| Purposes:This study was designed to elucidate the potential role of genetic polymorphisms of apolipoprotein E(APOE)in nonarteritic anterior ischemic optic neuropathy(NAION)and the association between APOE and NAION-induced ocular impairments.We also proposed to investigate the role of myelin degeneration in the pathological process of rat NAION(rAION)model and the neuroprotective effect of benztropine on rAION model and its mechanism.Methods:1.In this study,we collected demographic information and relevant medical history from NAION patients attending our outpatient clinic and age-sex matched healthy subjects at the same time,and performed a comprehensive eye examination(including visual acuity,intraocular pressure,visual field,optical coherence tomography imaging).We also collected the peripheral blood and extracted genomic DNA to amplify,sequence and analyze the two major single nucleotide polymorphism(SNP)loci(rs429358 and rs7412)of the APOE gene.The interaction between APOE polymorphisms and medical comorbidities was explored by using multifactor dimensionality reduction(MDR)method.The correlation between APOE polymorphisms and NAION phenotypes were further evaluated.2.The rat NAION model was constructed by photodynamic method and compared with normal healthy rats at the same time.The morphological changes of retinal tissue were observed by optical coherence tomography(OCT)and fundus color photography,and the morphological changes of myelin were observed by transmission electron microscopy.Flash visually evoked potentials(VEP)were used for the assessment of visual function.Retinal ganglion cells(RGCs)immunofluorescence staining and TUNEL assay were used to assess RGCs survival and apoptosis.Immunofluorescence staining tests of oligodendrocyte lineage cells,mature oligodendrocytes and myelin basic protein(MBP)on optic nerve were used to assess the number of oligodendrocytes and to indirectly assess their functional changes.3.After photodynamic construction of the rNAION model,the rats were randomly divided into benztropine treated group or untreated groups.The rats in the benztropine group received daily intraperitoneal injections of benztropine solution(10 mg/kg)prepared with PBS solution,and the rats in the untreated group received daily intraperitoneal injections of the same volume of PBS solution as the benztropine treated group.The differences in retinal histomorphology,optic nerve myelin structure,visual function,RGCs survival and apoptosis,oligodendrocyte number and myelin basic protein were compared between the two groups at multiple time points.In the current study,Optic nerve and retinal tissues were further collected from the two groups at 2nd week after the rAION induction for high-throughput transcriptome sequencing to compare the differences in gene expression and changes in signaling pathways at the transcriptional level between the two groups,which were verified by real-time fluorescence quantitative polymerase chain reaction method,protein blotting and immunofluorescence staining.Results:1.The differences in the three allele(P=0.022)and six genotype frequencies(P=0.044)of APOE were statistically significant in the NAION patient group compared with healthy controls,and ε4 carriers were significantly higher in NAION patients(P=0.016)than in healthy controls.After correcting for sex,age,hypertension,dyslipidemia,diabetes,cardiovascular disease and cerebrovascular disease,multifactorial analysis showed that the ε3/ε4 genotype(OR=3.86,95%CI=1.13 to 13.2 5,P=0.032),the ε4 allele(OR=3.55,95%CI=1.0 5 to 11.99,P=0.041)and the ε4/ε 4+ε3/ε4 genotype(OR=3.54,95%CI=1.04-12.05,P=0.043)were independent risk factors for NAION.The two best models analyzed by the MDR approach included APOE genotype,hypertension,hyperlipidemia orε4 carriers,hypertension and hyperlipidemia,but none of the model interactions were statistically significant.Compared with ε3/ε3+ε2/ε4 genotype,ε4/ε4+ε3/ε4 genotype had more severe damage in the inferior nasal quadrant of the visual field,thinner macula ganglion cell complex(mGCC)thickness in the inferior half of the retina,and greater focal volume loss and overall volume loss of mGCC.Visual field defects and mGCC loss were also more severe in ε4 carriers compared to ε4 non-carriers.2.Fundus photography showed that on the first day after rAION modeling,the rat’s optic nerve head was congested and edematous,with blurred borders,tortuous vascularity,and significantly reduced perfusion or even blocked flow in many vessels near the optic disc,followed by a gradual decrease in papilledema.At the fourth week post-rAION induction,a whitish atrophy of the optic papilla was seen.VEP showed a significant decrease in amplitude between P1 and N1 at 4th week after modeling,but the difference in latency was not statistically significant.The number of TUNEL-positive cells in the ganglion cell layer of rAION was significantly more than that of the normal control group at the 1st week,2nd week and 4th week,and the number of TUNEL-positive cells reached the maximum at 2nd week.The number of all oligodendrocyte lineage cells and mature oligodendrocytes were not significantly different between the two groups at the 1st week,while the number of oligodendrocytes and mature oligodendrocytes in rAION decreased significantly compared with the normal control group at 2nd and 4th week.In addition,the MBP fluorescence intensity of the optic nerve in the normal control group was significantly higher than that in the rAION model group at week 4.3.Compared with the untreated group,the NAION rat model after benztropine treatment showed improvement in the pathological process of early edema and late atrophy of GCC,reduction of VEP amplitude,loss of RGCs,damage to oligodendrocytes,and weakening of MBP fluorescence intensity.By functional enrichment analysis with high-throughput transcriptome sequencing,we found that the down-regulated genes in the differential genes of optic nerve specimens in the benztropine group were mainly associated with immune function and inflammatory response,while the up-regulated genes were mainly associated with axons and myelin sheaths compared with the untreated group.The enrichment results of retinal specimens showed that the differential genes showed mainly up-regulated changes and were mainly associated with axonal growth,development,neuronal guidance,and synaptic transmission processes.Further pathway analysis showed that Toll-like receptor signaling pathway and Wnt/beta-catenin signaling pathway were down-regulated based on high-throughput transcriptome sequencing analysis and validation of the optic nerve.Based on the transcriptome sequencing results of retinal tissues,we found that the expression of Tubb4a,Tubb3,Nefh,Tppp3,and Kif5a genes was significantly increased in the retinas of rats in the benztropine group compared with the untreated group.Conclusions:1.APOE polymorphisms conferred a significant risk of NAION and were significantly related to ocular impairments caused by NAION.2.Myelin degeneration is one of the important pathological processes in the rat NAION model.3.Benztropine has a neuroprotective effect on NAION rat model,and the possible mechanism is the down regulation of Toll-like receptor signaling pathway and Wnt/beta-catenin signaling pathway in optic nerve,which also directly or indirectly promotes the synthesis of raw materials for axonal cytoskeleton by neuron bodies and its transport to the distal axon. |
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