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

The essential requisites for the vaccine verification protocol are promptitude and accuracy. In this respect, the Evans blue assay result showed that BBB structure and function of ICR mice could be more vulnerable than those of B6 mice (Fig. 1) . And 4 h after LPS(S) injection into ICR mice, the ZO-1 mRNA and protein levels were thoroughly increased than 0 h PI, being compared with any other conditions (B6 mice, LPS(E) and 2 h, 24 h PI). qPCR method might be also suitable than WB method in terms of time requirement. Consequentially, the vaccine safety verification protocol for BBB and brain could be established by selecting optimum conditions and its results in Fig. 4 . Zonula occludens-1 (ZO-1) and occludin disruption and recovery processes in C57BL/6 and ICR mice. When lipopolysaccharide binds to a receptor (such as toll-like receptor 4), the nuclear factor kappa B (NF-B) pathway or other inflammatory pathways are activated and release various cytokines. ZO-1 are mislocated with occludin by cytokines, and ZO-1 or occludin proteins are degraded by proteasomes, followed by a recovery activity via protein synthesis from the mRNAs. However, this process might be different in B6 (A) or ICR (B) mice. Considering the low permeability of Evans blue dye and the low expressions of ZO-1 and occludin mRNA and protein, there may be a protective or inhibitory factor suppressing the inflammation activity or degradation of ZO-1 or occludin proteins. This process difference is thought to be related to the more than 24 h required for the protein restoration process of the proteins in brain epithelial cells in B6 mice (A). In ICR mice (B), however, disruption and restore pathways are rather more common than those in B6 mice. The arrow streams indicating translation activity show the different time-dependent patterns in ZO-1 and occludin protein synthesis from mRNA in brain endothelial cells of ICR mice (C).