Rod and cone photoreceptor cells produce ROS in response to stress in a live retinal explant system

[摘要] Purpose: The production of reactiveoxygen species (ROS) can lead to oxidative stress, which is a strongcontributory factor to many ocular diseases. In this study, the removalof trophic factors is used as a model system to investigate the effectsof stress in the retina. The aims were to determine if both rod andcone photoreceptor cells produce ROS when they are deprived of trophicfactor support and to demonstrate if the nicotinamide adeninedinucleotide phosphate (NADPH) oxidase (Nox) enzymes are responsiblefor this ROS production. Methods: Retinas were explanted frommice aged between postnatal days 8–10 and cultured overnight. Thefollowing morning, confocal microscopy combined with variousfluorescent probes was used to detect the production of ROS. Each timepeanut agglutinin (PNA), a cone photoreceptor marker, was used tofacilitate orientation of the retina. Dihydroethidium anddihydrorhodamine 123 (DHR123) were used to determine which cellsproduce ROS. Subsequently, western blots of retinal serial sectionswere used to detect the presence of Noxs in the different retinallayers. The Nox inhibitor apocynin was then tested to determine if italtered the production of ROS within these cells. Results: Live retinal explants, viewedat high magnifications using confocal microscopy, displayed an increasein the fluorescent products of dihydroethidium and DHR123 upon serumremoval when compared to controls. DHR123 fluorescence, once oxidized,localized to mitochondria and was found in the same focal plane as thePNA staining. This showed that cones and rods produced ROS whenstressed. Retinal serial sectioning established that the photoreceptorlayer expressed Nox4, dual oxidase (Duox) 1, and Duox2 at varyinglevels. Finally, the Nox inhibitor apocynin decreased the burststimulated by the stress of serum removal. Conclusions: Confocal microscopy and PNAstaining allowed differentiation of cell types within the outermostlayers of the retina, demonstrating that both rods and cones generatedROS in response to the stress of serum deprivation. Nox4 was the mostabundantly expressed Nox in the photoreceptor layer, but Duox1 andDuox2 were also present at detectable levels, and as apocynin reducedthe levels of ROS produced, this implied that these proteins may playsome role in this production.

[学科分类] 生物化学/生物物理