Establishment of minimal positive-control conditions to ensure brain safety during rapid development of emergency vaccines

Baek, Hyekyung; Kim, Kwang Ho; Park, Min Young; Kim, Kyeongryun; Ko, Bokyeong; Seo, Hyung Seok; Kim, Byoung Soo; Hahn, Tae-Wook; Yi, Sun Shin

Occludin is transmembrane protein, and ZO-1 is combined with the occludin C-terminal under the cell membrane in cytoplasm [4, 5, 8] . Under inflammatory conditions induced by LPS, it has been reported that TJs including occludin and ZO-1 are degraded along various pathways [21, 25] . In the present study, the qPCR results demonstrated that occludin and ZO-1 mRNA levels dynamically changed in accordance with exposure time after LPS injection, especially in ICR mice. The EB assay results showed that the BBB permeability was sufficiently damaged at 4 h PI. While developing the positive-control protocol we focused on the early PI period because we thought the less time spent determining the BBB damage point, the more the effective the vaccine safety verification protocol. The ZO-1 mRNA level increased until 4 h PI and was decreased at 24 h PI, recovering to the same level as that at 0 h PI in ICR mice brain. Meanwhile, occludin mRNA level was only significantly increased at 24 h PI. In common with the mRNA results, ZO-1 and occludin protein expressions significantly increased over time in the ICR mice but not in the B6 mice. Interestingly, while ZO-1 mRNA level was downregulated at 24 h PI, ZO-1 protein expression progressively increased. It is thought that this difference is related to the time required to synthesize proteins from mRNAs during the recovery process after TJ impairment. Similar to the ZO-1 results, occludin protein expression also showed an upward tendency at 24 h PI.