Figure 5.

Immunohistochemical Analysis of α-SMA, Cell Proliferation, and Thickness after Wounding

(A–C) Frozen sections of corneas 6 weeks after wounding were immunostained for α-SMA (green), DAPI (blue). (A) UnWnd, (B) Wnd-NTC with magnified inset, and (C) Wnd-US09 with magnified inset. Scale bar, 0.5 mm. (D) Compared to UnWnd, Wnd-NTC demonstrated a 5.77-fold increase in α-SMA immunostaining (p < 0.05). Compared to Wnd-NTC, α-SMA immunostaining after Wnd-US09 treatment was reduced by 83.6% (p < 0.05). The comparison between UnWnd and Wnd-US09 was not significant. (E and F) Cell proliferation was analyzed by the object counter plugin in ImageJ software. “Inside the wound” is denoted by the anterior cornea demarcated by the collagen III scar. “Outside the wound” is the posterior cornea beneath the scar. These counts were normalized by the total area of each portion to generate a nuclei density measurement. (E) Compared to UnWnd, Wnd-NTC demonstrated a 1.61-fold increase in cell proliferation (p < 0.01). Compared to Wnd-NTC, cell proliferation after Wnd-US09 treatment was reduced by 29.9% (p < 0.05). The comparison between UnWnd and Wnd-US09 was not significant. (F) Cell proliferation below the scar, in the stroma down to the endothelium. All relationships were not significant. (G) Corneal thickness was measured at pixel resolution in these thresholded images as the distance across the nonzero region, and thickness is averaged across the entire cornea. Wnd-NTC trended toward a slight decrease in thickness (p = 0.05). Wnd-US09 treatment restored corneal thickness to non-wounded parameters. N = 6 rabbits per condition.