The Experimental And Clinical Study Of Modified Riboflavin/UVA Corneal Collagen Crosslinking In Keratoconus

Part I The evaluation of riboflavin density in rabbit corneal stroma using standard or modified riboflavin solution under different riboflavin/UVA corneal collagen crosslinking modelsBackground As a new treatment method,riboflavin/UVA(370nm)corneal collagen cross-linkling(CXL)had been successfully used for keratoconus and corneal ectasia diseases.CXL mainly depends on the photochemical reaction between ultraviolet A and riboflavin.The riboflavin content of corneal stroma directly affect the final effect of crosslinking.The current standard riboflavin solution is the riboflavin dissolved in 20%dextran solution.20%dextran solution is relatively viscous and prone to gather on the surface of cornea,which interfere the UVA penetration during CXL.Present study made the modified riboflavin solution using 0.9%saline solution instead of 20%dextran solution.Studies have reported the different effect in CXL using riboflavin solution with different concentration,osmotic pressure,or composition.With the continuous improvement of corneal crosslinking technique,the corrsponding optimization of the riboflavin solution formula is needed.Present study used saline solution as the solvent of riboflavin,and evaluated the clinical feasibility of the modified riboflavin solution.Objective To evaluate the riboflavin density in corneal stroma using standard and modified riboflavin solution under 3 CXL models,including corneal epithelium-off,corneal epithelium-on combined with penetration enhancer(EDTA),and corneal epithelium-on.To evaluate the enhancing effect of EDTA on riboflavin penetrating into corneal stroma.,Methods Sixty adult New Zealand rabbits(F/M)were randomly classified into 3 groups:epithelial-off group,EDTA group(epithelial-on,preoperative 0.5%EDTA-Na topical administration,once every 3 minutes for 1 hour),and epithelial-on group,with 20 rabbits in each group.All treatments were applied on right eyes.In each group,10 rabbits were received 0.1%riboflavin dextran solution administration once every 3 minutes for 30 min,and another 10 rabbits received 0.1%riboflavin saline once every 3 minutes for 30 min.Comeal epithelium at the other epithelial-on groups were removed with the epithelial shovel,and residual riboflavin on corneal surface were rinsed with balanced salt solution.Central corneal tissue with 9mm diameter were cut off with microophthalmic scissors.Corneal tissue homogenate was prepared and supernatant was obtained for the evaluation of riboflavin content in corneal stroma with high performance liquid chromatography(HPLC)method.The effective riboflavin content in corneal stroma was 15 ?g/g tissue(15 ?g/g)during corneal crosslinking.Results 1.After the administration of 0.1%riboflavin dextran solution for 30 min,the riboflavin content in corneal stroma was(72.96± 5.82),(8.69 ± 1.10),(1.14± 0.89)?g/g in epithelial-off group,EDTA group,and epithelial-on group,respectively.There is significant difference among 3 groups(F= 1463.54,P=0.000).The riboflavin content in corneal stroma in epithelial-off group>EDTA group>epithelial-on group,with significant differences(t=64.27?71.82?7.55,P<0.05).2.After administration of 0.1%riboflavin saline solution for 30 min,the riboflavin content in corneal stroma was(35.85 ±7.00),(7.57±0.89),(1.01±0.33)?g/g in epithelial-off group,EDTA group,and epithelial-on group,respectively.There is significant difference among 3 groups(F=226.53,P=0.000).The riboflavin content in corneal stroma in epithelial-off group>EDTA group>epithelial-on group,with significant differences(t=28.28?34.85?6.56,P<0.05).3.The riboflavin content of corneal stroma in EDTA group and epithelial-on group is less than 15 ?g/g,while that in epithelial-off group is higher than 15 ?g/g.The difference of riboflavin content in corneal stroma in epithelial-off group using standard or modified riboflavin solution was statistically significant(t=13.515,p=0.000).The difference of riboflavin content in corneal stroma in EDTA group using standard or modified riboflavin solution was statistically significant(t=2.617,p=0.017).There was no statistically significant difference in epithelial-on groups using standard or modified riboflavin solution(t=1.111,p=0.280).Conclusion1.Under standard epithelial-off model,the usage of saline solution as riboflavin solvent improves the riboflavin penetration in corneal stroma,compared with the usage of 20%dextran as solvent.2.Using EDTA as penetration enhancer can increase the riboflavin content in corneal stroma,but can not reach the effective riboflavin content level that CXL required.The role of enhancers can not replace the role of epithelial-off for promoting riboflavin penetration in corneal stroma.Part ? Mini-corneal collagen crosslinking in the treatment of early-stage keratoconusBackground Keratoconus is essentially a bilateral eye disease,although the clinical manifestations are markedly asymmetrical.Clinical keratoconus is diagnosed,and corneal morphology in the other eye may change for several years.Early-stage,latent or subclinical keratoconus are waiting for disease progression,or early intervention,the treatment options is a difficult problem for clinicians.Standard CXL,which has the definite therapeutic effect in the treatment of advanced clinical keratoconus,can also apply with the early-stage keratoconus?With the widely application of CXL,clinical CXL parameters adjustment on the irradiation intensity,irradiation time and total irradiation energy appeared in dealing with different types and degrees of keratoconus,such as accelerated CXL.Accelerated CXL could improve visual acuity,reduce the value of Kmax,but the change of biomechanical properties of the cornea was inferior to standard CXL.Studies about the long-term safety and efficacy of CXL in the treatment of early-stage keratoconus are still less,so we put forward the concept of Mini-corneal collagen cross-linking(Mini-CXL).Under the other condition of standard corneal crosslinking,mini-CXL only reduce the irradiation time to 20 minutes.We followed early-stage keratoconus patients for 1 years after Mini-CXL,aimed at evaluating the long-term safety and efficacy of Mini-CXL in the treatment of early-stage keratoconus.Objective To evaluate the long-term safety and efficacy of Mini-CXL(3mW/cm2,20 minutes)in the treatment of early-stage keratoconus.Methods During October 2014 to October 2016,18 cases(20 eyes)of early-stage keratoconus patients were treated with Mini-CXL in the ophthalmology center of Shandong Provincal Hospital.Central cornea epithelium was removed under aseptic condition,0.1%riboflavin solution were applied at an interval of 3 minutes for 10 times.Irradiation parameters were as follow:wavelength of UVA was 370nm,irradiation energy density was 3mW/cm2,irradiation time was 20 minutes,total irradiation energy was 3.6 J/cm2,irradiation distance was 5cm.After Mini-CXL,patients wore soft contact lens,and applied antibiotics,non-steroidal anti-inflammatory eye drops four times a day for one week.To observe corneal epithelialization and surgery complications 1 weeks after operation.Record the uncorrected visual acuity(UCVA),best corrected visual acuity(BCVA),myopia,astigmatism and spherical equivalent,max-corneal curvature value(Kmax),thinnest corneal thickness(TCT),endothelial cell density(ECD),intraocular pressure(IOP)and the complications for each follow visit,the total follow time was one year.Results 1.The uncorrected visual acuity(UCVA)fell slightly after operation 1 month,increased gradually at 6 months and 12 months,then tended to be stable.There was no statistically significant difference in mean UCVA during the entire follow-up(p=0.093).The mean UDVA improved significantly 0.2LogMAR from baseline at 12 months(p=0.048),including three cases increased 3 lines,2 cases increased 4 lines.The best corrected visual acuity(BCVA)was 0.09±0.05 before operation,and the results were 0.09±0.06.0.07±0.04?0.06±0.03 after operation 1 months,6 months,12 months,respectively.BCVA tended to be stable after operation.There was no statistically significant difference in mean BCVA during the entire follow-up(p=0.167).2.There were no significant difference in mean spherical diopter of myopia,astigmatism and spherical equivalent during the entire follow-up,the overall trend was gradually reduced and tended to be stable at 6-12 months after operation.The mean myopia decreased 0.29D,astigmatism decreased 0.43D,spherical equivalent decreased 0.52D at 12 months.There were significant differences between preoperative astigmatism and postoperative astigmatism at 12 months(p=0.041).The maximum corneal curvature(Kmax)before operation was compared with the Kmax value at 6 and 12 months after operation,the difference was statistically significant(p=0.040,0.034).The mean Kmax tended to be stable at 6-12 months after operation,decreased 1.38D.The maximum decrease of Kmax was 3.5D,and 3 patients decreased over 2.0D at 12 months.3.There were no significant difference in mean thinnest corneal thickness(TCT)during the entire follow-up,the overall trend was gradually reduced and tended to be stable at 6-12 months after operation.The average decrease of TCT was 10um,there was no significant compared with the preoperative level.The endothelial cell count and intraocular pressure tended to be stable during the entire follow-up,which was similar to the preoperative level.4.Only one patient showed mild edema of corneal stroma on the first day after operation,and recovered clear at 3 days.The corneal epithelium was completely repaired at 2-3 days after operation.No corneal epithelium delayed healing and corneal infection.The corneal stroma opacity(Haze),corneal scar and other complications were not observed during the entire follow-up.ConclusionMini-CXL treatment of early-stage keratoconus patients,can effectively stabilize and improve UCVA and BCVA,and effectively reduce astigmatism and Kmax.The corneal thickness,corneal endothelial cells and intraocular pressure were stable in the preoperative baseline.There was no disease progression within 1 years after operation.Mini-CXL can effectively stabilize corneal morphology and achieve similar therapeutic effect compared with standard CXL,but the long term effects of Mini-CXL still needs further study.