| Objective: To observe the protection effect and study mechanism of single intravitreal administration of (human recombinant erythropoietin, rhEPO) on light-induced retinal degeneration in rats.Metheod:1. Animals were divided into three groups: normal, control and treatment group. Establish light-induced retinal damage in SD rats using a light exposure box, which was installed six white fluorescent lamp. The total intensity of the light is 2800lux. Animals was pretreatment with single intravitreal injection of different doses rhEPO (3μl) 24 hours before light exposure. After intravitreal injection of rhEPO and PBS (control), the animals were dark adapted for 24 hours and their pupils were dilated under dim red light 30 minutes before consistent light exposure of 5 hours. After light exposure, the animals was reared under dark environment until killed. Electroretinogram(ERG)was examined after light damage 5 and 10 days. After ERG recording the eye was taken for morphological analysis.2. Soluble erythropoietin receptor (sEPOR) was injected into the rats vitreous 24 hours before light exposure, and ERG was recorded and the eyes was taken for morphological analysis. Some simple light damage rats injected with rhEPO intravitreally after light exposure within 40 minutes were also analyzed.3. Rats in every group were killed 3 days after light exposure, the eyes and retina were taken respectively for TUNEL staining and transmission electromicroscope (TEM) and also tested for caspase-3 and Bcl-XL by Western blot analysis.4. Simple light damage rats were sacrificed on different time point of 0,6,12,24,48,72 hour and 7, 14 days after light exposure. The retina was taken and tested for EPO/EPOR protein and mRNA expression by Western blotting and RT-PCR. EPO/EPOR expression was also analyzed by immunohistochemistry.5. Rats in every group were sacrificed on 0, 6 hours after light exposure and the retina was tested for signal transduction protein of Akt/p-Akt, ERK1,2/p-ERK1,2, STAT5/p-STAT5 by Western blotting.Result:1. Five days after light exposure the ERG results indicated that the amplitude of b-wave was higher in the 5U rhEPO intravitreal group, and it followed a bell-shaped doses-response curve. The structure of the retina in this group was also better than other groups.2. There also showed a better protection effect in the rats on whom accepted intrvitreal administration of rhEPO after light exposure. And pre-treatment with sEPOR can exacerbated the retina both in the function and the structure. This effect also dose dependent.3. TUNEL staining results showed that there lack positive cells in the rhEPO treatment group and the apoptosis cell also minor in the outer nuclei layer examined by TEM. And expression of caspase-3 was reduced and the Bxl-XL was increased in this group.4. After simple light damage, the EPO/EPOR protein and mRNA were both increased and reached top 48 hours later and decreased thereafter. The EPOR was localized in the outer/inner segment of photoreceptor, outer nuclei layer, inner nuclei layer and ganglion cell layer. After light damage, EPOR expression was increased in the outer/inner segment of photoreceptor.5. Signal-transduction protein tests results showed that the expression of p-ERK1,2 was reduced in the rhEPO treatment group. Conclusion:1. Single intravitreal administration of 5U rhEPO can protect the rat's retina of light-induced damage both in function and morphology. The therapy window is from 24 hours before light exposure to 5.5 hours after light exposure.2. Endogenous EPO/EPOR system participate in the intrinsic recovery mechanism of the light-induced retinal damage in rats.3. rhEPO have an anti-apoptosis effect on this model and it may interfere with caspase-3 activation and upregulate Bcl-Xl expression.4. These data support a potential role for EPO as a therapeutic molecule against predominantly apoptotic neuronal cell death in the context of degenerative diseases and delineate the ERK1,2 pathway as the predominant mediator of EPO neuroprotection in this in vivo paradigm.
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