| Objective: To establish a rat model of acanthamoeba keratitis and investigate the influence of antiamebic drugs combined with doxycycline on expression of matrix metalloproteinase-8(MMP-8)and monocyte chemotactic protein-1(MCP-1)in corneas of rat with acanthamoeba keratitis.Methods: 1.Sixty healthy SD rats were randomly divided into three groups,twenty in each group.The blank control group was granted with no disposal.In conditional control group,scraped central corneal epithelium of right eye in rat and covered by aseptic lens then dropped in PBS between the cornea and lens.In experimental group,scraped central corneal epithelium of right eye in rat and covered by ameba-laden contact lens then dropped in acanthamoeba trophozoites suspension between the cornea and lens.The palpebral fissure was sutured under aseptic condition and took out stitches then removed the contact lens after 24 h.At the 1st,3rd,5th,7th and 10 th day,cornea scraping,cultivation of the amoeba and histopathological examination were used.2.Sixty-four healthy SD rats were randomly divided into four groups,sixteen in each group.Scraped corneal epithelium of right eye in rat.Surgical control group was covered by aseptic contact lens and dropped in PBS between the cornea and lens.Model group,chlorhexidine-treated group and treatment group were covered by ameba-laden contact lenses and dropped acanthamoeba trophozoites suspension between the cornea and lens.The palpebral fissure was sutured under aseptic condition and took out stitches then removed the contact lens after 24 h.The chlorhexidine-treated group was given 0.02% chlorhexidine acetate eye drop.The treatment group was given 0.02% chlorhexidine acetate eye drop combined with 0.02% doxycycline eye drop.The surgical control group and model group were given to menstruum eye drop.The inflammatory index of corneal disease was calculated at the 3rd,5th,7th and 10 th day.Then corneal tissue were taken for histopathology to count the number of inflammatory cells and the quantitative real-time PCR detection of MMP-8 and MCP-1.Results: 1.The right eye of rat in the experimental group was infected with acanthamoeba keratitis and the result can be confirmed by cornea scraping,cultivation of the amoeba and histopathological examination.The corneal pathological sections by staining with hematoxylin and eosin showed infected eyes were infiltrated by inflammatory cells on the first day,and the quantity of inflammatory cells had an upward trend on the third day and the fifth day,with a decline in the seventh to the tenth day.2.After the model of acanthamoeba keratitis was established,the inflammatory index of cornea in model group is 2.33±0.27 at the 3rd day,2.92±0.17 at the 5th day,2.75±0.17 at the 7th day,2.33±0.27 at the 10 th day;the inflammatory index of cornea in chlorhexidine-treated group is 1.92±0.17 at the 3rd day,2.59±0.17 at the 5th day,2.50±0.20 at the 7th day,2.17±0.19 at the 10 th day;the inflammatory index of cornea in treatment group is 1.42±0.17 at the 3rd day,2.25±0.17 at the 5th day,2.08±0.17 at the 7th day,1.84±0.19 at the 10 th day.At the 3rd,5th and 7th day,the inflammatory indexs in the treatment group were lower than those in the chlorhexidine-treated group(all P <0.05),which the difference was significant.The number of inflammatory cells in model group is 158.00±6.60 at the 3rd day,177.75±6.85 at the 5th day,139.50±8.96 at the 7th day,42.00±4.97 at the 10 th day;the number of inflammatory cells in chlorhexidine-treated group is 138.25±7.97 at the 3rd day,151.25±5.80 at the 5th day,108.25±11.09 at the 7th day,39.50±3.42 at the 10 th day;the number of inflammatory cells in treatment group is 120.25±6.55 at the 3rd day,127.75±7.63 at the 5th day,86.25±5.38 at the 7th day,29.50±8.96 at the 10 th day.At the 3rd,5th and 7th day,the number of inflammatory cells in the treatment group were lower than those in the chlorhexidine-treated group(all P <0.05),which the difference was significant.The relative amount of MMP-8 mRNA in model group is 2.10±0.31 at the 3rd day,3.39±0.34 at at the 5th day,1.76±0.44 at the 7th day,0.43±0.10 at the 10 th day;the relative amount of MMP-8 mRNA in chlorhexidine-treated group is 1.03±0.29 at the 3rd day,2.35±0.49 at the 5th day,1.49±0.34 at the 7th day,0.21±0.04 at the 10 th day;the relative amount of MMP-8 mRNA in treatment group is 0.29±0.16 at the 3rd day,0.83±0.23 at the 5th day,0.79±0.16 at the 7th day,0.19±0.03 at the 10 th day.The relative amount of MMP-8 mRNA in the treatment group were significantly smaller than that in the chlorhexidine-treated group at the 3rd,5th and 7th day(all P <0.05).There was no significant difference in the expression of MMP-8 mRNA between two groups at the 10 th day(P >0.05).The relative amount of MCP-1 mRNA in model group is 1.43±0.37 at the 3rd day,1.87±0.09 at the 5th day,0.48±0.05 at the 7th day,0.22±0.01 at the 10 th day;the relative amount of MCP-1 mRNA in chlorhexidine-treated group is 1.02±0.20 at the 3rd day,1.29±0.37 at the 5th day,0.26±0.05 at the 7th day,0.06±0.01 at the 10 th day;the relative amount of MCP-1 mRNA in treatment group is 0.35±0.07 at the 3rd day,0.29±0.12 at the 5th day,0.11±0.05 at the 7th day,0.05±0.02 at the 10 th day.The relative amount of MCP-1 mRNA in the treatment group were significantly smaller than that in the chlorhexidine-treated group at the 3rd,5th and 7th day(all P <0.05).There was no significant difference in the expression of MCP-1 mRNA between two groups at the 10 th day(P >0.05).Discussion:1.Covered with ameba-laden contact lens and dropped in acanthamoeba trophozoites suspension in the defective corneal epithelium,can successfully establish a stable rat model of acanthamoeba keratitis.2.It can alleviate the corneal inflammatory damage by inhibiting the inflammatory cell infiltration and down-regulating MMP-8 and MCP-1 expression during antiamebic drugs therapy together with topical application of doxycycline in treating acanthamoeba keratitis in rat. |
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