Riboflavin-Mediated Axonal Degeneration of Postnatal Retinal Ganglion Cells In Vitro is Related to the Formation of Free Radicals

It is well known that glial cells produce several neurotrophic factors. We detected a neurogedegenerative/neurite growth inhibiting activity in serum-free astrocyte-conditioned medium (ACM). After high performance liquid chromatography (HPLC)-purification, spectral analysis and test of biologic activity in tissue cultures of postnatal retinal explants we isolated a fraction containing a riboflavin-(vitamin B2)-like compound which caused the neuronal degeneration. We therefore investigated the influence of pure riboflavin on axonal regeneration in vitro. Riboflavin is a normal compound of Dulbecco’s modified Eagle medium (DMEM) and other tissue culture media in various concentrations. The removal of riboflavin from ACM by reversed phase chromatography abolished the neurite growth inhibiting effect and enhanced the regenerative response of axonal outgrowth from postnatal rat retinal explants. However, doubling of the normal medium concentration (1 μM) of riboflavin lead to strong degenerative alteration of the outgrowing axons in a dose-dependent manner, even under maximal growth stimulation by cultivating the explants in astrocyte-conditioned medium. To check the possibility that riboflavin-mediated cytotoxicity is related to the production of free radicals through photoabsorption from daylight, we irradiated culture medium with UV light, and induced radical stress by incubating the explants with Fe2+/3+. In an other set of experiments, we proofed, if antioxidants/free radical scavengers like pyruvate or vitamin C and E are able to overcome the neurite growth inhibiting influence of riboflavin or the radical stress. Our findings suggest an involvement of riboflavin-mediated formation of free radicals/reactive oxygen species and subsequent neurite degeneration in in vitro-assays of neuronal regeneration or neuronal cell cultures. How far the riboflavin/free radical-induced axonal degeneration could be an explanation for neurological degenerative disorders has to be elucidated.