Abstract
Purpose :
To determine the functional effects of EphrinB2, a member of the largest known receptor tyrosine kinase family, in corneal epithelium healing and nerve regeneration.
Methods :
The endogenous expression of EphrinB2 was evaluated by immunofluorescent staining (IFS) in mice cornea and trigeminal ganglia (TG) and human corneas. Its level of expression after corneal epithelium injury was determined by IFS and RT-qPCR. For in vitro assays, human corneal limbal epithelial (HCLE) cells and freshly isolated TG neurons were treated with recombinant EphrinB2 in a dose-response manner. HCLE cells were cultured until confluency and healing from scratch assays were measured by time-lapse microscopy. TG neurons isolated from Thy1-YFP mice were treated with medium only, NGF, or EphrinB2. Neuronal growth and neurite extension were quantified with Neurolucida software. The in vivo effects of EphrinB2 were tested on mice (N=5/condition) using a corneal epithelial debridement model. Immediately after injury, mice received subconjunctival injections or pellet implantation of EphrinB2 or vehicle. Corneal epithelium healing was imaged with a slit lamp microscope and analyzed in Fiji. Corneal nerve regeneration was evaluated in flat-mounted corneas stained with B-3 tubulin on day 10, using Neurolucida. Student’s t-test and ANOVA were used for statistical analyses.
Results :
EphrinB2 and the receptors EphB2 and EphB4 were highly expressed in the mouse and human corneal epithelium and the neuronal cell bodies of TG. After injury, EphrinB2 was upregulated in the corneal epithelium for 2 weeks. The scratch assays on HCLE cells show that EphrinB2 at 25 ng/mL induced significantly faster wound closure than control. Similarly, the in vivo data showed that mice receiving EphrinB2 had accelerated wound healing compared to control mice. The average neurite length was greater in isolated TG neurons treated with 50 ng/mL EphrinB2 (p=0.02) compared to the control. In vivo, the average length of subbasal nerves within the debrided area was greater for mice treated with EphrinB2 than for those treated with vehicle.
Conclusions :
EphrinB2 is normally expressed in the corneal epithelium, and after injury, its expression is highly upregulated. Endogenous addition of EphrinB2 increased corneal epithelial proliferation and neuron growth both in vitro and in vivo
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.