BALB/c小鼠单纯疱疹病毒性角膜炎复发感染模型的建立及鉴定

目的探索单纯疱疹病毒性角膜炎(HSK)复发模型的建立及鉴定方法。方法选取12只BALB/c健康小鼠,采用随机数字表法按照紫外灯照射时间不同分为紫外灯照射3 min组、2 min45 s组和2 min30 s组,每组4只,观察角膜损伤情况以确定最适紫外灯照射参数。另取72只BALB/c健康小鼠,采用随机数字表法将其分为空白对照组、模型组和复发组,每组24只。所有小鼠均用手术刀片在右眼角膜划开#字,空白对照组滴入5μl生理盐水,模型组和复发组均滴入Ⅰ型单纯疱疹病毒(HSV-1)悬液,感染后不予腹腔内注射HSV-1异种血清抗体。初次感染病毒后5周,对复发组采取紫外灯照射法诱导小鼠模型眼疾病复发;评价眼表病变症状评分和角膜擦拭液相对应的非洲绿猴肾(Vero)细胞培养后细胞的形态学改变。结果照射时长2 min45 s为紫外灯照射最适条件。模型组和复发组小鼠在初次感染后1周内出现HSK表现,1周后症状逐渐消失。诱导复发前裂隙灯显微镜检查模型组和复发组小鼠HSK均未自发性复发,经紫外灯照射后复发组小鼠1周内复发,出现HSK表现。诱导复发后1 d,空白对照组、模型组和复发组眼表病变症状评分分别为0.333±0.471、1.500±0.764和2.667±0.943;3 d时分别为0.000±0.000、0.833±0.373和5.167±2.267;7 d时分别为0.167±0.373、1.000±0.577和3.000±1.155;诱导复发后3 d,复发组眼表病变症状评分均高于空白对照组和模型组,差异均有统计学意义(均P<0.05)。Vero细胞培养小鼠角膜擦拭液后发生漂浮、聚拢等形态学改变(CPE病变),复发组小鼠角膜擦拭液细胞培养CPE病变阳性率为71%(17/24),明显高于模型组的8%(2/24),差异有统计学意义(P<0.01)。综合2个指标计算建模复发成功率可达71%。结论紫外灯照射可成功诱导未注射中和血清抗体的HSK小鼠复发。

Establishment and evaluation of a recurrent infection model of herpes simplex keratitis in BALB/c mice

Objective To explore the establishment and evaluation method of recurrent infection model of herpes simplex keratitis(HSK).Methods To determine the optimal duration of the ultraviolet light irradiation,12 healthy BALB/c mice were randomized into 3 minutes group,2 minutes and 45 seconds group,and 2 minutes and 30 seconds group based on the duration of the ultraviolet light irradiation according to random number table method,with 4 mice in each group.Another 72 healthy BALB/c mice were randomized into blank control group,model group and recurrence group according to random number table method,with 24 mice in each group.The 72 mice were scratched a#symbol on their right corneas with scalpel.Then the eyes in the blank control group were treated with 5μl of normal saline solution,while the eyes in the model group and recurrence group were treated with 5μl of herpes simplex virus I(HSV-1)suspension.All the 72 mice were not intraperitoneally injected with HSV-1 xenogeneic serum antibody.Five weeks after the initial infection,the mice in the recurrence group were irradiated at ultraviolet B-302 nm for the optimal duration.The feasibility of establishing HSK recurrence model induced by this method was evaluated by the score of ocular surface symptoms,and the cytopathic effect(CPE)lesion analysis of Vero cells cultured in corneal wiping fluid from the mice in the model group and recurrence group.The use and care of the animals complied with the Statement of the Association for Research in Vision and Ophthalmology(ARVO).The study protocol was approved by the Ethics Committee of Shanghai University of Traditional Chinese Medicine(No.PZSHUTCM190222039).Results The optimal duration of the ultraviolet light irradiation was 2 minutes and 45 seconds.Within one week,HSK syndromes appeared in the mice injected with HSV-1 virus in the model group and recurrence group,and then gradually disappeared after one week.Before inducing recurrence,slit lamp examination was performed,and there was no spontaneous recurrence of HSK in the model group and recurrence group.The mice in the recurrence group relapsed within one week after ultraviolet light irradiation.The symptom scores of ocular surface lesions in the blank control group,model group and recurrence group were 0.333±0.471,1.500±0.764 and 2.667±0.943 at one day after ultraviolet light irradiation,0.000±0.000,0.833±0.373 and 5.167±2.267 at three days after ultraviolet light irradiation,and 0.167±0.373,1.000±0.577 and 3.000±1.155 at seven days after ultraviolet light irradiation,respectively.The symptom score of ocular surface lesions was higher in the recurrence group than the blank group and the model group at three days after ultraviolet light irradiation,and the differences were statistically significant(both at P<0.05).Morphological changes such as floating and gathering were found in the Vero cells after being cultured in corneal wiping fluid.The positive rate of CPE lesion was 71%(17/24)in the recurrence group,which was significantly higher than 8%(2/24)in the model group,and the difference was statistically significant(P<0.01).Combining the two indicators,the success rate of recurrence in established models could reach 71%.Conclusions Ultraviolet light irradiation can successfully induce the recurrence of viral keratitis in HSK mice without injection of neutralizing serum antibody.