[Early histological changes detected by confocal microscopy in patients with advanced keratoconus receiving collagen cross-linking therapy].

Objective: To investigate the early histological changes by confocal microscopy of patients with advanced keratoconus receiving collagen cross-linking therapy. Methods: In this prospective case series study, confocal microscopy was used to observe 23 patients (32 eyes) who were diagnosed with advanced keratoconus and treated with collagen cross-linking at the Department of Ophthalmology, Chinese PLA General Hospital from September 2017 to March 2019, aged (26±10) year. All patients were examined before and at 1 week, 1 month and 3 months after the therapy. The tissue structure changes, the density of nerve fibers, stromal cells and endothelial cells, and the depth of the corneal stroma were recorded and compared. The overall differences at different times were compared by repeated measurement analysis of variance or Friedman test, and the pairwise comparison was corrected by LSD-t test or Bonferroni test. Results: One week after collagen cross-linking, the epithelial cells were in the repair stage, showing an increased nucleolar size and an enhanced reflection, and the activated cells could be detected under the epithelium. The superficial corneal stroma was swollen and spongiform, while the deep corneal stroma was patchy or cord-like, scattered and with a strong reflection. One month after the therapy, epithelial cells recovered, subepithelial nerves began to grow, the superficial corneal stroma still showed a spongy structure, and the reflection was further enhanced. The activation of the deep corneal stroma exhibited as thicker plaques or cord-like structure. Three months after the therapy, the continuous elongation of single nerve fibers could be detected occasionally. There was statistically significant difference in the density of nerve fibers before and early after the therapy (F=233.30, P<0.001). Compared with the preoperative value [(14.60±2.57) mm/mm2], the density of subepithelial nerve fibers decreased significantly in the early postoperative period, which was (0.51±0.31), (3.65±2.21) and (8.50± 4.02) mm/mm2, respectively, at 1 week, 1 month and 3 months, and there were significant differences between different time points (all P<0.05). There was also statistically significant differences in the density of anterior stromal cells before and early after the therapy (χ2=92.48, P<0.001). Compared with the preoperative value [347.00(345.00,395.75) cells/mm2] the density of anterior stromal cells decreased significantly in the early postoperative period, which was 2.00(1.00,5.75), 2.50(1.00,5.75) and 79.00(64.25,94.00) cells/mm2, respectively, at 1 week, 1 month and 3 months, and there were significant differences between different time points (all P<0.05). Within 3 months after the therapy, the depth of the corneal stroma observed by confocal microscopy ranged from 245 to 536 μm, with an average of (400.56±86.12) μm. Histologically, the depth of the corneal stroma ranged from 245 to 536 μm [average, (402.13±89.20) μm], from 251 to 527 μm [average, (399.88±85.92) μm] and from 259 to 530 μm [average, (399.69±85.94) μm] at 1 week, 1 month and 3 months, respectively, with no significant difference (F=0.797, P=0.455). There was no significant difference in the density of posterior stromal cells [(260.6±33.2) cells/mm2 preoperatively, (264.4±44.5) cells/mm2 at 1 week, (263.9±37.6) cells/mm2 at 1 month and (266.3±40.2) cells/mm2 at 3 months] and endothelial cells [(2 707±152.6) cells/mm2 preoperatively, (2 704±148.5) cells/mm2 at 1 week, (2 705±152.6) cells/mm2 at 1 month and (2 704±150.1) cells/mm2 at 3 months] between different time points (F=1.380, 1.011; P=0.259, 0.351). Conclusions: Confocal microscopy is able to clearly document the early morphological characteristics after collagen cross-linking in the treatment of keratoconus, including the epithelial and subepithelial nerve injury repair, the spongiform superficial corneal stroma, the patchy or cord-like deep corneal stroma, and the relatively stable stromal depth change.