Fig 2: Representative images of the clinical MC group and control group with (A) HE staining (scale bar = 1.25 mm), (B) immunofluorescence of GFAP and Iba-1 (scale bar = 200 µm), and (C) immunofluorescence of Tuj-1 and NeuN (scale bar = 200 µm); images in the lower right corner are magnified from the boxed area of the corresponding images, with target protein indicated by coloured arrows. (d)–(f) show quantitative measurement of GFAP/Iba-1/NeuN-positive cells (means ± SD of a single slice, * indicates P < 0.0.5). MCs: Modic changes.

Fig 3: A375 cell-derived exosomes caused BBB damage and glial activation. (A) Side views of the BBB chip 24 h later following exposure of A375 cell-derived exosomes (green) to the vascular side (n = 3). (B) Representative confocal images of glial cells 24 h later following exposure of A375 cell-derived exosomes labeled by PKH67 dye to the vascular side (n = 3). (C) BBB permeability assay of 10 kDa FITC-dextran following exposure of A375 cell-derived exosomes to the vascular side (n = 4). Data are presented as mean ± SEM, and are analyzed using unpaired Student’s t-test (*: p < 0.05). (D) Representative confocal images of brain endothelial cells (VE-cadherin) 72 h later following treatment of A375 cell-derived exosomes (n = 3). The areas indicated by the white boxes were enlarged on the right. (E) Western blotting showing the protein level of VE-cadherin and ZO-1 in brain endothelial cells 72 h later following treatment of A375 cell-derived exosomes (n = 3). GAPDH was used as an internal control. (F,G) Representative confocal images of astrocytes (GFAP; F) and microglia (IBA1; G) 72 h later following treatment of A375 cell-derived exosomes (n = 3). (H) Bar graph showing IL-6 concentration in culture supernatant of vascular channel 72 h later following exposure of A375 or A549 cell-derived exosomes to the vascular side detected by ELISA kit (n = 3). (I) Bar graph showing IL-6 concentration in culture supernatant of glial channel 72 h later following exposure of A375 or A549 cell-derived exosomes to the vascular side detected by Elisa kit (n = 3). (H,I) Data are presented as mean ± SEM, and are analyzed using a one-way ANOVA with Bonferroni post-test (**: p < 0.01; ***: p < 0.001). (J) Representative confocal images of brain endothelial cells and glial cells stained with PI dye 72 h later following treatment of A375 cell-derived exosomes. Cells positive for PI were indicated by white arrowheads (n = 4). (K) Bar graph showing the percentage of PI+ cells for each group based on (J) (n = 4). Data are presented as mean ± SEM, and are analyzed using unpaired Student’s t-test (***: p < 0.001).

Fig 4: Establishing a human BBB chip. (A) A schematic description of human brain and blood–brain barrier (BBB). (B) A real image of a BBB chip. Red and blue inks were poured into two channels. (C) Schematic description of the BBB chip by co-culturing of human brain microvascular endothelial cells, astrocytes and microglial cells on the porous PET membrane. (D) Representative confocal images of brain endothelial cells immunostained for VE-cadherin, ZO-1 and Claudin-5 on chip device under flow culture condition for 3 days (n = 3). (E) A 3D confocal image showed the BBB barrier interface on chip device. The BBB barrier interface was formed by co-culture of human brain microvascular endothelial cells (hCMEC/D3 cells; Clauding-5 staining), human astrocytes (HA cells; GFAP staining) and microglial cells (HMC3 cells, IBA1 staining) on the porous membrane under fluid flow conditions (n = 3). (F) Side views of the BBB interface identified by Claudin-5 in human brain microvascular endothelial cells, GFAP in astrocytes and IBA1 in microglial cells (n = 3). The area indicated by the white box was enlarged on the right. The white dotted line indicated the porous membrane.

Fig 5: Representative images of the SD rat MC group and control group with immunofluorescence of GFAP/iba-1 (A and C, scale bar = 100 µm) and Tuj-1/NeuN (B and D, scale bar = 100 µm); images in the lower right corner are magnified from the boxed area of the corresponding images, and white arrows indicate the GFAP/Iba-1/NeuN-positive neurons. (E) and (F) show the location of GFAP/Iba-1/NeuN-positive neurons in the IVD (cells with positive immunofluorescence were marked as bright yellow dots (scale bar = 1 mm)); (g)–(i) show quantitative measurement of GFAP/Iba-1/NeuN-positive cells (means ± SD of a single motion segment, * indicates P < 0.0.5). MCs: Modic changes.