| Objective:An animal model of alkali injury was established to understand the effect of doxycycline on the expression of inflammatory cells,Vim and Fn in corneal tissues of alkali burn rats,and to further study the possible mechanism of doxycycline in reducing corneal scar.Methods:1.Thirty-six SPF SD rats were divided into 3 groups(group a group b,and group c),with 12 rats in each group.The right eye was used to make corneal alkali burn animal model,and the corneal burn time of group a,b and c was set at 20s,25s and 30s,respectively.The anterior chamber hemorrhage,corneal ulcer,perforation and turbidity were observed and recorded within 14 days of the natural course of corneal alkali burn.2.Sixty healthy SD rats were selected and the right eye was used to make 25S corneal model of alkali burn.Six rats with irregular modeling were excluded,and then the model rats were divided into doxycycline group and control group,with 27 rats in each group.After successful modeling,doxycycline group was treated with doxycycline eye drops of 1.0g/L,while the control group was treated with eye solution of the same composition as doxycycline eye solution except that the eye solution did not contain doxycycline.Nine rats in each group were selected for corneal transparency score and inflammation index score under the microscope on the 3rd,7th and 14th day after alkali injury,Subsequently,the rats were killed by intraperitoneal injection of chloral hydrate,and the corneal tissue was quickly cut off by aseptic microshear.Five of them were used to detect the relative expression of Vim and Fn mRNA in the corneal tissues of alkali burn rats in each group by RT-qPCR.The other 4 corneas were used for inflammatory cell count by HE staining in pathological sections and IF to detect the expression levels of Vim and Fn proteins in corneal tissues.Results:1.(1)Group a(alkali burn time was 20s):1 eye(8.3%)had anterior chamber hemorrhage,0 eye(0.0%)had corneal perforation,and 4 eyes(33.3%%)had corneal ulcer.Group b(alkali burn time was 25s):1 eye(8.3%)had anterior chamber hemorrhage,1 eye(8.3%)had corneal perforation,and 7 eyes(58.3%)had corneal ulcer.Group c(alkali burn time was 30s):anterior chamber hemorrhage in 7 eyes(58.3%),corneal perforation in 3 eyes(25.0%),corneal ulcer in 8 eyes(66.7%);(2)Corneal opacity in group a,b and c:On day 7 after alkali burn,the corneal opacity score of group b(3.17±0.58)was higher than that of group a(1.67±0.78),and the difference was statistically significant(P<0.05).There was no significant difference in the degree of corneal turbidity between group c(3.33±0.49)and group b(3.17 ± 0.58)(P>0.05).2.(1)Corneal opacity score:On day 3 after alkali burn,both groups showed obvious corneal edema and epithelial defect;Day 7:The corneal edema in the control group was more severe,the stroma was cloudy,and the pupil was faintly visible.The doxycycline group had less corneal edema and less clear iris texture;Day 14:In the control group,new blood vessels grew into the alkali burn area,and the pupils were not visible.In doxycycline group,corneal stromal edema subsided,corneal clouding was rare and iris texture was clear.On day 3 after alkali burn,there was no significant difference in corneal turbidity between the two groups.On day 7 and 14,the corneal turbidity score of doxycycline group(2.11±0.60,0.78 ± 0.67)was lower than that of control group(3.22 ±0.44,3.11±0.60),and the differences were statistically significant(P<0.05).(2)Inflanlmation index:On the 3rd and 7th day after alkali burn,limbic ciliary congestion and corneal foggy opacification were observed in both groups,while the doxycycline group was milder than the control group.On day 14,the corneal ciliary congestion and corneal edema were improved in the control group,while corneal transparency was almost restored in the doxycycline group.On the 3rd,7th and 14th day after corneal alkali burn in rats,the corneal inflammation index of the doxycycline group(0.43±0.07,0.45 ± 0.07,0.06±0.06)was lower than that of the control group(0.61±0.08,0.83±0.14 0.52±0.08),the differences were statistically significant(P<0.05).(3)He staining of pathological sections:On the 3rd day after alkali burn,there were 2-3 layers of edema corneal epithelial cells,and the matrix was loosely arranged.There were fewer inflammatory cells in doxycycline group than in the control group;On day 7,the corneal epithelial cells in the control group were edema,with 4-6 layers of cells.The fibers in the stromal layer were arranged irregularly and a large number of inflammatory cells were immersed in the stromal layer.The corneal epithelium in doxycycline group was slightly edema,with 4-6 layers of cells,relatively regular arrangement of fibers,and a small amount of inflammatory cells immersed in the stromal layer;On day 14,The corneal epithelial cells of the control group were 4-6 layers,edema was still visible in the stromal layer,the fibers were arranged disorderly,and a large number of inflammatory cells were still infiltrated;The corneal epithelium of doxycycline group was completely repaired with regular fiber arrangement and very little inflammatory cell infiltration.On day 3,7 and 14 after alkali burn,inflammatory cells in doxycycline group(118.67±15.30,160.42±13.69,76.75±8.36)were less than those in control group(159.75±19.82,308.42±28.62,157.33±14.22),and the differences were statistically significant(P<0.05).(4)Quantitative Real-time PCR:on day 3,7 and 14 after alkali burn,The relative expression level of Vim-mRNA in doxycycline group(0.52±0.18,0.54±0.22,0.29±0.10)was lower than that of control group(0.91 ± 0.26,2.01 ± 0.56,0.900±0.31);The relative expression level of Fn-mRNA in doxycycline group(0.43±0.18,0.48±0.09,0.24±0.17)was also lower than that in control group(1.02± 0.23,1.16±0.29,0.89± 0.11),with statistical significance(P<0.05).(5)Immunofluorescence staining:On day 3,7 and 14 after alkali burn,the area of Vim positive fluorescence(Area%)in doxycycline group(10.38±0.41,12.17±1.43,7.08±0.80)was lower than that in control group(16.45±0.73,19.97±1.56,14.90±1.3 8);the area of Fn positive fluorescence(Area%)in doxycycline group(4.54 ± 0.21,6.50±0.27,2.07 ±0.25)was lower than that in the control group(7.43±0.31,10.44 ±0.55,8.92±0.24),with statistical significance(P<0.05).Conclusion:1.When the mold was placed in the center of the cornea for 25s,the alkali burn cornea model had the best effect and could successfully induce the formation of corneal scar.Prolonging the alkali burn time would increase the incidence of corneal perforation and hyphema.2.Doxycycline can inhibit the expression of inflammatory cells,Vim and Fn in the cornea of alkali burn rats and reduce the degree of corneal turbidity.It is likely to reduce the deposition of extracellular matrix(Vim and Fn)to reduce the degree of corneal turbidity in alkali burn rats. |
