Ischemic Boundary Zone Research Articles

MicroRNA-150 regulates blood-brain barrier permeability via Tie-2 after permanent middle cerebral artery occlusion in rats.

The mechanism of blood-brain barrier (BBB) disruption, involved in poststroke edema and hemorrhagic transformation, is important but elusive. We investigated microRNA-150 (miR-150)-mediated mechanism in the disruption of BBB after stroke in rats. We found that up-regulation of miR-150 increased permeability of BBB as detected by MRI after permanent middle cerebral artery occlusion in vivo as well as increased permeability of brain microvascular endothelial cells after oxygen-glucose deprivation in vitro. The expression of claudin-5, a key tight junction protein, was decreased in the ischemic boundary zone after up-regulation of miR-150. We found in brain microvascular endothelial cells that overexpression of miR-150 decreased not only cell survival rate but also the expression levels of claudin-5 after oxygen-glucose deprivation. With dual-luciferase assay, we confirmed that miR-150 could directly regulate the angiopoietin receptor Tie-2. Moreover, silencing Tie-2 with lentivirus-delivered small interfering RNA reversed the effect of miR-150 on endothelial permeability, cell survival, and claudin-5 expression. Furthermore, poststroke treatment with antagomir-150, a specific miR-150 antagonist, contributed to BBB protection, infarct volume reduction, and amelioration of neurologic deficits. Collectively, our findings suggested that miR-150 could regulate claudin-5 expression and endothelial cell survival by targeting Tie-2, thus affecting the permeability of BBB after permanent middle cerebral artery occlusion in rats, and that miR-150 might be a potential alternative target for the treatment of stroke.-Fang, Z., He, Q.-W., Li, Q., Chen, X.-L., Baral, S., Jin, H.-J., Zhu, Y.-Y., Li, M., Xia, Y.-P., Mao, L., Hu, B. MicroRNA-150 regulates blood-brain barrier permeability via Tie-2 after permanent middle cerebral artery occlusion in rats.

Simvastatin combined with bone marrow stromal cells treatment activatesastrocytes to ameliorate neurological function after ischemic stroke in rats

Evidence indicates that reactive astrocytes play an important role in neurological functional amelioration after stroke. Bone marrow stromal cell (BMSC) transplantation-promoted astrocyte-derived neurotrophic factors' expressions resulted in decreasing the number of apoptotic neurons and improving neurogenesis and angiogenesis in the ischemic boundary zone. Recent studies found that simvastatin had neuroprotective activity in acute stroke. However, it is still ambiguous as to whether simvastatin combined with BMSCs can further activate astrocytes, which contribute to neurological functional recovery after ischemic stroke. In our present study, we established a middle cerebral artery occlusion model in rats and found that combination treatment of simvastatin and BMSCs could significantly increase neurological functional scores and induce activated astrocytes to enhance stromal cell-derived factor-1 α, vascular endothelial growth factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor expressions compared with BMSC alone (P < 0.05) by immunofluorescence staining on the 7th day after ischemia. Furthermore, western blot results showed that combination treatment could notably activate the AKT/mammalian target of rapamycin signaling pathway in oxygen-glucose deprivation-induced astrocytes. It was superior to BMSC treatment at the 5-min time point (P < 0.01). Therefore, we think that combination treatment should be a more effective therapy after stroke due to synergic functions of supplementary approaches.

Human neural stem cells promote proliferation of endogenous neural stem cells and enhance angiogenesis in ischemic rat brain.

Transplantation of human neural stem cells into the dentate gyrus or ventricle of rodents has been reportedly to enhance neurogenesis. In this study, we examined endogenous stem cell proliferation and angiogenesis in the ischemic rat brain after the transplantation of human neural stem cells. Focal cerebral ischemia in the rat brain was induced by middle cerebral artery occlusion. Human neural stem cells were transplanted into the subventricular zone. The behavioral performance of human neural stem cells-treated ischemic rats was significantly improved and cerebral infarct volumes were reduced compared to those in untreated animals. Numerous transplanted human neural stem cells were alive and preferentially localized to the ipsilateral ischemic hemisphere. Furthermore, 5-bromo-2′-deoxyuridine-labeled endogenous neural stem cells were observed in the subventricular zone and hippocampus, where they differentiated into cells immunoreactive for the neural markers doublecortin, neuronal nuclear antigen NeuN, and astrocyte marker glial fibrillary acidic protein in human neural stem cells-treated rats, but not in the untreated ischemic animals. The number of 5-bromo-2′-deoxyuridine-positive ⁄ anti-von Willebrand factor-positive proliferating endothelial cells was higher in the ischemic boundary zone of human neural stem cells-treated rats than in controls. Finally, transplantation of human neural stem cells in the brains of rats with focal cerebral ischemia promoted the proliferation of endogenous neural stem cells and their differentiation into mature neural-like cells, and enhanced angiogenesis. This study provides valuable insights into the effect of human neural stem cell transplantation on focal cerebral ischemia, which can be applied to the development of an effective therapy for stroke.

D-4F Decreases White Matter Damage After Stroke in Mice.

Stroke-induced neuroinflammation and white matter damage are associated with neurological deficits. Whether D-4F, an apolipoprotein A-I mimetic peptide, treatment of stroke decreases neuroinflammation and white matter damage and improves functional outcome has not been investigated. Adult male C57BL/6 mice were subjected to permanent middle cerebral artery occlusion (MCAo) and were orally administered saline as a vehicle control and different doses of D-4F (2, 4, 8, 16, or 32 mg/kg) starting at 2 h after MCAo and daily until euthanized at 7 days after MCAo. D-4F treatment did not alter the blood levels of high-density lipoprotein, total cholesterol, triglyceride, blood-brain barrier leakage, and infarction volume compared with control group. D-4F (16 mg/kg) treatment of stroke significantly improved functional outcome, increased the white matter density and the number of oligodendrocyte progenitor cells in the ischemic boundary zone of the ipsilateral striatum, and increased myelin basic protein, insulin-like growth factor-1 (IGF1), but decreased inflammatory factor Toll-like receptor-4 and tumor necrosis factor-α expression in the ischemic brain 7 days after MCAo (P<0.05, n=11/group). The neurite/axonal outgrowth in primary cultured neurons was significantly increased when treated with D-4F (100 ng/mL) and IGF1 (100 ng/mL) compared with the nontreatment control. Inhibition of IGF1 significantly attenuated D-4F or IGF1 treatment-induced axonal outgrowth. D-4F-treatment did not increase oligodendrocyte-progenitor cell proliferation but decreased oligodendrocyte-progenitor cell death. D-4F treatment initiated 2 h after MCAo decreases neuroinflammation and white matter damage and improves functional outcome after stroke. D-4F-induced increase in IGF1 may contribute to D-4F-induced neurite/axonal outgrowth after stroke.

The Effect of Donor-Dependent Administration of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells following Focal Cerebral Ischemia in Rats

Stroke is an ischemic disease caused by clotted vessel-induced cell damage. It is characterized by high morbidity and mortality and is typically treated with a tissue plasminogen activator (tPA). However, this therapy is limited by temporal constraints. Recently, several studies have focused on cell therapy as an alternative treatment. Most researches have used fixed donor cell administration, and hence, the effect of donor-dependent cell administration is unknown. In this study, we administered 3 types of donor-derived human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) in the ischemic boundary zone of the ischemic stroke rat model. We then performed functional and pathological characterization using rotarod, the limb placement test, and immunofluorescent staining. We observed a significant decrease in neuron number, and notable stroke-like motor dysfunction, as assessed by the rotarod test (~40% decrease in time) and the limb placement test (4.5 point increase) in control rats with ischemic stroke. The neurobehavioral performance of the rats with ischemic stroke that were treated with hUCB-MSCs was significantly better than that of rats in the vehicle-injected control group. Regardless of which donor cells were used, hUCB-MSC transplantation resulted in an accumulation of neuronal progenitor cells, and angiogenic and tissue repair factors in the ischemic boundary zone. The neurogenic and angiogenic profiles of the 3 types of hUCB-MSCs were very similar. Our results suggest that intraparenchymal administration of hUCB-MSCs results in significant therapeutic effects in the ischemic brain regardless of the type of donor.

MicroRNA-107 contributes to post-stroke angiogenesis by targeting Dicer-1.

Previous studies have suggested that microRNA-107 (miR-107) regulates cell migration in tumor and promotes Hypoxia Inducible Factor 1α (HIF1α) regulated angiogenesis under hypoxia. We found that miR-107 was strongly expressed in ischemic boundary zone (IBZ) after permanent middle cerebral artery occlusion (pMCAO) in rats and inhibition of miR-107 could reduce capillary density in the IBZ after stroke. Such finding led us to hypothesize that miR-107 might regulate post-stroke angiogenesis and therefore serve as a therapeutic target. We also found that antagomir-107, a synthetic miR-107 inhibitor, decreased the number of capillaries in IBZ and increased overall infarct volume after pMCAO in rats. We demonstrated that miR-107 could directly down-regulate Dicer-1, a gene that encodes an enzyme essential for processing microRNA (miRNA) precursors. This resulted in translational desupression of VEGF (vascular endothelial growth factor) mRNA, thereby increasing expression of endothelial cell-derived VEGF (VEGF165/VEGF164), leading to angiogenesis after stroke. This process might be a protective mechanism for ischemia-induced cerebral injury and miR-107 might be used as a novel tool in stroke treatment.

Neuroprotective effects of bisperoxovanadium on cerebral ischemia by inflammation inhibition

PTEN is a dual specificity phosphatase and is implicated in inflammation and apoptosis of cerebral ischemia and reperfusion (I/R) injury. Bisperoxovanadium (Bpv), a specific inhibitor of PTEN’s phosphatase activity, has demonstrated powerful neuroprotective properties. We investigated the neuroprotective roles of Bpv in the rat model of middle cerebral artery occlusion (MCAO) cerebral I/R injury, and explored the modulation of inflammatory mediators and PI3K/Akt/GSK-3β pathways by Bpv. Our results showed that treatment with Bpv (0.2mg/kg/day) significantly decreased neurological deficit scores at 7 days after MCAO and infarct volume at 4 days after MCAO. The IL-10 concentration was increased and TNF-α concentration was decreased in the ischemic boundary zone of the cerebral cortex at 4 days after MCAO by Bpv. Furthermore, Bpv (0.2mg/kg/day) treatment significantly reduced PTEN mRNA and protein levels and increased PI3K, Akt and p-GSK-3β proteins expression in the ischemic boundary zone of the cerebral cortex at 4 days after MCAO. In conclusions, Bpv treatment demonstrates neuroprotective effects on cerebral ischemia and reperfusion injury of ischemic stroke rats and is associated with its modulation of inflammatory mediator production and up-regulation of PTEN downstream proteins PI3K, Akt and p-GSK-3β.

Valproic acid ameliorates ischemic brain injury in hyperglycemic rats with permanent middle cerebral occlusion

Valproic acid (VPA) is widely used for the clinical treatment of epilepsy. Previous studies have demonstrated that VPA ameliorates brain injury following experimental stroke. However, the effect of VPA in stroke models featuring comorbid conditions has not been fully explored. In this study, we investigate the effects of VPA on permanent ischemic stroke with hyperglycemia. Hyperglycemia was induced by streptozotocin (STZ) injection 3 days before. Test animals received a single injection of VPA immediately after induction of ischemia. Control animals received occlusion and physiological saline injection, or STZ, occlusion, and saline. Magnetic resonance imaging of cerebral blood flow (CBF) and apparent diffusion coefficient (ADC) was performed 60min after ischemia. Infarct volume, neurological deficits, rotarod test performance, and immunohistological markers were assessed 3 days after ischemia. Hyperglycemia significantly expanded the area of decreased of CBF and ADC, and increased the number of myeloperoxidase-positive cells, ionized calcium binding adapter molecule 1-positive cells, inducible nitric oxide synthase-positive cells, von Willebrand factor-positive cells, and Fluoro-Jade C-positive cells in the ischemic boundary zone, which was accompanied by increased infarct volume and deteriorated neurological deficit and rotarod test compared with normoglycemia (P<0.05). VPA significantly alleviated the aggravation of functional outcome accompanied by suppressing these inflammation, endothelial injury, and neuronal degeneration compared with saline-treated group (P<0.05). A single injection of VPA following permanent ischemia in STZ-induced hyperglycemic rats ameliorates neurological deficits and reduces neuronal degeneration by inhibiting inflammation and endovascular injury. VPA may be promising as a candidate therapy for human stroke.

Combination effect of memantine and mesenchymal stem cells on the recovery of brain damage in a rat model of brain ischemia

Whether the transplantation of neural progenitor cells (NPCs) derived from mesenchymal stem cells combined with memantine enhanced neuroprotective effects in an animal model of stroke was investigated. In combination therapy groups, significant reduction of infarct volume and improvement of neurological functions were observed at 3 days after middle cerebral artery occlusion (MCAO) compared to monotherapy. Co-administration of memantine and NPCs enhanced the antiapoptotic effect and reduced the number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells compared to the NPCs alone group at 7 days after MCAO. Intracarotid transplanted NPCs were localized to the ischemic boundary zone. Moreover, the number of transplanted NPCs on day 7 with combination therapy was significantly higher than those of NPCs monotherapy. Memantine and the combination of memantine + NPCs induced the expression of endogenous neurotrophic factor genes brain-derived neurotrophic factor, nerve growth factor, and glia-derived neurotrophic factor and also upregulated Arc (activity-regulated cytoskeleton associated protein). These effects might be responsible for the induced survival of NPCs and improved functional behavior. These finding indicates that combination therapy of memantine and NPCs enhances neuroprotection against ischemic brain injury.

Abstract W P110: ABCA1 Partially Mediates D-4F Protection of White Matter After Stroke

Introduction: White matter (WM) damage is associated with neurological deficits after stroke. Neuroinflammation increases WM damage. The ATP-binding cassette transporter A 1 (ABCA1) mediates the efflux of cholesterol to ApoA-1 and ApoE and is antiinflammatory and promotes axonal remodeling. D-4F is an ApoA-I mimetic peptide. We tested the hypothesis that D-4F treatment of stroke regulates WM damage and improves neurological functional outcome. Methods: C57BL/6 white type mice were subjected to permanent middle cerebral artery occlusion (MCAo) and were orally administered different doses of D-4F (2, 4, 8, 16 or 32mg/kg) starting at 2h after MCAo and daily for 7 days. Functional outcomes, blood biochemistry, BBB leakage, infarct volume, and WM and axonal changes were evaluated. Results: D-4F treatment did not alter the blood levels of HDL and total cholesterol, infarction volume and BBB leakage compared to control group. Treatment of stroke with D-4F 16mg/kg 1) significant improved neurological functional outcome (mNSS: 5.2±0.9 vs control 3.9±0.6; Left-foot-fault: 26.7±2.5 vs control 21.4±1.6); 2) did not change the number of oligodendrocyte progenitor cells (OPCs) and oligodendrocytes in the ischemic boundary zone (IBZ) of the striatum; 3) significant increased the density of axons (30.1±1.8% vs control: 19.9±1.7%), phosphorelated neurofilament (39.4±3.5 vs control: 25.3±2.7) and myelin (40.4±2.4 vs control: 25.2±3.6) in the WM bundles in the IBZ of the striatum; 4) increased ABCA1 and decreased TNFα expression in the ischemic brain (n=11/group). In vitro, neurite outgrowth in primary cultured neurons (PCNs) derived from brain specific ABCA1 deficient (ABCA1-B/-B) mice were significantly decreased after hypoxic ischemia compared with PCNs derived from floxed-control (ABCA1fl/fl) mice; D-4F (200ng/ml) treatment significantly increased neurite outgrowth in the ABCA1fl/fl-PCNs, but failed in ABCA1-B/-B-PCNs; D-4F treatment did not increase OPC proliferation, but decreased OPC death of cultured OPCs. Conclusions: D-4F treatment initiated 2h after MCAo decreases WM damage and improves functional outcome after stroke. Increasing ABCA1 expression in the ischemic brain may contribute to D-4F induced WM and axonal protection after stroke.

Abstract 165: Microrna-126 Contributes to Human Umbilicus Cord Blood Cell Induced Neurorestorative Effects in Type Two Diabetes Mice

Introduction: Human umbilical cord blood cell (HUCBC) treatment of stroke induces neurorestorative effects in non-diabetic rats. Whether HUCBC induces neurorestorative effects in type two diabetes (T2DM) has not been investigated. In this study, we tested the neurorestorative therapeutic effects and underlying mechanisms of action of HUCBC treatment of stroke in T2DM mice. Methods: BKS.Cg-m+/+Lepr db /J (db/db)-T2DM mice were subjected to extraluminal permanent distal MCAo ( dMCAo ) and were treated with: 1) PBS control; 2) HUCBC (1x10 6 ) at 3 days after dMCAo. A battery of functional tests was performed. Mice were sacrificed at 14 days after dMCAo. Vascular and white matter (WM) changes and microRNA were measured. Results: HUCBC treatment of stroke in T2DM mice does not decrease lesion volume (12.7±3.0% vs control: 14.5±3.2%), but significantly improves functional outcome, increases axon (Bielschowsky silver), myelin (Luxol fast blue) and arteriolar (a-SMA) density in the ischemic boundary zone (IBZ) compared to T2DM-dMCAo control mice (p&lt;0.05). To test the mechanisms of HUCBC induced neurorestorative effects, serum and ischemic brain miR-126 expression were measured. T2DM mice exhibit significantly decreased miR-126 expression in serum and in the IBZ compared to db+ (no-DM) control mice (p&lt;0.05). While HUCBC treatment in T2DM mice significantly increased miR-126 expression compared to non-treatment T2DM mice. In vitro, we found that HUCBC secretes miR-126 enriched exosomes and also stimulates brain endothelial cell miR-126 expression. Co-culture of brain endothelial cells (BECs) with primary cortical neurons (PCNs) increases PCN axon outgrowth. Knockdown of miR-126 in BECs not only decreases capillary tube formation, but also decreases PCN axonal outgrowth compared to miR-126-/-Con-BECs (p&lt;0.05). Knockdown miR-126 in HUCBC (miR-126-/-HUCBC) treatment of stroke in T2DM mice significantly decreases miR-126 expression in blood serum and attenuates HUCBC induced functional outcome after stroke in T2DM mice. Conclusion: HUCBC treatment initiated 3 days after stroke improves functional outcome, increases WM and vascular remodeling in T2DM mice. Increasing miR-126 contribute to HUCBC induced neurorestorative effects in T2DM.

Abstract W P90: Valproic Acid Ameliorates Ischemic Brain Injury in Hyperglycemic Rats with Permanent Middle Cerebral Occlusion

Background: Valproic acid (VPA) is widely used for the treatment of epilepsy in clinic. Some previous studies demonstrated that VPA ameliorated brain injury following experimental stroke. However, the effect of VPA in stroke model with comorbid conditions has not been fully studied. In this study, we investigated the effects of VPA in permanent ischemic stroke with hyperglycemia. Methods: Male Sprague-Dawley rats were subjected to permanent middle cerebral artery occlusion according to a modification of the Tamura method. Hyperglycemia was induced by streptozotocin (STZ) injection 3 days before ischemia induction. The animals received a single injection of VPA immediately after the induction of ischemia. Vehicle-treated animals underwent the same procedure with physiological saline. Infarct volume, neurological deficits and immunohistological assessments were performed 3 days after ischemia. Results: Hyperglycemia significantly enhanced the number of myeloperoxidase-positive cells, ionized calcium binding adapter molecule 1-positive cells, inducible nitric oxide synthase-positive cells, von Willebrand factor-positive cells, and Fluoro-Jade C-positive cells in the ischemic boundary zone, which was accompanied by increased infarct volume and neurological deficit compared with normoglycemia (p &lt; 0.05; Figure). VPA significantly alleviated the aggravation of neurological deficit by suppressing these inflammation, endothelial injury, and neuronal degeneration compared with saline-treated group (p &lt; 0.05; Figure). Conclusions: Our results showed that VPA ameliorated neurological deficits through modulating the inflammatory responses and protecting endothelial damage, leading to reduced neuronal cell death following ischemia in STZ-induced hyperglycemic rats.

Neuroprotective effects of cilostazol are mediated by multiple mechanisms in a mouse model of permanent focal ischemia

The phosphodiesterase (PDE) 3 inhibitor cilostazol, used as an anti-platelet drug, reportedly can also ameliorate ischemic brain injury. Here, we investigated the effects of cilostazol in a permanent focal ischemia mice model. Male Balb/c mice were subjected to permanent middle cerebral artery occlusion. Mice were then treated with either cilostazol (10 or 20mg/kg) or vehicle administered at 30min and 24h post-ischemia, and infarct volumes were assessed at 48h post-ischemia. Mice treated with 20mg/kg of cilostazol or vehicle were sacrificed at 6h or 24h post-ischemia and immunohistochemistry was used for brain sections. Treatment with 20mg/kg of cilostazol significantly reduced infarct volumes to 70.1% of those with vehicle treatment. Immunohistochemistry results for 8-hydroxydeoxyguanosine (OHdG) expression showed that some neurons underwent oxidative stress around the ischemic boundary zone at 6h post-ischemia. Cilostazol treatment significantly reduced the percentage of 8-OHdG-positive neurons (65.8±33.5% with vehicle and 21.3±9.9% with cilostazol). Moreover, NADPH oxidase (NOX) 2-positive neurons were significantly reduced with cilostazol treatment. In contrast, immunohistochemistry results for phosphorylated cyclic-AMP response element binding protein (pCREB) showed that there were significantly more pCREB-positive neurons around the ischemic boundary zone of cilostazol-treated mice than in those of vehicle-treated mice at 24h post-ischemia. These results suggested that cilostazol might have multiple mechanisms of action to ameliorate ischemic tissue damage, by attenuating oxidative stress mediated by suppressing NOX2 expression by ischemic neurons and an anti-apoptotic effect mediated through the pCREB pathway.

Empowering sonic hedgehog to rescue brain cells after ischemic stroke.

Ischemic stroke occurs when blood supply to the brain is interrupted. This can cause irreversible injury to the central nervous system (CNS) tissue. Each year in the United States almost 800,000 people experience a new or recurrent stroke. 15% of stroke patients die shortly after insult and only 10% recover completely, leaving the majority of surviving stroke patients with disabilities. Tissue-type plasminogen activator (tPA) is the only available therapy for stroke but its clinical use is limited because of associated danger of intracranial hemorrhage. Therefore, there is an emergent need for stroke therapeutics that are safe and effective when administered at a later time point after insult.

Biochanin A protects against focal cerebral ischemia/reperfusion in rats via inhibition of p38-mediated inflammatory responses

Biochanin A, an O-methylated natural isoflavonoid classified as phytoestrogen, has been reported to show anti-tumorigenesis, anti-oxidation, and anti-inflammatory properties. However, little is known about the effects of biochanin A on cerebral ischemia/reperfusion. In this study, the neuroprotective and anti-inflammatory effects of biochanin A against ischemia/reperfusion injury, as well as the related molecular mechanisms, were investigated in rat models. Male Sprague–Dawley rats were subjected to middle cerebral artery occlusion (MCAO) for 2h, followed by 24h of reperfusion. Then neurological deficits, infarct volume and brain edema were evaluated. The MPO activity and TNF-α and IL-1β levels in ischemic boundary zone were determined by a spectrophotometer and the enzyme-linked immunosorbent assay (ELISA). The expressions of TNF-α, IL-1β, and phosphorylation of p38 were measured by RT-PCR or Western blotting. Consequently, our findings showed that biochanin A treatment for 14days had significantly reduced infarct volume and brain edema, and improved neurological deficits in focal cerebral ischemia/reperfusion rats. The MPO activity and TNF-α and IL-1β levels were greatly increased after ischemia/reperfusion injury, while treatment with biochanin A dramatically suppressed these inflammatory processes. Furthermore, biochanin A attenuated the increase in p-p38 level in the ischemia/reperfusion brain tissue. Taken together, biochanin A has been shown to have neuroprotective effects in cerebral ischemia/reperfusion, and the mechanisms may correlate with inhibiting inflammatory response, as well as the inactivation of p38 signaling pathway.

Effect of bone marrow stromal cell transplantation on neurologic function and expression of VEGF in rats with focal cerebral ischemia.

There is evidence that the transplantation of mesenchymal stem cells into rat models of cerebral ischemia reduces ischemic damage; however, the mechanism remains to be elucidated. The present study aimed to assess the effect of transplantation of human bone marrow stromal cells (hBMSCs) on neurologic function and the expression of vascular endothelial growth factor (VEGF) in a rat model of focal cerebral ischemia. The left middle cerebral artery of adult Wistar rats was occluded for 90 min using a nylon thread, followed by reperfusion for 1 h. hBMSCs labeled with 5-bromo-2-deoxyuridine (BrdU) were stereotaxically injected into the ischemic boundary zone. Behavioral analysis using the Neurological Severity Score (NSS) was conducted on days 1, 3, 7 and 28, and a histologic evaluation was performed simultaneously. VEGF was detected by immunofluorescence staining and western blot analysis. Fifty rats were divided equally into five groups: Normal control, sham-operated, operated (no transplantation), Dulbecco’s medium Eagle’s medium (DMEM)-injected (received only serum-free DMEM), and hBMSC-transplanted. The hBMSC-transplanted group showed significantly improved behavioral recovery compared with the operated and DMEM-transplanted groups on days 3, 7 and 28. Histological examination showed that transplanted cells migrated from the injection site into nearby areas including the cortex. Expression of VEGF was significantly greater in the hBMSC group compared with the other four groups on each assessment day. The expression of VEGF was found to be beneficial for functional recovery following cerebral ischemic injury and hBMSC transplantation stimulated the expression of VEGF. Transplantation of BMSCs may be a promising therapeutic strategy for treating cerebral infarction.

Beneficial effects of gfap/vimentin reactive astrocytes for axonal remodeling and motor behavioral recovery in mice after stroke.

The functional role of reactive astrocytes after stroke is controversial. To elucidate whether reactive astrocytes contribute to neurological recovery, we compared behavioral outcome, axonal remodeling of the corticospinal tract (CST), and the spatio-temporal change of chondroitin sulfate proteoglycan (CSPG) expression between wild-type (WT) and glial fibrillary acidic protein/vimentin double knockout (GFAP(-/-) Vim(-/-) ) mice subjected to Rose Bengal induced cerebral cortical photothrombotic stroke in the right forelimb motor area. A foot-fault test and a single pellet reaching test were performed prior to and on day 3 after stroke, and weekly thereafter to monitor functional deficit and recovery. Biotinylated dextran amine (BDA) was injected into the left motor cortex to anterogradely label the CST axons. Compared with WT mice, the motor functional recovery and BDA-positive CST axonal length in the denervated side of the cervical gray matter were significantly reduced in GFAP(-/-) Vim(-/-) mice (n = 10/group, P < 0.01). Immunohistological data showed that in GFAP(-/-) Vim(-/-) mice, in which astrocytic reactivity is attenuated, CSPG expression was significantly increased in the lesion remote areas in both hemispheres, but decreased in the ischemic lesion boundary zone, compared with WT mice (n = 12/group, P < 0.001). Our data suggest that attenuated astrocytic reactivity impairs or delays neurological recovery by reducing CST axonal remodeling in the denervated spinal cord. Thus, manipulation of astrocytic reactivity post stroke may represent a therapeutic target for neurorestorative strategies.

IL-8 enhances the angiogenic potential of human bone marrow mesenchymal stem cells by increasing vascular endothelial growth factor.

The beneficial effects of mesenchymal stem cells (MSCs) are mediated partly by the paracrine production of cytoprotective and trophic factors. Vascular endothelial growth factor (VEGF) is released from MSCs as a paracrine trophic factor and contributes to the therapeutic effects of the stem cell by regulating angiogenesis and promoting revascularization in injured tissues. Interleukin-8 (IL-8), an inflammatory chemokine with potent proangiogenic properties, is upregulated in the ischemic brain and has been shown to promote homing of bone marrow-derived cells to injured sites. However, the effect of IL-8 on MSCs paracrine function remains unknown. We found that IL-8 induced VEGF production and phosphorylation of Akt and ERK. Both effects could be blocked by inhibitors (LY294002, PD098059) or siRNA-mediated silencing of Akt and ERK in human bone marrow MSCs (hBM-MSCs). IL-8-induced VEGF production in hBM-MSCs significantly increased tube formation on Matrigel compared with basal secreted VEGF. In a rat stroke model, administration of IL-8-treated hBM-MSCs decreased the infarction volume and increased angiogenesis in the ischemic boundary zone compared with hBM-MSC treatment alone. In conclusion, IL-8 stimulates VEGF production in hBM-MSCs in part via the PI3K/Akt and MAPK/ERK signal transduction pathways and that administration of IL-8-treated hBM-MSCs increases angiogenesis after stroke. This approach may be used to optimize MSC-based therapies for numerous diseases including stroke, myocardial ischemia, and spinal cord injury.

Omega-3 polyunsaturated fatty acids enhance cerebral angiogenesis and provide long-term protection after stroke

Stroke is a devastating neurological disorder and one of the leading causes of death and serious disability. After cerebral ischemia, revascularization in the ischemic boundary zone provides nutritive blood flow as well as various growth factors to promote the survival and activity of neurons and neural progenitor cells. Enhancement of angiogenesis and the resulting improvement of cerebral microcirculation are key restorative mechanisms and represent an important therapeutic strategy for ischemic stroke. In the present study, we tested the hypothesis that post-stroke angiogenesis would be enhanced by omega-3 polyunsaturated fatty acids (n−3 PUFAs), a major component of dietary fish oil. To this end, we found that transgenic fat-1 mice that overproduce n−3 PUFAs exhibited long-term behavioral and histological protection against transient focal cerebral ischemia (tFCI). Importantly, fat-1 transgenic mice also exhibited robust improvements in revascularization and angiogenesis compared to wild type littermates, suggesting a potential role for n−3 fatty acids in post-stroke cerebrovascular remodeling. Mechanistically, n−3 PUFAs induced upregulation of angiopoietin 2 (Ang 2) in astrocytes after tFCI and stimulated extracellular Ang 2 release from cultured astrocytes after oxygen and glucose deprivation. Ang 2 facilitated endothelial proliferation and barrier formation in vitro by potentiating the effects of VEGF on phospholipase Cγ1 and Src signaling. Consistent with these findings, blockade of Src activity in post-stroke fat-1 mice impaired n−3 PUFA-induced angiogenesis and exacerbated long-term neurological outcomes. Taken together, our findings strongly suggest that n−3 PUFA supplementation is a potential angiogenic treatment capable of augmenting brain repair and improving long-term functional recovery after cerebral ischemia.

Valproic acid enhances the effect of bone marrow-derived mononuclear cells in a rat ischemic stroke model

Bone marrow derived mononuclear cell (MNC) transplantation is a potential therapy for ischemic stroke. Here, we hypothesized that valproic acid (VPA) would modulate transplantation effects of MNCs in a rat ischemic stroke model. Male Sprague–Dawley rats were subjected to transient 90min middle cerebral artery occlusion. Infarct volume, neurological outcome, and immunohistological assessments were performed 7 days after ischemia. MNCs injected 6 or 24h but not 48 or 72h after ischemia significantly reduced infarct volume and improved neurological deficits. We then tested whether the therapeutic window of MNC transplantation could be expanded through combination therapy with VPA. MNC transplantation at 48h combined with VPA injection three times at 47, 53, and 72h after ischemia significantly ameliorated infarct volume and neurological deficits compared to a vehicle group. Combination therapy reduced the number of myeloperoxidase-positive cells, ionized calcium binding adapter molecule 1-positive cells, tumor necrosis factor-α-positive cells, and von Willebrand factor-positive cells in the ischemic boundary zone. The number of engrafted MNCs that were fluorescently labeled with PKH 26, on day 7, was significantly higher after combination therapy than after that MNC transplantation alone. Our results demonstrated that combination therapy with VPA enhanced the anti-inflammatory and vasculo-protective effects against endothelial damage following ischemia, and increased the survival of transplanted cells, leading to expansion of the therapeutic time window for MNC transplantation. Together, these findings suggest that VPA may be an appropriate partner for cell-based treatment of ischemic stroke.