intensity in myocardial locations that are perpendicular to the direction of insonification. Treatment and treatment duration show a significant interaction, with controls showing little or no change in intensity over 30 min, while collagenase treatment increases backscatter intensity of septal and lateral wall positions (triangles) for each timepoint.

FIGURE 2. Anisotropy Index. (a) The anisotropy index was determined from four combinations from the difference in backscatter at the four cardinal positions (Figure 1b). Symbols, with gray lines connecting the originating data positions: ♦‐anterior minus lateral; ▲‐posterior minus lateral; ▼‐ posterior minus septal, ★‐ anterior minus septal. (b) The Average Anisotropy Index was calculated from the mean absolute difference of the anisotropy index, that is, approximately the magnitude of the gap between the anterior or posterior walls and the lateral and septal walls. The anisotropy index shows no change over time in the control but is reduced as the tissue is treated with collagenase solution.

FIGURE 3. Histological Analysis of collagen. (a) Example circularly polarized light micrographs of hearts in the three treatment groups. Visible tinseling after 10 min of collagenase treatment. (b) Collagen to tissue ratio for the three treatment groups showing no change in collagen content, suggesting the amount of collagen is not changing over the course of the treatment. (c) Hue was used to estimate fiber size (orange thick, green thin). The relative percent of the hues differed, but hue was not dependent on treatment. (d) Angular deviation shows a dependence on treatment, suggesting that disarrangement of the collagen throughout the course of study treatment. (e) Myocyte (Muscle) Birefringence for the three treatment groups, showing no change, indicating no damage to myocytes.

FIGURE 4. Hydroxyproline content in the three treatment conditions showing a reduction in crosslinking after collagenase treatment, significant after 10 min.

FIGURE 5. Effect of collagen orientation and crosslinking on ultrasonic backscatter. (a) Increased ultrasonic backscatter is generated by collagen oriented perpendicular to the angle of insonification. When ultrasound is insonified parallel to the collagen fibers, the reflection back to the ultrasound source (backscatter) is reduced. (b) In the myocardium as described with the data in this study, collagenase treatment corresponds with increased angular deviation of the collagen fibers, and possibly the myocytes, and a reduction in collagen crosslinking. This is related to an increase in ultrasonic backscatter, likely generated by the fibers that are no longer aligned with the angle of insonification. A reorientation of fibers toward perpendicular (compared to the angle of insonification) would result in increased ultrasonic backscatter.