| ObjectiveTo explore the changes of retinal nerve fiber layer(RNFL)thickness,pepipapillary vascular morphology and optic disc perfusion in non-arteritic anterior ischemic optic neuropathy(NAION)from acute to chronic stage using optical coherence tomography angiography(OCT/OCTA);to observe the changes of ganglion cell complex(GCC)thickness,macular vascular morphology and flow;to analyze the pathogenic factors of NAION and the correlations and associations between visual function with vascular and structural measurements in different periods;SWAH-MS proteomic analysis was adopted to identify candidate biomarkers for NAION.MethodsThe eyes diagnosed with NAION were included in this study.The following vascular morphological parameters were assessed in pepipapillary and macular region:vascular dilatation with tortuosity,capillary dropout and vascular thinning;The following parameters were measured: RNFL thickness,GCC thickness,whole image vessel density of disc(wi VD),inside disc vessel density(id VD),pepipapillary vessel density(pp VD),superficial vessel density(SVD),deep vessel density(DVD),choroidal capillary layer density(CC)and foveal avasular zone(FAZ)parameters.Generalized Estimating Equations(GEE)analysis and paired sample t test were used to compare the parameters.Data of gender,age,body mass index(BMI),visual acuity(log MAR),visual defect(MD)and OCT/OCTA parameters was collected;logistic regression analysis was used to analyze the pathogenic factors of NAION;correlation analysis and multivariate linear regression analysis were performed to evaluate the correlations and associations between visual function with vascular and structural measurements.The r NAION model was established by Rose Bengal and laser.Twenty rats were randomly divided into 4 groups,the normal control group,the laser control group,the RB injection control group,and the r NAION model group,with 5 rats in each group.The right eye was used as the experimental eye.The retina was dissected at the third day after modeling.Enzyme digestion method was used for sample preparation,and data collection was performed in a non-dependent collection mode.The data were quantitatively analyzed by SWATH quantitative mass spectrometry,searching for differential proteins and performing function and pathway analysis.p<0.05 was considered statistically significant.ResultsPart Ⅰ: A total of 50 NAION eyes were included in the analysis of RNFL thickness in the long-term longitudinal study.Compared with the contralateral eyes,the RNFL thickness increased in the acute phase(p<0.001),returned to the normal level in the subacute phase(p>0.05),and decreased progressively in the chronic phase(p<0.001).The results of follow-up among groups showed that the average RNFL thickness of the > 24 months group was lower than that in the acute phase group,3 weeks-3months group,3-12 months group,and 12-24 months group(p<0.001,p<0.001,p<0.001,p=0.077);the RNFL thickness in the temporal,upper,nasal,and inferior quadrant was lower than that in the acute phase,3 weeks-3 months group and 3-12 months group(all p values <0.001),but showed no significant lower than the 12-24 months group(p=0.2,p=0.148,p=0.128,p=0.077).In comparison between different parts,at >24 months group,the upper RNFL thickness was only 51.48% of that of the contralateral eye,52.85% in the lower part,63.29% in the temporal side,and 59.78%in the nasal side.The most significant changes were in the superior quadrant,followed by the lower part,the nasal side and the temporal side.2.57 eyes in the acute phase and 69 eyes in the chronic phase were enrolled to observe the vascular morphology.In the acute phase,all of eyes showed vascular dilatation,38.6% showed capillary dropout,15.8 % showed large vessels submerged and 12.2% showed large vessels dilatation with tortuosity;while in the chronic phase,all of eyes were noted capillary dropout and 42% showed large vessels thinned.3.A total of 52 NAION eyes and 21 contralateral eyes in the acute phase,53 NAION eyes and 23 contralateral eyes in the chronic phase,and 24 normal eyes were included in the quantitative analysis.Compared with the normal control group,wi VD,wi CVD,pp VD,pp CVD,SH-VD and IH-VD of in the acute phase decreased,and the differences were statistically significantly(all p values <0.001);but wi LVD,id VD,id CVD,id LVD and pp LVD had no significantly differences(all p values=1.00);wi VD,wi CVD,wi LVD,pp VD,pp CVD,pp LVD,SH-VD and IH-VD were decreased in the chronic phase,and the difference was statistically significant(all p <0.001);id VD,id CVD,id LVD had no significant difference(all p=1.00);there was no significant difference between the contralateral eyes with the normal eyes both in the acute and chronic phases(p>0.05).Compared with the contralateral eyes,wi VD,wi CVD,id VD,id CVD,pp VD,and pp CVD in the acute phase decreased,and the difference was statistically significant(p<0.01);wi LVD,id LVD,pp LVD had no significant difference(p>0.05).Wi VD,wi CVD,id VD,id CVD,pp VD,pp CVD,and pp LVD was all decreased in the chronic phase(p<0.02);wi LVD and id LVD was not different(p =0.062,p =0.379).Longitudinal study showed wi VD,wi CVD,pp VD,pp CVD all decreased,and the difference was statistically significant(p<0.001);wi LVD,id VD,id CVD,id LVD,pp LVD did not change significantly(p>0.05).The dramatic change happened in the superior quadrant,which was decreased by 17.28%.Part Ⅱ: 1.A total of 42 eyes were included in the long-term longitudinal study of the GCC thickness.Compared with the contralateral eyes,there was no significant difference in all the GCC parameters in the acute phase group(p>0.05);the GCC thickness decreased,and FLV and GLV increased in the remaining groups(p<0.05).The results of the comparison among groups showed that the GCC thickness of the >24 months group in the average,superior and inferior quadrant was lower than that in the acute phase group,3 weeks-3 months group and 3-12 months group(all p values <0.001)but comparable to the 12-24 months group(p>0.05);FLV in the >24months group was significantly higher than in the acute phase group(p<0.001),but comparable to the 3 weeks-3 months group,3-12 months group and 12-24 months group(p>0.05);GLV in the >24 month group was much higher than that in the acute phase group,3 weeks-3 month group and 3-12 month group(p<0.01),but comparable to the 12-24 month group(p=1.00).2.A total of 39 eyes in the acute phase were included in the observation of vascular morphology surrounding the macular area.Among them,11 eyes(28.2%)in the SCP and 24 eyes(61.5%)in the DCP showed capillary dropout;61 eyes were included in the chronic phase,53 eyes(86.9%)performed capillary dropout in the SCP,while there were no obvious abnormal changes in the DCP.3.Quantitative analysis of macular perfusion including 42 NAION eyes and 24 contralateral eyes in the acute phase,42 NAION eyes and 24 contralateral eyes in the chronic phase,and 29 eyes in the normal group.In the acute phase,compared with the normal eyes,wi SVD and SH-SVD decreased(p=0.011,p=0.001),whereas IH-SVD,Fovea-SVD and Para-SVD also decreased,but there was no statistical significance(p=0.186,p=1.000,p=0.061);wi DVD,SH-DVD,IH-DVD,Fovea-DVD,Para-DVD,CC,FAZ area,perimeter and AI have no significant difference(p>0.05);in the chronic phase,wi SVD,SH-SVD,IH-SVD,Fovea-SVD and Para-SVD decreased(p<0.01);wi DVD,SH-DVD,IH-DVD,Fovea-DVD,Para-DVD,CC,FAZ area,perimeter and AI had no difference(p>0.05);there was no significant difference between the contralateral eyes with the normal eyes both in the acute and chronic phase(p>0.05).Compared with the contralateral eyes,wi SVD,SH-SVD,wi DVD,SH-DVD,IH-DVD,Para-DVD,CC decreased in the acute phase(p<0.02),IH-SVD,Fovea-SVD and Para-SVD also decreased but not statistically significant(p>0.05),Fovea-DVD,FAZ area,perimeter and AI had no difference(p>0.05).Wi-SVD,SH-SVD,IH-SVD,Fovea-SVD and Para-SVD in the chronic phase dramatically decreased,and the difference was statistically significant(p<0.02);wi DVD,SH-DVD,IH-DVD,Fovea-DVD and Para-DVD had no significant difference(p>0.05);FAZ area and perimeter increased(p=0.011,p=0.007),but there was no significant difference in AI(p=0.078).Longitudinal study showed wi SVD,SH-SVD,IH-SVD,Fovea-SVD and Para-SVD all decreased(p<0.03);whereas wi DVD,SH-DVD,IH-DVD,Para-DVD increased significantly(p<0.03);Fovea-DVD had no significant difference(p=0.124);AI increased and the difference was statistically significant(p=0.037);FAZ area,circumference and CC also increased,but the difference was not statistically significant(p>0.05).Part Ⅲ: 1.A total of 86 NAION eyes and 28 normal eyes were included in the analysis of risk factors of NAION.The univariate analysis showed that the related factors were disc area,C/D and sleep apnea.Multivariate regression analysis showed that C/D and sleep apnea were the important risk factors(p=0.001,p=0.015).2.66 eyes in the acute phase and 48 eyes in the chronic phase were included to analyze the correlation and regression between the visual function and fundus perfusion in different periods.The results of correlation analysis showed that log MAR was positively correlated with age(r=0.348,p=0.005)and negatively correlated with MD and SVD in the acute phase(r=-0.425,p=0.002;r=-0.383,p=0.031).In the chronic phase,the variables positively correlated with log MAR were FLV,GLV and C/D(r=0.361,p=0.021;r=0.356,p=0.022;r=0.414,p=0.009),and the negatively correlated variables were pp VD,SVD and GCC thickness(r=0.391,p=0.036;r=-0.535,p=0.004;r=-0.393,p=0.011).MD was positively correlated with gender,pp VD,SVD,GCC thickness and disc rim area(r=0.601,p=0.001;r=0.662,p<0.001;r=0.522,p=0.001;r=0.338,p=0.015)and negatively correlated with age,log MAR,FLV and GLV in the acute phase(r=-0.381,p=0.005;r=-0.425,p=0.002;r=-0.490,p=0.002;r=-0.499,p=0.001);in the chronic phase,MD was positively correlated with pp VD,SVD,DVD,GCC thickness and RNFL thickness(r=0.637,p=0.001;r=0.717,p<0.001;r=0.429,p=0.041;r=0.343,p=0.038;r=0.702,p<0.001)and negatively correlated with GLV(r=-0.367,p=0.026).Pp VD was positively correlated with MD,SVD,GCC and RNFL thickness(r=0.601,p=0.001;r=0.504,p=0.012;r=0.621,p<0.001;r=0.405,p=0.029),and negatively correlated with age,BMI,FLV and GLV in the acute phase(r=-0.454,p=0.009;r=-0.490,p=0.008;r=-0.442,p=0.021;r=-0.539,p=0.004);in the chronic phase,pp VD was positively correlated with MD,SVD,GCC thickness and RNFL thickness(r=0.637,p=0.001;r=0.61,p<0.001;r=0.621,p<0.001;r=0.825,p<0.001),and negatively correlated with FLV and GLV(r=-0.552,p=0.001;r=-0.628,p<0.001).The variables that were positively correlated with SVD were MD and pp VD(r=0.662,p<0.001;r=0.504,p=0.012),the negatively correlated variables were age and log MAR in the acute phase(r=-0.510,p=0.003;r=-0.383,p=0.031);the variables that were positively correlated with SVD were pp VD,DVD,GCC thickness and RNFL thickness(r=0.61,p<0.001;r=0.481,p=0.008;r=0.615,p<0.001;r=0.62,p<0.001),and the negatively correlated variables were log MAR,FLV and GLV in the chronic phase(r=-0.535,p=0.004;r=-0.388,p=0.037;r=-0.537,p=0.003;r=-0.61,p<0.001).The results of multivariate regression analysis showed that log MAR was associated with GLV in the acute phase(p=0.029),and associated with SVD in the chronic phase(p=0.003);MD was associated with SVD and GLV in the acute phase(p=0.01,p=0.001),and associated with pp VD in the chronic phase(p<0.001);pp VD was associated with age and GLV in the acute phase(p=0.044,p=0.029),and associated with RNFL in the chronic phase(p<0.001);SVD was associated with MD and C/D in the acute phase(p=0.001,p=0.011),and associated with GCC thickness in the chronic phase(p<0.001).Part Ⅳ: Compared with the other three control groups,a total of 184 differential proteins were detected in the r NAION group(expression fold greater than 1.5 times and p < 0.05),including 99 up-regulated proteins and 85 down-regulated proteins.The expressions of Clusterin(CLU),alpha-2-macroglobulin(A2M),alpha-1-antiproteinase(SERPINA1),glial fibrillary acidic protein(GFAP),guanine nucleotide binding protein 4(GNG4),laminin 1(LAMC1),14-3-3γ protein YWHAG were increased.Whereas the expressions of Leucine-rich glioma-inactivated protein 1(LGI1),Secretory carrier-associated membrane protein 5(SCAMP5),and Clathrin coat assembly protein AP180(SNAP91)were decreased.The differential proteins were mainly involved in biological processes such as coagulation function,energy metabolism,vesicle-mediated transport,the regulation of synaptic plasticity,apoptosis and inflammation.Pathway enrichment analysis showed that PI3K-Akt signaling pathway and complement and thrombin reaction pathway were related to the disease.ConclusionFrom the acute to chronic stage,the thickness of RNFL and GCC progressively decreased.Capillary dropout was detectable in the optic disc and macular area,which was more obvious in the chronic stage.Pp VD and SVD also performed gradually decreasing,while DVD decreased in the acute stage but returned to normal in the chronic stage.The C/D and sleep apnea were the most important pathogenic factors of NAION.The factors affecting the visual acuity,visual field and fundus perfusion were various in different periods.Proteomics analysis showed that fundus perfusion and neuronal apoptosis could be regulated by the protein expressions of coagulation function,energy metabolism,synaptic vesicle transport and PI3K-Akt signaling pathway in NAION. |
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