GSK-3β Inhibition Induced Neuroprotection, Regeneration, and Functional Recovery After Intracerebral Hemorrhagic Stroke

Abstract

Hemorrhagic stroke is a devastating disease that lacks effective therapies. In the present investigation, we tested 6-bromoindirubin-3'-oxime (BIO) as a selective glycogen synthase kinase-3β (GSK-3β) inhibitor in a mouse model of intracerebral hemorrhage (ICH). ICH was induced by injection of collagenase IV into the striatum of 8- to 10-week-old C57BL/6 mice. BIO (8 μg/kg, IP) was administered following either an acute delivery (0-2 h delay) or a prolonged regimen (every 48 h starting at 3 days post-ICH). At 2 days post-ICH, the acute BIO treatment significantly reduced the hematoma volume. In the perihematoma regions, BIO administration blocked GSK-3β phosphorylation/activation, increased Bcl-2 and β-catenin levels, and significantly increased viability of neurons and other cell types. The prolonged BIO regimen maintained a higher level of β-catenin, upregulated VEGF and BDNF, and promoted neurogenesis and angiogenesis in peri-injury zones at 14 days after ICH. The BIO treatment also promoted proliferation of neural stem cells (NSCs) and migration of nascent DCX+ neuroblasts from the subventricular zone (SVZ) to the lesioned cortex. BIO improved functional outcomes on both the neurological severity score and rotarod tests. The findings of this study corroborate the neuroprotective and regenerative effects of BIO and suggest that the Wnt/GSK-3β/β-catenin pathway may be explored for the treatment of acute or chronic ICH.

Figure 1.

Inhibitory effect of BIO on GSK-3β in the mouse brain. (A-C) Western blot analysis of 6-bromoindirubin-3′-oxime (BIO) target glycogen synthase kinase-3β (GSK-3β) was performed using tissue lysate from the perihemorrhagic regions following mouse intracerebral hemorrhage (ICH). (A) Representative immunoblots of all groups are shown. (B) Quantification of immunoblot intensities of total GSK-3β. (C) Quantification of immunoblot intensities of phospho-GSK-3β (Y216 residue). (D) Comparison of relative volumes of hematoma (mm³) in ICH animals treated with BIO. Mean+standard error of the mean (SEM). ^∗p < 0.05 compared to ICH-only group. n = 6–8 per group.

Figure 2.

Regulatory effects of BIO on β-catenin expression and other factors in the ICH brain. (A) Representative Western blot images of perihematoma tissue lysates collected at 2 days post-ICH. (B) Quantification of immunoblots of β-catenin and the antiapoptotic factor B-cell lymphoma 2 (Bcl-2). Samples are normalized to β-actin loading control, and groups are normalized to sham. (C) Representative Western blot data of perihematoma tissue lysates at 14 days post-ICH to evaluate long-term BIO effects. (D) Quantification of immunoblot intensities of β-catenin, vascular endothelial growth factor (VEGF), and brain-derived neurotrophic factor (BDNF). Samples are normalized to β-actin loading control, and groups are normalized to ICH + dimethyl sulfoxide (DMSO) group. Mean+SEM. ^∗p < 0.05 compared to ICH + DMSO group. ^#p < 0.05 compared to sham. n = 3 mice for sham group and n = 6–8 for other groups.

Figure 3.

BIO-enhanced viability of neurons in vitro and in the ICH brain. (A) Lactate dehydrogenase (LDH) release correlated with neuronal cell death following oxygen-glucose deprivation (OGD). A dose-dependent neuroprotective effect was observed in BIO-treated cultures. (B) Relative cell viabilities determined by the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay in OGD with and without BIO treatments. Mean+SEM. ^∗p < 0.05 compared to OGD + DMSO group. #p < 0.05 compared to OGD + 0.1 μM BIO group. (C) Representative images of multiplexed immunofluorescence of terminal deoxynucleotidyl transferase 2′-deoxyuridine 5′-triphosphate (dUTP) nick-end labeling (TUNEL, green), neuronal nuclei (NeuN, red), and 4′,6-diamidino-2-pheylindole (DAPI, blue). (D) Quantification of stereological counts of total cell numbers (DAPI⁺), numbers of cell death (TUNEL⁺ cells), and numbers of neuronal cell death (TUNEL⁺/NeuN⁺ colabeled cells, yellow signal). Mean + SEM. ^∗p<0.05 compared to ICH + DMSO group. n = 4–5 culture batches/group.

Figure 4.

BIO-enhanced neurogenesis in the perihematoma region at 14 days after ICH. (A) Immunofluorescence staining showed 5-bromo-2′-deoxyuridine (BrdU, red) and NeuN (green) colabeled cells (white arrow) in sham, vehicle group, and BIO treatment group. (B) Three-dimensional image confirmed BrdU and NeuN colabeling. (C) Quantification data for NeuN⁺/BrdU⁺-colabeled cell counting. BIO significantly increased the numbers of NeuN⁺/BrdU⁺-colabeled cells in the perihematoma region compared to the control group. NeuN: green; BrdU: red; Hoechst 33342: blue. Mean + SEM. ^∗p < 0.05. n = 8–12 animals/group.

Figure 5.

BIO-enhanced angiogenesis in perihematoma regions at 14 days after ICH. (A) Immunofluorescence staining showed BrdU (red) and glucose transporter 1 (GLUT1, green) colabeled cells (white arrow). (B) Three-dimensional image confirmed BrdU- and GLUT1-colabeled cells. (C) Quantification of GLUT1⁺/BrdU⁺-colabeled cells. BIO treatment resulted in greater numbers of GLUT1⁺/BrdU⁺-colabeled cells in the perihematoma region compared to the control group. GLUT1: green; BrdU: red; DAPI: blue. ^∗p < 0.05. Mean ± SEM. n = 8–12 animals/group.

Figure 6.

BIO-enhanced proliferation and migration of neuroblasts from the SVZ. (A) Immunofluorescence staining for doublecortin (DCX, green), BrdU (red), and DAPI (blue). (B–D) Quantification for DCX⁺, BrdU⁺, and DCX⁺/BrdU⁺ colabeling. BIO treatment resulted in greater numbers of DCX⁺ cells, proliferating cells (BrdU⁺ cells), and newly divided migratory neuroblasts (DCX⁺/BrdU⁺). Mean ± SEM. ^∗p < 0.05. ^∗∗p < 0.01. n = 8–12 animals/group.

Figure 7.

BIO-improved functional recovery after ICH. (A) Neurological severity (NSS) evaluation showed moderate neurological recovery at 3, 7, and 14 days after surgery, with significant improvement at 7 days. (B) Rotarod test at 3, 7, and 14 days after ICH. BIO significantly improved the motor function at 7 and 14 days. Mean+SEM. ^∗p < 0.01. ^∗∗p < 0.05. n = 12–16 animals/group.