Epo Inhibition Of Diabetic Retinal Glial Reactivity And To Increase Neurotrophic Factor Expression In M¨¹ller Cells

Diabetic retinopathy (DR), which results from neurodegeneration, microangiopathy, and glial dysfunction, is the leading cause of blindness in working age adults. It has been recognized that changes in retinal neurons and glial cells precede the development of overt microvascular pathology during diabetes. Therefore, the present study is focusing on glial dysfunction.Muller cells, one of the principal macroglial cells of the retina, extend from the inner limiting membrane to the outer limiting membrane. Their somas locate within the inner nuclear layer (INL) and the processes envelop all neurons and synapses. Muller cells participate in maintenance of retinal homeostasis through synthesis of neurotrophic factors, uptake and metabolism of neurotransmitters, spatial buffering of ions during retinal activity, and are required for the blood-retinal barrier (BRB). Thus, any malfunction of Muller cells could have deleterious effects on neurons and vascular cells in the diabetic retina. Reactive gliosis, which is a consistent feature of wound healing after retinal injury, is one of the characteristics of glial dysfunction in DR. Excessive gliosis leads to scar formation which ultimately causes fibrovascular membrane and tractional retinal detachment. Therefore, a strategy for controlling excessive gliosis is critical.Neurodegeneration is another early feature in DR, prompting investigations on neuroprotective agents in animal models of DR. Erythropoietin (EPO) has drawn great attention recently. For instance, its effectiveness as a neuroprotective agent has been proved in central nervous system diseases and in some retinal diseases. Recent work in our laboratory has demonstrated that a single intravitreal injection of EPO at the onset of diabetes in rats prevented retinal neuron death and protected the integrity of the BRB. The protective function is mediated by EPO/EpoR system. Binding of EPO to EpoR activates multiple down-stream signaling pathways in neurons. Among many possible signaling pathways, the MAPK/ERK1/2 pathway has been reported to play a critical role in EPO neuroprotection in early diabetic retinas. Recent studies have proposed that neurotrophic rescue by EPO may involve in interactions between neurotrophic factors and glial cells. EPO has been shown to promote neurogenesis in rat brain by inducing brain-derived neurotrophic factor (BDNF) expression by astrocytes. However, the possibility that EPO plays a neurotrophic role mediated through Muller cells has not bee explored.To characterize Muller cell-mediated neuroprotective and neurotrophic functions c the erythropoietin (EPO)/EPO receptor (EpoR) system in diabetic rat retina, a single intravitreal injection of EPO (8 mU/eye) was given 4 or 24 weeks after induction of diabetes in rats. Reactive gliosis was evaluated by immunofluorescence and Western blotting to detect glial fibrillary acidic protein (GFAP) and vimentin. EpoR in Muller cells was studied by immunohistochemistry. Brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) were quantified in the retina by real-time PCR and ELISA. The effects of EPO on primary rat retinal neurons and rMC-1 cells were studied in terms of neurite outgrowth and expression of BDNF, CNTF, cyclic AMP response element binding protein (CREB), and EPO/EpoR downstream signaling pathways.The present study has demonstrated that intravitreal EPO ameliorated the increased expression of GFAP and vimentin in the diabetic retina; EPO also up-regulated BDNF and CNTF after 24 weeks of diabetes. The expression of EpoR was up-regulated in Miiller cells in the diabetic retina, and exogenous EPO reduced this up-regulation. In rMC-1 cells, BDNF and CNTF were stimulated by EPO through MAPK/ERK1/2 and PI3K/Akt pathways, respectively. BDNF was involved in EPO-induced neurite outgrowth.In conclusions, exogenous EPO exerts neuroprotective and neurotrophic functions that attenuate reactive gliosis and promote neurotrophic factors in Miiller cells in the diabetic retina. The neuro-regenerative function of EPO, as indicated by promotion of neurite outgrowth, was corroborated in vitro study. Signaling pathways that are responsible for these Miiller cell-mediated EPO/EpoR functions may be therapeutic targets for diabetic neuronopathy.