| Objective:Research the effective concentration of the drug and dosage regimen about FK506 eye drops on corneal alkali burns ; evaluate the inhibition of immune and inflammatory response in corneal alkali burns, improve the effectiveness of corneal microenvironment and approach its mechanism.Methods:filter paper soaked NaOH solution was placed on the corneal to form moderate corneal alkali burn model. Than 96 wistar rats were randomly divided into A, B, C, D four groups. A, B, C group was treated by drug and was treated the patients eye with concentrations of 0.05%, 0.1%, 0.5% FK506 eye drops, respectively, four times/day. D group was as negative control group. Each group rats were killed after treating with FK506 in different time (3 days, 7 days, 14days,30days) and removed the corneas. HE staining detected corneal morphological changes and compared inflammatory cells of models. Immunofluorescence assayed the expression of CD4/CD8 + T cells in time and space law.Results The alkali burned cornea that was burned NaOH filter paper was haze edema; corneal burn areas lack the matrix and melting ulcers, limbal blood vessels to dilate, the late formation of new blood vessels, cornea was lack of transparent. The FK506 treatment group corneal ulcer area, corneal opacity level and the degree of corneal neovascularization were significantly better than the control group.①. Corneal melting result of quantitative analysis: The treatment groups: corneal melting area was A group (3.14±0.25) mm2, B group (2.65±0.28) mm2, C group (2.27±0.47) mm2, while the control group, D group (4.06±0.41) mm2 on the 3th day after corneal burned, the difference was significant (P<0.05);compared low concentrations of FK506 treatment group with medium concentration ,high concentration group, the difference was significantly(P<0.05), while 0.1% and 0.5% concentration group, there were not significant between difference. Corneal melting area of the 7 th days: A group (1.82±0.51) mm2, B group (1.34±0.41) mm2, C group (0.90±0.39) mm2, respectively, all less than D group (2.47±0.54) mm2,the difference was significantly(P<0.05).②. Corneal opacity of quantitative analysis: corneal opacity scores in the treatment groups all less than the control group (P<0.05 ), the change of the law and the corneal melting curve is consistent, but medium concentration and high concentration was not significant differences(P>0.05).③. Corneal neovascularization of quantitative analysis in treatment groups: the burn area of CNV was A group (4.26±0.36) mm2, B group (3.88±0.33) mm2, C group (3.54±0.46) mm2 respectively ,were smaller than the control group D group (5.00±0.12) mm2 for 3 days after corneal burned, the difference was statistically significant(P<0.01 ).There was als o statistically significant difference when compared A with B, C, but the difference was not significant between B and C groups(P>0.05); Such differences remain to 14d side decreases. HE staining showed the number of inflammatory cell infiltration burned corneal matrix in the treatment group was less than the control group. The results showed that when the inflammatory cell count 3d, A group (77.67±4.51)cells/ 0.04mm2, B group (51.33±3.21)cells/ 0.04mm2 ,C group (41.67±3.06) cells/ 0.04mm2 ,but synchronization the control group D group (97.00±6.25) cells/ 0.04mm2 , the differences were statistically significant(P < 0.05 ). Immunofluorescence prompt that the number of CD4 positive cells in the treatment groups are less than the control group, by optical density analysis, the low concentration of the treatment group for the moderate-intensity fluorescent, high concentrations of the treatment group was low fluorescence, while the control group showed diffuse intense fluorescence. There was not significant differences between the treatment groups and the control group that the infiltration and expression of CD8 molecules in the pathological process of corneal alkali burn.Conclusion: FK506 eye drops could significantly reduce specific immune response mediated by T lymphocytes after corneal alkali burned, but also indirectly reduce non-specific inflammatory response, and showed dose-dependent. FK506 could prevent the degree of corneal stromal melting after alkali burned, inhibit the growth of corneal neovascularization and reduce the structural damage and improve the prognosis of the cornea, so provide a new immune therapeutic model in the acute phase of corneal alkali burn.
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