Experimental Study Of Corneal Collagen Cross-linking Combined With 440nm Blue Light And Riboflavin For Bacterial Corneal Ulcer

Bacterial keratitis is the inflammation of the cornea caused by bacterial infection,which can cause corneal epithelial defects and stroma necrosis.It is an eye disease that seriously endangers patient' s eyesight.Keratitis caused by Staphylococcus aureus and Pseudomonas aeruginosa are the most common and most serious ones among all.Current treatment relies mainly on local use of antibacterial eye drops.However,due to multiple external factors and the impact of antibiotic abuse,some bacteria can mutate and develop certain antibiotic resistance,resulting in poor treatment effects.Chang retrospectively analyzed the Staphylococcus keratitis during 20 years and found the antibiotic resistance rate to be as high as 30.7%.For protracted course of corneal ulcers,a conjunctival flap or amniotic membrane covering surgery was tried.However,since it reduces the permeability of eyedrops,this surgery could only be done when infection was controlled.When severe infection and corneal melt occur,cornea transplantation is the only treatment.But limited resource of corneal material and frequent transplantation rejection or recurrence of inflammation after surgery result in enucleation of eyes in a large number of patients.Corneal collagen cross-linking(CXL)generates reactive oxygen species through riboflavin irradiation by ultraviolet A and induces photochemical cross-linking reaction between amino groups of corneal collagen fibers,thereby increasing the mechanical strength of collagen fibers and the resistance against ectasia.In 2000,Schnitzler et al.first reported the effectiveness of CXL for treating corneal ulcers.Later a large number of clinical reports confirmed that CXL is a new and effective treatment for infectious keratitis,which brought a new dawn for corneal disease treatment.Most antibiotics treat keratitis only by killing pathogens.But CXL can treat corneal ulcer in three ways:killing pathogens,increasing the anti-enzyme activity of cornea and alleviating the associated inflammatory reaction.There are three light absorption peaks of riboflavin at the wavelength of 270nm UVB,370nm UVA and 445nm blue light respectively.So they all can irradiate riboflavin and generate the same effect.In addition,the penetrating power of light is directly related to its wavelength.The longer wavelength,the deeper penetrating depth.So we infer that the action spot of CXL excited by 440nm blue light should be more deeper than 370nm light.The 370nm UVA penetrates only 300um of the cornea.But the cornea will thicken because of edema caused by bacterial corneal ulcer,and ulcer depth is often more than 300um.Accordingly,we propose that 440nm mediated blue light cross-linking should be superior to 370nm UVA in the treatment of severe corneal ulcers.In 1998,Spoerl E et al reported that CXL combined with riboflavin and 465nm blue light can enhance the porcine corneas in vitro.In 2008,Iseli reported that CXL by blue light could increase the mechanical strength of scleral collagen fibers.Yet,no report of treating corneal ulcer using CXL combined with riboflavin and 440 nm blue light has been found.In the first part of this study,440 nm blue light combined with riboflavin was used against S.aureus corneal ulcer in rabbits,and the results were compared with 370nm UVA to verify its effect on bacterial corneal ulcer.In the second part,we observed the treatment of Staphylococcus aureus corneal ulcer with 440nm blue light combined with riboflavin cross-linking,to evaluate the possible cytotoxic effect on the eye tissues such as corneal endothelium,retina,choroid and so on.Part 1 The Treatment Efficacy of Corneal Cross-linking Combined 440nin Blue Light and riboflavin for Bacterial Keratitis in Rabbits Objective:Study the effect of corneal collagen cross-linking combined with 440 nm blue light and riboflavin on bacterial corneal ulcer using animal experiments.Observe the therapeutic effect of the treatment group by comparing with the control group and with the corneal collagen cross-linking combined with 370nm UVA and riboflavin.Methods:1.Experimental animal model54 New Zealand white rabbits that developed Staphylococcus aureus corneal ulcer were randomly divided into three groups.18 eyes were used as blank control group;18 eyes were treated with corneal collagen cross-linking combined with 440 nm blue light and riboflavin;18 rabbits were treated with corneal collagen cross-linking combined with 370nm ultraviolet A light and riboflavin.One day,three days,seven days and fourteen days after cross-linking,the changes of corneal ulcer of each group was observed and compared to evaluate the treatment results of CXL combined with 440 nm blue light and riboflavin?2.Corneal cross-linking treatmentChloral hydrate(10%)3 mL/kg was injected via abdomen to anesthetize the rabbit.Corneal thickness was measured by an ultrasonic thickness meter to ensure all corneal thicknesses were greater than 400 ?m.Loose ulcer tissues and residual epithelia were scraped within 9mm diameter from the central cornea.The riboflavin solution(lg/L)was dropped on the cornea every 2-3 min,a total of 30 min.After 30 min,the 440 nm blue light therapy device and the 370 nm ultraviolet A light therapy device were then used to irradiate the two groups of cornea for 30 min,respectively,with a working distance of 50mm,power of 3mw·cm-2,and beam diameter of 9 mm.During treatment,riboflavin drops were given every 2-3 min,preventing cornea from drying.3.ObservationOne day,three days,seven days and fourteen days after cross-linking,eye secretions,conjunctival hyperemia,hypopyon and corneal infiltration were observed and rated as inflammation scores for statistical analysis.At the same time,the corneal ulcer area were analyzed by Image J software.Meanwhile,necrotic tissues or secretions from ulcer surface of all model eyes were taken for bacterial culture.One day,seven days and fourteen days after CXL,confocal microscopy to observe the inflammatory cell changes,and 2 eyes were removed respectively for observation of cornea pathological changes.Results:1.The ocular inflammation scores of three groups:Before CXL,there were no statistically significant differences among the three groups(P=0.814,P>0.05).One day,three days,seven days and fourteen days after CXL,there were statistically significant difference among the inflammation scores of three groups(P = 0.000,0.000,0.000,0.000;P<0.05).The differences of the scores between the 440 group and the control group was statistically significant(P=0.001,0.000,0.000,0.000;P<0.05).The scores of 370 group were significantly lower than that of control group(P=0.000,0.000,0.000,0.000;P<0.05).But there was no significant difference between the scores of 440 and 370 group(P=0.557,0.567,0.488,0.537;P>0.05).All the corneas of the control group had been perforated by the end of the observation.2.The changes of ocular inflammation scores in each group:The scores of 440 and 370 group were statistically different at the four time points respectively(P= 0.000,0.002;P<0.05),showing a downward trend.On the other hand,the score of ocular inflammation in the control group at the four time points showed an upward trend(P= 0.000,P<0.05),and they were statistically different.3.The corneal ulcer area of the three groups:Seven days and fourteen days after CXL,the corneal ulcer area of the control group was significantly greater than that of the 440 group and the 370 group(q=5.895,5.571;P<0.05),but there was no statistical difference in the area of corneal ulcer between the 440 groups and the 370 groups(q=0.323,0.582;P>0.05).4.Bacterial cultures:After CXL,bacterial cultures of 440 group and 370group both turned to be negative while control group remained positive.5.Pathological results of three groups:One day after CXL,pathology pictures of three groups all showed loss of cornea epithelia with many inflammatory cells in deep stroma.Seventh days after CXL,abscess formed in almost all corneal area in the control group,while in 440 group and 370 group there were multi-layer healing of corneal epithelia,neovascularization within ulcers and proliferation of a small amount of fibroblast.Fourteen days after CXL,abscess formed in all corneal area in the control group,almost perforating.Descemet's membrane was only partially visible.On the contrast,there are multilayer corneal epithelial healing,neovascularization,scar tissue and fibroblast proliferation in the 440 group and 370 group while inflammatory cells decreased.6.Confocal microscopy results of three groups:One day after CXL,a large number of inflammatory cells were seen in the corneal stroma of three groups.Seven days after CXL,a large number of inflammatory cells were seen still in 440 group and 370 group,but less than that of the control group.Fourteen days after CXL,the inflammatory cells in the stroma of the 440 and 370 groups were less than before,however,a large number of inflammatory cells were still observed in the control group.Conclusion:1.Corneal collagen cross-linking combined with 440 nm blue light and riboflavin is effective to treat Staphylococcus aureus corneal ulcer.2.There is no significant difference between CXL combined with 440nm blue light and 370nm UVA in the treatment of bacterial corneal ulcer.3.The long-term effect and safety of CXL combined with 440 nm blue light and riboflavin in the treatment of bacterial corneal ulcer needs further study.4.440nm blue light combined with riboflavin corneal cross-linking is prone to be a treatment for bacterial corneal ulcer.Part 2 Possible Cytotoxic Effect of Corneal collagen cross-linking combined with 440 nm blue light and riboflavin for Treatment of Bacterial Keratitis in RabbitsObjective:To observe the cytotoxic effects of 440nm blue light combined with riboflavin corneal cross-linking on corneal endothelium,retina,choroid,sclera and optic nerve in rabbits.Methods:The corneal ulcer models of Staphylococcus aureus were made by coeneal stroma injection method in 8 healthy New Zealand rabbits.Each eye was injected with 0.08ml,and the other steps were the same as the part 1.After 24 hours,the model was successful.Under general anesthesia,Corneal collagen cross-linking combined with 440 nm blue light and riboflavin was performed as the part 1 too.Four rabbits were killed by gas embolism after one day and seven days after cross linking and extracted the eye balls.Two of the eye balls were soaked in 40%poly Formaldehyde Solution for pathological section respectively,and the other four corneas were removed immediately for dying corneal endothelium by benzo blue and alizarin red staining method.Results:One day and seven days after CXL,There were no Cytotoxic Effect in corneal endothelial,retina,choroid,sclera and optic nerve.Conclusion:Corneal collagen cross-linking combined with 440 nm blue light and riboflavin is safe in the treatment of bacterial corneal ulcer.But its effect on the ultrastructure of the eye tissue needs further study.