Journal: bioRxiv

Article Title: Light-driven repair: Photobiomodulation restores blood–brain barrier function following hypoxic injury

doi: 10.64898/2026.02.15.706027

Figure Lengend Snippet: A) Experimental workflow illustrating the transwell-based BBB model, hypoxic exposure (6 h, 1% O₂), PBM treatment schedule, and downstream functional and molecular analyses (created with BioRender). (B–E) TEER expressed as relative change (%) from baseline. (B) Normoxic controls at 48 h. (C)TEER immediately following hypoxia. (D)TEER at 24 h post-hypoxia. (E)TEER at 48 h post-hypoxia. PBM significantly restored endothelial barrier resistance under hypoxic conditions. Statistical comparisons were performed using multiple unpaired t-tests with Holm–Šídák correction; N=4 independent biological replicates; *p<0.05. (F)ZO-1 mRNA expression in HBMECs at 48 h, normalised to RPL13A. (G)Quantification of ZO-1 protein levels relative to control, measured as ZO-1-positive area normalised to Hoechst nuclear area. (H)Representative ICC images showing ZO-1 (magenta) and nuclei (Hoechst, blue) in normoxic and hypoxic HBMECs with and without PBM. For mRNA and protein analyses, statistical significance was determined using one-way ANOVA with Šídák’s post hoc correction; N=3–4 independent biological replicates. Data are presented as mean±SEM.

Article Snippet: The cell lines used were purchased from Innoprot: human brain microvascular endothelial cells (HBMECs) (Innoprot; P10361-IM), human astrocytes (HAs) (Innoprot; P10251-IM), and human brain vascular pericytes (HVPCs) (Innoprot; P10363-IM).

Techniques: Functional Assay, Expressing, Control

Journal: bioRxiv

Article Title: Light-driven repair: Photobiomodulation restores blood–brain barrier function following hypoxic injury

doi: 10.64898/2026.02.15.706027

Figure Lengend Snippet: (A)vWF mRNA expression in HBMECs 48 h after normoxia or hypoxia (6 h, 1% O₂), with or without PBM treatment, normalised to RPL13A. (B)Representative ICC images showing vWF (yellow) and nuclei (Hoechst, blue) in normoxic and hypoxic endothelial cells ±PBM. (C)Quantification of vWF protein levels, expressed as mean nuclear-normalised fluorescence intensity relative to normoxia −PBM controls. (D)Validation of vWF knockdown efficiency in siRNA-transfected endothelial cells, shown as relative vWF mRNA expression normalised to RPL13A. (E-F) Relative TEER changes (%) in endothelial monocultures and BBB tri-cultures at 24 h and 48 h under normoxic (E) and hypoxic (F) conditions following vWF silencing. Statistical comparisons for mRNA and protein expression were performed using one-way ANOVA with Šidák’s post hoc test (N=3–4 biological replicates). siRNA validation was analysed using an unpaired two-tailed t-test (N=8 biological replicates). TEER data were analysed using multiple unpaired t-tests with Holm–Šidák correction (N=4 biological replicates). Data are presented as mean±SEM.

Article Snippet: The cell lines used were purchased from Innoprot: human brain microvascular endothelial cells (HBMECs) (Innoprot; P10361-IM), human astrocytes (HAs) (Innoprot; P10251-IM), and human brain vascular pericytes (HVPCs) (Innoprot; P10363-IM).

Techniques: Expressing, Fluorescence, Biomarker Discovery, Knockdown, Transfection, Two Tailed Test

Journal: bioRxiv

Article Title: Urban PM 2.5 at Realistic Environmental Concentrations Impairs Blood–Brain Barrier Integrity and Enhances LOX-1 Expression in Human Brain Endothelial Cells

doi: 10.64898/2026.01.29.702473

Figure Lengend Snippet: A . Physiological rationale: Ambient PM2.5 exposure is epidemiologically linked to increased ischemic stroke risk. This in vitro model simulates the real-life scenario of pre-existing PM2.5 exposure followed by ischemic stroke and subsequent reperfusion. B . Primary adult male HBMEC were exposed to 5, 15, 75, or 300 μg/m 3 PM 2.5 for 48h in total. To compare with the effects of physiological ischemic-like injury, some plates were exposed to hypoxia (1% O 2 ) and glucose deprived media (HGD) for 3h after the initial 24h incubation. Following HGD or normoxia, cells were reperfused with nutrient-enriched media and incubated with PM 2.5 at normoxic (21% O 2 ) conditions as a reference for resolution of ischemia. Barrier integrity, cell viability, reactive oxygen species (ROS), inflammation and LOX-1 expression was assessed. Figure created in BioRender.

Article Snippet: Primary adult male HBMEC were purchased from Innoprot (Spain, Catalog number: P10361, Lot number: 111224CS).

Techniques: In Vitro, Incubation, Expressing

Journal: bioRxiv

Article Title: Urban PM 2.5 at Realistic Environmental Concentrations Impairs Blood–Brain Barrier Integrity and Enhances LOX-1 Expression in Human Brain Endothelial Cells

doi: 10.64898/2026.01.29.702473

Figure Lengend Snippet: Adult male HBMEC were exposed to vehicle or PM 2.5 (5, 15, 75, or 300 μg/m 3 ) for 24h and incubated for 3h in normoxia- or hypoxia and glucose deprivation (HGD) followed by 24h reperfusion. A . Live cell count (CyQUANT nuclear stain) decreased when exposed to ≥75 μg/m 3 PM 2.5 compared to vehicle. HGD treatment reduced live cell count compared to normoxia but did not differ between particle treated groups. B . Reactive oxygen species (ROS) signal (DCHF-DA) normalized to live cell count. Relative ROS levels increased dose-dependently with PM 2.5 concentration, with significant increase observed at PM 2.5 ≥75 μg/m 3 , in comparison to normoxia vehicle. ROS levels were uniformly elevated following HGD across all doses in comparison to normoxia vehicle and significantly higher than untreated HBMEC. (n=12 technical replicates for vehicle and 5, n=8 technical replicates for 15, 75 and 300) C . Analysis of crystal violet-stained HBMEC shows a longer maximum cellular length when treated with ≥15 μg/m 3 PM 2.5 . (n=21-37 individual cells) D . Representative images of crystal violet-stained HBMEC visualizing a differentiated morphology in cells treated with higher PM 2.5 concentration, where cells appear more elongated and expanding towards neighbouring cells. Data presented as mean ± SD. Statistical significance assessed through Kruskal-Wallis test within treatment groups (Normoxia/HGD) and Mann-Whitney test between groups with different treatment (300 normoxia/vehicle HGD). *p<0.05. ***p<0.001. ****p<0.0001.

Article Snippet: Primary adult male HBMEC were purchased from Innoprot (Spain, Catalog number: P10361, Lot number: 111224CS).

Techniques: Incubation, Cell Characterization, CyQUANT Assay, Staining, Concentration Assay, Comparison, MANN-WHITNEY

Journal: bioRxiv

Article Title: Urban PM 2.5 at Realistic Environmental Concentrations Impairs Blood–Brain Barrier Integrity and Enhances LOX-1 Expression in Human Brain Endothelial Cells

doi: 10.64898/2026.01.29.702473

Figure Lengend Snippet: Western Blot assessment of adult male HBMEC exposed to vehicle, 5, 15, 75, or 300 μg/m 3 PM 2.5 during normoxia or ischemic-like injury with hypoxia, glucose deprivation and reperfusion (HGD). A . Representative Western Blot image of IL-6 and β-actin band migration. B . Signal quantification of 25kDa IL-6 shows no difference between PM 2.5 exposure or HGD treated group. C . Signal quantification of 17kDa IL-6 shows dose-dependency with higher IL-6 expression from higher PM 2.5 exposure, with significant increase ≥75 μg/m 3 and from HGD treatment compared to vehicle. D . Representative Western Blot image of LOX-1 and β-actin. E . Signal quantification of LOX-1 displays a dose-dependent increase in LOX-1 with exposure to ≥15 μg/m 3 PM 2.5 or HGD. (n=4-7 technical replicates). Data presented as mean +-SD. Statistical significance assessed by Kruskal-Wallis test. *p<0.05, **p<0.01.

Article Snippet: Primary adult male HBMEC were purchased from Innoprot (Spain, Catalog number: P10361, Lot number: 111224CS).

Techniques: Western Blot, Migration, Expressing