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. 2022 Jul 21;25(8):104813. doi: 10.1016/j.isci.2022.104813

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© 2022 The Authors

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

Brain-Chip response to TNF-α perfused through the vascular channel

(A–C) Representative immunofluorescent staining for microglia (CD68, red), neurons (MAP2, green), astrocytes (GFAP, magenta), nuclei (DAPI, blue), and pericytes (NG2, red) in TNF-α-treated of control chips (bar, 100 nm).

(D) Quantification of the CD68-positive events/field of view in n = 4 randomly selected different areas/chip, n = 3 Brain-Chips; data are represented as mean ± SEM, ∗∗∗∗p < 0.0001 compared to the untreated control group, statistical analysis by Student’s t-test.

(E) (Left) Quantification of the number of GFAP-positive events/field of view in n = 4 randomly selected different areas/chip, n = 3 Brain-Chips; data are represented as mean ± SEM, ∗∗∗p < 0.001 compared to the untreated control group; statistical analysis with Student’s t-test. (Right) Quantification of GFAP fluorescent intensity in n = 3 randomly selected different areas/chip, n = 3 Brain-Chips, ∗∗∗∗p < 0.0001 compared to the untreated control group, statistical analysis with Student’s t-test.

(F) Quantification of fluorescent intensity of NG2 in, n = 4, randomly selected different areas/chip, n = 3 Brain-Chips; data are represented as mean ± SEM, ∗∗∗∗p < 0.0001 compared to the untreated control group; statistical analysis with Student’s t-test.

(G) The nuclei counts based on DAPI staining were similar between the control and treated groups (n = 4 Brain-Chips, data are represented as mean ± SEM, NS: Not Significant compared to the untreated control group). Statistical analysis with Student’s t-test.

(H–J) Secreted levels of the proinflammatory cytokines IL-6, IL-1β, and IFNγ, in the brain channel of control or TNF-α-treated Brain-Chips. n = three to four independent chips, data are represented as mean ± SEM, ∗p < 0.05, P∗∗<0.01, ∗∗∗∗p < 0.0001, statistical analysis with Student’s t-test.

(K) Quantification of the number of MAP2 events/field of view in n = 4 randomly selected different areas/chip, n = 3 Brain-Chips, data are expressed as mean ± SEM, NS: Not Significant compared to the untreated control group, statistical analysis with Student’s t-test.

(L) Levels of secreted glutamate in the brain channel on culture day 7 (n = 6 independent chips; data are represented as mean ± SEM, NS: Not Significant compared to the untreated control group). Statistical analysis with Student’s t-test.

(M) Immunofluorescent staining of cell nuclei (DAPI, blue), intercellular adhesion molecule 1 (ICAM-1, red), tight junction protein 1 (ZO-1, green), and a merged image of all three markers (bar, 100 nm).

(N) Quantitative barrier function analysis via apparent permeability to 3kDa fluorescent dextran, upon 48 h of exposure to TNF-α via the vascular channel. n = 3–4 independent chips, NS = Not Significant, ∗p < 0.05, control group compared to TNF-α-treated group after 24 and 48 h of treatment; data are represented as mean ± SEM, statistical analysis by two-way ANOVA with Tukey’s multiple comparisons test.