| Retinal degenerative diseases are a group of serious blindness diseases of hereditary tendency or unknow aetiology,including retinitis pigmentosa,age-related macular degeneration,and glaucoma.The pathologies of retina and optic nerve degenerations are characteristically associated with neuron degeneration and glial cell over-activation.The changes of retina and optic nerve microenvironment that is characteristic of abnormal immune reaction and glial scar formation caused by glial cell over-activation play a key role in the development and injure recovery of the degenerations.In the normal retina,there are three types of glial cells to maintain retinal homeostasis,Müller cells,astrocyte cell(AC)and microglia.Müller cells go through the whole retina,from outer segment membrane to inner segment membrane,and enwrap all the bodies and the processes of neuron cells.Resting stated astrocytes are mainly located in the inner molecular layer and outer molecular layer.They are not only the framework of retina,but also related to the metabolism of retina,the phagocytosis of photoreceptor and neuronal debris,immunity reaction,the release of neurotransmitters and neurotrophic factors,and regulating immunity.In retinal degeneration,when neurons apoptosis,the released neurotransmitter increase,glia cell activation,the released inflammatory factor and cytokines increase,all of changes have affect on retinal degeneration and inner neuron apoptosis.Olfactory ensheathing cell(OEC)is a special type of macroglia cell isolated from the olfactory bulb and olfactory nerve,which has both the biological characteristics of astrocytes and Schwann cells.In recent years,a large number of experimental studies have been carried out on the regeneration of OECs in the degenerations of central and peripheral nervous system through regulating colloid microenvironment.Researches show that OECs transplantation can enwrap axons,secrete hydrolytic enzyme to degrade extracellular matrix close to axons,secrete nutrition factors,and promote axons across the damaged zone.OECs transplantation can help astrocytes hyperplasia to form the bridge,regulate microglia activation,and promote phagocytosis of cellular debris and pathogens and provide a suitable microenvironment for neuron regeneration.Our previous studies showed that OECs transplantation could regulate Müller cell microenvironment in the retinal degeneration.OECs transplantation could inhibit Müller cell proliferation through Notch pathway,and phagocytose outer segment membrane through MEG-F8 pathway in the RP model.In the glaucoma model,we found OECs transplantation could increase the number of optic nerve axons and AC numbers of optic nerve area.However,in addition to modulate macroglia microenvironment in the retinal and optic nerve degenerations,do OECs have effect on microglia microenvironment? What is the mechanism of OECs transplantation? What regulating effect do OECs have on abnormal AC in the optic disc and sieve plate area of glaucoma model and what is the mechanism? They are not clear yet.Based on our previous laboratory researches and current researches,we propose the following hypothesis: OECs transplantation improves the degenerative glial microenvironment by regulate microglia and astrocyte,improve visual function.This study consists of two parts:Part 1: OECs transplantation regulates the retinal microenvironment of microglia and delay retinal degenerationIn the development of retinitis pigmentosa,activation of microglia and changes of immune microenvironment occur in the early stage,probably duo to activation of primary or secondary photoreceptor apoptosis.Activated microglia releases a large number of inflammatory factors and free radicals in neurons,and activates microglia,including Müller cells and AC,in addition to changes in morphology,quantity,and up-regulation of a variety of immune active molecules,leading to deterioration of photoreceptor and neuron damages.In the activated macroglial cells,Müller cells produce glial proliferation reaction,and with the Müller cells continued proliferation,glial scar formation in the subretinal space further hinder the nutrients from choroid to retina transport,and accelerates the ischemic and anoxia of the retina and neuron.We choose the retinal degeneration model of RCS rat and controlled normal rats were detected with retinal immunofluorescence staining,q RT-PCR and Western blot.The results showed that in the process of RCS rat retinal degeneration,microglia activated,the retina showed M2 type inflammatory microenvironment in the early stage,advanced persistent present of M1 microenvironment in the middle and late stage,and the JAK2/STAT3 pathway,which was related with inflammatory factors,continuously activated in RCS rat retina.OECs reduced endogenous microglia activation in the retina,M1-phenotype cytokine expression in the retina decreased,the JAK2/STAT3 pathway was down-regulated,degeneration of photoreceptors and bipolar cells delayed,and retinal function improved after OECs transplanted into the subretinal space of RCS rats.With the establishment of in vitro BV2/OECs co-culture model and by lipopolysaccharide(LPS)induced activation of BV2,we found that OECs could inhibit the activation of BV2,promote the BV2 immune microenvironment of the transformation from M1 to M2,and down-regulate JAK2/STAT3 pathway.When we pretreated OECs with JAK2 inhibitor AG490,the role of OEC immune modulation directed against BV2 was inhibited.These results suggest that both OECs in vitro and in vivo can inhibit the activation of microglia,improve the immune microenvironment,and delay the degeneration of visual function by down regulating the JAK/STAT pathway.The second part: OECs transplantation regulates the retinal microenvironment of astrocyte and delay retinal degenerationIn the development of glaucoma,a kind of optic nerve degeneration,high intraocular pressure causes changes of AC colloid microenvironment in the optic nerve,and cell processes fallback.The fallback of close enwrapped radial glial processes of axons lead to axon uncovered and apoptosis of axons duo to lack of nutrition and support.Axonal apoptosis further induces neuronal cell apoptosis by reducing axonal transport.The colloid microenvironment plays a key role in maintaining function of retinal neuron homeostasis.Magnetic beads were injected into the anterior chamber of Long-Evans rats to establish a rat model of glaucoma,with normal Long-Evans rats without injection as control.Immunofluorescence staining,qRT-PCR,retinal glutamate content determination techniques were used.The results showed that anterior chamber injection of magnetic beads could stably and persistently elevate intraocular pressure of the Long-Evans rats,caused optic glial proliferation,apoptosis of neuron axon,and visual function injury.OECs transplantation into the subretinal space near the optic disc,the visual function of glaucoma rats were improved,and the gliosis of optic nerve and the apoptosis of optic nerve axons were alleviated.By establishing glutamate acid induced apoptosis of AC in vitro,we found that 200 m M glutamic acid could induce apoptosis of AC;however,co-cultured with OECs could protect AC by metabolizing excess glutamate acid.By comparison of primary cultured Müller cells,optic nerve AC and OECs,we found that the glutamate metabolism ability of OECs was the strongest among the three kinds of cells.Further in vivo study,we found that the ability of glutamate metabolism in the rat optic nerve area was weak compared with that of the retina,and during the glaucoma model development,glutamate metabolism in the optic nerve area decreased,and glutamate content increased.The ability of glutamate metabolism in the optic nerve tissue after transplantation of OECs was significantly increased.These results suggest that OECs can improve the AC microenvironment,protect axon,and delay the degeneration of visual function through accelerating glutamate metabolism both in vitro and in vivo.According to the above experimental results,this study draws the following conclusions:1.OECs transplantation down-regulate JAK2/STAT3 pathway in the RCS rat retina,reduce the expression level of M1 type inflammatory cytokines,regulate retinal immune microenvironment,protect retinal photoreceptors and bipolar cells,and delay the degeneration of visual function of RCS rat.2.OECs transplantation can improve the AC microenvironment,inhibit AC proliferation,protect neuron axon and visual acuity,and delay the degeneration of visual function of glaucoma rat through accelerating glutamate metabolism both in vitro and in vivo.We studied the two kind of glia microenvironment change in retinal degenerative diseases.The results show that transplanted OECs have abilities of regulating immune microenvironment of degenerative retina,and play an important role in optic glutamate metabolism of glaucoma model rats.OECs can reduce the expression level of M1 type inflammatory cytokines,improve microglia microenvironment,protect photoreceptor cells and bipolar cells,and delay the degeneration of visual function of RCS rat through down-regulation of JAK2/STAT3 pathway in the RCS rat retina.OECs transplantation can accelerate glutamate metabolism,decrease the glutamate content of optic nerve area,improve the AC microenvironment,reduce the proliferation of astrocytes,protect the optic nerve axons,and alleviate visual function degeneration of the glaucoma model rats.The results of our study provide new ideas for better understanding of microenvironment in retinal and optic nerve degenerative disease and provide theoretical basis for potential therapeutic targets of the diseases. |
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