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Tiffany C. Russ, Jiucheng He, Nicolas G. Bazan, Haydee E. Bazan; NPD1 and PEDF + DHA Enhance Neurite Outgrowth and Projection of Trigeminal Ganglion Neurons towards Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1813.
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Nerve fibers from the trigeminal ganglion neurons enter the stroma and divided in branches that penetrate the epithelium, giving rise to an intense array of fine fibers and nerve terminals. Alterations in corneal innervations result in epithelial damage. Increased nerve regeneration occurs when nerve growth factor (NGF) or pigment epithelial derived factor (PEDF), in conjunction with the ω-3 fatty acid docosahexaenoic acid (DHA), is applied to rabbit corneas after refractive surgery (IOVS, 2005, 46: 3121; IOVS, 2010, 51:804). Both NGF and PEDF in combination with DHA stimulate the synthesis of neuroprotectin D1 (NPD1). In this study, we used co-cultures of mice trigeminal ganglion neuronal cells and epithelial cells to test the effects of these neurotrophic factors on axon growth towards corneal epithelium.
Trigeminal ganglia of three- to five-day-old Swiss Webster mice were harvested, triturated, and cultured in DMEM F-12 plus 10% Fetal Bovine serum in the co-culture’s inner chamber (8 mm cloning cylinder). CnT-20 was used in the outer chamber. 5-Fluoro-2’-deoxyuridine and Uridine, mitotic inhibitors, were added to DMEM F-12 plus 10% Fetal Bovine serum, and 10 µM cytosine arabinoside was added to Cnt-20. After 24 h, the outer chamber was rinsed with CnT-20 without cytosine arabinoside and corneal epithelial cells were plated. Two days later, epithelial cells were supplemented with 50nM NGF, 50nM DHA plus 50nM PEDF, and 50nM NPD1. Images were taken of each condition at 1 and 7 days after treatment using MetaVue, and differences in neurite outgrowth towards the epithelial cells were measured.
Neurites grew out from the cloning cylinder in the direction of the epithelial cells. One day after stimulation, an increase in axonal growth was observed towards corneal epithelial cells treated with PEDF plus DHA or NPD1 in comparison with the control.
Stimulation of corneal epithelial cells with PEDF in the presence of DHA or with NPD1 increase axon outgrowth towards the epithelium. This approach will allow us to investigate the corneal signaling mechanisms involved in the interaction between the corneal epithelium and nerves.
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