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Zan Pan, Shima Fukuoka, Natalia Karagianni, Victor H. Guaiquil, Mark I. Rosenblatt; Effect of VEGF on Corneal Nerve Growth and Corneal Wound Healing. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3561.
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To evaluate whether VEGF stimulates corneal nerve regeneration and corneal epithelial wound healing.
Trigeminal ganglia (TG) were harvested from Thy1-YFP mice. TG neurons were isolated and cultured. Following pretreatment of either VEGF receptor type 2 (VEGFR2) inhibitors SU1498 and Ki8751, neutralizing antibodies for VEGFR1 and VEGFR2, or soluble FLT1 (sFLT1), neurons were coincubated with 50ng/ml VEGF. Neuron images were taken with fluorescence microscopy and analyzed using Neurolucida. To evaluate whether VEGF stimulates corneal nerve regeneration in vivo, two approaches of VEGF delivery were used. First, VEGF impregnated sucralfate/hydron pellets were implanted into a corneal stromal micropocket midway between the central cornea and limbus in Thy1-YFP mice. Mice then received corneal epithelium debridement 1 day after pellet implantation. Cornea nerves were imaged via fluorescence microscopy and were analyzed with Neurolucida. Corneal angiogenesis was monitored via slit lamp. The second approach is to administrate 5 ul of 100 ng/ml VEGF subconjunctivally at the superior temple area of bulbar conjunctiva after 2-mm central corneal epithelium debridement made in C57BL/6 mice. Epithelial defect areas were recorded with fluorescence camera, and corneal nerve regeneration was assessed in wholemount corneas with β3-tubulin antibody. VEGF expression in the corneal epithelium and trigeminal nerve in corneal wound mice were assessed by real-time PCR. To determine if VEGF directly influences corneal epithelial cell wound healing, the scratch assay was used. A linear scratch was made in confluent human corneal limbal epithelial (HCLE) cell culture. Cells were then cultured in media supplemented with VEGF, or EGF. Recovery of epithelial defect was photographed by microscopy and evaluated by Photoshop.
VEGF enhanced TG neuronal growth, which was suppressed by either SU1498 and Ki8751, neutralizing antibodies for VEGFR1 and VEGFR2, or soluble FLT1 (sFLT1). Following corneal epithelial debridement, there was an increase in VEGF and its receptors expression in corneal epithelium and TG nerve. Mice that received VEGF pellets showed an increase in corneal nerve regeneration. Mice that received VEGF subconjunctivally, however, did not demonstrated changes in corneal epithelial and nerve recovery. In both VEGF delivery models, no angiogenesis was observed. VEGF failed to directly promote cultured HCLE wound healing.
VEGF enhances TG neuronal cell growth and regeneration in vitro and in vivo following corneal epithelial debridement when delivered as pellets in corneal micropocket. However VEGF does not concomitantly accelerate the corneal epithelial recovery.
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