| Objective By establishing a rat model of fungal keratitis to observe the characteristics of natural course of fungal keratitis,and histopathological examination was performed at all time points to observe the tissue destruction in the course of fungal keratitis.To investigate the effect of doxycycline on the inflammatory cells and expression of monocyte chemoattractant protein-1（MCP-1）and matrix metalloproteinase-8（MMP-8）in rats with fungal keratitisMethods The first part,32 healthy SD rats were randomly divided into the operation model group and control group,with 16 rats in each group.A rat model of aspergillus fumigatus keratitis was established by soft corneal contact lens.In the experimental group,20?l fungal spores suspension was injected into the space between cornea and self-made corneal contact lens and the 20?l PBS buffer was injected in the control group.Pathological characteristics was observed at the third,fifth,seventh and tenth days after operation.A rat model of fungal keratitis was proved by corneal scraping to perform 10%potassium hydroxide wet preparation,bacterial culture and fungal culture.All rats were sacrificed for histologic examination to observe histopathological changes of corneal tissue injury.The second part,32 healthy SD rats were randomly divided into the control group and doxycycline-treated group,with 16 rats in each group.The models of aspergillus fumigatus keratitis in 32 healthy SD rats were established by soft corneal contact lens.The doxycycline-treated group was intervened 0.3g·L-1doxycycline eye drop,while the control group was intervened the solution of eye drop without doxycycline All rats were scored for corneal involvement at the3rd,5th,7th,10th day and sacrificed for histologic examination to record the number of inflammatory cells.The third part,64 healthy SD rats were randomly divided into control group and doxycycline-treated group,with 32 rats in each group.the models of aspergillus fumigatus keratitis were established by soft corneal contact lens.The doxycycline-treated group was intervened 0.3g·L-11 doxycycline eye drop,while the control group was intervened the solution of eye drop without doxycycline.All rats were scored for corneal involvement at the 3rd,5th,7th,10th day and sacrificed for expression of monocyte chemoattractant protein-1 and matrix metalloproteinase-8 by fluorescent quantitative RT-PCR and and enzyme-linked immunosorbent assay.Results 1.The aspergillus fumigatus filaments and spores were found with the corneal scraping,fungus cultivation is characterized of fungal colony.The characteristic of aspergillus fumigatus keratitis:the first day after operation showed mild conjunctival hyperemia,corneal edema,white corneal ulcer on surgical site;the third day showed that conjunctival hyperemia was worse,corneal surface was covered a large number of purulent secretions;corneal edema was alleviated,the area of corneal ulcer began to diminish on the fifth day.Some could lead to anterior chamber bleeding or descemetocele and microperforation.Around the necrosed tissue was the infiltrative PMNs.The seventh day corneal ulcer gradually diminished;Corneal epithelium proliferation were observed at the late stage,it gradually formed corneal leucoma or corneal neovascularization.The results of histopathological examination showed that a small amount of leukocytes were diffused in corneal stroma of aspergillus fumigatus keratitits on the 3rd day after inoculation;The inflammatory reaction was severse with corneal edema and corneal ulcer on the5th day after inoculation;Around the necrosed tissue was the infiltrative PMNs on the 7th day;Corneal epithelium proliferation and neovascularization were observed at the late stage.2.The normal corneal tissue in rats lack neutrophils.Early stage rats infected with Aspergillus fumigatus:the number of neutrophils was significantly increased,reached the peak on the fifth day,then decreased gradually.Compared with control group,the inflammatory index was lower in the doxycycline-treated group at the 3rd,5th,7th day,neutrophils count was significantly lesser at all time points（P<0.05）.3.The detection results of fluorescent quantitative RT-PCR and ELISA showed that the expression level of MCP-1 and MMP-8 was few,the expression of MCP-1 and MMP-8 increased after the operation,the late course gradually decreased,but the expression was still maintained at a high level.The detection results of fluorescent quantitative RT-PCR and ELISA showed that the relative amount of MCP-1 decreased in the doxycycline-treated group at the 3rd,5th 7th day（P<0.05）,and that the relative amount of MMP-8mRNA was lower at 3rd,5th day than that in the control group（P<0.05）.Conclusion 1.The rat model of aspergillus fumigatus keratitis can be successfully established by soft corneal contact lens.2.Doxycycline can decrease the infiltration of neutrophils in rats with aspergillus fumigatus keratitis to relieve the inflammatory response.3.Doxycycline can inhibit the expressions of MCP-1 and MMP-8 in rats with aspergillus fumigatus keratitis,that might be related to one of the mechanisms to relieve the inflammatory response. |
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