Nitric oxide increases in the retina after continuous illumination.

LOPEZ-COSTA, J,J; PIEHL, L.; CAPANI, F.; FACORRO, G.B.; LOPEZ, E.M.; RUBIN DE CELIS, C.; PUSTOVRH, C.; HAGER, A.A; COIRINI, H.

Georgia World Congress Center, Atlanta. GA. USA.

Congreso; 36th Annual Meeting of the Society for Neuroscience; 2006

Society for Neuroscience

Continuous illumination (CI) of the retina induces an oxidative stress followed by the degeneration of photoreceptors. The excessive production of nitric oxide (NO) may be involved in the degenerative phenomenon. The aim of this work was to determine NO levels during the illumination of the retina by electron paramagnetic resonance (EPR), and to obtain additional evidence by NADPH diaphorase technique and Western Blot. Sprague Dawley rats were continuously illuminated with white light (12,000 lux) for 1, 2, 5 and 7 days while control rats were maintained at light/dark cycles of 12/12 hs. Rats were pretreated with sodium nitroprusiate and sodium diethyldithiocarbamate (DETC) followed by an intravitreous injection of DETC and ferrous sulfate. The signal of Fe-DETC-NO complex in the retinas was recorded using a Bruker ECS 106 spectrometer. For NADPH diaphorase staining, eyes were fixed in a 4% paraformaldehyde solution. Cryostat sections were incubated in a solution containing 0.1 % â-NADPH (1mg/ml) and 0.02 % (0.2 mg/ml) nitroblue tetrazolium (NBT) chloride for 1 h at 37 °C. Sections were observed with a Zeiss Axiophot microscope and analyzed with a computerized image analysis. For Western Blot, retinal tissues were homogenized and centrifuged. Tissue protein extracts were subjected to SDS gel electrophoresis and transferred onto nitrocellulose membrane which were incubated with a monoclonal mouse IgG antibody to iNOS (1:500) or nNOS (1:500, Calbiochem). Using EPR, an increase of NO signal was observed in the exposed retinas after 2 hours peaking at 24 hours of CI. NADPH diaphorase showed an increase in the optical density of reactive amacrine cells during the illumination period and also an increase in the number of reactive amacrine cells after 1 and 2 days of CI. Western blot showed the expression of iNOS in the illuminated retinas with a peak after 24 hs of illumination, but nNOS did not show significant differences among illuminated and control retinas. The observed increase of NO during CI could be involved in photoreceptor degeneration.â-NADPH (1mg/ml) and 0.02 % (0.2 mg/ml) nitroblue tetrazolium (NBT) chloride for 1 h at 37 °C. Sections were observed with a Zeiss Axiophot microscope and analyzed with a computerized image analysis. For Western Blot, retinal tissues were homogenized and centrifuged. Tissue protein extracts were subjected to SDS gel electrophoresis and transferred onto nitrocellulose membrane which were incubated with a monoclonal mouse IgG antibody to iNOS (1:500) or nNOS (1:500, Calbiochem). Using EPR, an increase of NO signal was observed in the exposed retinas after 2 hours peaking at 24 hours of CI. NADPH diaphorase showed an increase in the optical density of reactive amacrine cells during the illumination period and also an increase in the number of reactive amacrine cells after 1 and 2 days of CI. Western blot showed the expression of iNOS in the illuminated retinas with a peak after 24 hs of illumination, but nNOS did not show significant differences among illuminated and control retinas. The observed increase of NO during CI could be involved in photoreceptor degeneration.