| Erythropoietin (EPO) has been viewed traditionally as a hematopoietic cytokine produced by the fetal liver and adult kidney in response to hypoxia. Results of recent studies now support a physiological role for EPO within the central nervous system. The expression of EPO and EPO receptors (EPO-Rs) in the central nervous system and the up-regulation of EPO by hypoxia-ischemia in vitro and in vivo suggest that this cytokine is an important mediator of the brain's response to injury. Consistent with this hypothesis, pretreatment with exogenous EPO protects cultured neurons from hypoxia, glutamate excitotoxicity, and growth-factor withdrawal. When administered systemically, EPO can cross the blood-brain barrier and reduce neuronal injury in animal models of focal ischemic stroke, traumatic brain injury, inflammation, kainate toxicity, and spinal cord injury. EPO rescues neurons from acute injury at least in part by inhibiting apoptosis via activation of specific protein kinase pathways and the recruitment of NF-kB. Prior studies of EPO in different models of brain injury raise the possibility that this cytokine may participate in the recovery and survival of retinal neurons from ischemia. Hence, the attentions to it is being paid increasingly.Retinal ischemia is a serious and common clinical problem. It occurs as a result of acute vascular occlusion and leads to visual loss in a number of ocular diseases such as acute glaucoma, diabetic retinopathy, and hypertensive vascular disease. Transient global retinal ischemia, for example, shares manysimilarities with transient global cerebral ischemia. Both cause selective damage of specific subpopulations of neurons. Pyramidal neurons in the CA-1 zone of the hippocampus are selectively vulnerable to transient cerebral ischemic injury. Similarly, neurons in the inner nuclear layer (INL) of the retina show significantly enhanced susceptibility to transient retinal ischemia as compared with outer nuclear layer (ONL) neurons. Both types of injury are associated with delayed neuronal death, which arises in part by apoptosis. Further, many of the same signaling pathways are activated in retinal and cerebral ischemia Given the similarities between these two types of neuronal injury, we conducted a study of the protective effect of EPO on rats retinal ganglion cells in a model of retinal ischemia-reperfusion injury.RIR can be seen at glaucoma caused by hypertension and in the process of retina vessel blocking diseases. During RIR, tissue of retina result in a series of change on metabolisms and structure, which lead to death of ganglion cells and finally cause loss of visual capacity permanently. It's mechanism may regard with increasing of oxygen free radical, calcium overloaded and releasing of activating amino acids. Some gene expression concerning apoptosis are important as well.EPO affords protection on injured retinal photoreceptor cells induced by axotomy. But its function in ganglion cells are unclearly after introducing RIR. Till now there is few report about it's relationship. No result has been reported on the function of ion channel of ganglion cells. Ganglion cells culture was applied in vitro. Excitability of ganglion cells and ion channel current were determined by electronic approaches. Combined with laser confocal technique, the concentration of calcium was assayed to study the relation between decreasing calcium overloading and apoptosis of ganglion cells.I .Effects of EPO on the calcium currents of rat retinal ganglion cells.The whole cell patch clamp technique was used to record the calcium currents of rat retinal ganglion cells, which was L-type voltage-dependent calcium channel currents from holding potential -70 mV stepping to +10 mV.The calcium current of 7 retinal ganglion cells isolated from rats were recorded. After hypoxia and ischemia treatment, the amplitude of calcium current was enhanced, 287.17±14.69,pA (n=6). The percentage of enhancement compared with normal control group were 12% ( P<0.01). Application of EPOC K l(h 100uM)decreased the amplitude of calcium current to 279.83±9.41 (n=6) > 271.50±11.89(n=6) #1 252.17±12.98(n=6) PA, respectively. The amplitude of calcium current in lOOuM EPO group is statiscally significant compared with that in ischemic RGCs ,P<0.01(n=6). Results suggest reduces L-type voltage-gated calcium currents in retinal ganglion cells.II .Effects of EPO on the spontaneous ganglion cell spikes.200 mM glutamate increased the spontaneous ganglion cell spikes (31.5±6.5 spikes /s). 100 uM Epo-mediated spontaneous ganglion cell spikes was 20.7±3.9 spikes /s and reduced these increases by approximately 34.3%, P<0.01(n=6). Results suggest Epo Reduces glutamate-Induced increase of spontaneous spiking in Retinal Ganglion Cells.III.Effects of EPO on the glutamate-gated cation currents of rat retinal ganglion cells.To determine how Epo affects glutamate-induced spontaneous spikes, we examined the actions of Epo on glutamate-induced current in ganglion cells under voltage-clamp conditions. Puff application of 200uM glutamate on a ganglion cell in the retinal slice induced postsynaptic currents C 289.21± 15.13 PA) o In the presence of 100 uM Epo , the glutamate-induced currents were reduced C261.66±10.88,pA) (n=6), The difference was statistically significant, P<0.05(n=6).. Results suggest Epo reduces significantly glutamate-gated cation currents in retinal ganglion cells.IV .Effects of EPO on the sodium currents of rat retinal ganglion cells.The whole cell patch clamp technique was used to record the sodium currents of rat retinal ganglion cells, which was from holding potential -70 mV stepping to +60 mV. The sodium current of 7 retinal ganglion cells isolatedfrom rats was recorded.The amplitude of calcium current in hypoxia retinal ganglion cell was 4.6±1.31(n=6);After application of EPO 1,10. lOOMm, the amplitude of sodium current was statistically decreased, P<0.01(n=6). Results suggest Epo reduces dose-dependently sodium currents in retinal ganglion cells.V .Measurement of intracellular free calcium concentration of retinal ganglion cells by using furo-3-AM fluorescence dye.Supervise the dynamic change of intracellular free calcium concentration of retinal ganglion cells under LSCM continuous scanning. First, intracellular free calcium concentration of normal retinal ganglion cell is 1081.136tl00.585. Glutamate increased the [Ca2+]i of Fluo-3/AM-loaded retinal ganglion cells isolated from rat retina to 2026.687±81.333. In the presence of EPO (l,10,100uM) the glutamate-induced increase of fluorescence was decreased to 1818.458±86.052,1521.004±68.580, and 1173.390±105.755, it is significant stastistically(p<0.05). Results demonstrate EPO inhibits the increase of [Ca2+]i produced by treatment with 10 mM glutamate in dose-dependent manner. The conclusion is consistent with those in ion current results we showed above.With elevating the intraocular pressure to 120 mm Hg for 60 min resulted in the typical histopathologic features expected subsequent to acute retinal ischemia with reperfusion, soluble EPO at 20 or 200 ng was given intravitreally just after the induction of ischemia, and rats were killed at day 1,3,7 after reperfusion . To investigate the effect and the mechanisms of antiapoptotic action of EPO on ischemic retinal ganglion cells, we use electroretinograms (ERGs), retinal flat-mounting cell counts , terminal Deoxynucleotidyltransferase-Mediated dUTP End Labeling(TUNEL), immunohistochemistry, half-quantity RT-PCR, and Western Blotting techniques. EPO may represent a therapeutic agent for several retinal diseases including acute glaucoma, acute retinal vascular occlusion, diabetic retinopathy, and hypertensive vascular disease.I .The effect of EPO on b-wave amplitude of electroretinography(ERG) in rats after ischemia-reperfusion.B-wave amplitude of ERG in rats of IR and IR-NS groups was decreased increasingly at I,3,7dto32..24±6.98, 24.04±2.8k 15.98±8.01uV;32.13±7.28> 23.91±2.77. 16.51±8.38uV, respectively.The difference between the two groups is unstatistically significant, P>0.05. After intravitrous administration of EPO, B-wave amplitude were elevated in correspond times: 57.24±4.54^V\ 73.77±9.33|^v\ 89.23±6.75uV, it is statistically significant comparing with IR and IR-NS, P<0.05. The results suggest EPO can improves retinal function by increasing b-wave amplitude of electroretinography.II .The effects of EPO on the flat-mounted retinal ganglion cells counts after rats ischemic-reperfusion.In the 10 unoperated normal rat eyes, the mean RGC counts were 12,619 ± 310 for the right eyes (mean density 2075 per mm2) and 12,519 ± 311 for the left eyes(mean density 2059 per mm2). There were no significant bilateral differences RGC counts and densities from the same animal (p = 0.72, paired / test). In the treated eyes of the IR rats and IR-NS rats, the mean RGC counts at the end of the 7-day period were 9116±273 (mean density 1499 per mm2) and 9489±293 (mean density 1561 per mm2),respectively, which represented significant decreases when compared with the 12,619±310 mean RGC counts of eyes of the normal control rats (p<0.005).By contrast, the mean RGC count of the eyes of the IR-EPO rats was 11,212±414 (mean density 1844 per mm2) representing a not statistically significant (p = 0.051).The results do suggest that EPO may well have played a beneficial role in preserving RGC survival after rats ischemia-reperfusion injury.III. The effects of EPO on TUNEL-positive expression RGCs.TUNEL-positive cells predominantly locate in the nucleus of ganglion cells. No TUNEL-positive cells were seen in the normal control retinas. However, 1,3, and 7d after retinal ischemia, a number of TUNEL-positive cells were noted in the IR group,the difference of TUNEL-positive cells between the two groups is statistically significant (PO.05) . However,TUNEL-positive cells expression in Low doses EPO-treated retinas was stronger than that in the high doses statistically (P<0.05) . Protreatment with rhEPO resulted in significantly fewer TUNEL-positive RGCs compared with the vehicle-treated groups, suggesting that rhEPO affords protection by inhibiting apoptosis.Morever, the efficiency of high doses is exceeded to low doses.IV. Measurement of immunohistochemistry for Bcl-2 and BDNF. Immunohistochemistry for Bcl-2 and BDNF were seen predominantly inretinal ganglion layers, Immunohistochemistry for Bcl-2 and BDNF shows that Bcl-2 and BDNF positive expression in IR model groups decreased at day 1,3,7;After intravitrous administration of EPO(high doses and low doses), the Bcl-2 and BDNF positive RGCs significantly increased stastistically compared with those in IR model groups at corresponding periods , P <0.05. Morever , the Bcl-2 and BDNF positive RGCs in low doses groups were fewer than those in the high doses groups, P <0.05. Results suggest EPO affords protection by upregulating the Bcl-2 and BDNF expression.V. The histochemical results of Bax and Caspase-3There is no positive cells staining Bax and Caspase-3 in control group. At the days of 1,3,7, Bax and caspase-3 positive cells increased in IR group. Compared with IR group, Bax and caspase-3 positive cells decreased remarkably at the various time. (P<0.01). In the IR-EPO low dose group Bax and caspase-3 positive cells decreased at the day of 1 compared with IR(P<0.05). While no significant difference at the days of 3 and 7(P>0.05).It has been shown that bax and Caspase-3 expression were down regulated after applying EPO administration.VI. The change of ratio of Bcl-2/Bax demonstrated that Bcl-2 was prevailed and decreased RGCs apoptosis. These results are the same as the TUNEL results.W. The RT-PCR and western blotting results of Bcl-2 and Caspase-3 At the level of expression of nucleic acid and protein, Bcl-2 gene was not expressed in the group of control and sham operation group. Compared with these two group, Bcl-2 was expressed in model group but notsignificant(P>0.05). Bcl-2 gene transcription and expression were triggered significantly in high dose group.(P<0.01). So was low dose group(P<0.05). At the level of expression of nucleic acid and protein, Caspase-3 gene was not expressed in the groups of controls and sham operation group. Compared with these two group, Caspase-3 gene transcription and expression were triggered significantly in control group.(P<0.01). Compared with model group, Caspase-3 gene expression was inhibited in high dose group in some extent(P<0.05). While, Caspase-3 gene transcription and expression were inhibited in low dose group, but not significant difference compared with model group(P>0.05). It has been shown that Caspase-3 expression in single RGC in high dose group is lower than that of low dose group(P<0.05), which suggesting inhibiting capability is stronger than that of low dose group.Our results suggest intravitrously administered recombinant human EPO(rhEPO) posttreatment is associated with both histopathological and functional protection of retinal neurons subjected to ischemic injury. We further show that rhEPO inhibits apoptosis after acute retinal ischemia. The present study provides further evidence for a neuroprotective and antiapoptotic effect of EPO and suggests that up-regulating and down-regulating apoptosis-related factors system and recruitment of brain-derived nutrious factors may enhance recovery from ocular diseases involving acute neuronal injuries. Our findings also demonstrate that agents that inhibit apoptosis can preserve neuronal function after acute ischemic injury. rhEPO may represent a therapeutic agent for several retinal diseases including acute glaucoma, acute retinal vascular occlusion, diabetic retinopathy, and hypertensive vascular disease. Future work using EPO that promote neuroprotective effects may be enlightening.
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