Mouse retina explants after long-term culture in serum free medium.

The neonatal mouse retina remains viable as an explant in serum-supplemented growth media for more than 4 weeks. Interpretation of drug effects on this tissue is compromised by the enigmatic composition of the serum. We sought to remove this ambiguity by culturing neonatal as well as late postnatal mouse retina in serum-free nutrient medium. In this study three important observations were made, (1) there is histotypic development of neonatal as well as preservation of late postnatal mouse retinal structure during long-term culture in serum-free medium, although the late postnatal tissue tends to show some loss of cells in the outer nuclear layer. (2) Protein expression in explant photoreceptor cells was similar to that in the litter-matched ones, except for green cone opsin and interphotoreceptor retinoid-binding protein, although mRNA of the latter is present at similar amounts as in age-matched in vivo controls. (3) Cells of the inner retina stained by antibodies to calcium-binding proteins display some novel sprouting of processes. The results show that the mouse retina can be cultured as an explant for more than 4 weeks in a serum-free medium. This represents an important step forward because, (1) the possibility of interference of drug effects by unknown serum factors has been eliminated; and (2) the spent culture medium can be analyzed to investigate biomolecules released by the retina in vitro.

Lens epithelium-derived growth factor (LEDGF) delays photoreceptor degeneration in explants of rd/rd mouse retina.

Lens epithelium derived growth factor (LEDGF) has been shown to rescue embryonic chick photoreceptor cells from serum starvation and heat stress, light damaged photoreceptor cells in Lewis rats, and photoreceptor cells in RCS rats. The aim of our study is to study the rescue effect of LEDGF on photoreceptor cells in the rd/rd mouse using our long-term serum free organ culture. At the end of this culture period of 21-26 days LEDGF treated rd mouse retina showed an increased photoreceptor survival compared to the untreated controls. LEDGF has no effect on expression and localization of opsin and arrestin in the rod photoreceptor cells when RPE is present. The protective potency of LEDGF on the retinal photoreceptor cells is similar to that of BDNF. LEDGF is known to activate heat shock proteins (Hsps) and the elevated Hsps are also reported to suppress apoptosis.

A combination of CNTF and BDNF rescues rd photoreceptors but changes rod differentiation in the presence of RPE in retinal explants.

PURPOSE: To gather information regarding the combination of ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF), compared with the individual factors when used as a treatment to retard photoreceptor cell loss in rd mouse retina explants and to investigate the observation that the retinal pigment epithelium (RPE) influences rod differentiation by this treatment. METHODS: Postnatal day (PN)2 or PN7 control and rd mouse retinas were grown with attached retinal pigment epithelium (RPE). The explants were kept in culture up to PN28. During this culture period CNTF, BDNF, CNTF+BDNF, or vehicle were continuously administered to the culture medium. The nontrophic factors cyclosporin A and N:-CBZ-aspartic acid-glutamic acid-valine-aspartic acid-fluoromethyl ketone (z-DEVD-fmk) were also used. The number of photoreceptor nuclei remaining in the outer nuclear layer (ONL) was analyzed in hematoxylin and eosin-stained sections. Rod- and cone-specific antibodies were used to determine identity and state of differentiation of the photoreceptors. RESULTS: Compared with vehicle treatment, BDNF or CNTF resulted in 1.4- or 2-fold more surviving cell rows in the ONL, respectively. However, when CNTF and BDNF were applied together, surviving ONL cell counts in the rd explants were approximately 3 times those in vehicle-treated explants. In the presence of CNTF or CNTF+BDNF, opsin and arrestin expression in rods was decreased compared with rods without attached RPE. Cyclosporin A and z-DEVD-fmk did not show rescue of rd photoreceptor cells. CONCLUSIONS: CNTF or BDNF treatment of rd retinal explants delays photoreceptor cell loss to some extent. However, when these agents are combined, photoreceptor rescue is much more effective. The quenching of opsin and arrestin expression caused by treatment suggests that simultaneous with rod rescue, rod differentiation is depressed. Regarding retinal degeneration, the results from the selective inhibitors of apoptosis rank the CNTF+BDNF combination treatment as the most consistent and effective experimental pharmacologic intervention currently available.