Progesterone Regulates Microglia Activation And Protects Retinal Ganglion Cells After Optic Nerve Crush

The pathological basis of optic nerve injury is apoptosis of retinal ganglion cells（RGCs）,which leads to irreversible damage to visual function.Retinal microglia,as an important immune cell in the retina,actively shrink and extern their processes to maintain the dynamic balance of the retina under normal conditions.They are activated when stimulated by external inflammation and then turn into ameboid and polarize into two cell phenotypes of pro-inflammatory（M1）and anti-inflammatory（M2）,which can phagocytose and clear the apoptotic neurons and play roles in inflammation inhibition,tissue remodeling and immune regulation.However,the over-activated microglia also abnormally engulf dying neurons and cause secondary damage to the nervous system.Regulating the activation phenotype of microglia can slow down retinal ganglion cell（RGC）apoptosis,prevent secondary damage to the nervous system by microglia,and be beneficial to the recovery of visual function.As a high-potential drug with multiple efficacies,Progesterone has neuroprotective effects on damaged central and peripheral nervous systems and can protect photoreceptors by inhibiting the activation of harmful microglia.In order to further explore the protective mechanism of progesterone on RGC after optic nerve crush（ONC）,we used confocal imaging technology according to the transgenic mice,whose microglia express the green fluorescent protein,to study the effect of progesterone on the retinal microglia in ONC model.At the same time,high-throughput analysis was used to determine the expression changes of microglia-related genes,and the role was further determined by selectively eliminating M1 microglia or macrophages after ONC.This study observed the effects of progesterone on the retina at 3 and 7 d after ONC.Histomorphological staining showed that progesterone significantly inhibited the apoptosis of the cells and the proliferation of astrocytes in the retinal ganglion cell layer（GCL）,but the repair effect on the morphology of single neurons was not pronounced.Confocal imaging results displayed that the density of microglia in the retina increased significantly after ONC,and the microglia in GCL became ameboid and arranged radially in the center of the retina.Although progesterone could reduce the density of microglia at 3 d after ONC,there was no significant difference;the density of microglia in GCL and the inner plexiform layer（IPL）was extremely significantly reduced at 7 d after ONC（P<0.01）.The results of skeletonization analysis revealed that the microglial morphology began to change at 3 d after ONC in IPL.Progesterone promoted the recovery of the total number of microglial process endpoints/cell at 3 d after ONC and significantly contributed to the change of microglial total process length/cell at 7 d after ONC.The total microglial processes length/cell in OPL changed significantly only at 7 d after ONC,and progesterone significantly affected the change of the total microglial process length/cell in the retinal central only at 3 d after ONC.Furthermore,changes in microglia density were highly negatively correlated with those in the total number of microglial process endpoints/cell and length/cell.The results of high-throughput sequencing analysis indicated that progesterone affected the changes in chemokine receptors,proliferation and inflammatory factors at 7 d after ONC.The results of q RT-PCR showed that the expression of M1 marks was significantly down-regulated by progesterone,while the expression of M2 markers was significantly up-regulated.It was preliminarily concluded that progesterone could prevent the retina from secondary damage by inhibiting the activation of retinal microglia and regulating microglia typing.To further determine the protective mechanism of progesterone on RGC,we used gadolinium chloride（GdCl₃）to eliminate M1 microglia/macrophages selectively after ONC.The result showed that the inhibitory effect of eliminating M1 on the change of retinal microglia density was basically the same as that of progesterone treatment,and GdCl₃ could not only significantly affect the change of the microglial total process length/cell,but also increase the total number of microglial process endpoints/cell at 7d after ONC.By analyzing the expression of microglial markers,it was found that GdCl₃ could significantly inhibit the expression of M1 markers and increase the expression of M2 markers.Therefore,eliminating M1 microglia after ONC was conducive to regulating the proliferation and morphological changes of microglia,thereby protecting RGCs.It was further confirmed that progesterone protected RGCs through the regulation of microglia.In summary,progesterone can inhibit the activation of microglia after ONC by regulating the proliferation,morphological changes and cell typing of microglia and protect RGC to slow down the loss of visual performance.It provides a particular reference for the treatment of eye diseases.