Molecular Mechanisms Of Retina Damage After Hypoxic-ischemic Damage

Part One Upregulation of Homer1 a Promoted Retinal Ganglion Cell Survival after Retinal Ischemia and Reperfusion via Interacting with Erk pathway 1 BackgroundRetinal ischemia-reperfusion(I/R) injury leads to evident pathological changes of retinal ganglion cells(RGCs), which can cause irreversible damage to the optic nerve, and result in vision loss and even blindness. Current studies found that, retinal I/R injury occurs in many ocular diseases, including retinal vein occlusion, immature retinopathy, diabetic retinopathy, glaucoma and other retinal vascular occlusion caused by ischemic disease, which mainly manifests disappearing of cell structure of RGCs and function destruction. Therefore, these studies on protecting the optic nerve and promoting RGCs survival after I/R injury are of great significance. Previous investigations suggest that, Homer1 a, as an important postsynaptic density, has an important neuroprotective effect on traumatic brain injury by regulating Erk signaling pathway. Additionally, studies also find that Homer1 is involved in ocular diseases. However, the expression and role of Homer1 a in retinal I/R injury is unclear and need to further be elucidated. Therefore, the purposes of this study was to:(1) clarify the change of expression of Homer1 a after retinal I/R injury;(2) explore the role and related mechanisms of Homer1 a in I/R induced RGCs injury.2 Methods(1) Cell model: using oxygen and glucose deprivation(OGD) / reperfusion model on primary culture of RGCs from mice;(2) Animal model: saline-induced mice retinal I/R model;(3) Protein expression: detecting the expression of Homer1 a, Erk, phosphorylated Erk(p-Erk), cleaved-Caspase3, and Caspase3 in RGCs and retinas after I/R by using Western blot;(4) RGCs injury assay: using TUNEL staining and the cleavage level of Caspase3 to detect I/R induced RGCs apoptosis and using β-III-Tublin staining to detect the RGCs survival in retina after I/R;(5) Inhibitor intervention: using Erk inhibitor(PD98059) to inhibit the activation of Erk;(6) Gene RNA interfere(RNAi)/overexpression: using lentivirus-mediated RNAi/overexpression to regulate the expression of Homer1 a in RGCs;(7) Gene knockout(KO) mice: using Homer1 a KO mice to verify the neuroprotection of Homer1 a in vivo.3 Results and conclusions The present study investigated the changes in expression and the effect of Homer1 a on RGCs both in vitro and in vivo after I/R injury using Western blot, TUNEL assay, gene interference and overexpression, and gene knockout procedures. The levels of Homer1 a and p-Erk increased in RGCs and retinas after I/R injury. Upregulation of Homer1 a in RGCs after I/R injury decreased the level of p-Erk, and mitigated RGCs apoptosis. Conversely, downregulation of Homer1 a increased the level of p-Erk, and augmented RGCs apoptosis. Furthermore, inhibition of the p-Erk reduced RGCs apoptosis, and increased the expression of Homer1 a after I/R injury. These results suggested that: both Homer1 a and p-Erk were involved in I/R induced RGCs injury; Homer1 a played an neuroprotective role in I/R induced RGCs injury; however, activation of p-Erk had a detrimental role in I/R induced RGCs injury. Finally, the retinas of Homer1 a knockout(KO) mice treated with saline-induced high-IOP had significantly less dendrites and RGCs, compared with Homer1 a wildtype(WT) mice. These findings demonstrated that Homer1 a may contribute to RGCs survival after I/R injury by interacting with Erk pathway.Part Two The roles of NLRP3 inflammasome in the retinal ischemia-reperfusion injury 1 BackgroundThere is considerable evidence that the initial injury in glaucoma occurs in the optic nerve head(ONH), where activated astrocytes produce mediators that damage axons and trigger inflammatory cell recruitment. We discovered that ONH astrocytes in human and mouse eyes constitutively express high levels of a critical regulator of inflammation called NLRP3, which is part of a multi-protein complex that contains the adapter protein ASC and activates Caspase1 to produce IL-1β and IL-18. We hypothesize that activation of the NLRP3 inflammasome in ONH astrocytes initiates inflammation and axonal damage in glaucoma.2 Methods Using immunofluorescence and PCR to assesse NLRP3 expression in human and mouse ONH. Intracameral microbead injection was used to elevate IOP in WT C57/BL6 J mice and different B6 KO mice(NLRP3 KO, ASC KO, IL-1R KO, and IL-18 KO). Controls were uninjected and saline-only injected eyes. IOP was monitored by rebound tonometry. At designated time points, mice were euthanized and the ONH was processed for:(1) RGCs counts(?-III tubulin),(2) axon counts(PPD),(3) immunostaining(GFAP, IL-1β, Iba1), and(4) RT-PCR.3 Results and conclusions Normal human and mouse ONH possessed astrocytes with high levels of NLRP3 but no evidence of inflammasome activation. Mice with elevated IOP displayed additional increases in NLRP3 and ASC, and activated Caspase1. To demonstrate a functional role of the activated inflammasome in glaucoma, microbead-induced elevated IOP was induced in a series of KO mice that targeted inflammasome components(NLRP3 and ASC), or products(IL-1β and IL-18). All WT and KO mice displayed a similar increase in IOP for 21 days as compared with controls. The absence of NLRP3 or ASC resulted in a significant reduction in RGCs and axon loss, as compared to WT controls. In addition, loss of IL-1R or IL-18 also produced a significant reduction in RGCs and axon loss. The reduced RGCs loss in NLRP3 KO mice correlated with a reduced infiltration of Iba1+ cells into the ONH as compared to WT controls.The components of the NLRP3 inflammasome are constitutively expressed in normal ONH astrocytes but remain inactive. Elevated IOP triggers activation of the inflammasome and production of IL-1β/IL-18, which are critical mediators of inflammation and necessary for development of glaucoma. Blocking inflammasome activation leads to neuroprotection in the presence of elevated IOP.