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Tiffany C. Russ, Jiucheng He, Donna Neumann, Nicholas G. Bazan, Haydee E. Bazan; Stimulation of Neurite Outgrowth in Cultured Trigeminal Ganglion Cells by Neuroprotectin D1. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4618.
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Previous studies from our laboratory have shown an increase in nerve regeneration 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 (Esquenazi et al, IOVS, 2005, 46: 3121; Cortina et al, IOVS, 2010, 51:804). Both NGF and PEDF in combination with DHA stimulate the synthesis of neuroprotectin D1 (NPD1). Trigeminal ganglion sensory neurons are the main source of corneal innervations. In this study, we used primary cultures of mice trigeminal ganglion neuronal cells to test the effects of different neurotrophins and their combinations with DHA on axon growth.
Trigeminal ganglia of five-day-old Swiss Webster mice were harvested, triturated and cultured in DMEM F-12 plus 10% Fetal Bovine serum. 24 h after plating the cells, the media was changed to DMEM F-12 plus 0.5% Horse Serum. 5-Fluoro-2’-deoxyuridine and Uridine, mitotic inhibitors were added to both types of media. Neurons were supplemented with 50ng NGF or 50ng PEDF plus 50nM DHA or with 50ng NPD1. Images were taken of each condition at 1 and 2 days after treatment using MetaVue, and measurements of soma and neurite outgrowth were recorded. Immunofluorescence using βIII-tubulin antibody was performed to identify the neurons and their axons.
No differences with respect to the controls in the cell bodies were observed after the different supplementations. One day after stimulation, a 50% increase in axonal outgrowth was found in cultures stimulated with NGF+DHA and PEDF+DHA. Combination of NGF+PEDF+DHA does not further increase axon growth. NPD1 stimulates axon growth with a 40% increase with respect to the control at 1 day and a 50% increase at 2 days after supplementation.
We established an in vitro model that responded to DHA in combination with PEDF and NGF as in our in vivo studies. Using this system, we will be able to investigate the signaling mechanisms involved in corneal nerve regeneration as well as make comparisons to the action of metabolites of the arachidonic acid cascade.
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