Changes Of MMPs In Corneal Stroma After Corneal Crosslinking And Clinical Observation Of Iontophoresis-assisted Corneal Crosslinking For Progressive Keratoconus

Background:Matrix metalloproteinases(MMPs)play a key role in the progress of degradation and thinning of keratoconus.Corneal crosslinking(CXL)can cause injury and repair of corneal epithelial cells,apoptosis,proliferation and migration of keratocyte.MMPs are mainly secreted by corneal epithelial cells and keratocyte in the cornea;The case report of corneal melting after CXL was reported.It is speculated that CXL may affect the synthesis and secretion of MMPs in the cornea.Standard epitheliual-off CXL(S-CXL)has the disadvantages of postoperative pain,risk of infection,long recovery time and so on.Iontophoresis-assisted epithelium-on corneal crosslinking(I-CXL)has become a hot spot of current research.I-CXL using 0.1%riboflavin in distilled water can obtain similar intrastromal riboflavin concentration compared to epitheliual-off imbibition.However,the clinical effect of I-CXL using 0.1%riboflavin in distilled water was almost not reported.Objective:To observe the changes of MMPs in corneal stroma after corneal cross-linking,and to further explore the corneal pathophysiological process after cross-linking;To evaluate the effectiveness and safety of I-CXL using 0.1%riboflavin in distilled water for progressive keratoconus.Methods:1.Forty-two rabbits(42 eyes)were randomly divided into 7 groups.One group served as control group and the other 6 groups were treated with CXL.The concerntrations of MMPs in corneal stroma were evaluated with parallel reaction monitoring at 3,7,15,30,90,180 days after treatment,respectively.2.In this clinical study,we examined 94 eyes of 75 patients with progressive keratoconus who were treated with I-CXL using 0.1%riboflavin in distilled water.Best correct visual acuity(BCVA),Pentacam anterior segment analyzer,anterior segment optical coherence tomography(AS-OCT),intraocular pressure(IOP),and endothelial cell density(ECD)were evaluated at baseline and 1,3,6,12,and 24mo after I-CXL.3.Fifteen eyes of 12 adolescent patients with progressive keratoconus[mean age 15.8±2.08 years;range,12?18 years]were treated.After 0.1%riboflavin-distilled water solution was administered by iontophoresis-assited(current 1mA)transepithelial method for 5min in total,standard surface ultraviolet A(UVA)irradiation(370nm,3mW7cm2)was performed for 30min.The uncorrected visual acuity(UCVA),BCVA,Kl,K2,Kmax,Kmean,corneal refractive astigmatism,ECD,IOP,the corneal apex and the thinnest point were measured preoperatively and 1 year postoperatively.4.Consecutive 85 patients(110 eyes)with progressive keratoconus were enrolled and treated with CXL.Twenty-one patients of 25 eyes underwent S-CXL treatment,aged 15-31(19.8 ± 4.1)years;Fourteen patients of 22 eyes underwent I-CXL using 0.1%ribiflavin-sodium lactate Ringer's solution(I-CXLa),aged 13-35(20.9 ± 6.4)years;Fifty patients of 63 eyes,underwent I-CXL using 0.1%riboflavin in distilled water(I-CXLb),aged 16?33(20.0 ± 4.1)years.Preoperative and postoperative TCTs were measured by Pentacam anterior segment analyzer.The differences of TCT decrease after treatment were compared among the three CXL protocols.Results:1.The levels of MMP-2 in corneal stroma of rabbits were 0.76 ± 0.07,2.78 ±1.39,4.12 ± 0.69,2.00 ± 0.29,2.00 ± 0.30,1.22 ± 0.18,1.35 ± 0.18(10-9mo1/g)at 3,7,15,30,90 and 180 days after treatment respectively;The contents of tissue inhibitor of metalloproteinase-1(TIMP-1)were 1.83 ± 0.26,7.94 ± 0.58,6.95 ± 2.64,3.81 ± 0.48,3.07± 0.92,1.72 ± 0.19,1.69 ± 0.74(10-9mol/g).The ratios of MMP-2/TIMP-1 were 0.42 ±0.33,0.36 ± 0.20,0.62 ± 0.10,0.54 ± 0.15,0.68 ± 0.13,0.71 ± 0.10,0.68 ± 0.09,respectively.After CXL,the expression of MMP-2 and TIMP-1 in rabbit corneal stroma increased first and then decreased.MMP-2 was still significantly higher than the baseline at 180 days after treatment,and TIMP-1 recovered to the baseline at 90 days after treatment.The ratio of MMP-2/TIMP-1 was significantly higher than the baseline throughout the followup.MMP-1,-3,-7,-9,-13,and TIMP-2 were not detected.2.BCVA significantly improved 6 months after I-CXL compared to the baseline level and further improved during the subsequent observation period.Flattest K values(K1),steepest K values(K2),and mean keratometry(Kmean)significantly decreased 3,6,12,24 months after I-CXL compared to the baseline level.Maximum keratometry(Kmax)significantly reduced 1 month after I-CXL and was lower than baseline level throughout the follow-up period.Index of surface variance(ISV)decreased significantly 6 months after treatment and continued to decrease.Central keratoconus index(CKI)significantly reduced 24 months after I-CXL.Minimum radius of curvature(Rmin)improved significantly 1 month after treatment and continued to improve.Other morphological parameters and corneal astigmatism remained stable throughout the follow-up period.At 1 month after treatment,the thinnest-point corneal thickness(TCT)decreased significantly and then gradually increased,but it did not return to preoperative levels at 24 months after treatment.There was no significant change in IOP and ECD.The demarcation line was visible in 83.1%of eyes at 1 month after treatment with a depth of 298.95±51.97 ?m,and gradually indistinguishable over time.One eye had repeat treatment.3.At 1 year after treatment,there were no significant changes in visual acuity,corneal curvature,corneal astigmatism,ECD and IOP(P>0.05).The TCT significantly decreased from 468.08±33.40 to 447.46±40.20(t=4.379,P=0.001).4.The differences of TCT from baseline after 3,6 and 12 months were(-14.93±27.16)?m,(-31.94±22.89)?m,(-27.71±26.01)?m in the S-CXL group respectively.The data in the I-CXLa were(-20.14± 19.09)?m,(-10.10±24.28)?m,(-7.11 ±22.26)?m respectively.The data in the I-CXLb were(-28.08±26.14)?m,(-21.08±25.62)?m,(-15.91±19.19)?m respectively.Three months after treatment,the differences of TCT decrease in the three groups were not statistically significant(F = 1.704,P = 0.188);Six and 12 months after treatment,the difference between S-CXL and I-CXLa was statistically significant(P<0.05),but the differences between S-CXL and I-CXLb,between I-CXLb and I-CXLa showed no significant difference(P>0.05).Conclusions:1.CXL lead to changes in the content of MMP-2 and TIMP-1 in the corneal stroma of rabbits.The ratio of MMP-2/TIMP-1 remained higher level indicating that MMP-2 was involved in the corneal pathophysiological process after CXL.2.I-CXL using 0.1%riboflavin in distilled water halts keratoconus progression within 24 months,resulting in a significant improvement in visual and topographic parameters.Moreover,the depth of the demarcation line is similar to that previously reported in S-CXL procedures.3.I-CXL in adolescent patients with progressive keratoconus is effective and safe which can halt deterioration of keratoconus within 1 year.4.The TCT decrease after CXL for progressive keratoconus may reflect the intensity of crossinking.The clinical effect of I-CXL using 0.1%riboflavin in distilled water may be close to S-CXL.