Experimental Study On Iontophoresis-Assisted Accelerated Riboflavin-UVA Scleral Cross-Linking As A Treatment For Myopia

Myopia is a major public health problem worldwide.The localized ectasia/ protrusion of the sclera is one of the typical characteristics of myopia,which is related to the decrease of the biomechanical strength caused by the impaired collagen cross-linking in the sclera.The riboflavin-ultraviolet A(UVA)collagen cross-linking treatement could effectively enhance the collagen crosslinks and improve the biomechanical properties of the sclera.In clinical practice,scientific researchers and medical workers are very concerned about whether riboflavin-UVA collagen cross-linking is effective in controlling the development of stable or progressive myopia.In this study,a relatively stable form deprivation animal model of myopia was developed by monocular eyelid suturing in New Zealand White(NZW)rabbits.The iontophoresis and an accelerated irradiation procedure were applied to modify the riboflavin-UVA scleral collagen cross-linking treatment,named iontophoresis-assisted accelerated riboflavin-UVA scleral cross-linking(i-ASXL).The effectiveness,safety of i-ASXL and its effect on scleral collagen metabolism were explored at four post-surgery time points.The main work and results are as follows:(1)One hundred 10-day-old NZW rabbits were used for monocular eyelid suturing.After form deprivation of 80 days,the right eyes(sutured)generally developed a relative myopia of about-2.28 D(P<0.01),and the average axial length(AXL)of the right eyes was about 0.82 mm longer than that of the left ones(P<0.01),which indicated the successful establishment of a form deprivation animal model of myopia in NZW rabbits with a relatively large sample(n=96).(2)The riboflavin-UVA scleral collagen cross-linking treatment was modified with the iontophoresis system and accelerated procedure and then was applied to the myopia animal model.The effectiveness of i-ASXL was evaluated at 4 post-surgery time points(1-day,10-day,1-month and 3-month groups).The abnormal elongation of the i-ASXL treated eyes was effectviely controlled in 1-month post-surgery group;the AXL disparity between the i-ASXL treated eyes and their contralateral eyes(left)was not significant in the 3-month group,which indicated that the excessive AXL elongation due to myopia was effectively slowed and controlled after i-ASXL treatment.The uniaxial mechanical test of scleral samples showed that the Young's modulus,physiological Young's modulus,and ultimate stress notably increased in the i-ASXL treated samples in all 4 post-surgery groups compared with the untreated ones,which demonstrated that the i-ASXL procedure could significantly enhance the biomechanical properties of sclera tissue in myopic eyes.(3)The safety of i-ASXL sugery was assessed.The result of H&E staining showed that no obvious structural or degenerative alteration in the sclera,choroid,and retina of the i-ASXL treated eyes in 4 post-surgery groups.The TUNEL apoptosis assay show no pronounced positive signals in the sclera,choroid,and retina tissue except in the outer and middle sclera of 1-day and 10-day post-treatment groups.These evidences showed the biological safety of the i-ASXL protocol.(4)The effect of i-ASXL on scleral collagen metabolism was investigated from multipule aspects including the gene/protein expression,cell ultrastructure and tissue level.The transmission electron microscopy observation showed that the collagen fibril diameter of the i-ASXL treated sclera was generally significantly larger than that of the myopia sclera.The ultrastructure of scleral fibroblasts demonstrated vigorous collagen metabolism in the samples from the i-ASXL treated eyes and the contralateral(left)ones.In contrast to this,a certain number of resting fibroblasts were found in the samples from myopic eyes.Relative expressions of genes related to collagen metabolism were evaluated by real-time quantitative PCR in scleral samples at 4 post-surgery time points.The relative expression levels of COL1A1?COL1A2?MMP-1?MMP-3?MMP-9?TIMP-1 and Lumican genes were prominently higher in the i-ASXL treated samples than in the myopia ones in different post-surgery stages;MMP-2,TIMP-2 and TIMP-4 genes had relatively low expression levels in the i-ASXL treated scleral samples;the relative expression of MMP-14 gene showed no significant difference at all post-surgery time points.The results of picrosirius red staining showed a significant higher collagen content in the i-ASXL treated sections.The IOD analysis of immunohistochemical images demonstrated that the expressions of collagen I and MMP-1 protein were notably higher in the i-ASXL treated samples than in the myopia ones at 4 post-surgery time points.The above results showed that the collagen metabolism changed in the i-ASXL treated sclera region and the change was in the direction of increasing collagen synthesis.To summarize,i-ASXL treatment could effectively enhance the biomechanical properties of the sclera in the experimental myopia animal and coule slow and control the elongation of the myopic eyes with relatively high biological safety;the collagen metabolism of the i-ASXL treated sclera was influenced and the collagen synthesis became more active in a considerable period after surgery;the treatment is likely to trigger the scleral remodeling process.