| Part 1:The changes of retinal morphology in retinal ischemic reperfusion injury animal modelObjective To analysis the changes of retinal morphology in retinal ischemic reperfusion injury animal model. Methods:Transient ischemia was induced in SD mice by raising intraocular pressure to 110 mmHg for 60 min followed by retinal reperfusion by restoring normal pressure. At various time points post I/R, retinal changes were monitored by histological assessment with H&E staining and by SD-OCT scanning. The retinal thickness were measured. Results:OCT of IRI 2h experimental group showed the relatively normal retina.12h,24h and 48h groups showed retina edema and increased thickness of the retina, The boundary layers of the structure can not been shown clearly. In the 168h group, the thickness of the neurosensory retina was thin, the structure of layers show is unclear. Pathological examination showed in 2h group, retinal edema, and occasionally retinal ganglion cell layer and inner nuclear layer degeneration.12h and 24h retinal nerve fiber layer, ganglion cell layer were a large vacuoles, thickness increases,48h edema peaked. Progress with the disease retinal ganglion cell layer began nuclear enrichment,168h nuclear enrichment significantly increased, retinal is thinner. Conclusion:In the early stage of ischemia reperfusion injury, nerve fiber layer and ganglion cell layer were edema, in the late stage of the injury, atrophy was observed.Part 2:The activation of microglia in different phase of retinal ischemia-reperfusion injuryObjective To analysis activation of microglia in different phase of retinal ischemia-reperfusion injury. Methods:Retinal ischemia reperfusion animal model was established, immunohistochemical staining was used to detect the expression of CD68 after 6h,12h,24h,48h,72h of the injury, the morphological and volume changes of activated microglia was observed. Results:The microglia are mainly located in the ganglion cell layer in normal control group, in the group of ischemia-reperfusion 2h, the distribution and the expression of the microglia is similar with the normal control group. In the group of 12h after retinal ischemia-reperfusion injury, the number of CD68+ cells increased, positive cells can be observed in the inner plexiform layer. In the group of 24h group CD68+ cells was significantly increased and migrated to the outer retina. In the group of 48h CD68+ cells can be observed. in the inner nuclear layer, outer plexiform layer of the retina. Ischemia-reperfusion injury after 72h, the number of activated microglia reached the highest level. Ultrastructure damage began to appear after 12h of the injury, the gap between the RGC cells expanded, the outer segment membrane of the photoreceptor became loose, some microglia can be observed. In the group of 24h, the gap between the RGC cells continues to expand and the number reduced, the outer segment membrane of photoreceptor deformed, fractured, swelled, the number of microglia significantly increased. In the group of 72h the lesions continue to increase, the number of RGCs significantly reduced, membrane swelled and organelles dissolved. Photoreceptor cell membrane became incomplete and dissolved, the apoptotic bodies can be observed in the layer of retinal ganglion cells, phagosome can be observed in the microglia is a direct proof of the damage of the microglia to light receptors.Conclusion:The activated microglia, showed morphological changes, location migration, CD68 upregulation in retinal ischemia-reperfusion injury.Part 3:The protective effects on retinal ganglion cell via inhibited the expression of cx3crl and microglial activationObjective To analysis the protective effects on retinal ganglion cell via inhibited the expression of cx3cr1 and microglial activation, explore the safety of intravitreal injection anti-cx3crl. Methods The rats were divided into normal control group, blank intervention group, positive control group and antibody intervention group. Antibody intervention group received intravitreal injection of anti cx3crl, retinal ganglion cell apoptosis was detected, microglial activation was detected by immunohistochemistry, and the expression of related inflammatory cytokine secretion was detected by PCR. Compared with the normal control group and the positive intervention group. The effects of anti cx3crlon retinal tissue was analysed in the intervention group. Results:Compared with the positive control group, the expression of cx3crl was downregulated and the apoptosis of ganglion cell was significantly inhibited in the antibody intervention group. Its mechanism including inhibition of microglial activation resulted in phagocytosis of ganglion cells decreased, and inflammatory cytokine secretion inhibited to avoid damage caused by excessive inflammation. While no significant differences was observed between the normal intervention group and control group, intravitreal injection cx3crl is safe and feasible.Conclusion Inhibition of cx3crl significantly inhibited the expression of retinal microglia activation, thereby protected the the retinal ganglion cells in. ischemia-reperfusion injury. |
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