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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.


Related in: MedlinePlus

HMGB1 mobilization under ICH and effect of anti-HMGB1 mAb on HMGB1 dynamics and BBB permeability after ICH.(a) Cerebral bleeding was induced by injection of 0.03 U bacterial type IV collagenase into the striatum, and the resultant bleeding areas with a volume of 3 × 3 × 3 mm3 (as indicated by the white square in the picture) were sampled at 24 h after ICH for western blotting to determine brain HMGB1 levels. The representative results of western blotting are shown. (b) Quantitative analyses of the western blotting results were performed using NIH Image J software. F(2,7) = 23.419, p < 0.001. (c) Determination of plasma levels of HMGB1 by ELISA in rats with ICH. Blood samples were collected 24 h after the induction of bleeding. F(2,20) = 4.576, p = 0.023. (d) Evaluation of the hemorrhagic volume of rats subjected to the striatal ICH. (e,f) The permeability of brain capillary vessels was determined by Evans blue leakage at 6 h (e) and 3 days (f) after ICH. Representative images of Evans blue leakage at 3 h after dye injection are shown for each group. Bar graphs (lower panels) represent the results of quantification of Evans blue dye in the hemorrhagic hemisphere of the anti-HMGB1 mAb- or control IgG-treatment group. (g) Assessment of brain water content induced by ICH. At 3 days after ICH onset, brain water content was determined in the ipsilateral hemisphere. F(2,9) = 216.059, p < 0.001. Results are shown for the sham group (Sham, n = 3, 7, 3 in (b,c,g), respectively), the control IgG-treated group (Con IgG, n = 3, 8, 5, 5, 5, 4 in (b–g), respectively), and anti-HMGB1 mAb-treated group (α-HMGB1, n = 4, 8, 5, 5, 5, 5 to (b–g), respectively). Values represent the means ± SEM. *p < 0.05, **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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f1: HMGB1 mobilization under ICH and effect of anti-HMGB1 mAb on HMGB1 dynamics and BBB permeability after ICH.(a) Cerebral bleeding was induced by injection of 0.03 U bacterial type IV collagenase into the striatum, and the resultant bleeding areas with a volume of 3 × 3 × 3 mm3 (as indicated by the white square in the picture) were sampled at 24 h after ICH for western blotting to determine brain HMGB1 levels. The representative results of western blotting are shown. (b) Quantitative analyses of the western blotting results were performed using NIH Image J software. F(2,7) = 23.419, p < 0.001. (c) Determination of plasma levels of HMGB1 by ELISA in rats with ICH. Blood samples were collected 24 h after the induction of bleeding. F(2,20) = 4.576, p = 0.023. (d) Evaluation of the hemorrhagic volume of rats subjected to the striatal ICH. (e,f) The permeability of brain capillary vessels was determined by Evans blue leakage at 6 h (e) and 3 days (f) after ICH. Representative images of Evans blue leakage at 3 h after dye injection are shown for each group. Bar graphs (lower panels) represent the results of quantification of Evans blue dye in the hemorrhagic hemisphere of the anti-HMGB1 mAb- or control IgG-treatment group. (g) Assessment of brain water content induced by ICH. At 3 days after ICH onset, brain water content was determined in the ipsilateral hemisphere. F(2,9) = 216.059, p < 0.001. Results are shown for the sham group (Sham, n = 3, 7, 3 in (b,c,g), respectively), the control IgG-treated group (Con IgG, n = 3, 8, 5, 5, 5, 4 in (b–g), respectively), and anti-HMGB1 mAb-treated group (α-HMGB1, n = 4, 8, 5, 5, 5, 5 to (b–g), respectively). Values represent the means ± SEM. *p < 0.05, **p < 0.01 compared with the sham group. #p < 0.05, ##p < 0.01 compared with the control IgG-treated group.

Mentions: We confirmed that the size of the hematoma in the control and anti-HMGB1-treated rats was the same based on the measurement of hemoglobin content in each group at 24 h after ICH (Fig. 1d).


Anti-high mobility group box-1 (HMGB1) antibody inhibits hemorrhage-induced brain injury and improved neurological deficits in rats
HMGB1 mobilization under ICH and effect of anti-HMGB1 mAb on HMGB1 dynamics and BBB permeability after ICH.(a) Cerebral bleeding was induced by injection of 0.03 U bacterial type IV collagenase into the striatum, and the resultant bleeding areas with a volume of 3 × 3 × 3 mm3 (as indicated by the white square in the picture) were sampled at 24 h after ICH for western blotting to determine brain HMGB1 levels. The representative results of western blotting are shown. (b) Quantitative analyses of the western blotting results were performed using NIH Image J software. F(2,7) = 23.419, p < 0.001. (c) Determination of plasma levels of HMGB1 by ELISA in rats with ICH. Blood samples were collected 24 h after the induction of bleeding. F(2,20) = 4.576, p = 0.023. (d) Evaluation of the hemorrhagic volume of rats subjected to the striatal ICH. (e,f) The permeability of brain capillary vessels was determined by Evans blue leakage at 6 h (e) and 3 days (f) after ICH. Representative images of Evans blue leakage at 3 h after dye injection are shown for each group. Bar graphs (lower panels) represent the results of quantification of Evans blue dye in the hemorrhagic hemisphere of the anti-HMGB1 mAb- or control IgG-treatment group. (g) Assessment of brain water content induced by ICH. At 3 days after ICH onset, brain water content was determined in the ipsilateral hemisphere. F(2,9) = 216.059, p < 0.001. Results are shown for the sham group (Sham, n = 3, 7, 3 in (b,c,g), respectively), the control IgG-treated group (Con IgG, n = 3, 8, 5, 5, 5, 4 in (b–g), respectively), and anti-HMGB1 mAb-treated group (α-HMGB1, n = 4, 8, 5, 5, 5, 5 to (b–g), respectively). Values represent the means ± SEM. *p < 0.05, **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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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5385548&req=5

f1: HMGB1 mobilization under ICH and effect of anti-HMGB1 mAb on HMGB1 dynamics and BBB permeability after ICH.(a) Cerebral bleeding was induced by injection of 0.03 U bacterial type IV collagenase into the striatum, and the resultant bleeding areas with a volume of 3 × 3 × 3 mm3 (as indicated by the white square in the picture) were sampled at 24 h after ICH for western blotting to determine brain HMGB1 levels. The representative results of western blotting are shown. (b) Quantitative analyses of the western blotting results were performed using NIH Image J software. F(2,7) = 23.419, p < 0.001. (c) Determination of plasma levels of HMGB1 by ELISA in rats with ICH. Blood samples were collected 24 h after the induction of bleeding. F(2,20) = 4.576, p = 0.023. (d) Evaluation of the hemorrhagic volume of rats subjected to the striatal ICH. (e,f) The permeability of brain capillary vessels was determined by Evans blue leakage at 6 h (e) and 3 days (f) after ICH. Representative images of Evans blue leakage at 3 h after dye injection are shown for each group. Bar graphs (lower panels) represent the results of quantification of Evans blue dye in the hemorrhagic hemisphere of the anti-HMGB1 mAb- or control IgG-treatment group. (g) Assessment of brain water content induced by ICH. At 3 days after ICH onset, brain water content was determined in the ipsilateral hemisphere. F(2,9) = 216.059, p < 0.001. Results are shown for the sham group (Sham, n = 3, 7, 3 in (b,c,g), respectively), the control IgG-treated group (Con IgG, n = 3, 8, 5, 5, 5, 4 in (b–g), respectively), and anti-HMGB1 mAb-treated group (α-HMGB1, n = 4, 8, 5, 5, 5, 5 to (b–g), respectively). Values represent the means ± SEM. *p < 0.05, **p < 0.01 compared with the sham group. #p < 0.05, ##p < 0.01 compared with the control IgG-treated group.
Mentions: We confirmed that the size of the hematoma in the control and anti-HMGB1-treated rats was the same based on the measurement of hemoglobin content in each group at 24 h after ICH (Fig. 1d).

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.


Related in: MedlinePlus