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

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Abstract

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.

Introduction

There are a lot of examples of random disturbances in the growth and cloning efficiency of cell cultures or cell lines1, 2, 3which prompted to identify medium components responsible for this effects. Development of phototoxicity in DMEM was originally observed by Warburg et al.,[4]who showed that medium containing riboflavin became toxic to mammalian cells when exposed to light and oxygen. Other studies support these results in the last few years,1, 5indicating DNA as a cellular target for medium-mediated toxic photoproducts.6, 7This has to be considered especially in experiments with conditioned media from meningeal or glial cell cultures, which are widely used to detect neurite-promoting activities or neurotrophic factors.8, 9, 10, 11, 12, 13On the other hand, an increasing number of studies have indicated that cytotoxic free radicals may significantly contribute to the neural damage,14, 15, 16and oxidative damage may be responsible in a wide variety of neurodegenerative diseases, e.g. Alzheimer’s disease,[17]Parkinson’s disease[18]or in familial amyotrophic lateral sclerosis.19, 20The present study addresses two issues. The first regards the elimination of a riboflavin-containing compound from astrocyte-conditioned medium (ACM) by reversed phase chromatography and the subsequent influence on axonal regeneration of postnatal retinal explants.[13]A second issue addresses the possible neuroprotective role of free radical scavengers against riboflavin- or free radical-mediated scanty outgrowth/neurite degeneration.[21]

Section snippets

Preparation of Retinal Explants

Retinal explants were prepared as described.[13]In brief, the eyes of P9 (postnatal day 9) to P12 rats were removed from the skull under semi-sterile conditions after decapitation of the animals. The retinae were dissected from the surrounding tissues, pieces of retina with a diameter of 400 μm were punched out with a sharpened syringe needle and collected in L-15 medium (Biochrom, Berlin, Germany).

Bioassay of Retinal Explants

The retinal explants were cultured in a three-dimensional fibrin gel (fibrinogen from swine, 2,5

Neurite Outgrowth From Retinal Explants

After three days in vitro many regenerated, but short neurites were visible in control cultures (Fig. 2a). After addition of ACM, the regenerative response was greatly enhanced (Fig. 2b). This positive reaction was enhanced again after chromatography of the ACM on Sep-Pak-columns, indicating the binding of a possible inhibitory molecule for neurite outgrowth to the column (Fig. 2c).

HPLC-Purification, Spectral Analysis, Mass—and 1H-NMR-Spectroscopy of the Neurite Growth Inhibiting Activity in ACM

The eluate from the Sep-Pac RP-18-column (see above) was analyzed by reversed phase HPLC and yielded several

Discussion

Normally, mammalian CNS axons fail to regenerate after lesions in the adult brain and spinal cord,25, 26but if they are supplied with growth-promoting substrates and/or with neurotrophic factors, they are able to regenerate new processes and reinnervate their target regions,27, 28for review see.[29]We here describe the influence of the vitamin riboflavin, a normal component of various tissue culture media, on the axonal regeneration of postnatal retinal ganglion cells in vitro. At postnatal day

Acknowledgements

We thank Sibille Piontek and Rosemarie Sprang for their expert technical assistance. This work was supported by grant Lu 617/1-1 from the Deutsche Forschungsgemeinschaft and the Hensel-Foundation, University of Kiel.

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