F. Effect of commercial contact lens solution on the AK development in mice
V. CONCLUSION
Acanthamoeba castellanii causes acanthamoebic keratitis (AK). To decipher the exact pathogenesis of AK and development of therapeutic drugs, in vivo model needs to be established. In this study, BALB/c mouse cornea was scratched using a syringe needle and ophthalmic surgical blade under anesthesia. A. castellanii (trophozoites mixed equally with cysts) were serially diluted from 1 × 106 to 0.3 × 105, placed on a 2-mm contact lens, and put into the mouse eye. After the eyelid was sutured, the occurrence of keratitis symptoms was observed from day 1 to 7 post-inoculation. In addition, to confirm the AK development, the genomic DNA was extracted from the infected mouse ocular tissue. Amplification of acanthamoebic 18S-rRNA gene by PCR using P-FLA primers was performed. In conclusion, the experimental mouse AK, characterized by typical hazy blurring and melting of mouse cornea, was developed from day 1 to 7 post-inoculation with 0.5 × 105 amoeba. PCR products from DNA samples of mouse eyeballs on day 1 to 7 were amplified to be same as 18S-rRNA gene of A. castellanii used a positive control. Subsequently, the amplified bands were identified as 18S-rDNA of A. castellanii, which showed 91–97 % homology, by BLAST search. To confirm the effect of the commercial lens solution, 0.5 × 105 amoebas (trophozoites mixed equally with cysts) were treated with solutions A or B for 1, 6, 12 or 24 h, and inoculated into mouse eye (previously established AK mouse model). Thereafter, AK development was observed for 7 days post-inoculation. AK
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occurrence by A. castellanii treated with lens solution A or B for 1 or 6 h was not grossly observed on day 1 and 2, but the infection progressed gradually with a typical circulatory edema and keratitis on day 3, 5 and 7. Upon pre-treatments for 12 or 24 h, the development of AK was not observed for 1, 2 or 3 days, but progressed to corneal infection at 5 and 7 days. Finally, commercial contact lens solutions did not show the protective effect against AK pathogenesis, although they delayed the occurrence of AK.
In conclusion, it is suggested that the present AK mouse model is an important in vivo model for future studies.
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