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Anti-high mobility group box-1 (HMGB1) antibody inhibits hemorrhage-induced brain injury and improved neurological deficits in rats

View Article: PubMed Central - PubMed

ABSTRACT

As one of the most lethal stroke subtypes, intracerebral hemorrhage (ICH) is acknowledged as a serious clinical problem lacking effective treatment. Available evidence from preclinical and clinical studies suggests that inflammatory mechanisms are involved in the progression of ICH-induced secondary brain injury. High mobility group box-1 (HMGB1) is a ubiquitous and abundant nonhistone DNA-binding protein, and is also an important proinflammatory molecule once released into the extracellular space from the nuclei. Here, we show that treatment with neutralizing anti-HMGB1 mAb (1 mg/kg, i.v. twice) remarkably ameliorated ICH-injury induced by local injection of collagenase IV in the striatum of rats. Administration of anti-HMGB1 mAb inhibited the release of HMGB1 into the extracellular space in the peri-hematomal region, reduced serum HMGB1 levels and decreased brain edema by protecting blood-brain barrier integrity, in association with decreased activated microglia and the expression of inflammation-related factors at 24 h after ICH. Consequently, anti-HMGB1 mAb reduced the oxidative stress and improved the behavioral performance of rats. These results strongly indicate that HMGB1 plays a critical role in the development of ICH-induced secondary injury through the amplification of plural inflammatory responses. Intravenous injection of neutralizing anti-HMGB1 mAb has potential as a novel therapeutic strategy for ICH.

No MeSH data available.


Release of HMGB1 in astrocytes and microglia and the activation of microglia 24 h after ICH.(a) The astrocytes in the dentate gyrus were double-immunostained with anti-HMGB1 (red) and anti-GFAP (green). Arrow indicates the HMGB1-negative astrocytes in control IgG-treated rat. The scale bar represents 5 μm. The cells in which HMGB1 completely disappeared from nuclei were recognized as HMGB1-negative cells. The number of HMGB1-negative astrocytes (F(2,10) = 8.762, p = 0.006) and the ratio of these cells in total astrocytes (F(2,10) = 6.944, p = 0.012) are expressed as the means ± SEM. (b) Typical microglia in the peri-hematomal region are shown after the double immunohistochemical staining with anti-HMGB1 and anti-Iba1. The scale bar represents 5 μm. The ratio of HMGB1-positive microglia to total microglia (F(2,12) = 2.298, p = 0.143) are expressed as the means ± SEM. (c) The Iba1-positive cell numbers were counted at 24 h post-ICH in the peri-hematomal region of the sham, control IgG- and anti-HMGB1-treated groups. Iba1-positive cells in the control IgG-treated group had short processes and relatively larger cell bodies. The scale bar represents 100 μm. F(2,15) = 20.537, p < 0.001 for area of iba1+ cell. F(2,15) = 14.090, p < 0.001 for immunofluorescence intensity of iba1+ cell. Results are shown for the sham group (Sham, n = 3, 3, 5, 6, 6 in (a-c), respectively), the control IgG-treated group (Con IgG, n = 5, 5, 5, 6, 6 in (a-c), respectively), and anti-HMGB1 mAb-treated group (α-HMGB1, n = 5, 5, 5, 6, 6 to (a-c), respectively). *p < 0.01, **p < 0.01 compared with the sham group. #p < 0.05, ##p < 0.01 compared with the control IgG-treated group.
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f3: Release of HMGB1 in astrocytes and microglia and the activation of microglia 24 h after ICH.(a) The astrocytes in the dentate gyrus were double-immunostained with anti-HMGB1 (red) and anti-GFAP (green). Arrow indicates the HMGB1-negative astrocytes in control IgG-treated rat. The scale bar represents 5 μm. The cells in which HMGB1 completely disappeared from nuclei were recognized as HMGB1-negative cells. The number of HMGB1-negative astrocytes (F(2,10) = 8.762, p = 0.006) and the ratio of these cells in total astrocytes (F(2,10) = 6.944, p = 0.012) are expressed as the means ± SEM. (b) Typical microglia in the peri-hematomal region are shown after the double immunohistochemical staining with anti-HMGB1 and anti-Iba1. The scale bar represents 5 μm. The ratio of HMGB1-positive microglia to total microglia (F(2,12) = 2.298, p = 0.143) are expressed as the means ± SEM. (c) The Iba1-positive cell numbers were counted at 24 h post-ICH in the peri-hematomal region of the sham, control IgG- and anti-HMGB1-treated groups. Iba1-positive cells in the control IgG-treated group had short processes and relatively larger cell bodies. The scale bar represents 100 μm. F(2,15) = 20.537, p < 0.001 for area of iba1+ cell. F(2,15) = 14.090, p < 0.001 for immunofluorescence intensity of iba1+ cell. Results are shown for the sham group (Sham, n = 3, 3, 5, 6, 6 in (a-c), respectively), the control IgG-treated group (Con IgG, n = 5, 5, 5, 6, 6 in (a-c), respectively), and anti-HMGB1 mAb-treated group (α-HMGB1, n = 5, 5, 5, 6, 6 to (a-c), respectively). *p < 0.01, **p < 0.01 compared with the sham group. #p < 0.05, ##p < 0.01 compared with the control IgG-treated group.

Mentions: There were two types of GFAP-positive astrocytes in peri-hematomal regions, HMGB1-negative (Fig. 3a) and HMGB1-positive cells (data not shown), suggesting that HMGB1 was released from both astrocytes and neurons. The number of HMGB1-negative astrocytes was reduced by the treatment with anti-HMGB1 (Fig. 3a). Thus, anti-HMGB1 strongly inhibited the translocation of HMGB1 in astrocytes.


Anti-high mobility group box-1 (HMGB1) antibody inhibits hemorrhage-induced brain injury and improved neurological deficits in rats
Release of HMGB1 in astrocytes and microglia and the activation of microglia 24 h after ICH.(a) The astrocytes in the dentate gyrus were double-immunostained with anti-HMGB1 (red) and anti-GFAP (green). Arrow indicates the HMGB1-negative astrocytes in control IgG-treated rat. The scale bar represents 5 μm. The cells in which HMGB1 completely disappeared from nuclei were recognized as HMGB1-negative cells. The number of HMGB1-negative astrocytes (F(2,10) = 8.762, p = 0.006) and the ratio of these cells in total astrocytes (F(2,10) = 6.944, p = 0.012) are expressed as the means ± SEM. (b) Typical microglia in the peri-hematomal region are shown after the double immunohistochemical staining with anti-HMGB1 and anti-Iba1. The scale bar represents 5 μm. The ratio of HMGB1-positive microglia to total microglia (F(2,12) = 2.298, p = 0.143) are expressed as the means ± SEM. (c) The Iba1-positive cell numbers were counted at 24 h post-ICH in the peri-hematomal region of the sham, control IgG- and anti-HMGB1-treated groups. Iba1-positive cells in the control IgG-treated group had short processes and relatively larger cell bodies. The scale bar represents 100 μm. F(2,15) = 20.537, p < 0.001 for area of iba1+ cell. F(2,15) = 14.090, p < 0.001 for immunofluorescence intensity of iba1+ cell. Results are shown for the sham group (Sham, n = 3, 3, 5, 6, 6 in (a-c), respectively), the control IgG-treated group (Con IgG, n = 5, 5, 5, 6, 6 in (a-c), respectively), and anti-HMGB1 mAb-treated group (α-HMGB1, n = 5, 5, 5, 6, 6 to (a-c), respectively). *p < 0.01, **p < 0.01 compared with the sham group. #p < 0.05, ##p < 0.01 compared with the control IgG-treated group.
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f3: Release of HMGB1 in astrocytes and microglia and the activation of microglia 24 h after ICH.(a) The astrocytes in the dentate gyrus were double-immunostained with anti-HMGB1 (red) and anti-GFAP (green). Arrow indicates the HMGB1-negative astrocytes in control IgG-treated rat. The scale bar represents 5 μm. The cells in which HMGB1 completely disappeared from nuclei were recognized as HMGB1-negative cells. The number of HMGB1-negative astrocytes (F(2,10) = 8.762, p = 0.006) and the ratio of these cells in total astrocytes (F(2,10) = 6.944, p = 0.012) are expressed as the means ± SEM. (b) Typical microglia in the peri-hematomal region are shown after the double immunohistochemical staining with anti-HMGB1 and anti-Iba1. The scale bar represents 5 μm. The ratio of HMGB1-positive microglia to total microglia (F(2,12) = 2.298, p = 0.143) are expressed as the means ± SEM. (c) The Iba1-positive cell numbers were counted at 24 h post-ICH in the peri-hematomal region of the sham, control IgG- and anti-HMGB1-treated groups. Iba1-positive cells in the control IgG-treated group had short processes and relatively larger cell bodies. The scale bar represents 100 μm. F(2,15) = 20.537, p < 0.001 for area of iba1+ cell. F(2,15) = 14.090, p < 0.001 for immunofluorescence intensity of iba1+ cell. Results are shown for the sham group (Sham, n = 3, 3, 5, 6, 6 in (a-c), respectively), the control IgG-treated group (Con IgG, n = 5, 5, 5, 6, 6 in (a-c), respectively), and anti-HMGB1 mAb-treated group (α-HMGB1, n = 5, 5, 5, 6, 6 to (a-c), respectively). *p < 0.01, **p < 0.01 compared with the sham group. #p < 0.05, ##p < 0.01 compared with the control IgG-treated group.
Mentions: There were two types of GFAP-positive astrocytes in peri-hematomal regions, HMGB1-negative (Fig. 3a) and HMGB1-positive cells (data not shown), suggesting that HMGB1 was released from both astrocytes and neurons. The number of HMGB1-negative astrocytes was reduced by the treatment with anti-HMGB1 (Fig. 3a). Thus, anti-HMGB1 strongly inhibited the translocation of HMGB1 in astrocytes.

View Article: PubMed Central - PubMed

ABSTRACT

As one of the most lethal stroke subtypes, intracerebral hemorrhage (ICH) is acknowledged as a serious clinical problem lacking effective treatment. Available evidence from preclinical and clinical studies suggests that inflammatory mechanisms are involved in the progression of ICH-induced secondary brain injury. High mobility group box-1 (HMGB1) is a ubiquitous and abundant nonhistone DNA-binding protein, and is also an important proinflammatory molecule once released into the extracellular space from the nuclei. Here, we show that treatment with neutralizing anti-HMGB1 mAb (1&thinsp;mg/kg, i.v. twice) remarkably ameliorated ICH-injury induced by local injection of collagenase IV in the striatum of rats. Administration of anti-HMGB1 mAb inhibited the release of HMGB1 into the extracellular space in the peri-hematomal region, reduced serum HMGB1 levels and decreased brain edema by protecting blood-brain barrier integrity, in association with decreased activated microglia and the expression of inflammation-related factors at 24&thinsp;h after ICH. Consequently, anti-HMGB1 mAb reduced the oxidative stress and improved the behavioral performance of rats. These results strongly indicate that HMGB1 plays a critical role in the development of ICH-induced secondary injury through the amplification of plural inflammatory responses. Intravenous injection of neutralizing anti-HMGB1 mAb has potential as a novel therapeutic strategy for ICH.

No MeSH data available.