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 ) Representative immunohistochemical images (A) and quantification (B) of ICAM-1 expression in cortical microvessels of WT, Nrf2⁻/⁻ , and ICAM-1⁻/⁻ mice 24 h after injury (n = 6/group). ( C ) Western blot analysis of ICAM-1 and β-actin in stretch-injured hBMVECs 24 h post-injury with control siRNA, Nrf2 siRNA, or CPUY192018 treatment (n = 10/group). ( D ) Western blot analysis of ICAM-1 and β-actin in stretch-injured hBMVECs 24 h post-injury with control siRNA, ICAM-1 siRNA, or A205804 (ICAM-1 inhibitor) treatment (n = 9/group). ( E ) Western blot expression level of ICAM-1, leukocyte adhesion receptors LFA-1 and Mac-1 and β-actin in pericontusional cortical tissue of WT, Nrf2⁻/⁻ , and ICAM-1⁻/⁻ mice 24 h after injury. ( F-H ) Bar graphs showing quantification of ICAM-1 (F), leukocyte adhesion receptors LFA-1 (G) and Mac-1 (H) in pericontusional cortical tissue of WT, Nrf2⁻/⁻ , and ICAM-1⁻/⁻ mice 24 h after injury (n = 9/group). ( I-J ) Double immunofluorescence showing co-localization of LFA-1 with ICAM-1 in cortical microvessels, particularly enhanced in Nrf2⁻/⁻ mice (I) and corresponding quantification (J) (n = 8/group). ( K-N ) Immunoprecipitation demonstrating direct ICAM-1 interactions with LFA-1 (K, L) and Mac-1 (K, N) in cortical tissue (n = 8/group). All values are expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA in B-D or two-way ANOVA in F-H, J, L-N, followed by Dunnett’s post hoc test . p < 0.05 statistically significant. ***P < 0.001 versus control in D. ‘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: Immunohistochemical staining, Expressing, Western Blot, Control, Immunofluorescence, Immunoprecipitation

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 ) Representative immunostaining (A) and quantification (B) of MMP-2 and MMP-9 in hBMVECs 24 h after stretch injury following treatment with control siRNA or Nrf2 siRNA (n = 6/group). ( C-D ) Western blot analysis (C) and quantification (D) of MMP-2 and MMP-9 in stretch-injured hBMVECs following treatment with control siRNA, Nrf2 siRNA, CPUY192018, ICAM-1 siRNA, ICAM-1 inhibitor (A205804), or recombinant ICAM-1 (n = 6/group). ( E-F ) Quantification of MMP-2 (E) and MMP-9 (F) expression in pericontusional cortical tissue from WT, Nrf2⁻/⁻ , and ICAM-1⁻/⁻ mice 24 h after injury (n = 6/group). All values are expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA in B and D or two-way ANOVA in F, followed by Dunnett’s post hoc test . p < 0.05 statistically significant. ***P< 0.001 versus control; ## P < 0.01 versus control siRNA; @@@ P < 0.001 versus Nrf2 siRNA; $$$ P < 0.001 versus injury in D. ‘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: Immunostaining, Control, Western Blot, Recombinant, Expressing

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 ) Representative immunofluorescence staining (A) and quantification (B) of tight junction proteins Occludin (green), Claudin-5 (red), and ZO-1 (green) merged with DAPI (blue) in human brain microvascular endothelial cells (hBMVECs) following 3.0 psi stretch injury with or without Nrf2 or ICAM-1 siRNA treatment (n = 6/group). ( C-E ) Western blot analysis of Occludin, ZO-1, and Claudin-5 with β-actin as a loading control, in cell lysates collected 24 h after 3.0 psi stretch injury in hBMVECs treated 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 densitometric quantification of Occludin and ZO-1 (D) and Claudin-5 (E) normalized to β-actin (n = 6/group). ( F-G ) Representative immunofluorescence staining (F) and quantification (G) of Claudin-5 (red) merged with the endothelial marker vWF (green) and DAPI (blue) in cortical brain sections from uninjured and injured groups of WT, Nrf2⁻/⁻ , and ICAM-1⁻/⁻ 24 hr after 25 psi fluid percussion injury (FPI) (n = 6/group). ( H-I ) Western blot analysis of Occludin, Claudin-5, and ZO-1, with β-actin as a loading control, in cortical brain tissue lysates from WT, Nrf2⁻/⁻ , and ICAM-1⁻/⁻ mice following 15 psi FPI. Bar graphs (I) show densitometric quantification of Occludin, Claudin-5, and ZO-1 normalized to β-actin (n = 8/group). All values are expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA in B, D, E, and G or two-way ANOVA in I followed by Dunnett’s post hoc test. p < 0.05 statistically significant. *P< 0.05, **P< 0.01, ***P < 0.001 versus control; # P < 0.05, ### P < 0.001 versus control siRNA; @@@ P < 0.001 versus Nrf2 siRNA; $ P < 0.05, $$ P < 0.01, $$$ P < 0.001 versus injury in D and E. ‘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, Staining, Western Blot, Control, Recombinant, Marker

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-B ) Representative immunofluorescence staining (A) and quantification (B) of N-cadherin (red) with nuclear counterstain DAPI (blue) in hBMVEC following 3.0 psi stretch injury with or without Nrf2 or ICAM-1 siRNA treatment (n = 6/group). Scale bar = 40 µm. ( C-D ) Western blot analysis of N-cadherin, Connexin-43, and β-actin in lysates from 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 densitometric quantification of N-cadherin (D) normalized to β-actin (n = 6/group). All values are expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA in B and D followed by Dunnett’s post hoc test. p < 0.05 statistically significant. ***P < 0.001 versus control; ### P < 0.001 versus control siRNA; $$$ P < 0.001 versus injury in D. ‘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, Staining, Western Blot, Control, Recombinant

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: 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 ) Measurement of myeloperoxidase (MPO) activity in human brain microvascular endothelial cells (hBMVECs) under control and injury conditions, with Nrf2 knockdown (siRNA), Nrf2 activation (CPUY192018), ICAM-1 knockdown (siRNA), ICAM-1 inhibition (A205804), and recombinant ICAM-1 protein treatment (n = 6/group). ( B ) Quantification of MPO-DNA complex formation across the indicated experimental conditions (n = 6/group). ( C ) Quantification of citrullinated histone H3 (H3Cit)-DNA complexes as a marker of neutrophil extracellular trap (NET) formation (n = 6/group). All values are expressed as mean ± SEM. Statistical analysis was performed using one-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, Knockdown, Activation Assay, Inhibition, Recombinant, Marker