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 were immortalised via the SV40 antigen T. Cells were used up to passage number 20 and maintained at 37°C and 5% CO 2 in the appropriate medium purchased from Innoprot, with a full medium change every two days: endothelial cell medium (EM) (Innoprot; P60104), astrocyte medium (AM) (Innoprot; P60101), or pericyte medium-PLUS (PM) (Innoprot; P60121-Plus).

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 were immortalised via the SV40 antigen T. Cells were used up to passage number 20 and maintained at 37°C and 5% CO 2 in the appropriate medium purchased from Innoprot, with a full medium change every two days: endothelial cell medium (EM) (Innoprot; P60104), astrocyte medium (AM) (Innoprot; P60101), or pericyte medium-PLUS (PM) (Innoprot; P60121-Plus).

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 were immortalised via the SV40 antigen T. Cells were used up to passage number 20 and maintained at 37°C and 5% CO 2 in the appropriate medium purchased from Innoprot, with a full medium change every two days: endothelial cell medium (EM) (Innoprot; P60104), astrocyte medium (AM) (Innoprot; P60101), or pericyte medium-PLUS (PM) (Innoprot; P60121-Plus).

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 were immortalised via the SV40 antigen T. Cells were used up to passage number 20 and maintained at 37°C and 5% CO 2 in the appropriate medium purchased from Innoprot, with a full medium change every two days: endothelial cell medium (EM) (Innoprot; P60104), astrocyte medium (AM) (Innoprot; P60101), or pericyte medium-PLUS (PM) (Innoprot; P60121-Plus).

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 were immortalised via the SV40 antigen T. Cells were used up to passage number 20 and maintained at 37°C and 5% CO 2 in the appropriate medium purchased from Innoprot, with a full medium change every two days: endothelial cell medium (EM) (Innoprot; P60104), astrocyte medium (AM) (Innoprot; P60101), or pericyte medium-PLUS (PM) (Innoprot; P60121-Plus).

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 were immortalised via the SV40 antigen T. Cells were used up to passage number 20 and maintained at 37°C and 5% CO 2 in the appropriate medium purchased from Innoprot, with a full medium change every two days: endothelial cell medium (EM) (Innoprot; P60104), astrocyte medium (AM) (Innoprot; P60101), or pericyte medium-PLUS (PM) (Innoprot; P60121-Plus).

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 were immortalised via the SV40 antigen T. Cells were used up to passage number 20 and maintained at 37°C and 5% CO 2 in the appropriate medium purchased from Innoprot, with a full medium change every two days: endothelial cell medium (EM) (Innoprot; P60104), astrocyte medium (AM) (Innoprot; P60101), or pericyte medium-PLUS (PM) (Innoprot; P60121-Plus).

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 were immortalised via the SV40 antigen T. Cells were used up to passage number 20 and maintained at 37°C and 5% CO 2 in the appropriate medium purchased from Innoprot, with a full medium change every two days: endothelial cell medium (EM) (Innoprot; P60104), astrocyte medium (AM) (Innoprot; P60101), or pericyte medium-PLUS (PM) (Innoprot; P60121-Plus).

Techniques: Expressing, Control, 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:

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