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Anti-high mobility group box-1 (HMGB1) antibody attenuates delayed cerebral vasospasm and brain injury after subarachnoid hemorrhage in rats

View Article: PubMed Central - PubMed

ABSTRACT

Although delayed cerebral vasospasm (DCV) following subarachnoid hemorrhage (SAH) is closely related to the progression of brain damage, little is known about the molecular mechanism underlying its development. High mobility group box-1 (HMGB1) plays an important role as an initial inflammatory mediator in SAH. In this study, an SAH rat model was employed to evaluate the effects of anti-HMGB1 monoclonal antibody (mAb) on DCV after SAH. A vasoconstriction of the basilar artery (BA) associated with a reduction of nuclear HMGB1 and its translocation in vascular smooth muscle cells were observed in SAH rats, and anti-HMGB1 mAb administration significantly suppressed these effects. Up-regulations of inflammation-related molecules and vasoconstriction-mediating receptors in the BA of SAH rats were inhibited by anti-HMGB1 mAb treatment. Anti-HMGB1 mAb attenuated the enhanced vasocontractile response to thrombin of the isolated BA from SAH rats and prevented activation of cerebrocortical microglia. Moreover, locomotor activity and weight loss recovery were also enhanced by anti-HMGB1 mAb administration. The vasocontractile response of the BA under SAH may be induced by events that are downstream of responses to HMGB1-induced inflammation and inhibited by anti-HMGB1 mAb. Anti-HMGB1 mAb treatment may provide a novel therapeutic strategy for DCV and early brain injury after SAH.

No MeSH data available.


Related in: MedlinePlus

Effects of anti-HMGB1 mAb on mobilization of HMGB1 in SAH rats.Effects of anti-HMGB1 mAb were examined at 48 hr after the SAH in rats treated with anti-HMGB1 mAb or control IgG at both 5 min and 24 hr after blood injection. (a) The whole brain including the BA was prepared as coronal sections, and subjected to immunostaining for HMGB1 (Alexa Fluor 555) or α-smooth muscle actin (α-SMA, a marker protein of vascular smooth muscle cell) (Alexa Fluor 488) in the BA. All sections were counterstained with DAPI. Representative images of the BA in each group are shown. Arrowheads indicate the nuclei staining negative for HMGB1 in VSMCs. (b) Representative images of the HMGB1 translocation (arrows) to the cytoplasm from the nucleus in the control IgG-treated group are shown. (c) Nuclear localization of HMGB1 in VSMCs was evaluated by the ratio of the nuclei staining negative for HMGB1 to whole nuclei on immunofluorescent staining. (d) Total RNA was extracted from the isolated BA and subjected to quantitative RT-PCR for analysis of the expression of HMGB1 mRNA. GAPDH was used as a housekeeping gene. (e) The plasma concentration of HMGB1 was measured by ELISA. The scale bars indicate 20 μm. Results are shown for the sham group (Sham, n = 3, 4, 5 in (a–e), respectively), the control IgG-treated group (Cont IgG, n = 5, 4, 9 in (a–e), respectively), and the anti-HMGB1 mAb-treated group (α-HMGB1, n = 5, 4, 11 to in (a–e), respectively). Values represent the means ± SE. *P < 0.05, **P < 0.01 compared with the sham group. #P < 0.05, ##P < 0.01 compared with the control IgG-treated group. N.S.: Not significant.
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f2: Effects of anti-HMGB1 mAb on mobilization of HMGB1 in SAH rats.Effects of anti-HMGB1 mAb were examined at 48 hr after the SAH in rats treated with anti-HMGB1 mAb or control IgG at both 5 min and 24 hr after blood injection. (a) The whole brain including the BA was prepared as coronal sections, and subjected to immunostaining for HMGB1 (Alexa Fluor 555) or α-smooth muscle actin (α-SMA, a marker protein of vascular smooth muscle cell) (Alexa Fluor 488) in the BA. All sections were counterstained with DAPI. Representative images of the BA in each group are shown. Arrowheads indicate the nuclei staining negative for HMGB1 in VSMCs. (b) Representative images of the HMGB1 translocation (arrows) to the cytoplasm from the nucleus in the control IgG-treated group are shown. (c) Nuclear localization of HMGB1 in VSMCs was evaluated by the ratio of the nuclei staining negative for HMGB1 to whole nuclei on immunofluorescent staining. (d) Total RNA was extracted from the isolated BA and subjected to quantitative RT-PCR for analysis of the expression of HMGB1 mRNA. GAPDH was used as a housekeeping gene. (e) The plasma concentration of HMGB1 was measured by ELISA. The scale bars indicate 20 μm. Results are shown for the sham group (Sham, n = 3, 4, 5 in (a–e), respectively), the control IgG-treated group (Cont IgG, n = 5, 4, 9 in (a–e), respectively), and the anti-HMGB1 mAb-treated group (α-HMGB1, n = 5, 4, 11 to in (a–e), respectively). Values represent the means ± SE. *P < 0.05, **P < 0.01 compared with the sham group. #P < 0.05, ##P < 0.01 compared with the control IgG-treated group. N.S.: Not significant.

Mentions: In the sham group, strong HMGB1 immunoreactivities were observed in the nuclei of vascular smooth muscle cells (VSMCs) of the BA (Fig. 2a upper panels). In the control IgG-treated group at 48 hr after SAH, some of the VSMCs were depleted of nuclear HMGB1. Generally, HMGB1 immunoreactivities in VSMCs of the BA were more faint than in the sham group and, unlike in the sham group, were present in the marginal zone of each nucleus (Fig. 2a middle panels). These results suggest the extracellular translocation of HMGB1 in VSMCs. We also observed cytoplasmic vesicle-like HMGB1 immunoreactivity in the VSMCs of the control IgG-treated group (Fig. 2b), which was considered to indicate the extranuclear translocation of HMGB1. On the other hand, the administration of anti-HMGB1 mAb restored most of the intranuclear localization of HMGB1 in VSMCs (Fig. 2a lower panels).


Anti-high mobility group box-1 (HMGB1) antibody attenuates delayed cerebral vasospasm and brain injury after subarachnoid hemorrhage in rats
Effects of anti-HMGB1 mAb on mobilization of HMGB1 in SAH rats.Effects of anti-HMGB1 mAb were examined at 48 hr after the SAH in rats treated with anti-HMGB1 mAb or control IgG at both 5 min and 24 hr after blood injection. (a) The whole brain including the BA was prepared as coronal sections, and subjected to immunostaining for HMGB1 (Alexa Fluor 555) or α-smooth muscle actin (α-SMA, a marker protein of vascular smooth muscle cell) (Alexa Fluor 488) in the BA. All sections were counterstained with DAPI. Representative images of the BA in each group are shown. Arrowheads indicate the nuclei staining negative for HMGB1 in VSMCs. (b) Representative images of the HMGB1 translocation (arrows) to the cytoplasm from the nucleus in the control IgG-treated group are shown. (c) Nuclear localization of HMGB1 in VSMCs was evaluated by the ratio of the nuclei staining negative for HMGB1 to whole nuclei on immunofluorescent staining. (d) Total RNA was extracted from the isolated BA and subjected to quantitative RT-PCR for analysis of the expression of HMGB1 mRNA. GAPDH was used as a housekeeping gene. (e) The plasma concentration of HMGB1 was measured by ELISA. The scale bars indicate 20 μm. Results are shown for the sham group (Sham, n = 3, 4, 5 in (a–e), respectively), the control IgG-treated group (Cont IgG, n = 5, 4, 9 in (a–e), respectively), and the anti-HMGB1 mAb-treated group (α-HMGB1, n = 5, 4, 11 to in (a–e), respectively). Values represent the means ± SE. *P < 0.05, **P < 0.01 compared with the sham group. #P < 0.05, ##P < 0.01 compared with the control IgG-treated group. N.S.: Not significant.
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f2: Effects of anti-HMGB1 mAb on mobilization of HMGB1 in SAH rats.Effects of anti-HMGB1 mAb were examined at 48 hr after the SAH in rats treated with anti-HMGB1 mAb or control IgG at both 5 min and 24 hr after blood injection. (a) The whole brain including the BA was prepared as coronal sections, and subjected to immunostaining for HMGB1 (Alexa Fluor 555) or α-smooth muscle actin (α-SMA, a marker protein of vascular smooth muscle cell) (Alexa Fluor 488) in the BA. All sections were counterstained with DAPI. Representative images of the BA in each group are shown. Arrowheads indicate the nuclei staining negative for HMGB1 in VSMCs. (b) Representative images of the HMGB1 translocation (arrows) to the cytoplasm from the nucleus in the control IgG-treated group are shown. (c) Nuclear localization of HMGB1 in VSMCs was evaluated by the ratio of the nuclei staining negative for HMGB1 to whole nuclei on immunofluorescent staining. (d) Total RNA was extracted from the isolated BA and subjected to quantitative RT-PCR for analysis of the expression of HMGB1 mRNA. GAPDH was used as a housekeeping gene. (e) The plasma concentration of HMGB1 was measured by ELISA. The scale bars indicate 20 μm. Results are shown for the sham group (Sham, n = 3, 4, 5 in (a–e), respectively), the control IgG-treated group (Cont IgG, n = 5, 4, 9 in (a–e), respectively), and the anti-HMGB1 mAb-treated group (α-HMGB1, n = 5, 4, 11 to in (a–e), respectively). Values represent the means ± SE. *P < 0.05, **P < 0.01 compared with the sham group. #P < 0.05, ##P < 0.01 compared with the control IgG-treated group. N.S.: Not significant.
Mentions: In the sham group, strong HMGB1 immunoreactivities were observed in the nuclei of vascular smooth muscle cells (VSMCs) of the BA (Fig. 2a upper panels). In the control IgG-treated group at 48 hr after SAH, some of the VSMCs were depleted of nuclear HMGB1. Generally, HMGB1 immunoreactivities in VSMCs of the BA were more faint than in the sham group and, unlike in the sham group, were present in the marginal zone of each nucleus (Fig. 2a middle panels). These results suggest the extracellular translocation of HMGB1 in VSMCs. We also observed cytoplasmic vesicle-like HMGB1 immunoreactivity in the VSMCs of the control IgG-treated group (Fig. 2b), which was considered to indicate the extranuclear translocation of HMGB1. On the other hand, the administration of anti-HMGB1 mAb restored most of the intranuclear localization of HMGB1 in VSMCs (Fig. 2a lower panels).

View Article: PubMed Central - PubMed

ABSTRACT

Although delayed cerebral vasospasm (DCV) following subarachnoid hemorrhage (SAH) is closely related to the progression of brain damage, little is known about the molecular mechanism underlying its development. High mobility group box-1 (HMGB1) plays an important role as an initial inflammatory mediator in SAH. In this study, an SAH rat model was employed to evaluate the effects of anti-HMGB1 monoclonal antibody (mAb) on DCV after SAH. A vasoconstriction of the basilar artery (BA) associated with a reduction of nuclear HMGB1 and its translocation in vascular smooth muscle cells were observed in SAH rats, and anti-HMGB1 mAb administration significantly suppressed these effects. Up-regulations of inflammation-related molecules and vasoconstriction-mediating receptors in the BA of SAH rats were inhibited by anti-HMGB1 mAb treatment. Anti-HMGB1 mAb attenuated the enhanced vasocontractile response to thrombin of the isolated BA from SAH rats and prevented activation of cerebrocortical microglia. Moreover, locomotor activity and weight loss recovery were also enhanced by anti-HMGB1 mAb administration. The vasocontractile response of the BA under SAH may be induced by events that are downstream of responses to HMGB1-induced inflammation and inhibited by anti-HMGB1 mAb. Anti-HMGB1 mAb treatment may provide a novel therapeutic strategy for DCV and early brain injury after SAH.

No MeSH data available.


Related in: MedlinePlus