Subconjunctival VEGF-A and VEGF-B treatment post-corneal epithelial debridement influences neuronal VEGFR1 and VEGFR2 tyrosine phosphorylation in Thy1-YFP mice

Abstract

Purpose : We have previously characterized VEGFR1-R2 heterodimers, VEGFR1-R1 and VEGFR2-R2 homodimers in neuronal cells with expression levels being different from that in endothelial cells. While differences in VEGF signaling between neurons and blood vessels have been characterized, the functional significance of VEGFR1-R2 heterodimers in regenerating corneal nerves is unknown. In these studies we have investigated the effect of subconjunctival VEGF-A and VEGF-B treatment after corneal epithelial debridement on VEGFR tyrosine phosphorylation in trigeminal ganglion neurons of Thy1-YFP mice.

Methods : All animal experiments were conducted according to guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Thy1-YFP mice were subjected to a 2 mm central corneal epithelium debridement using an Algier brush followed by fluorescein staining to determine the debrided epithelium area. Mice received a first subconjunctival injection of 5 µl of growth factors (either VEGF-A or VEGF-B; 10 µg/ml), vehicle control (PBS) or positive control (NGF; 10 µg/ml). On Days 2, mice received a second subconjunctival injection as described above. Epithelium recovery was imaged using a slit lamp at 0h, 24h, 30h and 48h. After this, mice were sacrificed and trigeminal ganglia were fixed in 4% paraformaldehyde and OCT frozen sections (10 µm thickness) were prepared and processed for immunostaining with anti-VEGFR1 and anti-VEGFR2 antibodies as well as specific anti-phosphotyrosine antibodies directed against VEGFR1 and VEGFR2 and fluorescent confocal microscopic visualization.

Results : VEGF-A and VEGF-B treated mice demonstrated faster corneal epithelium recovery at 48h versus 0h, 24h and 30h post-corneal epithelial debridement as compared to the control group. The NGF treatment group demonstrated fastest corneal epithelium recovery at 48h versus all other groups. VEGF-B treated mice showed slightly faster epithelium recovery as compared to VEGF-A. Confocal microscopy demonstrated differences in tyrosine phosphorylated VEGFR1 and VEGFR2 in VEGF-A and VEGF-B treatment groups.

Conclusions : Changes in VEGF receptor tyrosine phosphorylation after growth factor addition following injury could be related to corneal wound healing mechanisms involved in nerve regeneration and patterning of nerve fibers in the corneal epithelium.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.