Multipotent mesenchymal stromal cells (MSCs) have potential therapeutic benefit for the treatment of neurological diseases and injury. MSCs interact with and alter brain parenchymal cells by direct cell-cell communication and/or by indirect secretion of factors and thereby promote functional recovery. In this study, we found that MSC treatment of rats subjected to middle cerebral artery occlusion (MCAo) significantly increased microRNA 133b (miR-133b) level in the ipsilateral hemisphere. In vitro, miR-133b levels in MSCs and in their exosomes increased after MSCs were exposed to ipsilateral ischemic tissue extracts from rats subjected to MCAo. miR-133b levels were also increased in primary cultured neurons and astrocytes treated with the exosome-enriched fractions released from these MSCs. Knockdown of miR-133b in MSCs confirmed that the increased miR-133b level in astrocytes is attributed to their transfer from MSCs. Further verification of this exosome-mediated intercellular communication was performed using a cel-miR-67 luciferase reporter system and an MSC-astrocyte coculture model. Cel-miR-67 in MSCs was transferred to astrocytes via exosomes between 50 and 100 nm in diameter. Our data suggest that the cel-miR-67 released from MSCs was primarily contained in exosomes. A gap junction intercellular communication inhibitor arrested the exosomal microRNA communication by inhibiting exosome release. Cultured neurons treated with exosome-enriched fractions from MSCs exposed to 72 hours post-MCAo brain extracts significantly increased the neurite branch number and total neurite length. This study provides the first demonstration that MSCs communicate with brain parenchymal cells and may regulate neurite outgrowth by transfer of miR-133b to neural cells via exosomes.

Exosomes are 30–150 nm vesicles secreted by a wide range of mammalian cells that can contain microRNA (miRNA). To test if marrow stromal cell (MSC) exosomes could be used as a vehicle for delivery of anti-tumor miRNAs, we transfected MSCs with a miR-146b expression plasmid, and harvested exosomes released by the MSCs. Intra-tumor injection of exosomes derived from miR-146-expressing MSCs significantly reduced glioma xenograft growth in a rat model of primary brain tumor.

MicroRNAs are small RNAs that attenuate protein expression by complementary binding to the 3′‐UTR of a target mRNA. Currently, very little is known about microRNAs after cerebral ischemia. In particular, microRNA‐21 (miR‐21) is a strong antiapoptotic factor in some biological systems. We investigated the role of miR‐21 after stroke in the rat. We employed in situ hybridization and laser capture microdissection in combination with real‐time RT‐PCR to investigate the expression of miR‐21 after stroke. In situ hybridization revealed that miR‐21 expression was upregulated in neurons of the ischemic boundary zone, and quantitative real‐time RT‐PCR analysis revealed that stroke increased mature miR‐21 levels by approximately threefold in neurons isolated from the ischemic boundary zone by laser capture microdissection as compared with homologous contralateral neurons 2 days (n = 4; P < 0.05) and 7 days (n = 3; P < 0.05) after stroke. In vitro, overexpression of miR‐21 in cultured cortical neurons substantially suppressed oxygen and glucose deprivation‐induced apoptotic cell death, whereas attenuation of endogenous miR‐21 by antisense inhibition exacerbated cell death after oxygen and glucose deprivation. Moreover, overexpression of miR‐21 in neurons significantly reduced FASLG levels, and introduction of an miR‐21 mimic into 293‐HEK cells substantially reduced luciferase activity in a reporter system containing the 3′‐UTR of Faslg. Our data indicate that overexpression of miR‐21 protects against ischemic neuronal death, and that downregulation of FASLG, a tumor necrosis factor‐α family member and an important cell death‐inducing ligand whose gene is targeted by miR‐21, probably mediates the neuroprotective effect. These novel findings suggest that miR‐21 may be an attractive therapeutic molecule for treatment of stroke.

Blockage of Tie2 with siRNA-Tie2 and a Tie2-neutralizing antibody did not suppress ANG2-enhanced migration. However, inhibition of matrix metalloproteinases with GM6001 blocked ANG2-enhanced migration. Collectively, our data suggest that interaction of ANG2, a proangiogenic factor, with its receptor Tie2 promotes neural progenitor cell differentiation into neuronal lineage cells, whereas ANG2 regulates neural progenitor cell migration through matrix metalloproteinases, which do not require its receptor Tie2.The mammalian brain contains neural stem and progenitor cells in the sub-granular zone of the dentate gyrus and the subventricular zone (SVZ) 2 of the lateral ventricles to generate new neurons throughout lifetime (1-5). Neuroblasts generated in the SVZ migrate in chains rostrally toward the olfactory bulb where they differentiate into olfactory interneurons (6 -8).Cerebral ischemia increases neurogenesis in the SVZ (9 -12), and many SVZ neuroblasts migrate laterally toward the ischemic boundary zone (11,13,14). Upon arrival, some neuroblasts exhibit markers of striatal neurons (9, 10, 15). However, the molecules that mediate stroke-induced neurogenesis have not been fully investigated.The angiopoietins, including angiopoietin 1 (ANG1) and angiopoietin 2 are a family of structurally related proteins that bind with similar specificity and affinity to a common endothelial cell-specific receptor-tyrosine kinase (Tie2) (16). ANG1 and ANG2/Tie2 signaling play important roles in the angiogenic process and hematopoiesis (17, 18). The function of ANG2 is context-dependent. When acting in the absence of angiogenic inducers (such as vascular endothelial growth factor), ANG2 induced endothelial cell apoptosis with consequent vascular regression (19). When acting in concert with vascular endothelial growth factor, ANG2 may stimulate endothelial cell migration and proliferation, thus serving as a permissive angiogenic signal (19,20). ANG2 is also a critical effector of hypoxia-induced neovasculature and is involved in cerebral angiogenesis in the ischemic brain (21-24).Emerging evidence indicates that angiogenesis is coupled with neurogenesis under physiological and pathophysiological conditions (25-32). Neuroblasts in the SVZ could use cerebral blood vessels as a scaffold to migrate to the ischemic striatum (25). Cerebral endothelial cells activated by stroke promote neural progenitor cell differentiation into neurons, whereas ischemic neural progenitor cells facilitate angiogenesis (29). Vascular endothelial growth factor mediates the coupling of angiogenesis and neurogenesis in ischemic brain (27,29). In addition to vascular endothelial growth factor, stroke up-regulates ANG2 expression in SVZ neural progenitor cells (26). In the present study we investigated the effect of ANG2 on differentiation and migration of adult SVZ progenitor cells. There is no single marker to identify SVZ stem cells in the adult rodent brain. Therefore, in the present study the term "neural progen-* This work was supported, in whole or in par...

Oligodendrocytes are sensitive to ischemic damage. The Sonic hedgehog (Shh) pathway is critical in oligodendrogenesis; Gli1 is the principal effector of Shh signaling. We investigated oligodendrogenesis and Shh/Gli1 pathway activation after bone marrow stromal cell (BMSC) treatment of stroke in rats. Rats were subjected to the middle cerebral artery occlusion (MCAo). BMSCs have been shown to promote functional recovery post stroke. A therapeutic dose of BMSC (3times106 cells) treatment was initiated 1 day after MCAo. Immunohistochemistry was carried out to measure the oligodendrocyte progenitor cells, oligodendrocytes, myelin, and expressions of Shh and Gli1 at 14 days after MCAo. Gene expression of Shh and Gli1 was tested at 2 days after MCAo. An in vitro study was used to investigate the effects of BMSC on a premature oligodendrocyte cell line (N20.1 cells). BMSC treatment significantly increased 04+ oligodendrocytes, MBP+ area, and bromodeoxyuridine (BrdU)+, NG2+, BrdU+-NG2+ cells, and mRNA and protein expressions of Shh and Gli1 in the ipsilateral brain of the MCAo rats than that in phosphate buffered saline (PBS)-treated rats. BMSCs promoted N20.1 cell proliferation and Gli1 mRNA expression, and these effects were abolished by the Shh pathway inhibitor cyclopamine. These data indicate that the BMSC treatment stimulates oligodendrogenesis by activation of the Shh/Gli1 pathway post stroke.

Background and objective We investigated axonal plasticity in the bilateral motor cortices and the long term therapeutic effect of Niaspan on axonal remodeling after stroke in type-1 diabetic (T1DM) rats. Experimental approaches T1DM was induced in young adult male Wistar rats via injection of streptozotocin. T1DM rats were subjected to 2 h transient middle cerebral artery occlusion (MCAo) and were treated with 40 mg/kg Niaspan or saline starting 24 h after MCAo and daily for 28 days. Anterograde tracing using biotinylated dextran amine (BDA) injected into the contralateral motor cortex was performed to assess axonal sprouting in the ipsilateral motor cortex area. Functional outcome, SMI-31 (a pan-axonal microfilament marker), Bielschowsky silver and synaptophysin expression were measured. In vitro studies using primary cortical neuron (PCN) cultures and in vivo BDA injection into the brain to anterogradely label axons and terminals were employed. Results Niaspan treatment of stroke in T1DM–MCAo rats significantly improved functional outcome after stroke and increased SMI-31, Bielschowsky silver and synaptophysin expression in the ischemic brain compared to saline treated T1DM–MCAo rats (p<0.05). Using BDA to anterograde label axons and terminals, Niaspan treatment significantly increased axonal density in ipsilateral motor cortex in T1DM–MCAo rats (p<0.05, n=7/group). Niacin treatment of PCN significantly increased Ang1 expression under high glucose condition. Niacin and Ang1 significantly increased neurite outgrowth, and anti-Ang1 antibody marginally attenuated Niacin induced neurite outgrowth (p=0.06, n=6/group) in cultured PCN under high glucose condition. Conclusion Niaspan treatment increased ischemic brain Ang1 expression and promoted axonal remodeling in the ischemic brain as well as improved functional outcome after stroke. Ang1 may partially contribute to Niaspan-induced axonal remodeling after stroke in T1DM-rats.

Background and Purpose Treatment with a selective proteasome inhibitor, VELCADE, in combination with tissue plasminogen activator (tPA) extended the therapeutic window to 6 hours in young rats after stroke. However, stroke is a major cause of death and disability in the elderly. The present study investigated the effect of VELCADE in combination with a low-dose tPA on aged rats after embolic stroke. Methods Male Wistar rats at the age of 18 to 20 months were treated with VELCADE (0.2 mg/kg) alone, a low-dose tPA (5 mg/kg) alone, combination of VELCADE and tPA, or saline 2 hours after embolic middle cerebral artery occlusion. To test the contribution of endothelial nitric oxide synthase to VELCADE-mediated neuroprotection, endothelial nitric oxide synthase knockout and wild-type mice were treated with VELCADE (0.5 mg/kg) 2 hours after embolic stroke. Results Treatment with VELCADE significantly reduced infarct volume, whereas tPA alone did not reduce infarct volume and aggravated blood–brain barrier disruption in aged rats compared with saline-treated rats. However, the combination treatment significantly enhanced the reduction of infarct volume, which was associated with an increase in endothelial nitric oxide synthase activity compared with saline-treated rats. Additionally, the combination treatment promoted thrombolysis and did not increase the incidence of hemorrhage transformation. VELCADE significantly reduced lesion volume in wild-type mice but failed to significantly reduce lesion volume in endothelial nitric oxide synthase knockout mice. Conclusions Treatment with VELCADE exerts a neuroprotective effect in aged rats after stroke. The combination of VELCADE with the low-dose tPA further amplifies the neuroprotective effect. Endothelial nitric oxide synthase at least partly contributes to VELCADE-mediated neuroprotection after stroke.

Analysis of Affymetrix Probe data from glioma patient samples in conjuction with patient Kaplan-Meier Survival Plot indicate that expression of a glioma suppressor gene doublecortin (DCX) favors glioma patient survival. From neurosphere formation in culture, Time-Lapse Microscopy video recording and tumor xenograft, we show that DCX synthesis significantly reduces self-renewal of brain tumor stem cells (BTSCs) in human primary glioma (YU-PG, HF66) cells from surgically-removed human glioma specimens and U87 cells in vitro and in vivo. Time-Lapse Microscopic video recording revealed that double transfection of YU-PG, HF66 and U87 cells with DCX and neurabin II caused incomplete cell cycle with failure of cytokinesis, i.e. endomitosis by dividing into three daughter cells from one mother BTSC. Activation of c-jun NH2-terminal kinase 1 (JNK1) after simvastatin (10nM) treatment of DCX+neurabin II+ BTSCs from YU-PG, HF66 and U87 cells induced terminal differentiation into neuron-like cells. TUNEL staining data demonstrated that JNK1 activation also induced apoptosis only in double transfected BTSCs with DCX and neurabin II, but not in single transfected BTSCs from YU-PG, HF66 and U87 cells. Western blot analysis showed that procaspase-3 was induced after DCX transfection and activated after simvastatin treatment in YU-PG, HF66 and U87 BTSCs. Sequential immunoprecipitation and Western blot data revealed that DCX synthesis blocked protein phosphatase-1 (PP1)/caspase-3 protein-protein interaction and increased PP1-DCX interaction. These data demonstrate that DCX synthesis induces apoptosis in BTSCs via a novel JNK1/neurabin II/DCX/PP1/caspase-3 pathway.