| ObjectiveOptic nerve (ON) injury is one of the common reasons of retinal and optic nerve diseases, which finally developed to vision loss. To date, no effective treatment has been discovered to restore visual function. Progressing death of retinal ganglion cells (RGCs) is a major cause of irreversible impairment of visual function caused by ON injury. Recently, many researches have focused on how to promote and induce regeneration of injured RGCs, thus restore ON function. However, an effective, safe and applicable treatment for RGCs survival and axonal regeneration causey by ON injury still has not been discovered clinically.Recombinant human erythropoietin (rhEPO) is a widely used drug on clinical and has been confirmed to have neuroprotective effect. As a part of central neuron system, ON injury may also be combined with neuroprotective effect of rhEPO. However, a research on protective effective of rhEPO on RGCs in ON injured diseases has not been carried out and the accurate mechanism needs to be further determined.Our study is designed to clarify whether rhEPO can promote RGCs survival and regeneration in vitro and in vivo, and then restore visual function. For in vitro study, we observed the influence of rhEPO on cultured RGCs survival and axonal regeneration of rats, and detected the expression of growth associated protein 43 (GAP-43, a marker for neuron regeneration). For in vivo study, ON crash models of rats were used to mimic clinically incomplete ON injury. The application of rhEPO was performed locally (intravitreal injection) to avoid side effects of general use. The purpose of this part in our study is to evaluate the effects of rhEPO on RGCs survival and regeneration, investigate the restoration of visual function by flash visual evoked potential (F-VEP) examination, and explore an optimal dosage of rhEPO in vivo.Methods1. In vitro RGCs cultivation: RGCs were cultured in DMEM (control group) or DMEM containing rhEPO (rhEPO group). Cell morphology and axonal growth were observed under phase-contrast microscope. The length of the longest processes of RGCS were measured and compared between two groups. The GAP-43 expression was detected by Western blot and the gray value scales of GAP-43 were measured by imaging analysis system. All results were compared using t test.2. Establishing ON crush model: Adult Wistar rats were randomly divided into rhEPO group and control group (saline group). Both rhEPO group and control group were divided into 3 groups according to different time points (1w, 2w and 3w). The rhEPO group was also divided into 3 groups according to different dosage (5U, 15U and 50U). The rhEPO with different dosage or 5μl saline were injected into vitreous cavity immediately after ON injury.3. The rats were sacrificed after 1w, 2w and 3w post-operation. Eyeballs were enucleated for retinal paraffin sections. The expression of Thy1.1 and GAP-43 were detected by immunohistochemistry staining. Five visual frames under microscope were randomly chosen for collecting images, counting Thy1.1-positive cells, and measuring mean gray value scale of GAP-43. All results were compared using ANOVA.4. The F-VEP were detected pre-injury, immediately after injury, 1w, 2w and 3w post-injury, respectively. The latency and amplitude of P1 wave were measured and compared using ANOVA.Results1. Cell morphology: The RGCs were observed to extend processes under microscope after culturing for 3 days. The cells in rhEPO group were bigger and the processes were longer compared to the control group (P<0.05).2. The expression of GAP-43 in vitro detected by Western blot: The expression of GAP-43 is in rhEPO group was higher than that in control group (P<0.05).3. RGCs survival in vivo: The number of surviving RGCs in ON crush models was decreased gradually along with time. In rhEPO group with different dosage, the number of surviving cells at different time points was more than that in control (P<0.05). The surviving RGCs in 15U and 50U rhEPO group were much more than those in 5U rhEPO group (PO.05), however there was no significant difference between 15 U and 50U rhEPO group (P>0.05).4. The expression of GAP-43 in vivo detected by immunohistochemistry: No detectable expression of GAP-43 was observed in normal retina. In ON injury rats, the expression of GAP-43 was detected in retinal ganglion cell layer. The level of GAP-43 expression was higher after 1w post-injury, but it was decreased at 2w and almost undetectable at 4w. In rhEPO group with different dosage, the expression of GAP-43 was also observed in retinal ganglion cell layer. The level of GAP-43 expression was notably higher both at 1w and at 2w time points, and decreased after 4w post-injury. In each time point post-injury, the expression of GAP-43 in rhEPO group with different dosage was significantly higher than that in control (P<0.05). The level of GAP-43 expression in 15U and 50U rhEPO group were significantly higher compared to 5U rhEPO group (P<0.05). However, there was no significant difference between 15 U and 50U rhEPO group (P>0.05).5. F-VEP examination: The classical wave pattern of NPN was observed in normal Wistar rats, which exhibited a short latency and sharp peek of P1 wave. Obvious changes were detected immediately after ON injury, including significantly prolonged latency and decreased amplitude of P1 wave. In control group, the latency of P1 wave was shorter and the amplitude was slightly recovered after 4w post-injury. In rhEPO groups, a significant reduction in the latency and increase in the amplitude were observed at 2w post-injury, and showed a significantly more recovery compared to the control group at different time points (P<0.05). The recovery of P1 wave in 15U and 50U rhEPO group were more significant compared to 5U rhEPO group (P<0.05) Conclusion1. In vitro study shows that rhEPO can promote the axonal growth of cultured RGCs.2. The rhEPO can also increase the expression of GAP-43 in cultured RGCs, which may result in the promotion of rhEPO on the axonal growth of RGCs.3. In vivo study shows rhEPO can promote RGCs survival after ON injury.4. The rhEPO can prolong the expression of GAP-43 and increase its intensity, thus promote RGCs survival and regeneration.5. The rhEPO can restore function of injured ON.6. Using introvitreal injection, 5U rhEPO can only cause detectable changes in cellular level, but have less effect on restoration of visual function. To some extent, the neuroprotective effect is strengthened with the increasing dosage of rhEPO.
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