Study Of Experimental Fusarium Keratitis In BALB/c And C57BL/6 Mouse Models

BackgroundThe different type of Th immune responses has been understood for some timeas the critical determinant of outcome of the pulmonary aspergillosis:deflection intothe Th1 pathway produce a favorable outcome,whereas deflection into the Th2pathway results in asthma or allergic bronchopulmonary aspergillosis.However,theirrole played in the course and prognosis of fungal keratitis remains unknown.Idealmodel is needed urgently to facilitate those studies.BALB/c and C57BL/6 are two inbred mice with different genetic background andfavored different Th response.Thus,we induce corneal infection by F.solani in bothBALB/c and C57BL/6 mice,and examined the kinetics of corneal infection byclinical observation,histopathological examination,quantitation of viable fungi andpolymorphonuclear neutrophils(PMNs).We found that experimental fusarial keratitisin both mice was host dependent.Materials and MethodsAdult female inbred BALB/c and C57BL/6 mice were used at 6 to 8 weeks ofage.The wild-type strain of F.solani used for this study was isolated from the clinicalcase of fungal keratitis.The species identification was made by combining themacroscopic with microscopic criteria.The concentration of fungal suspension was 2 ×10~8 colony-forming units(CFU)/mL The cornea of the right eye of the mice was scarified under operative microscope and was applied 5-μl inoculum of F solani.After inoculation,all eyes were treated with 5-μl of the 0.1% dexamethasone and 0.3% tobramycin eye drops twice daily to a total of four times.Establishment of themodel was followed by three studies:1.Mice were scored and photographed with slit lamp biomicroscope twice dailyfor the first three days and daily afterwards for corneal involvements including thearea of corneal opacity,the density of corneal opacity and the surface regularity.2.At 1 day,2,3,4,5,7,10 and 15 days post infection(pi),5 infected eyes for each time point were enucleated for histopathological examination.The sections werestained with hematoxylin and eosin(HE),and periodic acid-Schiff(PAS)to evaluateinflammatory cell infiltration,corneal structural destruction and hyphal invasion.3.At 6 hours,1 day,2,3,5 and 7 days pi,6 infected eyes for each time point were enucleated for quantitation of viable fungi using method of plate count and quantitation of PMNs using a myeloperoxidase(MPO)assay.ResultsAll the mice show corneal infection since 1 day pi.1.Features of experimental Fusarium keratitis in BALB/c miceMost of the BALB/c mouse infected eyes exhibited marked corneal edema,which was regressed gradually and completely resolved at about 10 days pi without corneal scarring and neovascularization.2.Features of experimental Fusarium keratitis in C57BL/6 miceThe natural course of the experimental fusarial keratitis of C57BL/6 mice wasabout 15 days,which could be divided into three stages,including infiltrate enlarging,tissue necrosis,corneal neovascularization and scarring respectively.3.Histopathological findings of experimental Fusarium keratitis in BALB/cmiceAt 1 day,2 and 3 days pi,fusarial hyphae was invaded into the deep stroma and reach the Descemet's membrane(DM),but did not penetrated into the anteriorchamber(AC).From 4 days pi on,the amout of fungi in the stroma was graduallydecreased.Inflammatory cells,PMNs predominantly were observed since 2 days pi,and spread throughout the whole thickness of the corneal stroma on day 3,whichclustered into densely packed foci of cells.From 4 days pi on,inflammatory cells inthe stroma began to decrease,and were absent at 10 days pi.No necrotic tissue orvessel was observed in the stroma.4.Histopathological findings of experimental Fusarium keratitis in C57BL/6miceAt 1 day,2 and 3 days pi,fungi proliferated rapidly,spread throughout the wholethickness of cornea,and some of them even penetrated into the AC.From 4 days pi on,fusarial hyphae were gradually cleared from the cornea,and were absent from thecornea at 7 days pi.Inflammatory cells,PMNs predominantly were observed in theanterior half of the corneal stroma at 2 days pi,and diffused to the deep stroma andformed a dense immune ring surrounding the large foci of fungi on 3 days pi.From 4days pi on,the amout of inflammatory cells in the stroma gradually decreased.Necrotic tissue was first observed at 3 days pi within the immune ring,and increasedrapidly afterwards.Large area of DM deficiency was observed.From 7 days pi on,necrotic tissue gradually decreased and replaced by the fibroblast cells and vessels.5.The quantitative relationship between fungal proliferation and PMNsinfiltration of Fusarium keratitis in BALB/c miceThe results showed that there was positive relationship between the amount offungi and PMNs in the clinical course of Fusarium keratitis in BALB/c and C57BL/6mice respectively.The amount of fungi isolated from both BALB/c and C57BL/6infected eyes was similar(p=0.632)at 6 hours,as well as the number of the PMNs(p=0.850).However,the amount of the fungi was significant greater in C57BL/6 vsBALB/c mice at 2(p=0.002)and 3 days pi(p=0.009),as well as the amount of PMNsat 2(p=0.022),3(p=0.035)and 5 days pi(p=0.040). Conclusions1.From the results of this study,we could conclude that the wild-type of F.solani can induce corneal infection in both BALB/c and C57BL/6 mice by the route of corneal scarification.2.The features and outcome of experimental Fusarium keratitis of BALB/c micewere different from those of C57BL/6 mice.3.The histopathological findings showed that the amount and invasiveness offungi in C57BL/6 mice were greater than in BALB/c mice,and the pattern ofinflammatory cells infiltration was different between them.Furthermore,tissue necrosis did not occurred in most of the BALB/c mice,but it was severe in all theC57BL/6 mice.4.The results of quantitation of fungi and PMNs showed that there was positiverelationship between the amount of fungi and PMNs in the clinical course ofFusarium keratitis in BALB/c and C57BL/6 mice respectively,but the amount of thefungi and PMNs were both significantly greater in C57BL/6 than BALB/c mice.5.One to two days pi is critical for the development and prognosis of Fusarium keratitis in BALB/c and C57BL/6 mice.6.Fusarium keratitis in both BALB/c and C57BL/6 mouse models could be alternative experimental models for further study about the Th immune response offungal keratitis.