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 5 μl of normal saline solution, while the eyes in the model group and recurrence group were treated with 5 μl 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.

Herpes simplex keratitis; Recurrence model; BALB/c mice; Ultraviolet light irradiation; Non-serum antibody injection