The Protective Role And The Mechanism Of Microglial Exosomes In Oxygen-induced Retinopathy

Part One:Microglial density and activation determine the appearance of pathology neovascular tufts in oxygen induced retinopathyBackground:Retinopathy of prematurity(ROP)is a devastating disease in premature infants and a major cause of childhood vision impairment.Retinal neovascularization(RNV)is the main pathological characteristic of ROP.It has been identified that microglial cells,an inflammatory mediator may determine the appearance of retinal neovascularization.Objective:To investigate the effect of retinal microglial density or activation on neovascularization in an oxygen-induced retinopathy model.Methods:C57BL/6J mice,Balb/c mice and SD rats were placed in the volume fraction(75±2)%oxygen chamber on the 7th day after birth(P7)to P12 in order to establish oxygen-induced retinopathy animal model.Intraperitoneal injection of minocycline and intravitreal injection of clodronate liposomes were used to inhibit microglial activation or decrease microglial density in Balb/c OIR mice.Immunofluorescence,RT-qPCR and Western-blot were used to detect the density and activation of various glial cells in the retina,retinal flatmounts and Lectin staining were used to observe the effects of minocycline and clodronate liposomes on retinal angiogenesisResults:We found that there existed no avascular and neovascular area in Balb/c OIR mice at P17,which was very different from C57BL/6J OIR mice.After checking the density of glia cells,the results revealed that the density of microglia and astrocyte were both higher in Balb/c mice,while Muller gliosis seemed more obvious in C57BL/6J OIR mice at P17.With the decline of microglia by using the two drugs,avascular area was higher compared with Balb/c OIR mice,small area of neovascular tufts also appeared at P17.After checking the expression of Ibal,the marker of microglia,and GFAP,the marker of astrocytes and Muller cells,we found that minocycline and clodronate could inhibit the activation of microglia or decrease the density of microglia,while they had no significant effect on astrocytes and Muller cellsConclusions:These data suggest that the density and activation of microglia in retina may determine the result of the vasculopathy in OIR mice to some extentPart Two:Exosomes from microglia attenuate photoreceptor injury and neovascular formation in an animal model of retinopathy of prematurity Background:From the previous result,we found that the density of microglia in the retina can determine the degree of retinal vasculopathy in OIR mice to some extent,but the specific mechanism is still unclear.In recent years,it has been found that microglial exosomes of the central nervous system have protective effects on some diseases,but the role of retinal microglia exosomes in OIR is still unclearObjective:To investigate the role and mechanism of microglia exosomes in OIRMethods:Microglia were cultured in vitro,microglia exosomes were extracted,and intravitreal injection was performed to observe the avascular avascular zone and neovascularization state in C57BL/6J OIR mice,and the expression levels of pro-angiogenic factors Electroretinogram was used to detect retinal function.Photoreceptor cells were cultured in hypoxic environment.Apoptosis levels were detected by flow cytometry.Endoplasmic reticulum stress(ER stress)status of photoreceptors was detected by RT-qPCR and Western-blot.High-throughput sequencing was used to detect highly expressed miRNAs in microglia exosomes,and the relationship between miRNA and ER stress and apoptosis was analyzed by RT-qPCR and Western-blot.Finally,an in vitro tuber formation assay was performed to examine the effect of microglial exosomes photoreceptor cells on the tuber formation of vascular endothelial cellsResults:Exosome-treated OIR mice exhibited smaller avascular areas and fewer neovascular tufts,in addition to decreased vascular endothelial growth factor(VEGF)and transforming growth factor-?(TGF-?)expression.Moreover,photoreceptor apoptosis was suppressed by exosome injection.Mechanistically,exosomes from microglial cells were incorporated into photoreceptors and inhibited the inositol-requiring enzyme la(IREla)-X-box binding protein 1(XBP1)cascade,which contributes to hypoxia-induced photoreceptor apoptosis Furthermore,the exosomes also downregulated mRNA and protein levels of VEGF and TGF-? in hypoxia-exposed photoreceptors.A microRNA assay showed that miR-24-3p levels were extremely high in exosomes from microglial cells,suggesting that this could be the key molecule that inhibit the hypoxia-induced expression of IRE la in photoreceptorsConclusions:The exosomes purified from the supernatant of microglia alleviated vasculopathy and vision injury in an ROP animal model.Microglial-derived exosomes could be internalized into photoreceptors and inhibit hypoxia-induced photoreceptor apoptosis via the ER stress pathway and IRE1?-XBP1/JNK-CHOP signaling through the transfer of miR-24-3p Exosomes from microglia could inhibit the expression of pro-angiogenic factors in hypoxic photoreceptors,including VEGF and TGF-?,which suggest that microglia might play a beneficial role in ROP.