Inhibitor of NADPH oxidase slow photoreceptor cell death in the retinal degeneration of rd mice

Background Our previous study demonstrated that microglial activation is closely associated with photoreceptor apoptosis in rd mice.Recent studies on central nervous system (CNS) showed that activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays a key role in the microglia activation and neural cell death.However,the mechanism of NADPH oxidase during the retinal degeneration and the effect of NADPH oxidase inhibitor on photoreceptor apoptosis are concerned.Objective The aim of this study was to further explore the production of reactive oxygen species (ROS) by NADPH oxidase in the retinal degenerative process of rd mice and protection of NADPH oxidase inhibitor on photoreceptors.Methods Sixty rd mice at postnatal day 9 (P9) were randomized into the experimental group and the control group by throwing coins method.Apocynin,a NADPH oxidase inhibitor,was intraperitoneally injected in the dose of 10 mg/kg (0.01 ml/kg) once daily for 5 days (P13) in the experimental group,and the equal amount of PBS was used in the same way in the control group,and 10 C57BL/6N mice without injection of any drugs served as the wild type mice group.All the mice were sacrificed in P14 for the preparation of retinal sections.The expression of ROS in the retina was detected by dihydroethidium (DHE) fluorescence staining.Expression level of rhodopsin mRNA in the photoreceptor of the mice was determined by real-time PCR,and the thickness of retinal outer nuclear layer (ONL) in the mice of the experimental group and the control group was measured using hematoxylin & eosin staining.The use and care of the animals complied with the Statement of Association for Research in Vision and Ophthalmology.Results DHE staining showed that the ROS presented with the red fluorescence in the mouse retinas.In the rd mice of the experimental group,the ROS fluorescence intensity was dramatically enhanced in comparison with C57BL/6N mice,but weakened in comparison with the rd mice of the control group.Real-time PCR revealed that the relative expressing level of rhodopsin mRNA in the photoreceptor was (4.21±0.33) in the experimental group and (0.93±0.24) in the control group,showing a significant difference between them (t =2.360,P =0.000).The thickness value of retinal ONL was (35.95±1.63)μm in the mice of the experimental group,which was significantly higher than that in the mice of the control group ([23.17±1.38] μm) (t=3.850,P=0.016).Conclusions In the retinal degeneration of rd mice,activation of NADPH oxidase increases the ROS production.Apocynin can slow the apoptosis procedure of photoreceptor cells of rd mice. Key words: Retinal degeneration/genetics;  rd Mice;  Nicotinamide adenine dinucleotide phosphate oxidase/inhibitor;  Reactive oxygen species;  Microglia