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

doi: 10.64898/2026.02.15.706027

Figure Lengend Snippet:

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: Staining

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-C) Quantification of ROS levels measured by luminescence assay in endothelial cells (A), astrocytes (B), and pericytes (C) following hypoxia (6 h, 1% O₂) with or without PBM treatment. PBM significantly reduced hypoxia-induced ROS accumulation in astrocytes and pericytes, with a modest effect in endothelial cells. (D-F) Representative ICC images showing HIF-1α (cyan) and nuclei (Hoechst, blue) in endothelial cells (D), astrocytes (E), and pericytes (F) under normoxia (–PBM), hypoxia (–PBM), and hypoxia with PBM (+PBM). Insets display higher-magnification views illustrating nuclear localisation of HIF-1α. The mRNA expression of HIF-1α was normalised to RPL13A. Nuclear HIF-1α protein levels were quantified as mean nuclear fluorescence intensity normalised to Hoechst area and expressed relative to normoxic controls. Statistical comparisons were performed 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: Luminescence Assay, Expressing, Fluorescence

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,C,E) Average luminescence signal quantifying ROS levels in normoxic endothelial cells (A), astrocytes (C), and pericytes (E), comparing untreated and PBM-treated groups. (B,D,F) Representative ICC images of HIF-1α (cyan) with Hoechst nuclear counterstain (blue) in normoxic endothelial cells (B), astrocytes (D), and pericytes (F) under non-PBM (–PBM) and PBM-treated (+PBM) conditions. Quantitative comparisons were performed using one-way ANOVA with Šidák’s multiple comparisons test (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:

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–C) Mitochondrial oxygen consumption rate (OCR) profiles measured 24 hours after hypoxia (6 h, 1% O₂) in in endothelial cells (A), astrocytes (B), and pericytes (C), with or without PBM treatment. OCR traces are plotted as percentage change over time, normalised to protein content and baseline respiration. Sequential injections of oligomycin and FCCP were used to determine bioenergetic parameters. Basal respiration was calculated prior to oligomycin injection (pmol/min/µg protein). ATP-linked respiration was determined as the reduction in OCR following oligomycin and expressed relative to baseline OCR. Maximal respiration was calculated following FCCP treatment and normalised to OCR. Hypoxia significantly reduced mitochondrial function in endothelial cells and astrocytes, whereas PBM selectively increased maximal respiratory capacity in endothelial cells under hypoxic conditions. Statistical comparisons were performed using one-way ANOVA with Šídák’s post hoc correction; N=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: Injection

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–F) Oxygen consumption rate (OCR) measurements in endothelial cells (A,D), astrocytes (B,E), and pericytes (C,F) 48 hours post-treatment. OCR traces are plotted over time and normalised to protein content and baseline respiration. Basal respiration (pmol/min/µg protein) was calculated prior to oligomycin injection. ATP-linked respiration was determined as the difference between basal respiration and OCR following oligomycin, normalised to total OCR. Maximal respiration was calculated from OCR after FCCP injection and normalised accordingly. Proton leak was derived from OCR after oligomycin injection, and spare respiratory capacity was defined as the difference between maximal and basal respiration, both normalised to OCR. Statistical comparisons were performed using one-way ANOVA with Šidák’s multiple comparisons test (N=4 biological replicates; *p<0.05, **p<0.01). 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: Injection, Derivative Assay

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-C) Quantification of proton leak and spare respiratory capacity 24 hours after treatment in endothelial cells (A), astrocytes (B), and pericytes (C). Proton leak was calculated from OCR values following oligomycin injection and normalised to total OCR. Spare respiratory capacity was determined as the difference between maximal respiration (post-FCCP) and basal respiration, normalised to OCR. Statistical comparisons were performed using one-way ANOVA with Šidák’s multiple comparisons test (N=4 biological replicates; **p<0.01). 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: Injection

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–C) ECAR measurements in endothelial cells (A), astrocytes (B), and pericytes (C) at 24 and 48 hours across all experimental conditions. Basal glycolysis (mpH/min/µg protein) was calculated prior to oligomycin injection. Glycolytic reserve capacity was defined as the difference between ECAR following oligomycin and basal glycolysis, normalised to total ECAR. Statistical comparisons were performed using one-way ANOVA with Šidák’s multiple comparisons test (N=4 biological replicates; *p<0.05, **p<0.01). 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: Injection

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) Rgs5 mRNA expression in human vascular pericytes (HVPCs) at 48 hours across all experimental conditions, normalised to RPL13A. (B) Representative ICC images showing PDGFRβ (yellow) and nuclei (Hoechst, blue) in normoxic and hypoxic HVPCs, with and without PBM treatment. (C) Quantification of PDGFRβ protein expression relative to control, calculated as mean fluorescence intensity normalised to Hoechst area. (D) αSMA mRNA expression in HVPCs at 48 hours, normalised to RPL13A. (E) Representative ICC images showing filamentous actin (magenta) and nuclei (blue) under normoxic and hypoxic conditions ± PBM. (F) Quantitative analysis of actin protein expression relative to control, measured as mean fluorescence intensity normalised to Hoechst area. Statistical comparisons were performed using one-way ANOVA with Šidák’s multiple comparisons test (N=3–4 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, Control, Fluorescence

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