Figure 3. Effect of anti-CLEC5A mAb on dynamic changes in the BBB during JEV infection.

(A) Stat1^−/− mice were injected intravenously with ^99mTc-DTPA to enable brain SPECT/CT imaging, before (D0) or after JEV challenge (100 pfu/mouse), in the presence of anti-CLECA5 mAb (3E3G4) or an isotype matched control. Antibody (150 µg/mouse) was administrated intraperitoneally on days 0, 2, 4, 6 and 8 to determine any protection effects in reduction to BBB permeability. Image datasets were reconstructed using the ordered-subset expectation maximization algorithm with standard-mode parameters. (B) The extent of BBB breakdown was calculated as the ratio of the mean counts/pixel in the region of the brain with the greatest accumulation of radiotracer divided by the mean counts/pixel in the neck muscle (b/m ratio). (C) Changes in BBB permeability at day 7 post JEV infection (100 pfu/mouse) were determined by Evans Blue assay. (D) Brains of JEV-infected mice (n = 5) with isotype control or anti-CLEC5A Ab treatment were harvested at day 5 and day 9 post infection to analyze the expression of transcripts encoding tight junction proteins and adhesion molecules by quantitative real-time PCR. For tight junction proteins, y-axis units represent the expression level of each target gene relative to mock control after internal control normalization; the expression level of adhesion molecules is displayed as fold increase relative to mock control. Two-tailed Student's t-tests were performed. (E) H&E staining of murine cerebral cortex at day 5 after JEV infection revealed the inhibitory effect of anti-CLEC5A mAb on perivascular cuffing. Scale bars, 200 µm. Five random fields of views in medium power field (original magnification (OM)×200) were photographed, and the numbers of foci and vessel cross sections in each sample were counted, summed up and represented as mean ± s.e.m. (under each picture) of four independent experiments.