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.

Retinoic acid produces rod photoreceptor selective apoptosis in developing mammalian retina.

PURPOSE: All-trans retinoic acid (ATRA) or 9-cis retinoic acid (9CRA), added to dissociated developing neural retinal cells, induces progenitor cells to adopt the rod cell's fate. Retinoic acid (RA) also produces apoptotic cell death in developing tissues. The effects of retinoids on mouse retinal development were examined. METHODS: Retinas were explanted on postnatal day (PN)1 and cultured with or without the retinal pigment epithelium (RPE) attached. Retinas were cultured for 3 weeks in the absence or presence of 100 or 500 nM ATRA or 9CRA. Morphologic development and apoptotic cell death were examined using cell-specific immunocytochemical markers, the TdT-dUTP terminal nick-end labeling (TUNEL) method, and a caspase assay. RESULTS: Retinal explants, with and without RPE, had similar age-dependent increases in opsin expression. In contrast, explants with RPE had less apoptosis during the first week than retinas without RPE. In explants with RPE, ATRA or 9CRA produced rod-selective apoptotic cell death in which 20% to 25% were lost by PN7 with no further loss by PN21. 9CRA-treated explants without RPE had a decreased number of apoptotic cells and a higher number of (rhod)opsin-positive cells at PN3. CONCLUSIONS: Factors in RPE appear to regulate rod apoptosis in developing retina. Retinoids produce rod-selective apoptotic cell death during normal rod differentiation. In contrast, retinoids accelerate the expression of opsin in retinas without RPE. These differential effects of RA on rod photoreceptors-apoptosis and differentiation-are similar to those observed in other developing tissues and play an important role in both normal and pathologic development.

9-cis-retinoic acid in combination with retinal pigment epithelium induces apoptosis in cultured retinal explants only during early postnatal development.

Retinoic acid is one of the active metabolites of vitamin A and has profound effects on the development of the CNS including retina. Previously, we have shown that rod-specific apoptosis is induced in retinal explants from neonatal mice by exposure to 9-cis-retinoic acid (9CRA) when the retinal pigment epithelium (RPE) is present. In explants lacking RPE, it instead has a differentiation-promoting effect seen as an accelerated opsin expression on postnatal day 3. To investigate the long-term effect of 9CRA exposure, we have explanted retinas from neonatal C3H mice with or without RPE attached and placed in organ culture. After 19 or 48 h in culture or 7, 8 or 13 days in culture, the explants were either fixed for histochemical examination or frozen for assay of DEVDase activity. We found that long-term exposure to 9CRA caused a decrease in the number of cell layers in the outer nuclear layer (ONL) only in explants with the RPE attached. When explants with RPE attached were exposed to 9CRA only during the second postnatal week, neither an increase in DEVDase activity, TUNEL-positive cells, nor a decrease in cell layers of the ONL could be demonstrated, indicating that the retina was insensitive to the apoptosis-inducing effect of 9CRA after the first postnatal week. The absence of RPE in control explants resulted in a higher number of rosettes and the extrusion of cells into the subretinal space.

Characterization of the anticonflict effect of MK-801, a non-competitive NMDA antagonist.

Brain serotonergic, noradrenergic and GABAergic mechanisms are all involved in the regulation of conflict behaviour, and the GABAA/benzodiazepine receptor complex may play the most central role in this context. Since facilitation of GABAergic inhibitory transmission produces anticonflict effects, it has been suggested that antagonism of excitatory inputs may serve the same cause, and, indeed, blockade of excitatory neurotransmission mediated via N-methyl-D-aspartate (NMDA), receptors, produces anticonflict effects. In the present study, using a modified Vogel's rat conflict model, we have investigated whether the anticonflict effect of the non-competitive NMDA antagonist MK-801 can be linked to NMDA receptor blockade, and if stimulation of these receptors instead produces proconflict effects. The tentative involvement of noradrenergic, serotonergic or GABAergic effects in the MK-801-induced anticonflict effect was also studied. MK-801 produced a dose-dependent and specific anticonflict effect (maximal effect after 0.05 mg/kg, intraperitoneally, -90 min.). This anticonflict action was completely counteracted by NMDA in a dose (0.125 microgram, intracerebroventricularly) not affecting behaviour per se. The highest dose tested of NMDA alone (0.5 microgram) tended to produce a proconflict effect, but this action may be unspecific due to concomitant drug-induced motor-inhibition. Neither bicuculline and picrotoxin, antagonists at the GABAA/benzodiazepine receptor complex, nor the adrenoceptor antagonists propranolol and prazosin significantly altered the MK-801-induced anticonflict effect, whereas L-5-HTP (50 mg/kg, intraperitoneally, after inhibition of peripheral decarboxylation with benzerazide) completely abolished the anticonflict effect of MK-801.(ABSTRACT TRUNCATED AT 250 WORDS)