| ObjectiveThe purpose of this study was to establish animal models for graded optic nerve injury and observe the electrophysiological changes、histomorphology and the natural development.And then explore the effect of recombinant human erythropoietin(rhEPO)through intravitreal injection on the moderate and severe injured optic nerve.The expression of Caspase-3 、 growth associated protein 43(GAP-43)、 myelin basic protein(Mbp)were also examined.The recovery mechanism of rhEPO on graded optic nerve injury were investigated.Methods(1)O ne hundred healthy adult Wistar rats were randomly divided into four groups: control group,mild injury group,moderate injury group,severe injury group,each group with 25 rats.Micro vascular clamp was used to damage the optic nerve at the retrobulbar 1-2mm point,the injury time with 2s for mild injury group,15 s for moderate injury group,30 s for severe injury group,no injury for control group.The rats with mydriasis,the direct reaction disappearing,the undirect reaction existing were involved in the trial.Flash visual evoked potential(FVEP)was examined one hour after damage.It was workable with the wave descending or disappearing for the injured eyes and with normal wave for the control eyes.The rats were killed at 1d、4d、7d、14d、28d after FVEP examination.The optic nerve and retina of the experimental eyes and normal eyes were obtained for pathological section and HE staining.The morphology was observed and the change of FVEP was analysed.(2)Eighty healthy adult Wistar rats were randomly divided into four groups: moderate injury control group,severe injury control group,moderate injury with treatment group,severe injury with treatment group,each group with 20 rats.After the optic nerve injury models were established successfully,rhEPO(6ul/eye,60IU/eye)was injected intravitreal in the treatment groups and normal saline(6ul/eye)for the control groups.The rats were killed at 4d 、7d、14d、28d after FVEP examination.The optic nerve of the experimental eyes and normal eyes were obtained for pathological section and HE staining.(3)The pathological sections of optic nerve from moderate injury control group,severe injury control group,moderate injury with treatment group,severe injury with treatment group and normal optic nerve were obtained for immumohistochemical staining.The expression of Caspase-3、GAP-43、Mbp were observed and given the semiquantitative analysis and comparison among groups.Results(1)FVEP:There are no statistical significance(P﹥0.05)between the control group and the normal rats in incubation period and amplitude at every time point.Compared to the control group,incubation period of P2 wave was lengthened and amplitude decreased in all the injury groups at 1h after injury,the difference has statistical significance(P<0.05).There was obvious difference among all the injury groups about the incubation period and amplitude at 1h and 1d except the incubation period at 1h between mild injury group and moderate injury group.As the grade of injury increased,incubation period was lengthened and amplitude decreased.FVEP of mild injury group became right at 1d.Incubation period was lengthened and amplitude decreased within 14 days in the moderate injury group and the wave vanished at 4d in the severe injury group.HE staining of optic nerve:In the vertical section,nerve fiber was parallel aligned with balancing staining and sporadic neurogliocyte in control group and normal nerve.About mild injury group,the optic nerve was slightly edema a t 1d and 4d,the fiber was parallel and regular,endochylema of gliocyte was homogeneous,edema relieved at 7d and got right at 14 d and 28 d.About moderate injury group,except edema vacuolar degeneration existed at 1d and aggravated at 4d,inflammatory cell infiltration increased at 7d,necrosis appearing and a small amount fiber broken with karyorrhexis,cytolysis,gliocyte proliferation.About severe injury group,fiber arranged in disorder and partial fiber broken with much inflammatory cell infiltration.The extent of lesion enlarged and aggravated with time,necrosis appeared and partial nerve fiber bundle disappeared.HE staining of retina: In the control group and normal retina,the structure of retina was clear,retinal ganglion cells(RGCs)arranged in single layer neatly,cell nucleus was clear and large with thick staining.About mild injury group,the retina was slightly edema at 1d and 4d,the chang was slight,RGCs arranged neatly and got right at 7d、14d and 28 d.About moderate injury group,the retina became swollen and thicken,RGCs arranged anomaly with uneven staining at 1d.Karyopyknosis appeared with time.RGCs reduced obviously at 28 d.The lesion in the severe injury group is more serious,the retina atrophied and became thin without normal structure at 28 d.(2)FVEP:There are no statistical significance(P﹥0.05)between the moderate injury control group and the moderate injury group in incubation period and amplitude at 4d、7d、14d、28d time points.Compared to the moderate injury control group,incubation period of the moderate injury with treatment group was shortened and amplitude increased at 4d、7d、14d、28d time points,the difference has statistical significance(P﹤0.05).The difference was not evident among the severe injury group、the severe injury control group、the severe injury with treatment group,there was no wave in the control and treatment group at 4d、7d、14d、28d time points.HE staining of optic nerve:There are no obvious difference between the moderate injury control group and the moderate injury group.Nerve fiber in the moderate injury with treatment group was still swollen with inflammatory cell infiltration at 4d and 7d,but necrosis and broken fiber decreased compared to the moderate injury control group.The difference was not evident among the severe injury group、the severe injury control group、the severe injury with treatment group at 4d、7d、14d、28d time points.(3)Caspase-3 expression in the injured optic nerve:The normal optic nerve showed no expression of Caspase-3.In moderate and severe injury control groups,the expression was strengthened at 4d and reached to the peak at 7d and decreased gradually and was more than the normal level at 14 d and 28 d.The expression of Caspase-3 in severe injury control group was more than in moderate injury control group at every time point,the difference has statistical significance(P﹤0.05).GAP-43 expression in the injured optic nerve:The normal optic nerve showed no expression of GAP-43.In moderate and severe injury control groups,the expression was strengthened at 4d and reached to the peak at 7d and decreased gradually and was more than the normal level at 28 d,there was no significant difference(P>0.05)between moderate and severe injury control groups at every time point.Mbp expression in the injured optic nerve:Mbp was highly expressed in normal optic nerve.The expression of Mbp in moderate injury control group began to reduce gradually from 7d to 14 d and became stable.The expression of Mbp in severe injury control group began to reduce gradually from 4d to 14 d and and became stable.The expression of Mbp in severe injury control group was less than in moderate injury control group at every time point,the difference has statistical significance(P﹤0.05).(4)The effect of rhEPO on Caspase-3 expression:The expression of Caspase-3 in moderate and severe injury with treatment group at every time point was less than in the moderate and severe injury control group,the number of the positive cells decreased,the difference has statistical significance(P﹤0.05).The effect of rhEPO on GAP-43 expression:The expression of GAP-43 in moderate injury with treatment group at every time point was more than in the moderate control group,the number of the positive cells increased,the difference has statistical significance(P﹤0.05),there was no significant difference(P>0.05)between severe injury control and treatment groups at every time point.The effect of rhEPO on Mbp expression:There was no significant difference(P>0.05)between moderate injury control and treatment groups at 4d time point.The expression of Mbp in moderate injury with treatment group at 7d、14d、28d time points was more than in the moderate control group,the number of the positive cells increased,the difference has statistical significance(P﹤0.05),there was no significant difference(P>0.05)between severe injury control and treatment groups at every time point.Conclusions(1)The optic nerve with mild injury can repair by itself,the optic nerve with moderate or severe injury may develop to the irreversible lesion without timely intervention.(2)The expression of Caspase-3 is closely related to the injury extent,the more serious the injury and the higher the expression.GAP-43 promote the body,s self-repair after optic nerve injury,the expression can not rise unlimitedly with the injury extent increasing.The expression of Mbp is closely related to the injury extent,the more serious the injury and the less the expression,it reflects the injury extent and prognosis.(3)RhEPO can repair the moderate injured optic nerve and imp rove the visual function,the mechanism maybe inhibit the expression of Caspase-3 and enhance the expression of GAP-43 and Mbp,to promote the regeneration of axon and myelin sheath.(4)RhEPO can not repair the severe injured optic nerve. |
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