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

Figure 2.

Figure 2

Effect of hyperglycemic conditions on the brain microvasculature and neurons determined using the NV chip. A) Schematics of the hyperglycemic NV chip. Glucose concentrations under normal and hyperglycemic conditions are 5.5 and 15.5 mm, respectively. B) Immunofluorescence images of hBMECs on the NV chip under normal (NG) and high‐glucose (HG) conditions. VE‐cadherin (green) and F‐actin (red) were stained. The nucleus was counterstained with DAPI (blue). Scale bar = 100 µm. C) Blood vessel diameter calculated on the NV chip under NG and HG (n = 12 for NG, n = 20 for HG; ****, p < 0.0001). D) VE‐cadherin, CD31, ZO‐1, and SIRT1 expression in hBMECs on the NV chip under NG and HG conditions. Loading control is GAPDH. E) Ratio of normalized SIRT1 expression in hBMECs on the NV chip under NG and HG conditions. SIRT1 expression determined via western blotting was normalized to GAPDH, and the ratio was calculated based on NG conditions (n = 4; ****, p < 0.0001). F) SIRT1 immunofluorescence (green) of hBMECs on the NV chip. The nucleus was counterstained with DAPI (blue). Scale bar = 100 µm. G) Permeability of brain microvasculature on the NV chip under NG and HG conditions. Permeability to 4.4 kDa TRITC‐dextran within 5 min. Images were captured every 1 min. Scale bar = 500 µm. H) Accumulation and transport of amyloid‐beta (Aß, red) under NG and HG conditions. The nucleus was counterstained with DAPI (blue). Scale bar = 200 µm. I) Immunofluorescence of ReN cells on the NV chip with (w/) (+) or without (w/o) (−) Aß treatment under NG and HG conditions. Aß (red), pTau (green), and MAP2 (yellow) were stained. The nucleus was counterstained with DAPI (blue). Scale bar = 100 µm. J) Protein expression in ReN cells cultured using the Transwell method under NG and HG conditions w/ (+) or w/o (−) Aß treatment. ß‐actin was used as the loading control. K) Viability of ReN cells determined using the Transwell method w/ (+) or w/o (−) Aß treatment under NG and HG conditions (n = 4; *, p < 0.05; ****, p < 0.0001). L) SIRT1 immunofluorescence staining (green) of ReN cells on the NV chip under NG and HG conditions. Tuj1 (red) and nucleus (blue, DAPI) were co‐stained. Scale bar = 100 µm. M–P) Expression of total (M,N), cytoplasmic and nuclear (O,P) SIRT1 in ReN cells under NG and HG conditions. GAPDH was used as the loading control for total proteins, and ß‐tubulin and lamin A/C were used as loading controls for cytoplasmic and nuclear proteins, respectively. The ratio of normalized values (N,P) of SIRT1 expression in ReN cells under NG and HG conditions. SIRT1 expression determined using western blotting was normalized to GAPDH (N), ß‐tubulin, and lamin A/C (P), and the ratio was calculated based on NG (n = 3; **, p < 0.01; n.s., no significance). Uncropped blotting images in (D), (J), (M), and (O) with the exact protein size represented in Figure S11, Supporting Information. Box and whiskers plots in (C) 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 (E), (K), (N), and (P) represents the mean ± standard deviation (SD) with bars and error bars showing all points. Significance in (C), (E), (N), and (P) was calculated using an unpaired t‐test. Significance in (K) was calculated using an ordinary one‐way ANOVA Tukey's multiple comparisons test.