Fig 2: Characterization of the barrier in the Brain-Chip(A) Top Image: Immunofluorescence staining of the vascular channel stained for the tight junction marker ZO-1 (green) (bar, 1 mm). Bottom Images: Immunofluorescence micrographs of the human brain endothelium cultured on-chip for 7 days labeled with occludin (green), PECAM1 (magenta), and DAPI (blue) (bar, 100 µm).(B) Quantitative barrier function analysis by the apparent permeability to 3kDa fluorescent dextran, in two independent iPSC donor lines on culture days 5, 6, and 7 (n = four to six independent chips). NS: not significant. Data are represented as mean ± SEM, statistical analysis by Student’s t-test.(C) The apparent permeability of different size dextran molecules (3-70 kDa) across Brain-Chips correlated with previously reported in vivo rodent brain uptake data. (n = three to five independent chips). Data are represented as mean ± SEM).(D) Left: Exploded view of the chip. Interaction of primary human astrocyte end-feet-like processes (GFAP, red) with endothelial-like cells (ZO-1, yellow), MAP2 (green) (bar, 200um). Right: Representative scanning electron microscopy (SEM) image showing astrocytic endfoot passing through 7 µm pores into the vascular channel (bar, 30um). White arrows show the astrocytic endfoot.

Fig 3: Representative Images at Each Critical Step of the Dot Blot AssayA membrane was dotted with neuronal cell lysates (1 µg protein/dot) and imaged following staining with Revert® 700 Total protein stain (shown red) (A), after removal of the Revert® stain (B), and after incubation with primary and secondary antibodies (shown green) (C). Note the intensity of the dots following staining with Revert® are relatively uniform indicating that samples were accurately applied to the membrane. Primary antibody used was mouse-anti Map2 (Invitrogen, MA5-12826) at 1:1,000 dilution. Secondary antibody was goat-anti-mouse IRDye® 800 at a 1:10,000 dilution (LiCor). The quantification of these membranes and an example workflow is demonstrated in Table 2.

Fig 4: Effect of naringenin (Nar) on senescence of mouse neural stem cells (mNSCs). (A) p16ink4a mRNA expression was measured by qRT-PCR in 23M-NSCs with and without treatment of 6.8 µg/mL Nar for 48 h. (B) Cell cycle phase distributions of 23M-NSCs treated with Nar for 48 h and control cells. (C) The relative telomere length of 23M-NSCs increased significantly with Nar treatment. (D) Immunofluorescence Ki67 staining of mNSCs treated with Nar for 48 h, with DAPI nuclear labeling. (E) Quantification of (D). (F) Representative fields of MAP2 and GFAP immunofluorescence staining of cultured mNSCs after control and Nar treatment. (G) Quantification of (F). Data are presented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.0001 compared with untreated cells.

Fig 5: Changes in neuronal markers: (A,C) representative Western blot bands for each group are presented; (B) graphs showing quantification of BDNF expression in the sham and MCAO groups compared, respectively, in each group with an independent t-test. There was no significant change of BDNF expression after MIF injection in the sham group (p = 0.503); the expression of BDNF in the MCAO+MIF group was significantly higher than in the MCAO+veh group (* p < 0.042); (D) the MAP2 expression of the MCAO+MIF group was significantly increased compared to that of the MCAO+MIF group (* p < 0.0001); the expression level of BDNF and MAP2 in the sham and MCAO groups were analyzed using a t-test, respectively. Mean±SD values are presented from 12 independent experiments. MCAO: middle cerebral artery occlusion; MIF: macrophage migration inhibitory factor.