Visualization of corneal innervation after syngeneic corneal transplantation using in vivo multiphoton confocal microscopy.

Abstract

Purpose : Nerve regeneration after corneal transplants has typically been assessed by histological methods, which require mouse euthanasia. The goal of this study is to visualize corneal innervation in the donor button after syngeneic corneal transplantation using calcitonin gene-related peptide (CGRP)-GFP BAC transgenic (Tg) mice by in vivo corneal nerve imaging.

Methods : Syngeneic corneal transplantation was performed on C57BL/6-background CGRP-GFP BAC Tg mice, in which CGRP+ nerves are labeled with GFP, as recipients and wild-type C57BL/6 as donors. Corneal nerves were imaged at weeks 2 and 5 post-transplant using in vivo fluorescence microscopy. Afterward, the nerve and graft area were manually segmented using custom MATLAB software, and their ratio was calculated. At week 5, corneal nerves were imaged using in vivo multiphoton confocal microscopy (MCM) (Leica SP8 DIVE), and 3D nerve images were reconstructed using Imaris software. Cochet-Bonnet Esthesiometry was performed on the central corneal graft at week 5.

Results : CGRP-GFP BAC Tg mice underwent syngeneic corneal transplants (C57BL/6 to C57BL/6) and the grafts maintained clarity with partial opacity. While obtaining fluorescent microscopy images took less time than in vivo MCM, nerve tracing and quantification were limited by their low resolution. In vivo MCM demonstrated nerve re-growth inside the corneal graft by week 5 post-surgery (n=2 representative transplants). Importantly, 3D images showed a newly formed neural network within the donor stromal bed on week 5. However, these nascent stromal nerves were thinner than those found in an unmanipulated cornea. Corneal mechano-sensation was present in the central cornea at 5 weeks post-surgery, suggesting that the newly formed nerves are functional.

Conclusions : It was demonstrated that the innervation of syngeneic corneal graft in CGRP-GFP BAC Tg recipient mice can be readily monitored and analyzed using in vivo MCM. Interestingly, innervation of corneal grafts has a distinct pattern and correlates with graft clarity. This strategy will enable a better understanding of corneal nerve regeneration after corneal transplantation and will allow insight regarding how immune rejection will alter patterns of innervation in allogeneic corneal transplants.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.