Deep Corneal Nerve Plexus Selective Damage in Persistent Neurotrophic Corneal Epithelial Defects Detected by In Vivo Multiphoton Confocal Microscopy

Abstract

Purpose: To investigate the corneal nerve damage in neurotrophic corneal persistent epithelial defects by an in vivo imaging system using in vivo multiphoton confocal microscopy (MCM) and calcitonin gene-related peptide (CGRP):GFP Tg mice.

Methods: Corneal epithelium was scraped, followed by administering a single dose of benzalkonium chloride (BAK) to develop a neurotrophic persistent epithelial defect. The defect was imaged with fluorescein staining for up to 24 hours, and wound closure percentage (%, WCP) was calculated. CGRP:GFP Tg mice were used in combination with in vivo MCM to acquire in vivo images of corneal nerve before and 24 hours after the creation of a corneal epithelial defect. GFP signals from CGRP-positive nerves were reconstructed into three-dimensional (3D) images, and nerve volume was analyzed. Additionally, corneal mechanosensation was evaluated using Cochet-Bonnet esthesiometry.

Results: BAK-treated eyes showed a significant delay in WCP at 24 hours. In CGRP:GFP Tg mice, CGRP-positive nerves were successfully captured by in vivo MCM and reconstructed into 3D images. BAK-treated eyes showed a significant decrease in both stromal nerve volume and corneal mechanosensation compared to no BAK eyes at 24 hours after corneal scraping, suggesting that BAK impaired the stromal nerves in both structural and functional asides.

Conclusions: Our in vivo corneal nerve imaging system using the combination of in vivo MCM and CGRP:GFP Tg mice demonstrated a longitudinal observation of murine corneal nerves. This system revealed that corneal stromal nerves were selectively damaged in persistent neurotrophic corneal epithelial defects and offered outstanding potential for various applications.

Figure 1.

CGRP-positive nerves in CGRP:GFP Tg mouse imaged by fluorescence microscopy. Corneas of (a) wild-type (WT) and (b) CGRP:GFP Tg mice were imaged using fluorescence microscopy. CGRP-positive corneal nerves are visualized as bright green filaments only in the CGRP:GFP Tg mice. Corneal images obtained by fluorescence microscopy allow for in vivo visualization of the entire corneal nerve network.

Figure 2.

Slices of corneal images in CGRP:GFP Tg mice obtained by in vivo multiphoton confocal microscopy. Images obtained by in vivo multiphoton confocal microscopy depicted the unique distributions of the corneal nerves in CGRP:GFP Tg mice. (a) Terminals of intraepithelial nerve fibers were captured as numerous punctate signals (green) in the most superficial layer of the corneal epithelium. Within the epithelial layer, (b) nerves formed a dense meshwork, which represent the subbasal nerve plexus. (c) Nerves that appeared within the stroma (blue) were thicker than those in the epithelium and represented the subepithelial nerve plexus.

Figure 3.

Reconstruction of 3D images of corneal nerves and stroma. GFP (green) signals and second harmonic signals (blue) from the corneal stroma in CGRP:GFP Tg mice were stitched and processed by image software Imaris. Green and blue signals were isolated from each other and then reconstructed into 3D images.

Figure 4.

In vivo 3D image reconstruction of corneal nerves segmented into epithelial and parenchymal nerves based on corneal parenchymal signals. The 3D images of the CGRP-positive corneal nerves in CGRP:GFP Tg mice were successfully captured by in vivo multiphoton microscopy and processed by Imaris software. (a) Based on the 3D confines of corneal stroma revealed by its second harmonic–generated signal, GFP signals (green) were separated into two categories: extra-stromal (yellow: stromal nerves) and intrastromal (red: epithelial nerves) signals. Each category was then individually reconstructed into 3D images. In low magnification, the entire corneal nerve distribution can be observed. (b) Corneal epithelial nerves are distributed radially around the central corneal vortex, forming a subbasal nerve plexus. From a lateral view with high magnification, (c) the branches of epithelial nerves extending toward the corneal epithelium (intraepithelial terminals) are well represented. (d) Thick stromal nerves consisting of the subepithelial nerve plexus were identified from a posterior view.

Figure 5.

Delayed wound-healing process by benzalkonium chloride administration. Corneal epithelial defects were created with a 2-mm diameter and imaged with fluorescein staining using a blue-free filter every 6 hours, ranging from immediately after the creation of the defect up to 24 hours later. (a) Representative photos of cornea and corneal fluorescein staining showed that the control group showed quick and complete re-epithelialization at 24 hours while the BAK-treated group showed delayed wound healing and incomplete re-epithelialization at 24 hours. Wound closure percentage (WCP) was calculated as the percentage of area epithelialized over the baseline defect area. (b) Wound healing was significantly delayed in the BAK group compared to the control group at 12 hours after defect creation. (c) At 24 hours, the BAK group showed a significantly lower WCP than the control group (P = 0.007).

Figure 6.

Destructive corneal epithelial and stromal nerve structures after corneal debridement and following benzalkonium chloride administration. The 3D models of corneal nerves in CGRP:GFP Tg mice were obtained by in vivo multiphoton microscopy and Imaris at baseline (inset) and 24 hours after corneal epithelial debridement. (a, b) Images show a drastic decrease in epithelial nerve volume (yellow), and the dense subbasal plexus virtually disappeared after corneal epithelial debridement in both the control and BAK groups. Stromal nerves (red) were fragmented and lost their continuity in (b) the BAK-applied eye, while the continuity of the stromal nerves was relatively preserved in (a) the control group.

Figure 7.

Corneal nerves were damaged both functionally and structurally by benzalkonium chloride. Corneal nerves were evaluated from both structural and functional perspectives. (a, b) Based on nerve lengths and volumes obtained in Imaris, the ratios of length and volume between baseline and 24-hour postprocedure of epithelial and stromal nerves were calculated. (a) Nerve length did not show significant differences between the BAK and control groups in both epithelial and stromal nerve length at 24 hours. (b) Although there was no significant difference between the BAK and control groups in epithelial nerve volume, stromal nerve volume at 24 hours was significantly (P = 0.0095) less in the BAK group than in the control group. (c) Corneal mechanosensation was measured using Cochett–Bonnet at baseline and 24 hours after corneal debridement. The BAK group showed significantly (P = 0.0095) impaired mechanosensation compared to the control group at 24 hours.