Abstract
Purpose :
In the setting of human corneal transplants and refractive surgery, incomplete stromal reinnervation and/or axonal degeneration pose clinical concerns. Corneal Schwann cells (cSCs) are glial cells that ensheath and provide trophic support to stromal axons. To investigate cSCs during corneal tissue preservation, as well as to study how stromal injury impacts cSCs, we analyzed the expression of cSC markers using ex vivo mouse corneal and eye-globe models.
Methods :
We used adult wild-type (WT) C57LB6 mice, and PLP-eGFP transgenic mice (Bargagna-Mohan et al., J. Neurosci. Res., 2020). For the organ culture model, WT (n= 6) and PLP-eGFP (n=4) corneas including limbal tissue were excised, and cultured at 37oC in Opti-minimal essential medium containing 4% Dextran, 8% fetal bovine serum, and a cocktail of antibiotic/antimycotic for 6 days (6d). For the whole-eye model, after eye enucleation, using a 1.5-mm diameter trephine a circular incision was made on WT corneas to injure the stroma. Control (uninjured, n= 5) and injured eye-globes (n= 10) were cultured for 6d, and after fixation, corneas were isolated. All corneas were then processed for whole mount immunohistochemical analysis using the axonal marker b-III tubulin and cSC markers PLP-1 and DKK. Stained tissues were examined by epifluorescence microscopy.
Results :
In the organ culture model, 6d post-culture caused a loss of axonal density mostly in the central zone in both WT and PLP corneas. At the limbus/peripheral zone, b-III tubulin+ axons co-stained with PLP-1+ and DKK+ cSCs, whereas in the mid-central cornea b-III tubulin staining was fragmented, and showed some axons lacking PLP-1 and DKK staining. In the whole-eye model, axonal integrity was more evident at the proximal sites where viability of cSCs was higher. At the distal sites trephine injury caused b-III tubulin fragmentation and downregulation of PLP-1 and DKK expression, suggesting these severed axons are highly sensitive to cSC loss.
Conclusions :
Our results suggest that cSCs in the central cornea lose expression of PLP-1 and DKK after 6d in culture. This loss of expression appears to be associated with a certain degree of axonal degeneration, which is possibly due to a loss of trophic support from cSCs. Further studies will explore methods of how to best support the cSCs, and thus, the neural network that is crucial for corneal function.
This is a 2021 ARVO Annual Meeting abstract.