Journal: bioRxiv

Article Title: Nrf2 regulates ICAM-1–mediated neutrophil extracellular trap formation after traumatic brain injury

doi: 10.64898/2026.05.01.722360

Figure Lengend Snippet: (A) In vitro transmigration assay assessing monocyte adhesion and migration across injured human brain microvascular endothelial cell (hBMVEC) monolayers following Nrf2 knockdown, treatment with the Nrf2 activator CPUY192018, or ICAM-1 inhibition (n = 6/group). (B-C) Representative images and quantification of GFP-labeled monocyte adhesion (green) to injured hBMVEC monolayers (red). The nuclei were counter stained with DAPI (blue). Monocyte adhesion at endothelial junctions was evaluated under Nrf2-deficient conditions and after ICAM-1 silencing. Arrows show the adhered monocytes on the hBMVEC monolayer (n = 6/group). Scale bar = 10 µm. (D-E) Adhesion and transmigration of Calcein-AM–labeled macrophages in the perivascular space of wild-type (WT), Nrf2⁻/⁻ , and ICAM-1⁻/⁻ mice following injury. Bar graph (E) shows the quantification of adhered and transmigrated macrophages in the perivascular space (n = 8/group). Scale bar = 100 µm. ( F-H ) Transmigration of leukocyte analysis by infusing cultured GFP monocytes (green) through the common carotid artery of WT, Nrf2⁻/⁻ , and ICAM-1⁻/⁻ mice 24 h after injury and co-localized with immunostaining images of Mac-1 (red) and DAPI (blue). Panels in second through fourth rows are selected and enlarged areas of first row. Scale bar: 400 µm in the panels of first row and 80 µm in the panels of second through fourth rows. ( G-H ) Quantitative analysis of the number of infused GFP (G) and Mac-1 (H) positive cells. All values are expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA in A and C or two-way ANOVA in E, G, and H followed by Dunnett’s post hoc test. p < 0.05 statistically significant. ***P < 0.001 versus control; # P < 0.05, ## P < 0.01, ### P < 0.001 versus control siRNA; @ P < 0.05 , @@ P < 0.01 versus Nrf2 siRNA; $$$ P < 0.001 versus injury in A. ‘ns’ is not significant.

Article Snippet: Male and female C57/BL6 mice wild-type (WT), Nrf2 knockout ( Nrf2−/− ) mice and ICAM-1 knockout ( ICAM-1−/−) mice (9 weeks old, 20-25 g; Jackson Laboratory, Bar Harbor, ME) were used for this study.

Techniques: In Vitro, Transmigration Assay, Migration, Knockdown, Inhibition, Labeling, Staining, Cell Culture, Immunostaining, Control

Journal: bioRxiv

Article Title: Nrf2 regulates ICAM-1–mediated neutrophil extracellular trap formation after traumatic brain injury

doi: 10.64898/2026.05.01.722360

Figure Lengend Snippet: ( A ) In vitro assessment of blood–brain barrier (BBB) integrity using FITC-dextran permeability assay in stretch-injured hBMVEC cultures 24 h after treatment with control siRNA, Nrf2 siRNA, the Nrf2 activator CPUY, ICAM-1 siRNA, the ICAM-1 inhibitor A2015804, or recombinant ICAM-1 protein. Bar graphs represent fold change quantification of FITC-dextran-4 permeability normalized to control group (n = 6/group). ( B-C ) In vivo evaluation of BBB permeability following injury using sodium fluorescein (B) and Evans blue ( C ) tracer assays in wild-type (WT), Nrf2⁻/⁻, and ICAM-1⁻/⁻ mice (n = 8/group). All values are expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA in A or two-way ANOVA in B and C followed by Dunnett’s post hoc test. p < 0.05 statistically significant. **P < 0.01, ***P < 0.001 versus control; # P < 0.05, ## P < 0.01 versus control siRNA; @@ P < 0.01 versus Nrf2 siRNA; $$ P < 0.01, $$$ P < 0.001 versus injury in A. Statistical significance between groups is indicated on the graphs B and C.

Article Snippet: Male and female C57/BL6 mice wild-type (WT), Nrf2 knockout ( Nrf2−/− ) mice and ICAM-1 knockout ( ICAM-1−/−) mice (9 weeks old, 20-25 g; Jackson Laboratory, Bar Harbor, ME) were used for this study.

Techniques: In Vitro, FITC-Dextran Permeability Assay, Control, Recombinant, Permeability, In Vivo

Journal: bioRxiv

Article Title: Nrf2 regulates ICAM-1–mediated neutrophil extracellular trap formation after traumatic brain injury

doi: 10.64898/2026.05.01.722360

Figure Lengend Snippet: ( A-C ) Quantification of neuron-specific enolase (NSE) levels in brain tissue lysate (A), blood plasma (B), and cerebrospinal fluid (CSF) (C) from wild-type (WT), Nrf2⁻/⁻, and ICAM-1⁻/⁻ mice under control and injury conditions (n = 8/group).( D-F ) Quantification of S100β levels in brain tissue lysate (D), blood plasma (E), and CSF (F) across the same experimental groups (n = 8/group). All values are expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA in A or two-way ANOVA in B and C followed by Dunnett’s post hoc test. Statistical significance between groups is indicated on the graphs. p < 0.05 statistically significant.

Article Snippet: Male and female C57/BL6 mice wild-type (WT), Nrf2 knockout ( Nrf2−/− ) mice and ICAM-1 knockout ( ICAM-1−/−) mice (9 weeks old, 20-25 g; Jackson Laboratory, Bar Harbor, ME) were used for this study.

Techniques: Clinical Proteomics, Control

Journal: bioRxiv

Article Title: Nrf2 regulates ICAM-1–mediated neutrophil extracellular trap formation after traumatic brain injury

doi: 10.64898/2026.05.01.722360

Figure Lengend Snippet: Immunofluorescence analysis of NET formation in the pericontusional cortex of wild-type (WT), Nrf2⁻/⁻ , and ICAM-1⁻/⁻ mice following TBI. NETs were identified by co-localization of citrullinated histone H3 (H3Cit, a specific NET marker; red) and Ly6G (neutrophil marker; green). The nuclei were counterstained with DAPI (blue). Scale bar = 250 µm in the panels of first row, and 80 µm in the panels of second through fourth rows. Bar graphs show the quantification of H3Cit (B) and Ly6G (C) positive cells (n = 6/group). (D-F) Western blotting expression of PAD4, Ly6G, and H3Cit 24 hr after injury following genetic disruption of PAD4 (CRISPR/Cas9), pharmacological inhibition with GSK484, or DNase I, and the treatment with phorbol 12-myristate 13-acetate (PMA), or purified NETs in human brain microvascular endothelial cells (hBMVECs) exposed to injury. (E and F) Bar graphs represent densitometric quantification of H3Cit normalized to total H3 (E), and PAD4 and Ly6G normalized to β-actin (F) (n = 6/group). (G-I) Western blotting analysis of PAD4, Ly6G, and histone H3 citrullination in injured hBMVECs under Nrf2-deficient conditions and following inhibition of NET formation or ICAM-1 blockade. (H and I) Bar graphs represent densitometric quantification of PAD4 and Ly6G normalized to β-actin (H) and H3Cit normalized to total H3 (I) (n = 6/group). (J-L) Western blotting analysis of PAD4, Ly6G, and H3Cit expression in brain tissue from WT, Nrf2⁻/⁻ , and ICAM-1⁻/⁻ mice under control and TBI conditions. (K and L) Bar graphs represent densitometric quantification of PAD4 and Ly6G normalized to β-actin (H) and H3Cit normalized to total H3 (I) (n = 8/group). (M-N) Measurement of NET-associated myeloperoxidase (MPO) activity (M) and MPO–DNA complexes (N) in brain tissue lysates from WT, Nrf2⁻/⁻ , and ICAM-1⁻/⁻ mice following TBI (n= 8/group). All values are expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA in A, B, E, F, H, and I and two-way ANOVA in K-N, followed by Dunnett’s post hoc test. p < 0.05 statistically significant. ***P < 0.001 versus control; # P < 0.05, # P < 0.05 versus control siRNA; $$$ P < 0.001 versus injury in E, F, H, and I. ‘ns’ is not significant.

Article Snippet: Male and female C57/BL6 mice wild-type (WT), Nrf2 knockout ( Nrf2−/− ) mice and ICAM-1 knockout ( ICAM-1−/−) mice (9 weeks old, 20-25 g; Jackson Laboratory, Bar Harbor, ME) were used for this study.

Techniques: Immunofluorescence, Marker, Western Blot, Expressing, Disruption, CRISPR, Inhibition, Purification, Control, Activity Assay

Journal: bioRxiv

Article Title: Nrf2 regulates ICAM-1–mediated neutrophil extracellular trap formation after traumatic brain injury

doi: 10.64898/2026.05.01.722360

Figure Lengend Snippet: ( A-B ) Immunohistochemical analysis of CD68⁺ macrophage (red), and co-localized with von Willibrand Factor (vWF, a microvessel maker, green) and DAPI (nucleus, blue). Infiltration of CD68 + cells into the perivascular space in wild-type (WT), Nrf2⁻/⁻ , and ICAM-1⁻/⁻ mice under control and injury conditions are shown in the representative images (A) and corresponding quantification (B) (n = 8/group). Scale bar = 40 µm. All values are expressed as mean ± SEM. Statistical analysis was performed using two-way ANOVA followed by Dunnett’s post hoc test and statistical significance is indicated on the graph. p < 0.05 statistically significant.

Article Snippet: Male and female C57/BL6 mice wild-type (WT), Nrf2 knockout ( Nrf2−/− ) mice and ICAM-1 knockout ( ICAM-1−/−) mice (9 weeks old, 20-25 g; Jackson Laboratory, Bar Harbor, ME) were used for this study.

Techniques: Immunohistochemical staining, Control

Journal: bioRxiv

Article Title: Nrf2 regulates ICAM-1–mediated neutrophil extracellular trap formation after traumatic brain injury

doi: 10.64898/2026.05.01.722360

Figure Lengend Snippet: (A) Rotarod test assessing motor coordination and balance in wild-type (WT), Nrf2⁻/⁻ , and ICAM-1⁻/⁻ mice under control and injury conditions. Latency to fall was measured to evaluate motor performance (n = 8/group). (B) Representative swim trajectories from the Morris water maze showing search patterns during the probe trial in WT, Nrf2⁻/⁻ , and ICAM-1⁻/⁻ mice following injury. (C) Escape latency during the acquisition phase of the Morris water maze test assessing spatial learning (n = 8/group). (D-E) Probe trial analysis of spatial memory, including the number of platform crossings (D) and time spent in the target quadrant (E) (n = 8/group). (F) Representative open-field movement tracks illustrating exploratory behavior patterns in WT, Nrf2⁻/⁻ , and ICAM-1⁻/⁻ mice. (G-J) Quantification of open-field parameters including total distance traveled (G), time spent in the center (H), number of center entries (I), and thigmotaxis (J) (n = 8/group). All values are expressed as mean ± SEM. Statistical analysis was performed using two-way ANOVA followed by Dunnett’s post hoc test. p < 0.05 statistically significant. ***P < 0.001 versus control (WT); ### P < 0.001 versus control ( Nrf2⁻/⁻ ); @@@ P < 0.001 versus control ( ICAM-1⁻/⁻ ). $ P < 0.05, $$$ P < 0.001 versus injury (WT) in A and C.

Article Snippet: Male and female C57/BL6 mice wild-type (WT), Nrf2 knockout ( Nrf2−/− ) mice and ICAM-1 knockout ( ICAM-1−/−) mice (9 weeks old, 20-25 g; Jackson Laboratory, Bar Harbor, ME) were used for this study.

Techniques: Control

Journal: bioRxiv

Article Title: Nrf2 regulates ICAM-1–mediated neutrophil extracellular trap formation after traumatic brain injury

doi: 10.64898/2026.05.01.722360

Figure Lengend Snippet: ( A-B ) Quantification of citrullinated histone H3 (H3Cit)-DNA complexes by ELISA in brain tissue lysates (A) and plasma samples (B) from wild-type (WT), Nrf2⁻/⁻ , and ICAM-1⁻/⁻ mice under control and injury conditions (n = 8/group). All values are expressed as mean ± SEM. Statistical analysis was performed using two-way ANOVA followed by Dunnett’s post hoc test and statistical significance between groups is indicated on the graphs. p < 0.05 statistically significant.

Article Snippet: Male and female C57/BL6 mice wild-type (WT), Nrf2 knockout ( Nrf2−/− ) mice and ICAM-1 knockout ( ICAM-1−/−) mice (9 weeks old, 20-25 g; Jackson Laboratory, Bar Harbor, ME) were used for this study.

Techniques: Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Control

Journal: bioRxiv

Article Title: Nrf2 regulates ICAM-1–mediated neutrophil extracellular trap formation after traumatic brain injury

doi: 10.64898/2026.05.01.722360

Figure Lengend Snippet: ( A ) Measurement of myeloperoxidase (MPO) activity in brain tissue lysates from wild-type (WT), Nrf2⁻/⁻ , and ICAM-1⁻/⁻ mice under control and injury conditions (n = 8/group). ( B ) Quantification of MPO-DNA complexes in plasma samples as an indicator of NET-associated MPO release across the same experimental groups (n = 8/group). All values are expressed as mean ± SEM. Statistical analysis was performed using two-way ANOVA followed by Dunnett’s post hoc test and statistical significance between groups is indicated on the graphs. p < 0.05 statistically significant.

Article Snippet: Male and female C57/BL6 mice wild-type (WT), Nrf2 knockout ( Nrf2−/− ) mice and ICAM-1 knockout ( ICAM-1−/−) mice (9 weeks old, 20-25 g; Jackson Laboratory, Bar Harbor, ME) were used for this study.

Techniques: Activity Assay, Control, Clinical Proteomics