Treatment Of Fungal Keratitis By Ultraviolet A/Riboflavin Corneal Cross-linking In Rabbit

Objective:Rabbit models for studying Aspergillus fumigatus keratitis were established andthe pathological variations after treatment of fungal keratitis by corneal collagen crosslinking (CXL) with UV-A riboflavin were observed, genetic expression and proteindistribution of Matrix metalloproteinase-2,9(MMP-2,9) in corneal tissue with FKwere detected, with the hope of investigating effects and related mechanismassociated with CXL, and providing principle basis for treatment of FK with CXL.Methods:44healthy Japanese long-eared rabbits were randomized into4groups. The16right eyes in group A (n=16) were inoculated with Aspergillus fumigatus and no CXLwas performed. The16right eyes in group B (n=16) were also inoculated withAspergillus fumigatus and single CXL was performed at the0d (the day successfulinoculation was confirmed). The12right eyes in group C were inoculated withAspergillus fumigatus and single CXL was performed at0d and3d respectively. And4healthy corneas were randomly selected from the3groups to serve as blank control(group D). Clinical ulcer grading was performed for all the eyes at the0d,1d,4d,7dand14d after successful FK model establishment. Every4rabbits in group A and Bwere sacrificed to obtain corneal tissue at1d,4d,7d and14d. As the situation ingroup C was the same as in group B, no rabbits were sacrificed at d1and the left weresacrificed as those in group A and B. The degree of inflammatory cell infiltration andthe depth hyphae invaded into the cornea were observed histopathologically.Distributions of MMP-2and MMP-9and their derivations were observed usingimmunohistochemical methods and their expressions were semi-quantitativelyanalyzed. The concentrations of MMP-2and MMP-9m-RNAs within corneal tissueswere detected using Reverse transcription polymerase chain reaction (RT-PCR).Results:1.All the inoculated eyes were infected by Aspergillus fumigatus. Thedifferences in ulcer grading at0d among different groups were not statistically significant (P>0.05) and the grading scores at the14d in CXL intervene groups(group B and C) were significantly lower compared to untreated group (group A)(P<0.05). The differences in ulcer grading between group B and group C was notstatistically significant (P>0.05), but with lower score observed in group C. All the3groups showed significant difference in ulcer grading when compared with the blankcontrol group (group D)(P<0.05).2.Histopathological examination:(1) HE staining revealed purulent inflammationof the cornea caused dominantly by fungal hyphae and spores invasion andneutrophils infiltration.(2) Group B and group C had decreased depth ofinflammatory cell infiltration and degree of necrosis when compared to group A.After CXL, fungal hyphae showed decreased growth speed and invasion depth.3.Immunohistochemical staining showed (1) increased MMP-2and MMP-9expressions in corneal tissue with aspergillus-induced FK, and the two kinds ofproteins mainly distributed in epithelial basal cells, fibroblasts and inflammatory cells,and also with MMP-2expression in neovascular endothelial cells. Peripheral corneahad higher levels of MMP-2and MMP-9expression than central cornea.(2) Corneasin group A had highest MMP-2expression at the1d, and with slight decrease at the4d, then slight increase at the7d and finally the lowest at the14d. Corneas in thegroup A had increasing positive MMP-9expression since the0d, and showedcontinuous increase since the4d.(3) The differences in MMP-2expression HIC scoreat the1d,4d,7d and14d among the group A, B and C were not statisticallysignificant (P>0.05). Group B and C had significant higher MMP-2expression levelsat the14d compared to group A (P<0.05). Differences in MMP-2expression levels ateach time point between group B and group C were not statistically significant(P>0.05). After CXL, group B and group C had lower MMP-9expression levelscompared to group A, and the differences at4d and14d were statistically significant(P<0.05), but not at other time points (P>0.05). Differences at all the time pointsbetween group B and group C were not statistically significant (P>0.05).4.RT-PCR results indicated that:(1) Since successful inoculation for group A,MMP-2expression level at4d was lower than that at1d, and MMP-2expression levelgradually increased since4d. MMP-9expression levels increased after inoculation, and reached the highest at14d.(2) The difference in MMP-2expression at the14dbetween group A and group B were statistically significant (P<0.05). The differencesin MMP-2expression levels at1d,4d and7d between group A and group C werestatistically significant (P<0.05). The corresponding differences among the left groupswere not statistically significant (P>0.05). Group B and C had significantly higherMMP-2expression at14d when compared to group A (P<0.05). Group B and C hadsignificantly lower MMP-9expression compared to group A, and the differences at4d,7d and14d were statistically significant compared to group A (P<0.05). Butdifferences in MMP-9expression at all the time point between group B and group Cwere statistically significant (P>0.05).Conclusions:1.Ultraviolet A/riboflavin corneal cross-linking could lead to significant decreaseof clinical ulcer grading score and inflammatory cell necrosis degree, could reducethe invasion depth by lowering the speed of hyphae growth within cornea with FK.2.Ultraviolet A/riboflavin corneal cross-linking significantly inhibited expressionof MMP-9within cornea with FK, thereby reduced the damage to the dissolution ofcorneal collagen，may be one of the therapeutic mechanism of infectious keratitis.3.Ultraviolet A/riboflavin corneal cross-linking failed to achieve regulation onexpression of MMP-2at early stage, and may even increase the expression ofMMP-2.