Abstract
Purpose :
EphrinB2, a cell surface transmembrane ligand, can enhance corneal epithelial healing and neuronal growth in vitro. This study assesses the effect of exogenous EphrinB2 in promoting corneal wound healing in vivo using three distinct delivery methods.
Methods :
The neuro-epithelial role of EphrinB2 was verified in-vitro using human corneal limbal epithelial (HCLE) cells and trigeminal ganglia (TG) neurons. The healing progress of scratch assays on confluent HCLE cells was assessed via time-lapse microscopy. TG neurons from Thy1-YFP mice were treated with vehicle, NGF, or EphrinB2, and neuronal growth was quantified using Neurolucida software. In vivo, adult C57 male and female mice (6-8 weeks old, n=6) underwent central 2mm corneal epithelium debridement. Animals received three different eye delivery methods and control mice received only vehicle. The first group received 5 ul subconjunctival injection of recombinant EphrinB2 (R&D System cat # 7397-EB) in PBS. The second group received a pellet insertion with EphrinB2 embedded in HEMA:Sucralfate:GF (5:1:4). The third group was treated with topical EphrinB2 mixed in eye drops containing hyaluronidase, three times daily for five days. Corneal epithelial closure was assessed via slit lamp and analyzed with FIJI. Corneas were harvested at 14 days, immunostained with B-3 Tubulin, and imaged using a Zeiss AxioObserver fluorescent microscope. Nerve regeneration was traced and quantified using Neurolucida and treatments compared using student’s t-test.
Results :
EphrinB2 significantly accelerated wound closure in HCLE cells and induces potent neurite growth from TG neurons, exhibiting superior results than NGF treated neurons (p=0.02). In vivo, epithelial closure occurs faster in the subconjunctival group healing earlier than vehicle controls. No substantial differences were observed in the pellet or eye drop groups. The analysis of nerve regeneration showed that only pellet insertion demonstrated a significant effect on sub-basal nerve recovery (p=0.0317).
Conclusions :
Among the three delivery methods tested, pellet insertion exhibits the most promise in promoting sub-basal nerve recovery following eye injury, which is probably due to the slow release of EphrinB2. Future studies will incorporate the use of nanoparticles to provide longer permanency of the therapeutics in the injured cornea.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.