Guanghui Tang

CXCR4 Antagonist AMD3100 Protects Blood–Brain Barrier Integrity and Reduces Inflammatory Response After Focal Ischemia in Mice

Background and Purpose— Inflammatory response plays a critical role in propagating tissue damage after focal cerebral ischemia. CXCL12 is a key chemokine for leukocyte recruitment. However, the role of CXCL12 and its receptor CXCR4 in ischemia-induced inflammatory response is unclear. Here we use the pharmacological antagonist of CXCR4, AMD3100, to investigate the function of CXCL12/CXCR4 in regulating inflammatory response during acute ischemia. Methods— Adult male CD-1 mice (n=184) underwent permanent suture middle cerebral artery occlusion (MCAO). AMD3100 was injected for 3 days (1 mg/kg/day) after MCAO. Brain water content, infarct volume, neurological score, and myeloperoxidase (MPO) expression and activity were examined at 24, 48, and 72 hours after MCAO. Proinflammatory cytokine RNA and protein levels in brain tissue were measured by RT-PCR and enzyme linked immunosorbent assay. Results— Neurological score was greatly improved in AMD3100-treated mice compared with the control mice 3 days after MCAO (P<0.05). Brain edema–induced change of water content, IgG protein leakage, Evans blue extravasation, occludin, and ZO-1 expression in ipsilateral hemisphere were alleviated by acute treatment of AMD3100. MPO expression and activity revealed that AMD3100 profoundly reduced the number of MPO-positive cells in the ischemic region (P<0.05). It also attenuated proinflammatory cytokines including interleukin 6, tumor necrosis factor &agr;, and interferon &ggr;; their mRNA and protein levels changed accordingly compared with the controls (P<0.05). Conclusions— CXCR4 antagonist AMD3100 significantly suppressed inflammatory response and reduced blood–brain barrier disruption after MCAO. AMD3100 attenuated ischemia-induced acute inflammation by suppressing leukocyte migration and infiltration, in addition to reducing proinflammatory cytokine expression in the ischemic region.

Netrin-1 Hyperexpression in Mouse Brain Promotes Angiogenesis and Long-Term Neurological Recovery After Transient Focal Ischemia

Background and Purpose— Netrin-1 (NT-1) stimulates endothelial cell proliferation and migration in vitro and promotes focal neovascularization in the adult brain in vivo. This in vivo study in mice investigated the effect of NT-1 hyperexpression on focal angiogenesis and long-term functional outcome after transient middle cerebral artery occlusion (tMCAO). Methods— Adeno-associated viral vectors carrying either the NT-1 gene (AAV–NT-1) or GFP (AAV-GFP) were generated and injected into the brains of separate groups of 93 mice. Seven days later, tMCAO followed by 7–28 days of reperfusion were carried out. Histological outcomes and behavioral deficits were quantified 7–28 days after tMCAO. Small cerebral vessel network and angiogenesis were assessed 28 days after tMCAO, using synchrotron radiation microangiography and immunohistochemistry. Results— Western blot and immunohistochemistry showed that on the day of tMCAO, NT-1 hyperexpression had been achieved in both normal and ischemic hemispheres. Immunofluorescence imaging showed that NT-1 expression was primarily in neurons and astrocytes. Ischemia-induced infarction in the NT-1 hyperexpression group was attenuated in comparison to saline or AAV-GFP–treated groups (P<0.01). Similarly, neurological deficits were greatly improved in AAV–NT-1–treated mice compared with mice in saline or AAV-GFP–treated groups (P<0.05). In addition, angiogenesis was increased in AAV–NT-1–treated mice compared with the other 2 groups (P<0.05). In vivo synchrotron radiation microangiography 28 days after tMCAO revealed more branches in AAV–NT-1–treated mice than in other groups. Conclusions— AAV–NT-1 induced NT-1 hyperexpression before tMCAO reduced infarct size, enhanced neovascularization, and improved long-term functional recovery.

Mesenchymal Stem Cells Maintain Blood‐Brain Barrier Integrity by Inhibiting Aquaporin‐4 Upregulation After Cerebral Ischemia

Rationale: Cerebral ischemia upregulates aquaporin‐4 expression, increases blood‐brain barrier (BBB) permeability, and induces brain edema. Mesenchymal stem cells (MSCs) can repress inflammatory cytokines and show great potential for ischemic stroke therapy. However, the effect of MSCs regarding the protection of ischemia‐induced BBB break down is unknown. Objective: We test whether MSCs therapy protects BBB integrity and explore the molecular mechanisms of aquaporin‐4 on BBB integrity. Methods and Results: Two hundred and twenty‐eight adult CD1 male mice underwent 90 minutes transient middle cerebral artery occlusion and received 2 × 105 MSCs intracranial transplantation. The neurological severity score was improved and both ischemia‐induced brain edema and BBB leakage were reduced in MSC‐treated mice. MSCs therapy reduced astrocyte apoptosis and inhibited ischemia‐induced aquaporin‐4 upregulation. In addition, small‐interfering RNA knockdown of aquaporin‐4 after cerebral ischemia effectively reduced aquaporin‐4 expression, brain edema, BBB leakage, and astrocyte apoptosis. Conditional medium from lipopolysaccharide (LPS)‐activated microglia enhanced aquaporin‐4 expression, p38 and JNK phosphorylation, and apoptosis of cultured astrocytes. MSC treatment reduced the expression of inflammatory cytokines in LPS‐activated microglia, and subsequently reduced aquaporin‐4 expression and apoptosis of astrocytes. Knockdown of aquaporin‐4 in cultured astrocytes also reduced apoptosis. Treatment with p38 and JNK inhibitors showed that p38, but not the JNK signaling pathway, was responsible for the aquaporin‐4 upregulation. Conclusion: MSCs protected BBB integrity by reducing the apoptosis of astrocytes after ischemic attack, which was due to the attenuation of inflammatory response and downregulation of aquaporin‐4 expression via p38 signaling pathway. Stem Cells 2014;32:3150–3162

Metformin attenuates blood-brain barrier disruption in mice following middle cerebral artery occlusion

BackgroundMetformin, a widely used hypoglycemic drug, reduces stroke incidence and alleviates chronic inflammation in clinical trials. However, the effect of metformin in ischemic stroke is unclear. Here, we investigated the effect of metformin on ischemic stroke in mice and further explored the possible underlying mechanisms.MethodsNinety-eight adult male CD-1 mice underwent 90-minute transient middle cerebral artery occlusion (tMCAO). Metformin (200 mg/kg) was administrated for up to 14 days. Neurobehavioral outcomes, brain infarct volume, inflammatory factors, blood-brain barrier (BBB) permeability and AMPK signaling pathways were evaluated following tMCAO. Oxygen glucose deprivation was performed on bEND.3 cells to explore the mechanisms of metformin in inhibiting inflammatory signaling pathways.ResultsInfarct volume was reduced in metformin-treated mice compared to the control group following tMCAO (P < 0.05). Neurobehavioral outcomes were greatly improved in metformin-treated mice (P < 0.05). MPO+ cells, Gr1+ cells, MPO activity and BBB permeability were decreased after metformin administration (P < 0.05). In addition, metformin activated AMPK phosphorylation, inhibited NF- κB activation, down-regulated cytokine (IL-1 β, IL-6, TNF- α) and ICAM-1 expression following tMCAO (P < 0.05). Furthermore, metformin activated AMPK signaling pathway and alleviated oxygen-glucose deprivation-induced ICAM-1 expression in bEND.3 cells (P < 0.05). Compound C, a selective AMPK inhibitor, eliminated this promotional effect.ConclusionsMetformin down-regulated ICAM-1 in an AMPK-dependent manner, which could effectively prevent ischemia-induced brain injury by alleviating neutrophil infiltration, suggesting that metformin is a promising therapeutic agent in stroke therapy.

Postacute Stromal Cell–Derived Factor-1α Expression Promotes Neurovascular Recovery in Ischemic Mice

Background and Purpose— Acute interventions of stroke are often challenged by a narrow treatment window. In this study, we explore treatments in the postacute phase of stroke with wider windows of opportunity. We investigated the effects of stromal cell–derived factor (SDF-1&agr;) in neurovascular recovery during the postacute phase and downstream signaling pathways, underlying SDF-1&agr;–mediated neurovascular recovery. Methods— Adult male Institute of Cancer Research (ICR) mice underwent middle cerebral artery occlusion. One week after middle cerebral artery occlusion, the animals received stereotactic injection of adenoassociated virus (AAV) carrying SDF-1&agr; gene as treatment or AAV-green fluorescent protein as control and were monitored for 5 weeks. Neurobehavioral outcomes were evaluated, and brain atrophy was measured. Neurogenesis and angiogenesis were examined. The proliferation and migration of neural progenitor cells were evaluated. Downstream pathways of SDF-1&agr; were investigated. Inflammatory response was monitored. Results— Neurobehavioral outcomes were improved, and brain atrophy was greatly reduced for ⩽5 weeks in AAV-SDF-1&agr; groups when compared with the control. SDF-1 receptor CXCR4 was upregulated and colocalized with neural and endothelial progenitor cells. The number of nestin+ and doublecortin+/bromodeoxyuridine+ cells in the subventricular zone, doublecortin+ and neuron+/bromodeoxyuridine+ cells in the perifocal region, and cluster of differentiation (CD)31+ and bromodeoxyuridine+/CD31+ microvessels are also significantly increased in AAV-SDF-1&agr; groups. Administration of CXCR4 antagonist AMD3100 eliminated the beneficial effects of SDF-1&agr;. SDF-1&agr;/CXCR4 interaction activated AKT, extracellular signal-regulated kinases (ERK), and P38 mitogen-activated protein kinase (MAPK) signaling pathways but not the c-Jun N-terminal kinase (JNK) pathway. Conclusions— SDF-1&agr; promoted neurogenesis and angiogenesis during the postacute phase of ischemia without eliciting an inflammatory response. AAV-SDF-1&agr; expression represents a promising avenue for ischemic stroke therapy with a wider treatment window.

Silica-coated superparamagnetic iron oxide nanoparticles targeting of EPCs in ischemic brain injury

Intravenous transplantation of endothelial progenitor cells (EPCs) reduced ischemic brain injury. However, less cell homing to damaged sites limited its functions. In present study, we labeled EPCs with silica-coated superparamagnetic iron oxide nanoparticles (SiO4@SPIONs) and applied exterior magnetic field to guide SiO4@SPIONs-labeled EPCs (SiO4@SPIONs-EPCs) to the ischemic hemisphere of the brain. We optimized SiO4@SPIONs labeling dose, which did not affect proliferation, migration and tube formation of EPCs in vitro. SiO4@SPIONs-EPCs homing was greatly increased in ischemic hemisphere with magnetic field treatment in mice underwent transient middle cerebral artery occlusion (tMCAO). Injection of SiO4@SPIONs-EPCs and followed by magnetic field treatment showed improved neurobehavioral outcomes, reduced brain atrophic volume, increased microvessel density and VEGF expression in the ischemic perifocal region compared to groups without magnetic field treatment (p < 0.05). Our results demonstrated that exterior magnetic field could guide SiO4@SPIONs-EPCs to ischemic region and enhance therapeutic effect, suggesting that magnetic-guided SiO4@SPIONs-EPCs delivery is a promising approach in cerebral ischemic therapy.

Effect of HMGB1 on the Paracrine Action of EPC Promotes Post‐Ischemic Neovascularization in Mice

Transplantation of endothelial progenitor cells (EPCs) leads to better outcomes in experimental stroke, but the mechanism remains unclear. It was reported that astrocytic‐high mobility group box1 (HMGB1) promoted endogenous EPC‐mediated neurovascular remodeling during stroke recovery. It is unclear whether HMGB1 involves in exogenous EPC‐mediated stroke recovery. In this study, we aim to explore whether microglial HMGB1 contributes to human peripheral blood‐derived (hPB)‐EPCs‐mediated neurovascular remodeling by modulating the paracrine function of exogenous hPB‐EPCs. Coculturing hPB‐EPCs with lipopolysaccharides stimulated BV2 cells upregulated Interleukin‐8 expression in hPB‐EPCs; this was blocked by treating BV2 cells with HMGB1 inhibitor Glycyrrhizin. Conditioned medium (CM) of hPB‐EPCs cocultured with BV2 cells promoted the viability and tube formation of human umbilical cord vein cells. Inhibiting either HMGB1 or IL‐8 could block the effect of hPB‐EPCs CM. In vivo study showed hPB‐EPCs transplantation improved neurobehavioral outcomes, reduced brain atrophy volume, and enhanced neovascularization in transient middle cerebral artery occlusion (tMCAO) mice. Intraperitoneally administration of HMGB1 inhibitor glycyrrhizin blocked the beneficial effect of hPB‐EPC transplantation. We did not observe the integration of green fluorescent protein‐labeled hPB‐EPCs with microvessels in peri‐infarct areas at day‐14 after tMCAO. In summary, the result suggested that HMGB1 upregulation in postischemic brain could promote exogenous hPB‐EPC‐mediated stroke recovery by modulating paracrine function of hPB‐EPCs. Stem Cells 2014;32:2679–2689

MicroRNA-29b is a therapeutic target in cerebral ischemia associated with aquaporin 4

MicroRNA-29b (miR-29b) is involved in regulating ischemia process, but the molecular mechanism is unclear. In this work, we explored the function of miR-29b in cerebral ischemia. The level of miR-29b in white blood cells was evaluated in patients and mice after ischemic stroke. Brain infarct volume and National Institute of Health stroke scale (NIHSS) scores were analyzed to determine the relationship between miR-29b expression and the severity of stroke. The relationship of miR-29b and aquaporin-4 (AQP4) was further studied in mice. We found that miR-29b was significantly downregulated in stroke patients (P < 0.05). MiR-29b level negatively associated with NIHSS scores (r = −0.349, P < 0.01) and brain infarct volume (r = −0.321, P < 0.05). In ischemic mice, miR-29b in the brain and blood were both downregulated (r =0.723, P < 0.05). MiR-29b overexpression reduced infarct volume (49.50 ±6.55 versus 35.48 ±2.28 mm3, P < 0.05), edema (164±4% versus 108±4%, P < 0.05), and blood-brain barrier (BBB) disruption compared with controls (15 ±9% versus 7 ±3%, P < 0.05). Aquaporin-4 expression greatly decreased after miR-29b overexpression (28±7% versus 11 ±3%, P < 0.05). Dual-luciferase reporter system showed that AQP-4 was the direct target of miR-29b (P < 0.05). We concluded that miR-29b could potentially predict stroke outcomes as a novel circulating biomarker, and miR-29b overexpression reduced BBB disruption after ischemic stroke via downregulating AQP-4.

CXCL12 Gene Therapy Ameliorates Ischemia-Induced White Matter Injury in Mouse Brain

Remyelination is an important repair process after ischemic stroke‐induced white matter injury. It often fails because of the insufficient recruitment of oligodendrocyte progenitor cells (OPCs) to the demyelinated site or the inefficient differentiation of OPCs to oligodendrocytes. We investigated whether CXCL12 gene therapy promoted remyelination after middle cerebral artery occlusion in adult mice. The results showed that CXCL12 gene therapy at 1 week after ischemia could protect myelin sheath integrity in the perifocal region, increase the number of platelet‐derived growth factor receptor‐α (PDGFRα)‐positive and PDGFRα/bromodeoxyuridine‐double positive OPCs in the subventricular zone, and further enhance their migration to the ischemic lesion area. Coadministration of AMD3100, the antagonist for CXCL12 receptor CXCR4, eliminated the beneficial effect of CXCL12 on myelin sheath integrity and negatively influenced OPC proliferation and migration. At 5 weeks after ischemia, CXCR4 was found on the PDGFRα‐ and/or neuron/glia type 2 (NG2)‐positive OPCs but not on the myelin basic protein‐positive mature myelin sheaths, and CXCR7 was only expressed on the mature myelin sheath in the ischemic mouse brain. Our data indicated that CXCL12 gene therapy effectively protected white matter and promoted its repair after ischemic injury. The treatment at 1 week after ischemia is effective, suggesting that this strategy has a longer therapeutic time window than the treatments currently available.

Optical inhibition of striatal neurons promotes focal neurogenesis and neurobehavioral recovery in mice after middle cerebral artery occlusion

Striatal neurons regulate the activity of neural progenitor cells in the subventricular zone, but the effect of striatal neuronal activity on neurogenesis after ischemic stroke is unclear. In this study, we used optogenetic tools to investigate the impact of striatal neuronal activity on the neurogenesis and functional recovery after cerebral ischemia. We transfected striatal neurons with channelrhodopsin-2 or halorhodopsin from Natronomonas so that they can be excited by 473 nm laser or inhibited by 594 nm laser, respectively. Neural inhibition but not excitation at 4–7 days after middle cerebral artery occlusion resulted in reduced atrophy volume (6.8 ± 0.7 vs 8.5 ± 1.2 mm3, p < 0.05) and better performance represented by longer sustaining time on rotarod (99.3 ± 9 vs 80.1 ± 11 s, p < 0.01) and faster moving speed (7.7 ± 2 vs 5.7 ± 1.1 cm/s, p < 0.05) in open field tests. Furthermore, neural inhibition increased the number of nestin+, BrdU+/doublecortin+ and BrdU+/NeuN+ cells (p < 0.001) in the subventricular zone and peri-focal region, and the expression level of axon guidance factor Netrin-1 (0.39 ± 0.16 vs 0.16 ± 0.02, p < 0.05) in the peri-focal region. These data suggest that striatal neuronal activity plays an important role in regulating neurogenesis and neural-behavioral outcomes, and that inhibiting striatal neurons by optogenetics promotes the recovery after ischemic stroke in mice.