Research On The Protective Mechanism Of Rapamycin On Experimental Glaucoma

Background and purpose :Glaucoma is the leading cause of blindness worldwide characterized by both depressed atrophy of optic nerve and visual field defect.While the pathological increase in the intraocular pressure(IOP)is the main risk factor for glaucoma,retinal ischemia and hypoxia,excitotoxicity,inflammation,and optic nerve injury are all factors that cannot be ignored in the damage of retinal ganglion cells(RGCs).Because of the complex and unclear pathogenesis of glaucoma,researchers and clinicians are eager to explore new treatments to protect RGCs from damage,reduce optic nerve damage and promote axon regeneration.When the optic nerve is damaged,astrocytes and microglia in the retina are activated and produce a large number of inflammatory factors,and the injury may also induce retinal pyroptosis,which may cause further damage to RGCs.In addition,optic nerve injury may also cause damage to the RGC axons.This may be because the injury induces the formation of glial scar and the release of inflammatory factors in the optic nerve,and the activation of the ROCK pathway will inhibit regeneration of axons.Rapamycin is a specific inhibitor of the mammalian target of rapamycin(m TOR)signaling pathway,which not only inhibits the activation of glial cells,but also mediates the signaling pathways of small GTPases and Rho to regulate the actin cytoskeleton.Although a large number of studies have shown that rapamycin has protective effects against glaucoma,the specific mechanism remains unclear and further research is needed.Part Ⅰ: Study on the retinal protection of rapamycin in three experimental animal models Purpose:Glaucoma is a group of ocular diseases resulting from multiple factors.This study aimed to investigate whether intravitreal injection of rapamycin has protective effects in various glaucoma models.Methods:In this study,we first established three animal models of IOP-independent glaucoma,including the optical nerve crush(ONC),retinal ischemia?reperfusion(IR)and N-methyl-D-aspartate(NMDA)-mediated retinal excitotoxicity models.Then,rapamycin was injected into the vitreal cavity 0 and 3 days after the operation to observe the effect of the drug on glaucoma in these models.Then,the changes in retina thickness and injury of retinal cells were observed using paraffin sections stained with hematoxylin and eosin(H&E)and terminal deoxynucleotidyl transferase-mediated nick end labeling(TUNEL).Furthermore,retinal tiling and RNA binding protein with multiple splicing(RBPMS)staining were performed to specifically label RGCs in the retina,the effect of rapamycin on RGCs was observed.Results:In the normal group,the retinal structure was complete,and the layers were clear.There was no obvious change after drug treatment in the retina of normal SD rats.However,in the ONC,IR and NMDA-mediated excitotoxicity models,the retina exhibited obvious atrophy and thinning,and ganglion cell layer(GCL)cells were swollen and significantly decreased.Moreover,TUNEL staining revealed a large number of TUNEL-positive cells in the three injury groups.In addition,compared with the normal group,the number of RGCs in the center,middle,and periphery of the retina decreased by about 50% after ONC,IR,and NMDA injury.However,after treatment with rapamycin,the retinal structure was repaired to a certain extent,the degree of atrophy was decreased,the number of TUNEL-positive cells was reduced,and the survival rate of RGCs in the whole retina was significantly increased.Conclusion:Intravitreous injection of rapamycin can significantly alleviate the retinal thickness damage caused by ONC,IR,and NMDA-mediated excitotoxicity,but also significantly improve the survival rate of RGCs.Part Ⅱ: Effect of rapamycin on retinal inflammation after ONC injury Purpose:Retinal inflammation plays a crucial role in the damage process of RGCs.In the Part I,it has been demonstrated that rapamycin has a protective effect on RGCs after ONC injury.This experiment further explored whether the potential mechanism of rapamycin’s protection against RGCs is related to glial cell activation and pyroptosis.Methods:This experiment established ONC model and randomly divided the rats into four groups: control + vehicle(Con + Veh)group,control + rapamycin(Con + RAPA)group,ONC + vehicle(ONC + Veh)group and ONC + rapamycin(ONC + RAPA)group.Firstly,the protein expression of RBPMS,glial fibrillary acid protein(GFAP),ionized calcium binding adapter molecule 1(IBA1),and inducible nitric oxide synthase(i NOS)in the retina of each group was detected using western blotting.We also observed the morphological changes of astrocytes and microglia in the retina by immunofluorescence.Furthermore,changes in expression of pyroptosis-related proteins in the retina were evaluated.Results:The protein expression of RBPMS was significantly decreased in the ONC +Veh group compared with the Con + Veh group,while the protein expression of GFAP,IBA1 and i NOS was significantly increased.In addition,astrocytes were significantly activated in the retina,the cell body enlarged,and the branches of astrocytes crossed from the GCL into the outer nuclear layer(ONL).The activation of microglia increased,and microglia adopted an ameboid morphology and migrated to the GCL.On the other hand,inflammasome activation and the expression of inflammatory factors associated with pyroptosis increased significantly.After treatment with rapamycin,the activation of glial cells was significantly weakened,the protein expression of i NOS was also significantly reduced,and retinal pyroptosis was significantly inhibited compared with the ONC group without rapamycin.Conclusion:Rapamycin can protect RGCs from ONC injury,which may be due to weakening the activation of astrocytes and microglia induced by ONC.On the other hand,it may inhibit the activation of inflammasome and the release of pyroptosis-related inflammatory factors.Part Ⅲ: Study on the effect of rapamycin in protecting RGC axons from ONC injury Purpose:Optic nerve injury not only induces secondary loss of RGCs but also leads to direct damage to RGC axons.The purpose of this study was to explore whether rapamycin has a protective effect on axons after injury and its potential mechanisms.Methods:We first established an ONC model and randomly divided the rats into four groups according to the method in Part II.Then,immunofluorescence and transmission electron microscopy were used to assess the overall and local structural changes in the longitudinal and transverse sections of the optic nerve,while using cholera toxin B subunit(CTB)to specifically label the axons.To further explore the possible mechanisms,on the one hand,we observed the activation of astrocytes using immunofluorescence techniques;On the other hand,western blotting was used to examine the expression of proteins in the m TOR/Rho associated kinase(ROCK)pathway.Results:Neurofilament expression at the site of ONC was decreased in the model group compared with the Con + Veh group,the distribution of neurofilament 200(NF200),phosphorylated neurofilament(SMI-31)and nonphosphorylated neurofilament(SMI-32)in axons near the lesion was disordered,there was a large number of cavities and retracting axons,and the nucleus was disordered.Transmission electron microscopy showed that the number of axons was significantly reduced,and myelin sheaths were loose or even lost.There were almost no CTB-positive axons extending from the lesion to the brain.Moreover,the astrocytes around the injury were obviously activated,and the morphology became disordered.The p-m TOR and ROCK protein expression in the optic nerve increased significantly after injury.After intravitreal injection of rapamycin,NF expression around the lesion was significantly elevated,and the number of axons was also significantly increased.The activation of glial cells and the release of inflammatory factors in the optic nerve were also inhibited.In addition,the protein expression of ROCK1 and ROCK2 decreased significantly,but growth-associated protein 43(GAP43)expression increased significantly after drug treatment.Conclusion:Rapamycin can protect RGCs axons from damage,and its potential mechanism may be that rapamycin inhibits the scarring and related inflammatory damage of astrocytes on the one hand,and may also inhibit the activation of the ROCK pathway on the other hand,which promotes axon regeneration.