Figure 3.

Time course of corneal nerve regeneration after injury. (A) Entire view of corneal nerve architecture immediately following the surgery. All epithelial and anterior stromal nerve branches within a total area of about 3.14 mm² (Φ = 2 mm) were removed by using a corneal rust ring remover under an Evolution Zoom Microscope (63122; Seiler Instrument Inc., St. Louis, MO, USA), but deeper stromal nerves still were present. Representative images were labeled with monoclonal rabbit anti-PGP9.5 antibody and recorded at a magnification of 10× objective. A total of 35 images were merged together to reconstitute the entire view of corneal nerve architecture. Three representative videos showing the details of corneal nerve regeneration in the central corneas recorded on 1 and 3 days and 3 weeks after injury are provided in the supplementary materials (Supplementary Video S1, S2, S3). (B) Representative images labeled with PGP9.5 antibody and recorded from postoperative 12 hours to 4 weeks showing the nerve regeneration. Red arrows indicate the newly regenerated nerve buds. Yellow arrows indicate the new epithelial nerve bundles originated from the underlying stromal nerve branches. Yellow arrow at 4 weeks indicates the vortex running clockwise. (C) Central subepithelial PGP9.5-, SP-, and TRPM8-labeled nerve density at different time postinjury (pi). Data at each time point represent the average percentage of subepithelial nerve density postinjury versus preinjury (N). At each time point, 8 mice were euthanized, 4 corneas were double labeled with PGP + SP, and 4 corneas were double labeled with TRPM8 + SP. All the time points are significantly decreased respect to noninjury values (P < 0.05). (D) Changes in the number of PGP9.5, SP and TRPM8 nerve terminals pi. Significant difference at 8 to 15 weeks compared to noninjured (P < 0.001). Representative images showing the epithelial terminals at 1, 3, 5, and 7 days and at 3, 6, 8, and 12 weeks are provided in Supplementary Figure S1 (recorded at a magnification of 10× objective). E and F are representative images showing the entire subbasal nerves labeled with PGP9.5, SP, and TRPM8 antibodies at early (12–72 hours) and late (1–15 weeks) stages. At each time point, a total of 16 images recorded from the central cornea of 4 mice were calculated for each antibody. Data are expressed as average ± SD.