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. 2022 Sep 8;9(34):2201882. doi: 10.1002/advs.202201882

Figure 3.

Figure 3

Effect of glucose re‐stabilization for the functional and protein‐level recovery of the brain microvasculature and neurons on the NV chip. A) Immunofluorescence images of hBMECs on the NV chip under normal (NG), high (HG), and recovered glucose (HG→NG) conditions w/ (+TNFα) or w/o TNFα (−TNFα) treatment. ICAM‐1 (green) and F‐actin (red) were stained, and the nucleus was counterstained with DAPI (blue). Scale bar = 100 µm. B) Protein expression in hBMECs on the NV chip under NG, HG, and HG→NG conditions w/ or w/o TNFα (± TNFα) treatment. GAPDH was used as a loading control. C) Ratio of normalized SIRT1 expression in hBMECs on the NV chip under NG, HG, and HG→NG conditions w/ or w/o TNFα (± TNFα) treatment. SIRT1 expression determined via western blotting was normalized to GAPDH, and the ratio was calculated based on NG (n = 5 in −TNFα, n = 3 in +TNFα; **, p < 0.01). D) Blood vessel diameter calculated on the NV chip depending on NG, HG, and HG→NG conditions w/ or w/o TNFα (± TNFα) treatment (n = 8, **, p < 0.01; ****, p < 0.0001). E) Permeability of hBMECs to 4.4 kDa TRITC‐dextran determined using the Transwell assay under NG, HG, and HG→NG conditions with or without TNFα (± TNFα) treatment (n ≥ 12, *, p < 0.05; ***, p < 0.001; ****, p < 0.0001; n.s.; no significance). F) Transportation of Aß (red) in each condition. The nucleus was counterstained using DAPI (blue). Scale bar = 200 µm. G) Immunofluorescence staining of ReN cells on the NV chip under NG, HG, and HG→NG conditions w/ or w/o TNFα (± TNFα) treatment. Aß (red), pTau (green) and MAP2 (yellow) were stained, and the nucleus was counterstained with DAPI (blue). Scale bar = 100 µm. H) Protein expression in ReN cells cultured using the Transwell assay under NG, HG, and HG→NG conditions w/ or w/o TNFα (± TNFα) treatment. ß‐actin was used as the loading control. I) Expression of cytoplasmic and nuclear SIRT1 in ReN cells under NG, HG, and HG→NG conditions. ß‐tubulin and Lamin A/C were used as loading controls for cytoplasmic and nuclear proteins, respectively. J) Ratio of normalized SIRT1 expression in ReN cells under NG, HG, and HG→NG conditions w/ or w/o TNFα (± TNFα) treatment. Cytoplasmic and nuclear SIRT1 expressions determined using western blotting were normalized to ß‐tubulin and Lamin A/C, respectively, and the ratio was calculated based on NG (n = 3; ****, p < 0.0001; n.s., no significance). Uncropped blotting images in (B), (H), and (I) with the exact protein size represented in Figure S12, Supporting Information. Box and whiskers plots in (D) and (E) represent the median (horizontal bars), 25 to 75 percentiles (box edges), and minimum to maximum values (whiskers) including all points. The scatter dot plot in (C) and (J) represents the mean ± SD with bars and error bars showing all points. Significance in (C), (D), (E), and (J) was calculated using an ordinary one‐way ANOVA Tukey's multiple comparisons test.