Traditional Chinese Medicine Screening For Retinal Ganglion Cells Axon Growth

Aims:The common feature of glaucoma, optic neuritis, and some other optic nerve diseases is the irreversible apoptosis of retinal ganglion cells (RGCs) and its axons’ degeneration. Searching for methods that promote axonal growth and optic nerve regeneration makes the treatment for these diseases easy. The traditional chinese medicine is know as unique pharmacological effects and less side effects and is being paid more and more attentions in the field of optic nerve regeneration. The apply of the traditional chinese medicine is a good complementary to the treatment for these disease. In this study, we filtrate ingredients and extracts of herbs that possibly promote axonal growth and explore the mechanism of the effective ingredient.Methods:Retina explants were cultured in a three-dimensional tissue culture system, and after applying different ingredients the number and length of neurites were measured and analyzed under the phase-contrast light microscope to screen out the ingredient that promotes axonal growth effectively. For the effective ingredient, the number and length of neurites were analyzed again to define the optimal concentration. When axonal growth reached its peak, immunofluorescence staining was performed to confirm that the neurite observed was axon of RGCs. Retina explants at different time point were made into frozen sections. TUNEL staining was applied to observe apoptosis in retinal ganglion layer (GCL) and Caspase-3staining was applied to observe its activation in GCL. Retina explants were also tested by Western blot to analyze the activation of PKC-a. Furthermore, we measured the number and length of axons when PKC inhibitor was applied.Result:Neurites began to sprout one after another on1～2d and grew more numerous and longer until6d. Neurites could be observed in all different groups but the number and length differed from one to another. Neurites in ginkgolide B and scutellarin were more and longer than those in control. The optimal concentration of ginkgolide B was2mg/L and that of scutellarin was50umol/L. The neurites observed under the phase-contrast light microscope were co-labeled by GAP-43and Thyl.1(or β-tubulin Ⅲ), suggesting that the neurites were axons of RGCs. Compared to the control, both ginkgolide B and scutellarin reduced the cell apoptosis and the activation of Caspase-3in GCL. Western blot revealed that PKC-a was activated during the period of axonal growth and scutellarin enhanced the activation. After applying PKC inhibitor, the axonal promoting effect of scutellarin was inhibited.Conclusion:By filtrating possible ingredients, ginkgolide B and scutellarin was found to exhibit axonal promoting effect and neuroprotection effect. The axonal promoting effect of scutellarin was mediated by activation of PKC-a.