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M. Zhang, M. I. Rosenblatt; Expression of Semaphorin and VEGF Ligands and Receptors Following Superficial Corneal Injury. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1961.
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To investigate the expression of semaphorin and VEGF ligands and receptors in the corneal epithelium and trigeminal ganglia in a murine model of superficial corneal injury.
A 2 mm circular corneal epithelial defect was made in anesthetized mice using a scalpel blade. Injured corneas were evaluated for the degree of neuronal injury using immunostaining for anti-neurofilament (a neuronal marker). Epithelium from the injured ocular surface as well as the ipsilateral trigeminal ganglion from sacrificed mice who were either not injured or who were 1, 3, and 5 days post-injury were dissected and subjected to RNA extraction. RNA isolated from these tissues was analyzed by non quantitative PCR with primers specific for semaphorin 3A, 3B, 3C, 3D, 3E, 3F, plexin A3, plexinA4 neuropilin-1,neuropilin-2,VEGF164 and VEGFR2. Further quantitative PCR (qPCR) was performed on the same RNA using assays (Taqman) for semaphorin 3A and 3F, plexin A3, neuropilin1,VEGF-A and VEGFR2.
In addition to the obvious loss of corneal epithelium, superficial corneal scraping results in complete loss of the sub-basal neuronal plexus and intra-epithelial nerve endings. By two days post-injury, the epithelial defect had closed, but only minimal neuronal regeneration was observed. By non-quantitative PCR, semaphorin 3A, 3B, 3C, 3D, 3E, 3F, plexin A3, neuropilin-1,VEGF164 and VEGFR2 were expressed both in corneal epithelium and trigeminal ganglion (although yielding bands of different intensity when analyzed by agarose gel electrphoresis), while plexinA4 and neuropilin-2 were expressed only in trigeminal ganglion. Further investigation by qPCR revealed that at one day post-injury, semaphorin 3A and VEGF164 increased compared to control in epithelium samples, while levels of expression similar to control were present at days 3 and 5. Conversely, the expression of semaphorin 3B, plexinA3 and neuropilin-1 in epithelium were decreased at day 1 relative to control and then returned to baseline levels at days 3 and 5. The expression of semaphorin 3A,VEGF-A and VEGFR2 was increased in trigeminal ganglion at days 1, 3, and 5.
Following superficial corneal injury, the corneal epithelium and trigeminal ganglion display altered expression of semaphorin and VEGF ligands and receptors. The regulation of these axonal guidance genes after injury suggests a role for these molecules in the regeneration of corneal nerves.
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