Effect Of RhEPO On The Retinal Neurons After Incomplete Injury Of Optic Nerve In Rats

Objectives To evaluate the effects of recombinat human erythropoietin(rhEPO)on survival, processes'growth of retinal ganglion cells(RGCs) and expression of growth associated protein 43(GAP-43) in RGCs in vitro,and to investigate the possible mechanism of rhEPO'effects on RGCs in vitro.To examine the protective effects of rhEPO on retinal neurons and its mechanism after incomplete optic nerve(ON) injury in rats.Thereby, explore a new method and strategy for the treatment of clinical ON injury.Methods1. Experiment in vitro: RGCs were cultured in DMEM and DMEM containing rhEPO,then the expression of GAP-43 were detected by immunocytochemical staining. The growth regularities and survival time of RGCs in vitro were observed under phase-contrast microscope. The length of the longest processes of RGCs and the average gray scale of RGCs' GAP-43 were measured on the 2nd, the 4th, and the 6th day.2. Experiment in vivo: Male Long Evans rats were randomly divided into test groups(n=25,per group) and control groups(n=25,per group). Test groups and control groups were both divided into 5 groups according to the different time points (ld,3d,5d,7d,14d) after incomplete ON injury.The experimental model of incomplete ON injury was established using the same wounding time (15s) with constant wounding force by a defined forceps at 2.0mm behind eyeball.For each animal operation was performed on one eye,the fellow eye was taken as a normal control.rhEPO at 5000units/kg or normal saline control was administered i.p. immediately after ON injury. The injections were repeated on the 3rd, the 5th and the 7th day after injury. The rats were sacrificed on the day of post-operative 1,3,5,7 and 14. (1)Before sacrifice, flash visual evoked potential(F-VEP) were detected in order to observe the effect of rhEPO on the recovery of visual function.(2)Apoptosis of retina neurons were detected by TUNEL method.(3)Retinal paraffin sections were stained by immunohistochemistry to study the expression of apoptosis-relataed genes(Bax, Bcl-2).Results1. Survival time of RGCs in vitro: RGCs died out after cultured for 6 to 8d in DMEM. However, RGCs could survive for 12 to 14d in DMEM containing rhEPO, with the survival time of RGCs significantly longer than DMEM group (p<0.01).2. The length of the longest processes of RGCs: After cultured for the 2nd, the 4th and the 6th day, the length of the longest processes of RGCs of rhEPO group were 55.47±7.07um,100.16±7.78um and 118.63±11.5Oum,while DMEM group were 42.90±4.71uin, 79.74±8.49um and 110.02±10.79um.After cultured for 2d or 4d, the length of the longest processes of RGCs of rhEPO group were longer than that of DMEM group(p<0.01). After cultured for 6d, the length of the longest processes of RGCs of rhEPO group were longer than that of the DMEM group (p<0.05).3. The expression of GAP-43 : The level of expression of GAP-43 in RGCs of both groups were high on 2d.The level of expression of GAP-43 in RGCs reacheded the peak on 4d.While on 6d, the level of expression of GAP-43 in RGCs decreased notablely.In every time point after injury, the average gray scale of RGCs' GAP-43 in rhEPO group were more higher than those in DMEM group, and the difference was significant (P<0.01)o4. There were no apoptotic cells in normal retinas and in those retinas on the lst,the 3rd and the 5th day of postoperation. Apoptotic cells were shown in ganglion cell layer(GCL), inner plexiform layer,inner nuclear layer on the 7th day of postoperation.Compared with those on the 7th day of postoperation, apoptotic cells increased significantiy on the 14th day, apoptotic cells were intensively distributed in retinas.The number of aptopotic cells decreased notablely in rhEPO group on the 7th and the 14th day of postoperation, the difference of number of apoptotic cells in GCL between rhEPO group and control group was significant(P<0.01).5. On the 14th day after injury,the latency of F-VEP recovered nearly to normal. There were no differences of latency among these groups(P>0.05).On the day of postoperation 1,3,5 and 7,amplitude of P1-N2 wave in rhEPO group were not different from that of the control (p>0.05),but on the 14th day of postoperation,the difference was significant (p<0.05).Amplitude recovered to 51.11% of normal in rhEPO group, while only to 32.39% of normal amplitude in control.6. The level of expression of Bax in the retinas were high in every time point after injury. While the level of expression of Bcl-2 in retinas became low gradually after ONinjury.After injury for 7d or 14d,the level of expression of Bcl-2 of control group were low, while the level of expression of Bax in the retinas of rhEPO group were higher than that of the control.Conclusions1. Cell culture experiment showed that rhEPO can prolong the survival time and promote the growth of RGCs' processes in vitro significantly.2. rhEPO can increase the expression of GAP-43 in cultured RGCs in vitro,which suggest that the promotion of rhEPO on the growth of RGCs' processes in vitro may relate to up-regulation of the expression of GAP-43.3. Experiment in vivo showed that rhEPO can effectively inhibit apoptosis of retinal neurons,and can promote the recovery of visual function after incomplete injury of ON.4. The decline of the expression of Bcl-2 may be one of the reasons of inducing apoptosis of retinal neurons after incomplete injury of ON. Detection of Bcl-2 protein showed that rhEPO can increase the expression of Bcl-2 in retinas after injury.The effects of rhEPO on inhibiting apoptosis of retinal neurons may relate to up-regulation of the expression of Bcl-2 gene.