| Purpose: Erythropoietin (EPO), known for its role in erythroid proliferation and differentiation, has been suggested to have neuroprotective effect on retinal neurons in animal models of ischemia-reperfusion, light-induced retinal damage, axotomized ganglion cell, ocular hypertension, streptozotocin-induced diabetic retinopathy, premature retinopathy and experimental autoimmune encephalomyelitis. The aims of present study were to investigate the protective effects and the mechanisms of both endogenous and exogenous EPO on the survival of photoreceptor cells following retinal detachment (RD).Methods: To induce rat model of RD, 1.4% hyaluronic acid was slowly injected into the subretinal space, thus detaching the retina from the underlying retinal pigment epithelium. The expression levels of mRNA and protein of EPO and EPOR were measured by reverse transcription-polymersase chain reaction (RT-PCR) and Western-blot analysis. Meanwhile, the locations of EPO and EPOR were checked by immunohistochemistry. To observe the effect of soluble erythropoietin receptor (EPOsR) on retinal neurons at normal status, in other words, the endogenous EPO on the survival of normal retinal neurons, 2, 20 and 200 ng EPOsR was intravitreously injected, respectively. Flash electroretinograms (FERG) were recorded before intravitreal administration and repeated on day 3 after administration. At the same time-point, photoreceptor cell apoptosis was assayed. On day 7, histopathological examination was carried out using light microscope and tansmission electron microscope. To investigate the protective effect of endogenous EPO on photoreceptor cells survival of detached retina, once the RD model was induced, 2, 20 and 200 ng EPOsR was intravitreously injected, respectively. On day 3, apoptosis was assayed and the activity of Caspase-3 was measured by Western-blot analysis and immunofluorescence; on day 14, histopathologic examination was carried out and the thickness of outer nuclear layer (ONL) was measured. EPO at dosage of 400 ng was intraveally administrated to evaluate its toxicity on retina. To further testify the effect of EPO on survival of photoreceptor cells following RD, 100, 200 and 400 ng rat recombinant EPO was intravitreally administered, respectively and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, the activity of Caspase-3 and expression of Bcl-XL were all analyzed on day 3 after RD. Histopathologic examination and measurement of the thickness of ONL were processed on day 14 and month 2, respectively. Furthermore, the protein levels of JAK2, p-JAK2, Akt, p-Akt, ERK-1/2, p-ERK-1/2, STAT5, p-STAT5, NF-κB and p-NF-κB in neurosensory retina were detected by Western blot following EPO intravitreally injected into RD model.Results: The mRNA levels of EPO and EPOR were increased following RD, and peaked at 48 h; they were significantly higher than those in normal retinas (P<0.05) at 12 h and 6 h, respectively. Also, the protein levels of EPO and EPOR presented the same tendency, and significantly higher than those in normal retinas all at 3 h (P<0.05). Compared with the pre-injection, the latencies and amplitudes of FERG a- and b-waves of post-injection were not changed significantly (P>0.05). Also, there was no significant difference both in oscillatory potentials (OPs) P4 latency and in total amplitude between the pre- and the post-injection (P>0.05). No apoptotic cells were presented within retinas in any group after injection; or obvious edema or vacuolar degeneration was observed in retinas in any group under light microscope and transmission electron microscope. Meanwhile, intravitreal administration of EPOsR dose-dependently exacerbated photoreceptor cells apoptosis following RD, and the activity of Caspase-3 presented corresponding change. On day 14 following RD, the thickness of ONL in normal control, RD, RD+PBS, RD+EPOsR 2, 20, 200 ng groups was (47.39±3.39), (33.96±3.54), (31.83±5.21), (31.40±2.63), (24.99±2.06) and (19.30±3.71)μm, respectively; and the thickness of ONL in RD+EPOsR 200 ng group was significantly thinner than that in any group (P<0.05). Intravitreal administration of EPO did not change the morphology and function of retina. In addition, supplement of exogenous EPO could dose-dependently alleviate the photoreceptor cell death, and the activity of Caspase-3 presented corresponding change. On day 14 following RD, the thickness of ONL in normal control, RD, RD+PBS, RD+EPO 100, 200, 400 ng groups was (45.70±1.53), (35.00±1.66), (34.79±1.15), (34.60±1.02), (36.82±1.23) and (40.20±2.41)μm, respectively; and on month 2 was (46.74±1.97), (14.51±1.42), (14.70±1.19), (18.17±1.23), (26.28±4.25) and (34.00±2.40)μm, respectively. Furthermore, the length and the regularity of the rod inner and outer segments of photoreceptor were all better than those in RD group both on day 14 and on month 2. Western-blot analysis showed that supplement of exogenous EPO could not induce the up-regulation of EPOR within retina, but inhibit Caspase-3 activation and promote Bcl-XL expression. EPO did not change the inactive form levels of JAK2, Akt, ERK-1/2, STAT5 and NF-κB but increased phosphorylated form levels of JAK2, Akt and ERK-1/2.Conclusions: At the status of detachment, the expression of EPO and EPOR time-dependently increased in neurosensory retina because of hypoxia, and peaked at 48 h. Most layers of retina could express EPO and EPOR. Under normal condition, endogenous EPO could not play a crucial role in preserving the morphology and function of retinal neurons, but play an important role in protection of photoreceptor cells under hypoxia. It was showed that intravitreal administration of EPO was safe and that supplement of exogenous EPO could prevent photoreceptor cell death from hypoxic injury via decrease of the activity of Caspase-3 and increase of the Bcl-XL expression. Maybe, two intracellular signaling transduction pathways, including PI-3K/Akt and MAPK/ERK-1/2, participated in protection of photoreceptors by EPO. The present study also indicated that EPO could be considered as an important neuroprotective agent.
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