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A. Stett, H. Schmid, T. Herrmann, K. Kohler; Inhibition of Apoptosis of Retinal Cells by Prolonged Electrical Stimulation of Organotypic Cultures of Adult Rcs Rat Retina. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3705.
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© ARVO (1962-2015); The Authors (2016-present)
To explore effects of continuous electrical stimulation on retinal tissue.
We developed an organotypic retina culture that allows us to keep segments of adult rat retina over six days on a microelectrode array (MEA). The MEAs contained 60 Ir microelectrodes (size 50 x 50 µm²). Retinas were obtained from Royal College of Surgeon (RCS) rats (age: postnatal day 90). The culture system allowed for continuous electrical stimulation and monitoring of the electrical charge transfer from the microelectrodes to the retina. Rectangular monophasic voltage impulses were delivered to 30 electrodes of each MEA. The others remained unstimulated. After the stimulation period cryostat sections of 12µm were collected on silane-coated slices. Specific antibodies were used to stain resident peritoneal and activated microglia in the retina. Apoptotic cells were detected by TUNEL reaction performed on slices as well as on flat-mounted retinas directly on MEA. To determine thresholds for electrically evoked alterations of retinal morphology, voltage impulses up to 3 V (pulse duration 500 µs, frequency 20 Hz) were continuously applied to the cultured retinas for 2, 3 or 6 days. The average charge density at 3 V was 430 µC/cm².
A significant lower number of light-microscopically "dark" appearing cells was observed in the stimulated area compared to the non-stimulated area after 3 days in culture and at a minimum stimulus of 2 Volt. These cells were identified immunohistochemically as microglial cells by the use of ED1 and OX-42 antibodies. We found an increase of microglial cells during the duration of the organotypic cell culture. When stimulated with voltages higher than 1 V , the increase was already surpressed on the stimulated area after one day of stimulation.The TUNEL-staining showed a significantly reduced amount of TUNEL-positive cells on the stimulated area of the degenerated retinae of the RCS-/- rats when stimulated with at least 2 V compared to the non-stimulated area. This effect was also prominent after one day of stimulation. The non-degenerated control group of RCS+/+ rats did not show such an effect in the TUNEL-staining.
Our results indicate that prolonged electrical stimulation has an inhibitory effect on apoptosis of retinal cells in degenerating retinas. It remains to be shown whether this effect is related to neuroprotective effects or not.
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