Establishment Of Experimental Bacterial Keratitis In A Tree Shrew Infection Model And Expression Of Interleukin-17 In The Model

Objectives:Bacterial keratitis is a kind of serious ocular infection that is threatening to vision.Both pseudomonas aeruginosa and staphylococcus aureus are common pathogens of bacterial keratitis.In this study,pseudomonas aeruginosa and staphylococcus aureus keratitis infection models of tree shrews are established to observe the clinical feature and pathological characteristics of the models and to determine the expression of interleukin-17(IL-17)in the bacterial infection process of tree shrews cornea.Furthermore,the mechanism of IL-17 in bacterial keratitis of tree shrews will be discussed.Methods:(1)After activating and enriching the culture of pseudomonas aeruginosa and staphylococcus aureus,a single bacterial colony was selected and placed into tryptone soy broth medium,shaking culture at 37℃ for 24 hours.The bacterial concentration was adjusted to 1 × 109 CFU/ml.Thirty-six healthy adult tree shrews were randomly divided into 3 groups:PA group,SAU group and control group.Corneal injuries were caused by the corneal contact lens assisted by the scatch method,and bacterial suspensions were inoculated on the damaged corneas.Pseudomonas aeruginosa solution was added to the tree shrews’ corneas of the PA group,staphylococcus aureus solution was added to the tree shrews’ corneas of the SAU group,and the same amount of saline was added to the control group.After establishing models successfully,the infection symptoms of the model were evaluated by using the slit-lamp microscope and confocal microscopy on the 1st,4th,7th and 14th days after inoculation of the bacterial suspensions.The pathological and histological changes of corneal tissue were observed by pathological sections.Microorganisms in the infected corneal tissues were identified,and the models were evaluated.(2)After the modeling was bing successful,samples were collected at the corresponding time as pointed above,and the expressions of IL-17 mRNA in the corneal tissue of tree shrews were detected by real-time quantitative PCR,and the expressions of IL-17 protein were detected by ELISA and Western blot.Results:(1)The infected tree shrews which in the PA group and SAU group have shown typical clinical symptoms.At the same time,the clinical manifestations and inflammatory cell infiltration of the keratitis were consistent with the changing rules of the cornea in histopathology.The progression of the disease appeared to be similar in the two model groups.The symptom of infection would appear within 24-36 hours after modeling.The main clinical manifestation was corneal edema,inflammatory infiltration in the superficial stroma,and highly reflective inflammatory cells around the scratcheson confocal examination.Subsequently,the inflammatory response gradually increased,and the corneal ulcer reached its peak on the 4th day after infection.With increased inflammatory exudation and the formation of infiltrating lesions,the inflammatory cells in the stromal layer had a wider range of infiltration,which was primarily based on neutrophils.The confocal examination showed that the inflammatory cells in the ulcer area were more dense,accompanied by medium-high reflective necrotic tissue.On the 7th day after the infection,it was the recovery period.The inflammatory infiltration weakened,and the cornea gradually became transparent.The corneal structure was repaired,and the ulcer surface began to heal but it was easy to leave scars.It was confirmed by the sequencing result of corneal tissue DNA that the corneal inflammations of tree shrews in the PA group and SAU group were caused by pseudomonas aeruginosa and staphylococcus aureus.The success rates of modeling were 92%and 100%.(2)The expressions of the IL-17 gene and protein in corneas of successfully infected tree shrews were basically consistent with the severity of corneal inflammation basically.The expressions of IL-17 gradually increased after infection.On the 4th day,it reached the peak and then gradually decreased.However,in the same time period after the infection,the expressions of IL-17 between the PA group and the SAU group showed no significant statistical difference(P>0.05).Conclusions:(1)Without immunosuppressants,the models of pseudomonas aeruginosa and staphylococcus aureus keratitis in tree shrew were established by using a contact lens-assisted method,and their symptoms were similar to those of human bacterial keratitis.Those animal models were practical for the further study of the pathogenesis and drug research of bacterial keratitis.(2)IL-17 was expressed in the whole disease term after the cornea infected by pseudomonas aeruginosa and staphylococcus aureus.The expression was broadly in line with the development trend of the disease,especially in the early stage of infection and the peak of inflammation when the corneal inflammatory response was more serious and inflammatory cell infiltration was obvious.IL-17 participated in the occurrence and development of bacterial keratitis in tree shrews,and its specific function and mechanism still require further study.