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A. Namavari, C. Sano, M. Bakir, S. Jain; A Murine Model for Investigating Corneal Nerve Regeneration. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6215.
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Previous investigators looked at nerve regeneration in several corneal injury models including full- and partial-thickness incisions, cryodestruction, excimer laser ablation, and epithelial debridement. We report herein corneal nerve regeneration in a murine lamellar corneal flap.
A 2.0 mm lamellar corneal flap was created in murine corneas (n=48) using a preset 50 um diamond blade for initial incision and a Grieshaber UltraVit spatula for lamellar dissection. Mice (n=6) were sacrificed and corneas were stained with H&E to confirm the consistency of the surgical technique by measuring the flap/whole cornea thickness ratio. Mice (n=12/timepoint) were sacrificed at days 0, 3, 7 and 14. Corneal flap, underlying bed and surrounding uninjured cornea as well as ipsilateral trigeminal ganglion (TG)and contralateral trigeminal ganglion (TG) and cornea were analyzed by quantitative RT-PCR and quantitative Western blot (LiCor) for nerve regeneration markers (GAP-43), structural nerve proteins (beta-III tubulin and neurofilaments), neurotrophins (NGF, GDNF, BDNF, CNTF, NT-3, NT4/5) and Nerve guidance proteins (EphB1, EphrinB1).
The flap/whole cornea thickness ratio was 69% +- 1%. Quantitative Western blot for beta- III tubulin levels showed a decrease in the flap (25%), peripheral uninjured cornea (29%), and ipsilateral TG (29%) and an increasing pattern in the underlying bed (63%), whereas contralateral TG showed no change at day 14; GAP-43 and NGF levels were initially decreased and then increased in all groups except the flap. BDNF expression was differentially upregulated in the flap (15% increase) compared to the underlying bed (16% decrease) and the peripheral uninjured cornea (no change).
Murine model wherein lamellar corneal flap is fashioned for investigating nerve regeneration is feasible. Expression of neurotrophins and nerve guidance proteins is differentially upregulated in the corneal flap, bed and uninjured cornea.
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