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Azucena Kakazu, Nicolas Bazan, Haydee Bazan; Neuroprotectin D1 Stimulates the Expression and Secretion of Nerve Growth Factor in Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):546.
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© ARVO (1962-2015); The Authors (2016-present)
Nerve growth factor (NGF) is a neurotrophic factor expressed in the corneal epithelium that promotes cell proliferation and wound healing. It is responsible for the axonal growth and survival of sensory neurons. Neuroprotectin D1 (NPD1) is a lipid mediator derived from docosahexaenoic acid (DHA) with anti-inflammatory and neuroprotective actions. Synthesis of NPD1 is stimulated in corneal epithelial cells treated with pigment epithelial derived factor (PEDF) in conjunction with DHA. Recent studies in our laboratory showed that topical treatment of NPD1, applied to rabbit corneas after experimental surgery, increase corneal nerve regeneration and enhanced neurite outgrowth of trigeminal ganglion neurons in culture (ARVO 2012). The exact mechanism by which NPD1promotes nerve regeneration is not understood. The purpose of this study was to investigate if NPD1 and/or its precursors PEDF plus DHA stimulate NGF synthesis.
First passage rabbit corneal epithelial cells (RCEC) were used. The cells were grown in serum-free medium (CnT-20). Once they reached 70-80 % confluence, the cells were starved overnight and then stimulated with 50nM NPD1 or with the combination of 50ng/ml PEDF and 50nM DHA for different times. Changes in NGF mRNA expression were assayed by PCR performed using Taq PCR Master Mix Kit (Qiagen) with specific primers for rabbit. Secretion of NGF peptide was measured in the tissue culture supernatant by ELISA.
NGF gene expression increased significantly after 3h and 6h of NPD1 stimulation; at 16h gene expression started to decrease. There was an increase of NGF secreted into the medium from the cells in the presence of NPD1 or PEDF+DHA. After 48h, NGF stimulation increased between 40 and 50% when NPD1 was added to the medium; in presence of PEDF plus DHA, the NGF increment was around 30%.
The results suggest that NPD1 and its precursors PEDF plus DHA promote regenerative corneal innervation by modulating NGF gene expression, followed by its synthesis and secretion.
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