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