| Objective1.To establish a rat model of nonarteritic anterior ischemic optic neuropathy(rNAION)and evaluate the optic nerve(ON)damage.2.To preliminarily understand the effect of intravitreal injection of siCASP2 on optic nerve injury in rNAION.3.To investigate the mechanism of siCASP2’s protective effect on optic nerve in rNAION.Methods1.The rats were randomly divided into 3 groups:the photodynamic method was used to construct the rNAION as the NAION group(n=27),and the observation time points were 7 days,14 days and 28 days;the sham laser group was not injected with photosensitizer,only the optic disc was irradiated by laser(n=9);the rats in the normal control group were free of any intervention(n=9);the right eye was used as the experimental eye.The histopathological manifestations of rat optic nerve ischemia injury were observed by HE staining of retina and optic nerve,and toluidine blue staining of optic nerve,and the density and survival rate of RGCs were observed by immunofluorescence staining.2.Rats were randomly divided into 3 groups:PBS group(n=9)rats were not modeled,but only given intravitreal PBS;NAION+PBS group(n=9)rats were given intravitreal injection of PBS after modeling;Rats in the NAION+siCASP2 group(n=9)were injected with the same amount of siCASP2(20μg/4μL)into the vitreous cavity after modeling;the right eye was used as the experimental eye.The morphological changes of optic nerve,the density and survival rate of RGCs,and the number of apoptotic cells were compared between groups by histopathological examination,TUNEL staining and immunofluorescence staining.3.Rats were randomly divided into 3 groups:blank group(n=36)without any intervention;model group(n=72)with NAION modeling;treatment group(n=36)with intravitreal siCASP2 intervention after modeling;the right eye was used as the experimental eye.The expression of Caspase-2 mRNA in retina and optic nerve,the expression of Caspase-2 and related apoptosis proteins in retina were detected by Western Blot and RT-qPCR.Results1.HE staining results showed edema in the optic disc and RNFL on the 3rd day after the rNAION model,and the edema began to subside on the 7th day.After 28 days,optic nerve atrophy,RNFL thinning and RGCs lost were observed.Toluidine blue staining of the optic nerve showed that the optic nerve fiber bundles in the NAION group were partially disintegrated at 28 days,as well as a large number of axonal loss and glial scars.On the 14th and 28th day,the retinal RGCs of the NAION group were 1885±328/mm2 and 1269±269/mm2 in the central retina,and 1043±269/mm2 and 752±181/mm2 in the mid-peripheral retina.The decrease compared to sham laser group was statistically significant(P<0.0001).Central RGCs lost slower in the first 2 weeks and then accelerated,while mid-peripheral RGCs lost faster in the first 2 weeks and then decreased.2.The densities of RGCs by immunofluorescence staining at 4 weeks in the central and mid-peripheral retina in the NAION+PBS group were 1362±216/mm2 and 812±113/mm2,while those in the NAION+siCASP2 group were 1790+227/mm2 and 1081±140/mm2,respectively.The difference between the two group was statistically significant(P<0.0001).After siCASP2 treatment,the survival rate of RGCs increased from 58.74%to 77.20%in the central retina and from 56.91%to 75.75%in the midperipheral retina.Toluidine blue staining of optic nerve semi-thin sections showed that after 4 weeks of siCASP2 intervention,the structure of optic nerve fiber bundles was still partially disintegrated,but axonal loss and glial scars were reduced compared with those in the untreated group,and the optic nerve damage was alleviated.The results of TUNEL staining showed that the number of TUNEL positive cells in the NAION+PBS group was 10.67±2.08/HPF at 4 weeks,which was significantly higher than that in the PBS group(P<0.0001),but decreased to 5.33±1.53/HPF after siCASP2 intervention.The difference was also statistically significant(P<0.001).3.The measurement results of early apoptosis protein levels in the NAION model group showed that retinal Procaspase-2 and Procaspase-2L proteins were significantly and continuously up-regulated on the 3rd and 7th days after modeling,and BIM protein remained unchanged within 1 week after modeling.At 2 weeks and 4 weeks,the protein expressions of Procaspase-2,Procaspase-2L,BIM,PIDD1 and RAIDD in the model group were significantly up-regulated compared with those in the blank group,and the differences were statistically significant(P<0.001).After 4 weeks of intravitreal injection of siCASP2 in the treatment group,compared with the model group,the expression of the above proteins was significantly down-regulated except RAIDD at 2 weeks(P<0.01),The content of Caspase-2 mRNA in the model group increased at 2 weeks,but there was no significant difference compared with the blank group(P>0.05);at 4 weeks,the Caspase-2 mRNA in retina and optic nerve was up-regulated statistically(P<0.05).Conclusion1.The NAION rat model in this experiment simulates the pathophysiological changes of human eye NAION,and is appropriate as an animal model for subsequent experiments.The extended period of apoptosis of RGCs in the rNAION induction is longer than previously recognized,and this precious time window is a good opportunity to rescue damaged RGCs.2.After intravitreal injection of siCASP2,the survival rate of RGCs increased significantly,and the number of apoptotic cells in RGCs decreased.The optic nerve damage was also reduced.Therefore,siCASP2 could be neuroprotective against NAION injury for at least 28 days.3.Caspase-2 in RGCs was specifically activated after ischemic injury in NAION rats.Intravitreal siCASP2 can inhibit Caspase-2 expression to rescue RGCs death and reduce the number of apoptotic cells,thus siCASP2 may serve as a potential neuroprotective agent. |
