The Establishment And Identification Of A Herpes Simplex Keratitis Mouse Model

Objectives:To establish a stable,standard and repeatable mouse model of herpessimplex keratitis(HSK),which could provide basic model for the following research on pathogenesis and reference for other keratitis models.Methods:1).The part ? experiment:48 Balb/c mice aged six weeks with SPF level were randomly divided into A?B?C groups,with sixteen mice in each group.Making corneal epithelial defect in both eyes of each mice,then the rig ht eye of each mice was inoculated with viruses at different titers of 105PFU?104PFU and 103PFU,and the left eye was inoculated with equal volume DME M.The most appropriate titer of the virus will be determined according to the survival rate and corneal symptoms,2)The part ? experiment:68 Balb/c mic e aged six weeks with SPF level were randomly divided into the control group(n=6)and the experimental group(n=62).The method described in the prelim inary experiment was used for the model building.Parameters on mice survival rate of mice and use Slit lamp microscope to observe corneal symptoms,reco rd the score of Corneal fluorescein sodium staining score and corneal turbidity score,Co-culture of tear and vero cells,then analysis of the results at differe nt infection time points to draw a conclusion.Results:The results of part ? experiments:The survival rates of the three grou ps were 37.5,75,and 93.75 percent;Both group A and B mice were infected effective;group C were infected.On the basis of effective infection;group B guaranteed sufficient survival rate and typical progression of HSK,which was suitable for the establishment of mouse model.The results of part ? experiments:1)Clinical signs of mice shown The corneal defects were repaired third day af ter virus and DMEM inoculation;Dendritic lesions appeared in the corneal epit helium on the fourth to fifth day;On the sixth to seventh day,the mice were manic,but most of them had no obviously corneal lesions;The corneal ulcer gradually recovered after fourteenth day.The right eye showed typical progres sion of HSK infection,while the left eye remained asymptomatic three days af ter repair of the defect.2)The Statistical results of infected eye show at different times the score of c linical symptoms,corneal turbidity and fluorescein sodium staining were statisti cally significant(P<0.05),and the peak of clinical symptom score of the rig ht eye was on the seven days after infection,while the peak of corneal turbidi ty and fluorescein staining scores were around the 10th day after infection,and the scores at all time points were in the middle,and the infection was relativ ely mild.3)Bilateral mandibular lymph node statistical results show that bilateral lymph nodes of all mice increased gradually,the lymph nodes on the right are larger than those on the left,there was congestion in the lymph nodes in seventh d ay.The lymph nodes continued to increase until the fourteenth day,and gradua lly decreased to normal size after fourteenth day.4)Results of tear and vero cells co-culture show the cell pathological effects were observed on the third to fourteenth day after infection,all cells died on t he fifth day after infection,and seventy percent of the cells died after seventh to tenth days,only thirty percent of the cells died on third day and fourteent h day,no cells died after fourteenth day.5)PCR results showed that on the seventh day after infection,six mice's right trigeminal ganglia were randomly selected for DNA extraction,after Polymera se Chain Reaction all of which showed target bands,while the control group a nd negative control group did not show target bands.Randomly selected six m ice' double trigeminal ganglia samples at each time point for DNA extraction,after Polymerase Chain Reaction the right simples showed target bands,while t he left simple show target bands did not show target bands in fourteenth day after infection.6)The survival rate of mice was 75 percent,which means sample sampling ca n be satisfiedConclusions:1)The model of mouse HSK can be established effectively by c orneal ring drill;2)The mouse model established by this method is uniform a nd standardized,stable and effective,and can be applied to other model animal s;3)The mouse model established by this method has a high sample retention rate and good sample quality,which can be applied in other studies of HSK;4)This method can also be applied to establishing animal models of bacterial or fungal keratitis.