Blockade of cerebral ischemia-induced release of HMGB1 by amlexanox. (A–C) Mice were pretreated with amlexanox (Amx) at a dose of 10 μg/5 μl (i.c.v.) 30 minutes before 1-hour tMCAO and DAB immunostaining using coronal brain sections was performed at 3 hours after tMCAO. Following Amx post-treatment (10 μg/5 μl, i.c.v.) at 24 hours after 1-hour tMCAO, DAB immunostaining was performed at 27 hours after tMCAO. Representative images show immunoreactive HMGB1 in the somatosensory cortex (A), striatum (B), and hippocampus (Hip) (C). Immunoreactive HMGB1 in stratum radiatum (Str rad) in the Hip of untreated mice is indicated by black arrows. Red-colored arrows indicate HMGB1 immunoreactivity at 3 hours after tMCAO. Red-colored arrowheads indicate HMGB1 immunoreactivity in the Str rad at 27 hours after tMCAO. Insets in (A–C) indicate the higher magnification view of immunoreactive HMGB1 in the cortex (A), striatum (B) and cornu ammonis (CA1) of Hip (C) noted by red squares. n = 3 in each group.

Amlexanox-reversible blockade of HMGB1 release from neurons following ischemia. (A). Mice were pretreated with amlexanox (Amx) at a dose of 10 μg/5 μl (i.c.v.) 30 minutes before 1-hour tMCAO, and immunostaining of NeuN and HMGB1 (NeuN, green; HMGB1, red; merge, yellow) in the somatosensory cortex using coronal brain sections was performed at 3 hours after tMCAO. Following Amx post-treatment (10 μg/5 μl, i.c.v.) at 24 hours after 1-hour tMCAO, immunostaining of NeuN and HMGB1 was performed at 27 hours after tMCAO. The higher magnification views in the lower panels show HMGB1 localization in neurons noted by dotted squares in the upper panels. (B) Quantitative analysis of the number of HMGB1-positive neurons in the somatosensory cortex. Data are means ± S.E.M. One-way ANOVA, Dunnett’s multiple comparison tests, **P < 0.01 vs. untreated mice, ^##P < 0.01 vs. ischemic vehicle (3 hours). (C) Following Amx pretreatment (10 μg/5 μl, i.c.v.) 30 minutes before 1-hour tMCAO, immunostaining of NeuN and HMGB1 (NeuN, green; HMGB1, red; merge, yellow) in the striatum (upper panels) and CA1 of hippocampus (lower panels) was performed 3 hours after tMCAO. (D) Quantitative analysis of HMGB1-positive neurons in the striatum. Data are means ± S.E.M. One-way ANOVA, Dunnett’s multiple comparison tests, **P < 0.01 vs. untreated mice, ^##P < 0.01 vs. ischemic vehicle (3 hours). (E) Quantitative analysis of HMGB1-positive neurons in CA1 in the hippocampus (Hip). Data are means ± S.E.M. One-way ANOVA, Dunnett’s multiple comparison tests, **P < 0.01 vs. untreated mice, ^##P < 0.01 vs. ischemic vehicle (3 hours); n = 3 in each group.

Amlexanox blocks ischemia-induced release of HMGB1 from astrocytes. (A) Mice were pretreated with amlexanox (Amx) at a dose of 10 μg/5 μl (i.c.v.) 30 minutes before 1-hour tMCAO, and immunostaining of GFAP and HMGB1 (GFAP, green; HMGB1, red) in the somatosensory cortex using coronal brain sections was performed at 3 hours after tMCAO. Following Amx post-treatment (10 μg/5 μl, i.c.v.) at 24 hours after 1-hour tMCAO, immunostaining of GFAP and HMGB1 was performed at 27 hours after tMCAO. Insets indicate the higher magnification view of HMGB1 signals in astrocytes noted by dotted squares in (A). (B) Triple immunostaining (GFAP, green; HMGB1, red; Hoechst, blue) showed the higher magnification view of HMGB1 localization in astrocytes at 0 (untreated), 3 hours after tMCAO (ischemic vehicle or Amx-pretreated mice), and 27 hours after tMCAO (vehicle or Amx post-treated mice). Nuclear and cytoplasmic HMGB1 signals in astrocytes are indicated by arrow and arrowhead, respectively. (C) Quantitative analysis of HMGB1-positive astrocytes in the cortex at 3, 18, and 27 hours after 1-hour tMCAO. Data are means ± S.E.M. One-way ANOVA, Dunnett’s multiple comparison tests, **P < 0.01 vs. untreated mice, ^##P < 0.01 vs. ischemic vehicle (27 hours); n = 3 in each group. (D) Following Amx post-treatment at 24 hours after 1 hour tMCAO, HMGB1 signals were observed in the nuclei of cortical astrocytes at 48 or 96 hours after 1 hour tMCAO in both ischemic vehicle and Amx-treated mice. Insets indicate the higher magnification view of HMGB1 signals in astrocytes noted by dotted squares in (D); n = 3 in each group.

Cerebral ischemia-induced expression of HMGB1 in microglia. (A) Mice were pretreated with amlexanox (Amx) at a dose of 10 μg/5 μl (i.c.v.) 30 minutes before 1 hour tMCAO and immunostaining of Iba-1 and HMGB1 (Iba-1, green; HMGB1, red) in the somatosensory cortex using coronal brain sections was performed at 3 hours after tMCAO. Following Amx post-treatment (10 μg/5 μl, i.c.v.) at 24 hours after 1-hour tMCAO, immunostaining of Iba-1 and HMGB1 was performed at 27 hours after tMCAO. Insets indicate higher magnification view of immunoreactive-HMGB1 in Iba-1-positive microglia noted by dotted squares. (B) Triple immunostaining (Iba-1, green; HMGB1, red; Hoechst, blue) show the higher magnification view of HMGB1 localization in microglia at 0 (untreated) and 3 hours after tMCAO (vehicle- or Amx-pretreated mice), and 27 hours after tMCAO (ischemic vehicle or Amx post-treated mice). (C) Quantitative analysis of HMGB1-positive microglia in the somatosensory cortex. Data are means ± S.E.M. One-way ANOVA, Dunnett’s multiple comparison tests, **P < 0.01 vs. untreated mice. (D) Following Amx treatment (10 μg/5 μl, i.c.v.) at 24 hours after 1-hour tMCAO, immunoreactive HMGB1 was still observed in the nuclei of Iba-1-positive microglia in the cortex at 48 or 96 hours after 1-hour tMCAO. Insets in (D) indicate higher magnification view of HMGB1 expression in microglia noted by dotted squares; n = 3 in each group.

Amlexanox pretreatment exacerbates the ischemia-induced damage. (A and B) Following amlexanox (Amx) pretreatment (10 μg/5 μl, i.c.v.) 30 minutes before 1-hour tMCAO, TTC staining (A) and infarct volume measurement (B) were performed at 24 hours after tMCAO. Data are means ± S.E.M. Student’s t test (not significant); n = 3 in each group. (C–E) Mice were pretreated with Amx (10 μg/5 μl, i.c.v.) 30 minutes before 15-minute tMCAO. Nonischemic (NI) mice also received Amx (10 μg/5 μl, i.c.v.). TTC staining (C), infarct volume measurement (D), and clinical score (E) were performed at 24 hours after tMCAO. Data are means ± S.E.M. *P < 0.05 vs. ischemic vehicle. Untreated, n = 3; ischemic vehicle, n = 6; Amx + tMCAO, n = 6; Amx + nonischemic (NI), n = 3.

Late treatment with amlexanox protects the brain from ischemic damage. (A and B) Following amlexanox (Amx) post-treatment (10 μg/5 μl, i.c.v.) at 24 hours after 30-minute tMCAO, TTC staining (A) and infarct volume measurement (B) were performed at 48 hours after ischemia. Data are means ± S.E.M. Student’s t test, *P < 0.05 vs. ischemic vehicle; n = 3 in each group. (C) Following Amx post-treatment at 24 hours after 30-minute tMCAO, evaluation of clinical score was performed every day for 7 days after tMCAO. Data are means ± S.E.M. *P < 0.05 vs. ischemic vehicle; n = 6 in each group. (D–F) Following Amx post-treatment (10 μg/5 μl, i.c.v.) at 48 hours after 30-minute tMCAO, TTC staining (D), infarct volume measurement (E), and clinical score evaluation (F) were performed at 72 hours after tMCAO. Data are means ± S.E.M. Student’s t test, *P < 0.05 vs. ischemic vehicle; *P < 0.05 vs. ischemic vehicle; n = 4 in each group.