Combined treatment of neurotrophic factors and cytokines enhances survival of neonatal retinal ganglion cells in vitro

Abstract

Purpose: Neurotrophins, especially BDNF, are the best-studied neuroprotective molecules for retinal ganglion cells (RGCs). Recently, cytokines are shown to increase the survival of RGCs in culture. In this study we examined the effects of combined treatments of neurotrophins and cytokines on the survival of RGCs in vitro. Methods: RGCs were retrogradely labelled with Di-Asp from both superior colliculi in postnatal day one SD rat pups three days before the procedures used for cell culture. Following mechanical dissociation of the retinae, 50 µl of cell suspension were seeded at 4∼5X104 cells/well in poly-L-lysine/laminin precoated 96-well plates. Four hours later, after cells attached to the bottom of the well, 50 µl/well of fresh completed DMEM with or without addition of neurotrophins, cytokines or the combinations of the two was added. After 1, 2, 4, 7 days, fixed Di-Asp prelabeled RGCs were counted under inverted fluorescent microscope. Results: The percentage of survived RGCs decreased gradually to less than 20% after one week in culture. The results demonstrated that either BDNF, IL-4, IL-6 alone or a combination of BDNF with IL-2 plus IL-4, CNTF with IL-2 plus IL-4, BDNF with IL-6, CNTF with IL-6 increased the survival of RGCs (20%) after one-day treatment. A combination of IL-2, IL-4 and IL-6 provided almost 100% and 40% neuroprotection of RGCs after one-day and one-week treatment, respectively. One week later, the effect on RGCs survival could still be obtained from some treatment groups, especially combined treatments of neurotrophin with IL-2, IL-4 and IL-6. Conclusion: Not only neurotrophins but also cytokines can enhance RGCs survival in vitro. Synergistic effects of RGCs neuroprotection could be obtained after combined treatment of neurotrophins and cytokines.