Experimental Study Of Curative Effect Regarding Corneal Cross-Linking Treatment Of Riboflavin Combined With 360nmUVA Or 440nm Blue-Light For Fungal Keratitis Of The Rabbits

Part IBuilding rabbit model of fungal keratitisBackground:Fungal keratitis is an infective corneal disease with very high blind rate,and is an important cause of corneal blindness.The industrial injuries of the cornea provided opportunities for fungal infection;in addition,with the wide application of antibiotics,corticosteroids,and anti-viral drugs,as well as the increased application of corneal contact lens,the incidence of fungal keratitis increases by years.Fungi mainly include filamentous fungi(such as fusarium,aspergillus,penicillium,and dematiaceous fungi)and yeasts(mainly candida species).Candida albicans is the major pathogenic fungi in the developed countries and cold regions.The previously published studies about fungal keratitis also mainly focused on candida albicans,fusarium,aspergillus,and very few dematiaceous fungi.Inducing appropriate animal model of fungal keratitis is essential to investigate the effectiveness of the treatments for fungal keratitis.Rabbit is a very commonly used experimental animal for ophthalmic researches,and has been acknowledged as a better living animal model than other animals.The morphologyand anatomic features of the rabbit eyes are similar to human eyes,the eyeballs are relatively big,and the area of the cornea is about 30%of the eyeball area,which could facilitate the surgical processes and the observations.Moreover,rabbit is a tame animal that is easy to raise,and is very cost-effective,therefore the present study selected rabbit to induce the fungal keratitis model.Currently,the universally used method to induce fungal keratitis model is to inject fungal spores into the corneal stroma of the rabbits to induce the deep corneal fungal infection.Injecting fungal spores into the corneal stroma is with high successful rate of infection,while the rate of mixed corneal infection is very low,which is also very cheap and easy to perform.Objectives:To induce a rabbit model of superficial fungal keratitis,identify the key processes in the model induction,observe the corneal changes before and after the infection,and clarify the fungal infection and the type of fungi infected via corneal scraping followed by culture and confocal microscopy.Methods:Forty-eight healthy adult New Zealand White Rabbits with the body weight of 2.5-3.0 kg and either sex were obtained.After rabbits with active eye diseases of anterior segment were excluded,the rabbits were acclimated for 2 weeks.106 CFU/mL standard candida albicans solution was prepared,and then 0.1 mL of the solution was injected into the superficial corneal stroma to form a round gray-white change with the diameter of about 4 mm.and thus induce the rabbit model of candida albicans infection.Slit-lamp examination,corneal ulcer scraping followed by culture to clarify the infected fungus,and confocal microscope to observe the fungi were performed.Results:Slit-lamp examination of the corneal fungal infection showed the formation of corneal ulcer and corneal edema,with satellite lesions around.Corneal ulcer scraping followed by culture showed the fungal clone was round and relatively big,the center of the clone surface was rough and wrinkled,and the pseudohypha extended into the culture medium,which is the typical feature of yeast-like fungi clone.Confocal microscope observation revealed that corneal ulcer appeared at 3-5 days after the model induction.Repeated confocal microscope observations showed that all the rabbits were with hyphae or spores.Conclusion:I)The morphology and anatomic features of the rabbit eyes are similar to human eyes,the eyeballs are relatively large,and thus is the ideal animal for the induction of infective keratitis model;2)In the present study,fungus was injected into the superficial comeal stroma to induce the rabbit model,which successfully induced the rabbit model of candida keratitis.This method is simple,while the successful rate is very high,and the risk of corneal perforation is significantly reduced.Part ?Observation of curative effect regarding corneal cross-linking treatment of riboflavin combined with 360nmUVA or 440 nm blue-light for fungal keratitis of the rabbitsBackground:Fungal keratitis is a serious eye disease with high blind rate.Fungal keratitis is now the first leading infective keratitis in some regions in China,and the incidence of this disease increased evidently in the past 40 years.Currently,the major treatment method for fungal keratitis is still the local or combined with systemic application of antifungal drugs.However,as most of the antifungal drugs are with relatively high toxicity and low ocular tissue permeability,thus drug treatment for fungal keratitis is relatively difficult.In addition,scraping of the corneal ulcer in combination with iodine burning is also used,but the effectiveness is limited.The basic rational of corneal cross-linking is the unique absorption spectrum of riboflavin.the photosensitizer,which could absorb the photon energy under ultraviolet or visible light irradiation,induce the chemical groups in and between the collagens to form more covalent bond crosslinks,thus increase the biomechanical characteristics of the collagens,and improve the mechanical strength of the collagens and the ability to against the dilation of the cornea.Corneal collagen cross-linking has been widely used in the clinical practices in the Department of Ophthalmology,especially for the treatment of keratoconus and corneal dilation,of which the treatment efficacies have already been demonstrated.The efficacies of using 360 nm UVA combining riboflavin to induce corneal collagen cross-linking in treating fungal keratitis have already been demonstrated in both animal experiments and the clinical practices.It is well known that the absorption spectrum of riboflavin peaks at 360 and 440 nm.Currently,the effectiveness of inducing corneal cross-linking(CXL)with riboflavin combining 360 nm ultraviolet radiation A(UVA)has already been acknowledged,while the effectiveness of inducing CXL with riboflavin combining 440 nm UVA has not been investigated to date.Objective:To investigate the effectiveness of using 360 nm UVA or 440 nm blue light in combination with riboflavin to induce corneal collagen cross-linking in treating fungal keratitis in rabbit models.Methods:Forty-eight rabbit models of corneal keratitis were randomly divided into control,360 and 440 groups.The rabbits in the control group did not receive any treatment.while the ones in the 360 and 440 groups were treated with corneal crosslinking.For the rabbits in the 360 group.the rabbits were dripped with 0.1%riboflavin for 30 min.and then irradiated with 360 nm UVA for 30 min:while for the ones in the 440 group.the rabbits were dripped with 0.1%riboflavin for 30 min.and then irradiated with 440 nm blue licght for 30 min.Slit-lamp examinations.photographing of the cornea,and confocal microscopy were performed on the days 1,3.7and15 after the treatment,and the areas of the corneal ulcers were calculated.On the days 1,7,15 after the treatment,4 rabitts were executed respectively every group,then sniped the cornea and made pathological section.Results:No significant difference was found among the three groups on the day 1 after the treatment(P>0.05).On the day 3 after the treatment,the ulcer area in the control group increased by 6.94%,while the area in the 360 and 440 groups decreased by 35.65%(P=0.012)and 37.94%(P=0.015)as compared with the area before the treatment,respectively.On the day 7 after the treatment,the ulcer area in the control group decreased by 13.14%,while the area in the 360 and 440 groups decreased by 73.82%(P=0.00)and 70.00%(P=0.001)as compared with the area before the treatment,respectively.There is significant difference between the 360 and control group,between the 440 and control group.However,no significant difference between the 360 nm and 440 nm groups on the day 7 after the treatment was found(P=0.75).On the day 15 after the treatment,the ulcer area in the control group decreased by 83.16%,360 and 440 group corneal ulcers are recured.Confocal microscopy results showed no evident difference among the three groups on the day 1 after the treatment;while on the days 3 and 7 after the treatment,the numbers of the fungal hyphae in the 360 nm and 440 nm groups were evidently lower than that in the control group,the fungal hyphae changed to short-bar like morphology and then disappeared,the numbers of inflammatory cells were also lower than that in the control group.On the day 15 after treatment,there are a little numbers fungal hyphaes and corneal stroma cells;And there are not fungal hyphae in the 360 and 440 group.Pathological section report:On the day 1 after treatment,inflammatory reaction was serious in every group.On the day 7 after treatment,infammatory reaction in the control group was more serious,but it's better in the 360 nm and 440 nm groups.On the day 15 after treatment.there are more fibroblasts and small vessels in all groups.But epithelium corneae is not complete in the control group.Conclusion:Using both 360 nm UVA and 440 nm blue light in combination with riboflavin to induce corneal collagen cross-linking are effective in treating rabbit fungal keratitis,which showed no significant difference between these two methods.