A disintegrin and metalloproteinase-17 (ADAM17) is involved in proteolytic ectodomain shedding of several membrane-bound growth factors and cytokines. The expression and activity of ADAM17 increase under some pathological conditions such as stroke and cancer. ADAM17 promotes neural progenitor cell migration and contributes to neurogenesis after stroke and breast cancer growth and invasion. In the present study, we sought to elucidate whether ADAM17 contributes to glioma progression. To this end, we examined the role of ADAM17 in the proliferation, invasion, and tube formation of U87 human glioma cells in vitro and tumor growth in vivo. Stable transfection of the U87 cell line with either a plasmid for over-expression of human ADAM17, or a siRNA to ADAM17 was employed in this study to establish high or low ADAM17 expression in glioma cells, respectively. For study of mechanism, the ADAM17 inhibitor TAPI-2 and the PI3K-AKT inhibitor LY294002 were used to counteract high ADAM17 expression and the activated PI3K-AKT pathway, respectively.
Proliferation of glioma cells were tested by thiazolyl blue tetrazolium bromide (MTT) assay, Bromodeoxyuridine incorporation assay, growth curve, and sulforhodamine B assay. Matrigel invasion assays were used to assess the ability of U87 cells to penetrate the extra-cellular matrix (ECM). A Matrigel tube formation assay was performed to test capillary tube formation ability. EGFR-PI3K-Akt pathway activation in U87 cells under different ADAM17 expression levels were tested by Western blot.
Our data show that ADAM17 promotes the U87 malignant phenotype by increased proliferation, invasion, angiogenesis and in vivo tumor growth. Tumor growth in nude mice was significantly inhibited by ADAM17 inhibitor and A17-shRNA in vivo transfection. TGF-α, VEGF secretion and VEGF expression was increased by ADAM17 and counteracted by ADAM17 siRNA, TAPI-2, and LY294002 in U87 cells. ADAM17 activated, whereas ADAM17 siRNA, TAPI-2, and LY294002 deactivated the EGFR-PI3K-AKT signal pathway, which correlated with U87 cell malignant phenotype changes.
This study suggests ADAM17 contributes to glioma progression through activation of the EGFR-PI3K-AKT signal pathway.
ADAM17; TACE; Glioma; EGFR-PI3K-AKT; Proliferation; Invasion; Angiogenesis; Tumor growth; RNA interference
As a thrombolytic agent, application of recombinant tissue plasminogen activator (tPA) to ischemic stroke is limited by the narrow time window and side effects on brain edema and hemorrhage. This study examined whether tPA, administered by intranasal delivery directly targeting the brain and spinal cord, provides therapeutic benefit during the subacute phase after stroke. Adult male Wistar rats were subjected to permanent right middle cerebral artery occlusion (MCAo). Animals were treated intranasally with saline, 60 μg or 600 μg recombinant human tPA at 7 and 14 days after MCAo (n=8/group), respectively. An adhesive-removal test and a foot-fault test were used to monitor functional recovery. Biotinylated dextran amine (BDA) was injected into the left motor cortex to anterogradely label the corticorubral tract (CRT) and the corticospinal tract (CST). Naive rats (n=6) were employed as normal control. Animals were euthanized 8 weeks after stroke. Compared with saline treated animals, significant functional improvements were evident in rats treated with 600 μg tPA (p<0.05), but not in 60 μg tPA treated rats. Furthermore, 600 μg tPA treatment significantly enhanced both CRT and CST sprouting originating from the contralesional cortex sprouting into the denervated side of the red nucleus and cervical gray matter compared with control group (p<0.01), respectively. The behavioral outcomes were highly correlated with CRT and CST axonal remodeling. Our data suggest that delayed tPA intranasal treatment provides therapeutic benefits for neurological recovery after stroke by, at least in part, promoting neuronal remodeling in the brain and spinal cord.
functional recovery; middle cerebral artery occlusion; neuronal remodeling; tissue plasminogen activator
Human umbilical tissue-derived cells (hUTC) are a promising source of cells for regenerative treatment of stroke. In this study, we tested the efficacy of hUTC in experimental stroke and whether multiple injections of hUTC provide additional therapeutic benefits as compared to a single injection.
Adult male Wistar rats were subjected to 2 hours of middle cerebral artery occlusion (MCAo), and randomly selected animals were injected (i.v) with 3×106 hUTC or with vehicle control (at day: 1, 1&3 or 1&7 after MCAo, n = 8–9/group). A battery of functional outcome tests was performed at days 1, 7, 14, 21, 28, 35, 42, 49, 56 and 63 after MCAo. Rats were sacrificed at 63 days after MCAo and lesion volumes were measured. To investigate the underlying mechanism of hUTC treatment of stroke, Von Willebrand Factor (vWF), and Synaptophysin immunostaining were performed.
All hUTC treated groups, single or multiple injections, had better functional recovery compared to control (p<0.01). There was no statistically significant difference between a single and multiple injections of hUTC (p = 0.23) or between different multiple injections groups (p>0.07) in functional outcome. All hUTC treatment groups showed significant increases in Synaptophysin, vascular density and perimeter compared to the control group (p<0.05). There was no statistically significant difference between a single and multiple injections of hUTC or between the two groups of multiple injections in all immunohistochemical measurements (p>0.1).
hUTC treatment significantly improves long term functional outcome after stroke and promotes vascular density and synaptic plasticity. At the proscribed doses, multiple injections of hUTC were not superior to single injection therapy in both functional outcome and histological assessments.
MicroRNAs are important regulators of gene expression and have been suggested to play a key role in tumorigenesis. In this study, we show that miR-145 is significantly down-regulated in glioma cell lines as compared to normal brain tissue and negatively regulates tumorigenesis. Restoration of miR-145 in glioma cells significantly reduced in vitro proliferation, migration, and invasion. Also, overexpression of miR-145 reduced ADAM17 and EGFR expression. We then tested the hypothesis that the miR-145-mediated suppression of cell proliferation, migration and invasion is at least in part, due to silencing of ADAM17 and EGFR gene expression. Using luciferase reporters carrying the 3′-untranslated region of ADAM17 combined with Western blot, we identified ADAM17 as a direct target of miR-145. Taken together, these results suggest that as a tumor suppressor, miR-145 inhibits not only tumor proliferation, but also cell migration and invasion, and warrants further investigation.
miR-145; ADAM17; EGFR; glioma; migration; invasion
In order to increase understanding of how infused cells work, it becomes important to track their initial movement, localization, and engraftment efficiency following transplantation. However, the available in vivo cell tracking techniques are suboptimal. The study objective was to determine the biodistribution of intravenously administered Indium-111 (In-111) oxine labeled human umbilical tissue-derived cells (hUTC) in a rat model of transient middle cerebral occlusion (tMCAo) using single photon emission computed tomography (SPECT).
Rats received 3 million In-111 labeled hUTC (i.v.) 48 hrs after tMCAo. Following the administration of either hUTC or equivalent dose of In-111-oxine (18.5 MBq), animals underwent SPECT imaging on days 0, 1, and 3. Radioactivity in various organs as well as in the stroke area and contralateral hemisphere was determined, decay corrected and normalized to the total (whole body including head) radioactivity on day 0. Immunohistochemical analysis was also performed to confirm the beneficial effects of hUTC on vascular and synaptic density, and apoptosis.
Most of the radioactivity (43.36±23.07% on day 0) trafficked to the lungs immediately following IV administration of In-111 labeled hUTC (day 0) and decreased drastically to 8.81±7.75 and 4.01±4.52% on days 1 and 3 post-injection, respectively. In contrast, radioactivity measured in the lung of animals that received In-111-oxine alone remained relatively unchanged from day 0 to day 1 (18.38±5.45% at day 0 to 12.59±5.94%) and decreased to 8.34±4.25% on day 3. Significantly higher radioactivity was observed in stroke areas of animals that received In-111 labeled hUTC indicating the presence of cells at the site of injury representing approximately 1% of total administered dose. In addition, there was significant increase in vascular and synaptophysin immunoreactivity in stroke areas of rats that received In-111 labeled hUTC.
The present studies showed the tracking of In-111 labeled hUTC to the sites of stroke in a rat model of tMCAo using SPECT. Animals treated with In-111 labeled hUTC showed histological improvements, with higher vascular and synaptic densities observed in the ischemic boundary zone (IBZ).
Human umbilical tissue-derived cells (hUTC); In-111-oxine; SPECT; Cell tracking; Stroke rats
Background and purpose
Sildenafil provides restorative therapeutic benefits in the treatment of experimental stroke. The majority of experimental studies on treatment of stroke have been performed in young animals; however, stroke is primarily a disease of the aged. Thus, using MRI, we evaluated the effects of sildenafil treatment of embolic stroke in aged animals.
Aged male Wistar rats (18 months) were subjected to embolic stroke and treated daily with saline (n=10) or with sildenafil (n=10) initiated at 24h and subsequently for 7 days after onset of ischemia. MRI measurements were performed at 24h and weekly to 6 weeks after embolization.
MRI and histological measurements demonstrated that sildenafil treatment of aged rats significantly enhanced angiogenesis and axonal remodeling after stroke compared to saline treated aged rats. Local CBF in the angiogenic area was elevated and expansion of the ipsilateral ventricle and consequently brain atrophy was significantly reduced in the sildenafil treated rats.
Treatment of embolic stroke in aged rats with sildenafil significantly augments angiogenesis and axonal remodeling, which increased local blood flow and reduced expansion of the ipsilateral ventricle 6 weeks after stroke, compared to control aged rats. MRI can be employed to investigate brain repair after stroke in aged rats.
aged rat; embolic stroke; magnetic resonance imaging; neurorestorative treatment; sildenafil
Erythropoietin (EPO) improves functional recovery after traumatic brain injury (TBI). Here, we investigated the role of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) on EPO-induced therapeutic efficacy in rats after TBI. Young male Wistar rats were subjected to unilateral controlled cortical impact injury and then infused intracerebroventricularly with either a potent selective VEGFR2 inhibitor SU5416 or vehicle dimethyl sulfoxide. Animals from both groups received delayed EPO treatment (5,000 U/kg in saline) administered intraperitoneally daily at 1, 2, and 3 days post injury. TBI rats treated with saline administered intraperitoneally daily at 1, 2, and 3 days post injury served as EPO treatment controls. 5-bromo-2-deoxyuridine was administered to label dividing cells. Spatial learning and sensorimotor function were assessed using a modified Morris water maze test and modified neurological severity score, respectively. Animals were sacrificed at 4 days post injury for measurement of VEGF and VEGFR2 or 35 days post injury for evaluation of cell proliferation, angiogenesis and neurogenesis. EPO treatment promoted sensorimotor and cognitive functional recovery after TBI. EPO treatment increased brain VEGF expression and phosphorylation of VEGFR2. EPO significantly increased cell proliferation, angiogenesis and neurogenesis in the dentate gyrus after TBI. Compared to the vehicle, SU5416 infusion significantly inhibited phosphorylation of VEGFR2, cell proliferation, angiogenesis, and neurogenesis as well as abolished functional recovery in EPO-treated TBI rats. These findings indicate the VEGF/VEGFR2 activation plays an important role in EPO-mediated neurobehavioral recovery and neurovascular remodeling after TBI.
angiogenesis; erythropoietin; neurogenesis; traumatic brain injury; vascular endothelial growth factor
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.
MicroRNA 133b; Exosomes; Multipotent mesenchymal stromal cells; Neurite outgrowth; Stroke
Multipotent mesenchymal stromal cells (MSCs) increase tissue plasminogen activator (tPA) activity in astrocytes of the ischemic boundary zone, leading to increased neurite outgrowth in the brain. To probe the mechanisms that underlie MSC-mediated activation of tPA, we investigated the morphogenetic gene, sonic hedgehog (Shh) pathway. In vitro oxygen and glucose deprivation and coculture of astrocytes and MSCs were used to mimic an in vivo ischemic condition. Both real-time-PCR and western blot showed that MSC coculture significantly increased the Shh level and concomitantly increased tPA and decreased plasminogen activator inhibitor 1 (PAI-1) levels in astrocytes. Inhibiting the Shh signaling pathway with cyclopamine blocked the increase of tPA and the decrease of PAI-1 expression in astrocytes subjected to MSC coculture or recombinant mouse Shh (rm-Shh) treatment. Both MSCs and rm-Shh decreased the transforming growth factor-β1 level in astrocytes, and the Shh pathway inhibitor cyclopamine reversed these decreases. Both Shh-small-interfering RNA (siRNA) and Glil-siRNA downregulated Shh and Gli1 (a key mediator of the Shh transduction pathway) expression in cultured astrocytes and concomitantly decreased tPA expression and increased PAI-1 expression in these astrocytes after MSC or rm-Shh treatment. Our data indicate that MSCs increase astrocytic Shh, which subsequently increases tPA expression and decreases PAI-1 expression after ischemia.
astrocytes; multipotent mesenchymal stromal cells (MSCs); plasminogen activator inhibitor 1 (PAI-1); sonic hedgehog (Shh); stroke; tissue plasminogen activator (tPA)
Adult neural stem cells give rise to neurons, oligodendrocytes and astrocytes. Aging reduces neural stem cells. Using an inducible nestin-CreERT2/R26R-yellow fluorescent protein (YFP) mouse, we investigated the effect of Sildenafil, a phosphodiesterase type 5 (PDE5) inhibitor, on nestin lineage neural stem cells and their progeny in the ischemic brain of the middle-aged mouse. We showed that focal cerebral ischemia induced nestin lineage neural stem cells in the subventricular zone (SVZ) of the lateral ventricles and nestin expressing NeuN positive neurons and adenomatous polyposis coli (APC) positive mature oligodendrocytes in the ischemic striatum and corpus callosum in the aged mouse. Treatment of the ischemic middle-aged mouse with Sildenafil increased nestin expressing neural stem cells, mature neurons, and oligodendrocytes by 33, 75, and 30%, respectively, in the ischemic brain. These data indicate that Sildenafil amplifies nestin expressing neural stem cells and their neuronal and oligodendrocyte progeny in the ischemic brain of the middle-aged mouse.
MicroRNAs are important regulators of gene expression and have been suggested to play a key role in tumorigenesis. In this study, we show that miR-145 is significantly downregulated in glioma cell lines compared to normal brain tissue and negatively regulates tumorigenesis. Restoration of miR-145 in glioma cells significantly reduced in vitro proliferation, migration and invasion. Also, overexpression of miR-145 reduced ADAM17 and EGFR expression. In addition, we tested the hypothesis that the miR-145-mediated suppression of cell proliferation, migration and invasion is, at least in part, due to silencing of ADAM17 and EGFR gene expression. Using luciferase reporters carrying the 3′-untranslated region of ADAM17 combined with western blotting, we identified ADAM17 as a direct target of miR-145. Collectively, these results suggest that as a tumor suppressor, miR-145 inhibits not only tumor proliferation, but also cell migration and invasion, and warrants further investigation.
miR-145; ADAM17; EGFR; glioma; migration; invasion
Niaspan, an extended-release formulation of Niacin (vitamin B3), has been widely used to increase high density lipoprotein (HDL) cholesterol and to prevent cardiovascular diseases and stroke. We have previously demonstrated that Niaspan (40mg/kg) administered at 2 hours after stroke induces neuroprotection, while low dose Niaspan (20mg/kg) does not reduce infarct volume. Tissue plasminogen activator (tPA) is an effective therapy for acute stroke, but its use remains limited by narrow therapeutic window. We have previously demonstrated that intravenous administration of tPA 4 hours after stroke in rats does not reduce infarct volume. In this study, we tested whether combination treatment with low-dose Niaspan (20mg/kg) and tPA administered 4 hours after embolic stroke in a rat model reduces infarct volume and provides neuroprotection.
Adult male Wistar rats were subjected to embolic middle cerebral artery occlusion (MCAo) and treated with low-dose Niaspan (20mg/kg) alone (n=7), tPA (10mg/kg) alone (n=7), combination of low-dose Niaspan and tPA (n=7), or saline control (n=9), 4 hours after stroke. A battery of functional outcome tests was performed. Rats were sacrificed at 7 days after MCAo and lesion volumes were measured. To investigate the underlying mechanism of combination treatment neuroprotective effect, deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL), cleaved caspase-3, tumor necrosis factor alpha (TNF-alpha), and toll-like receptor 4 (TLR-4) immunostaining were performed.
Combination treatment with low-dose Niaspan and tPA significantly improved functional outcome compared to the saline control group (p<0.05), while treatment with Niaspan or tPA alone did not significantly improve functional outcome compared to saline control group. Additionally, combination treatment significantly reduced infarct volume compared to saline control group (p=0.006) and infarct volume was significantly correlated with functional outcome (p=0.0008; r=0.63). Monotherapy with Niaspan or tPA did not significantly decrease infarct volume compared to saline control group.
Combination treatment reduced apoptosis as measured by significant reduction in the number of TUNEL-positive cells and cleaved caspase-3 expression in the ischemic brain compared to saline control group (p<0.05). Combination treatment also significantly reduced the expression of TNF-alpha and TLR-4 in the ischemic brain compared to Niaspan, tPA and saline treatment groups (p<0.05). A significant interaction between Niaspan and tPA on the TNF-alpha expression was detected (p<0.05), indicating a synergy effect in the combination treatment group.
Treatment of stroke with combination of low-dose Niaspan and tPA at 4 hours after embolic stroke reduces infarct volume, improves neurological outcome and provides neuroprotection. The neuroprotective effects of combination treatment were associated with reduction of apoptosis and attenuation of TNF-alpha and TLR-4 expression.
Niaspan; tPA; Neuroprotection; Apoptosis; TNF-alpha; TLR-4
Peripheral neuropathy is a common and major complication of diabetes, the underlying mechanisms of which are not fully understood. Using a mouse model of type II diabetes, the present study investigated the role of phosphodiesterase-5 (PDE5) in peripheral neuropathy. BKS.Cg-m+/+Leprdb/J (db/db) mice were treated with sildenafil, a specific inhibitor of PDE5, at doses of 2 and 10 mg/kg or saline. Levels of PDE5 and morphometric parameters in sciatic nerve tissue as well as the motor and sensory function were measured in these mice. In diabetic mice, PDE5 expression in sciatic nerve tissue was significantly upregulated, while the myelin sheath thickness, myelin basic protein (MBP), and subcutaneous nerve fibers were significantly reduced. Treatment with sildenafil, significantly improved neurological function, assayed by motor and sensory conducting velocities and thermal and mechanical noxious stimuli, concomitantly with increases in myelin sheath thickness, MBP levels, and subcutaneous nerve fibers. In vitro, hyperglycemia upregulated PDE5 in Schwann cells, and reduced Schwann cell proliferation, migration and expression of brain-derived neurotrophic factor (BDNF). Blockage of PDE5 with sildenafil, increased cGMP, and completely abolished the effect of hyperglycemia on Schwann cells. Sildenafil upregulated cGMP-dependent protein kinase G1 (PKG1), whereas inhibition of PKG1 with a PKG inhibitor, KT5823, suppressed the inhibitory effect of sildenafil on Schwann cells. These data indicate that hyperglycemia substantially upregulates PDE5 expression and that the cGMP/PKG signaling pathway activated by sildenafil mediates the beneficial effects of sildenafil on diabetic peripheral neuropathy.
sildenafil; PDE5; peripheral neuropathy; function; diabetes; mice
The therapeutic efficacy of cell-based therapy after stroke can be enhanced by making the host brain tissue more receptive to the administered cells, which thereby facilitates brain plasticity. We hypothesized that simvastatin increases human umbilical cord blood cell (HUCBC) migration into the ischemic brain and promotes brain plasticity and neurological functional outcome after stroke. Rats were subjected to 2-h middle cerebral artery occlusion (MCAo) and administered subtherapeutic doses of simvastatin (0.5 mg/kg, gavaged daily for 7 days), HUCBCs (1 × 106, one time injection via tail vein), or combination simvastatin with HUCBCs starting at 24 h after stroke. Combination treatment of stroke showed an interactive effect in improvement of neurological outcome compared with simvastatin or HUCBC monotherapy groups. In addition, combination treatment significantly increased brain-derived neurotrophic factor/TrkB expression and the number of engrafted HUCBCs in the ischemic brain compared with HUCBC monotherapy. The number of engrafted HUCBCs was significantly correlated with functional outcome (modified neurological severity score). Combination treatment significantly increased neurogenesis and synaptic plasticity in the ischemic brain, and promoted neuroblast migration in cultured subventricular zone explants. Using primary cultured neurons (PCNs), we found that combination treatment enhanced neurite outgrowth compared with nontreatment control, simvastatin or HUCBC supernatant monotherapy. Inhibition of TrkB significantly attenuated combination treatment-induced neurite outgrowth. Our data indicate that combination simvastatin and HUCBC treatment of stroke increases BDNF/TrkB expression, enhances HUCBC migration into the ischemic brain, amplifies endogenous neurogenesis, synaptic plasticity and axonal growth, and thereby improves functional outcome after stroke.
Simvastatin; Human umbilical cord blood cells (HUCBCs); Neurogenesis; Synaptic plasticity; Stroke
Delayed (24 hours post injury) treatment with erythropoietin (EPO) improves functional recovery following experimental traumatic brain injury (TBI). In this study, we tested whether therapeutic effects of delayed EPO treatment for TBI are dose-dependent in an attempt to establish an optimal dose paradigm for the delayed EPO treatment.
Experimental TBI was performed in anesthetized young adult male Wistar rats using a controlled cortical impact device. Sham animals underwent the same surgical procedure without injury. The animals (8 rats/group) received 3 intraperitoneal injections of EPO (0, 1000, 3000, 5000 or 7000 U/kg body weight, at 24, 48 and 72 hours) after TBI. Sensorimotor and cognitive functions were assessed using a modified neurological severity score and foot fault test, and Morris water maze tests, respectively. Animals were sacrificed 35 days after injury and brain sections stained for immunohistochemical analyses.
Compared to the saline treatment, EPO treatment at doses from1000 to 7000 U/kg did not alter lesion volume but significantly reduced hippocampal neuron loss, enhanced angiogenesis and neurogenesis in the injured cortex and hippocampus, and significantly improved sensorimotor function and spatial learning. The medium dose at 5000 U/kg exhibited a significant improvement in histological and functional outcomes compared with the lower or higher EPO dose groups.
These data demonstrate that delayed (24 hours post injury) treatment with EPO provides dose-dependent neurorestoration which may contribute to improved functional recovery after TBI, implying that application of an optimal dose of EPO is likely to increase successful preclinical and clinical trials for treatment of TBI.
angiogenesis; cell proliferation; erythropoietin; neurogenesis; rat; sensorimotor; spatial learning; traumatic brain injury
Human umbilical tissue-derived cells (hUTC) represent an attractive cell source and a potential technology for neurorestoration and improvement of functional outcomes following stroke. Male Wistar rats were subjected to a transient middle cerebral artery occlusion (tMCAo) and were intravenously administered hUTC (N = 11) or vehicle (N = 10) 48 hrs after stroke. White matter and vascular reorganization was monitored over a 12-week period using MRI and histopathology. MRI results were correlated with neurological functional and histology outcomes to demonstrate that MRI can be a useful tool to measure structural recovery after stroke. MRI revealed a significant reduction in the ventricular volume expansion and improvement in cerebral blood flow (CBF) in the hUTC treated group compared to vehicle treated group. Treatment with hUTC resulted in histological and functional improvements as evidenced by enhanced expression of vWF and synaptophysin, and improved outcomes on behavioral tests. Significant correlations were detected between MRI ventricular volumes and histological lesion volume as well as number of apoptotic cells. A positive correlation was also observed between MRI CBF or cerebral blood volume (CBV) and histological synaptic density. Neurological functional tests were also significantly correlated with MRI ventricular volume and CBV. Our data demonstrated that MRI measurements can detect the effect of hUTC therapy on the brain reorganization and exhibited positive correlation with histological measurements of brain structural changes and functional behavioral tests after stroke. MRI ventricular volumes provided the most sensitive index in monitoring brain remodeling and treatment effects and highly correlated with histological and functional measurements.
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.
We investigated the changes and the molecular mechanisms of cerebral vascular damage after stroke in type-2 diabetic (T2DM) mice. Adult male db/db T2DM and wild-type (WT) mice were subjected to transient middle cerebral artery occlusion (MCAo) and sacrificed 24 hours after MCAo. T2DM-mice exhibited significantly increased blood glucose, brain hemorrhagic rate, mortality and cerebrovascular density, but decreased cerebrovascular diameter, arteriolar density and arterial mural cell numbers in the ischemic brain compared with WT-mice. The hemorrhagic rate was significantly correlated with the mortality (r=0.85). T2DM-mice also exhibited increased blood-brain barrier leakage and concomitantly, increased Angiopoietin2, but decreased Angiopoietin1, Tie2 and tight junction protein expression in the ischemic brain. Angiopoietin1 gene expression also significantly decreased in the common carotid artery (CCA) in T2DM-mice compared with WT-mice after stroke. To further test the effects of T2DM on cerebrovascular damage, we performed in vitro studies. The capillary-like tube formation of primary cultured mouse brain endothelial cells (MBECs) significantly increased, but artery cell migration in the primary CCA cultures significantly decreased both in Sham and MCAo T2DM-mice compared with the WT-mice. Angiopoietin1 treatment significantly increased artery cell migration in T2DMCCA after MCAo. Tie2-FC, a neutralized Tie2 antibody, significantly decreased artery cell migration in WT-CCA after MCAo. Therefore, decreased Angiopoietin1/Tie2 and increased Angiopoietin2 expression may contribute to diabetes-induced vascular damage after stroke.
diabetes; vascular damage; angiopoietin; stroke; mice
This study examines the effects of combination therapy of collagen scaffolds and human marrow stromal cells (hMSCs) on the expression of tissue plasminogen activator (tPA) after traumatic brain injury (TBI) in rats. Adult male Wistar rats (n=48) were injured with controlled cortical impact and treated either with scaffolds suffused with hMSCs (3×106) or hMSCs (3×106) alone transplanted into the lesion cavity 1 week after TBI. A control group was treated with saline. Neurological function was assessed using the Morris Water Maze test (MWM) and modified Neurological Severity Scores (mNSS). The rats were sacrificed 14 days after TBI and brain samples were processed for immunohistochemical analysis and quantitative Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) studies. Enhanced functional improvement was observed on both the mNSS and MWM tests in the scaffold+hMSC-treated group compared to the other two groups. Immunostaining with anti-human mitochondrial antibody (E5204) showed more hMSCs in the injury zone of the scaffold+hMSC group compared to the hMSC-alone group. Triple staining showed that more neurons were tPA-positive in the scaffold+hMSC group compared to the other two groups (p<0.05). Western blot analysis and qRT-PCR showed that scaffold+hMSC and hMSC-alone treatment enhanced the expression of tPA compared to controls (p<0.05), but tPA expression was significantly greater in the scaffold+hMSC group. The induction of tPA by hMSCs after TBI may be one of the mechanisms involved in promoting functional improvement after TBI.
collagen scaffolds; marrow stromal cells; tissue plasminogen activator; traumatic brain injury
We treated traumatic brain injury (TBI) with human bone marrow stromal cells (hMSCs) and evaluated the effect of treatment on white matter reorganization using MRI. We subjected male Wistar rats (n = 17) to controlled cortical impact and either withheld treatment (controls; n = 9) or inserted collagen scaffolds containing hMSCs (n = 8). Six weeks later, the rats were sacrificed and MRI revealed selective migration of grafted neural progenitor cells towards the white matter reorganized boundary of the TBI-induced lesion. Histology confirmed that the white matter had been reorganized, associated with increased fractional anisotropy (FA; p <0.01) in the recovery regions relative to the injured core region in both treated and control groups. Treatment with hMSCs increased FA in the recovery regions, lowered T2 in the core region, decreased lesion volume and improved functional recovery relative to untreated controls. Immunoreactive staining showed axonal projections emanating from neurons and extruding from the corpus callosum into the ipsilateral cortex at the boundary of the lesion. Fiber tracking (FT) maps derived from diffusion tensor imaging confirmed the immunohistological data and provided information on axonal rewiring. The apparent kurtosis coefficient (AKC) detected additional axonal remodeling regions with crossing axons, confirmed by immunohistological staining, compared with FA. Our data demonstrate that AKC, FA, FTand T2 can be used to evaluate treatment-induced white matter recovery, which may facilitate restorative therapy in patients with TBI.
MRI; DTI; traumatic brain injury; rat brain
There is a paucity of therapies for most central nervous system (CNS) disorders. Bone marrow stromal cells (MSCs) are a mixed cell population, including stem and progenitor cells, and are currently a strong candidate for cell-based therapy in “brain attack”, including stroke, and traumatic brain injury (TBI), since they are easily isolated and can be expanded in culture from patients without ethical and technical problems. Although it has been suggested that trans-differentiation of MSCs into cells of neural lineage may occur in vitro, no one has yet observed that MSCs give rise to fully differentiated and functional neurons in vivo. The overwhelming body of data indicate that bioactive factors secreted by MSCs in response to the local environment underlie the tissue restorative effects of MSCs. The MSCs that are employed in this therapy are not necessarily stem cells, but progenitor and differentiated cells that escape immune system surveillance and survive in the CNS even for transplantation of allogeneic or xenogeneic MSCs. The injured CNS is stimulated by the MSCs to amplify its intrinsic restorative processes. Treatment of damaged brain with MSCs promotes functional recovery, and facilitates CNS endogenous plasticity and remodeling. The current mini-review is mainly based on our data and focuses on possible cellular and molecular mechanisms of interaction of MSCs with glia, neurons and vessels after brain attack. The transplantation of MSCs opens up new avenues of cell therapy and may provide an effective treatment for various CNS diseases.
Bone marrow stromal cells; Stroke; TBI
Our previous studies demonstrated that simvastatin reduced neuronal death, increased neurogenesis, and promoted functional recovery after TBI. Objective: To investigate the effect of simvastatin on angiogenesis after TBI, and the related signaling pathways.
Saline or simvastatin (1 mg/kg) was administered orally to rats starting at day 1 after TBI or sham surgery and then daily for 14 days. Rats were sacrificed at 3 and 14 days after treatment. Brain sections and tissues were prepared for immunohistochemical staining, ELISA, and Western blot analysis, respectively. Cultured rat brain microvascular endothelial cells (RBMVECs) were subjected to oxygen-glucose deprivation (OGD) followed by immunocytochemical staining with phallotoxins and vascular endothelial growth factor receptor-2 (VEGFR-2). Western blot analysis was carried out to examine the simvastatin-induced activation of the v-akt murine thymoma viral oncogene homolog (Akt) signaling pathway. The expression of VEGFR-2 was detected by ELISA.
Simvastatin significantly increased the length of vascular perimeter, promoted the proliferation of endothelial cells, and improved the sensorimotor function after TBI. Simvastatin stimulated endothelial cell tube formation after OGD in vitro. VEGFR-2 expression in both brain tissues and cultured RBMVECs was enhanced after simvastatin treatment, which may be modulated by activation of Akt. Akt-dependent endothelial nitric oxide synthase (eNOS) phosphorylation was also induced by simvastatin in vivo and in vitro.
Simvastatin augments TBI-induced angiogenesis in the lesion boundary zone and hippocampus and improves functional recovery. Simvastatin also promotes angiogenesis in vitro. These beneficial effects on angiogenesis may be related to simvastatin-induced activation of the VEGFR-2/Akt/eNOS signaling pathway.
Angiogenesis; Simvastatin; Traumatic brain injury; VEGFR-2
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.
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.
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.
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.
Type-one diabetes rats; Stroke; Angiopoietin; Axonal remodeling; Niaspan
Erythropoietin (EPO) improves functional recovery after traumatic brain injury (TBI). This study was designed to investigate long-term (3 mo) effects of EPO on brain remodeling and functional recovery in rats after TBI. Young male Wistar rats were subjected to unilateral controlled cortical impact injury. TBI rats were divided into the following groups: 1) Saline group (n = 7); 2) EPO-6h group (n = 8); and 3) EPO-24h group (n = 8). EPO (5,000 U/kg in saline) was administered intraperitoneally at 6 h, and 1 and 2 days (EPO-6h group) or at 1, 2, and 3 days (EPO-24h group) post injury. Neurological function was assessed using a modified neurological severity score, footfault and Morris water maze tests. Animals were sacrificed at 3 mos after injury and brain sections stained for immunohistochemical analyses. Compared to the saline, EPO-6h treatment significantly reduced cortical lesion volume, while EPO-24h therapy did not affect the lesion volume (P<0.05). Both the EPO-6h and EPO-24h treatments significantly reduced hippocampal cell loss (P<0.05), promoted angiogenesis (P<0.05) and increased endogenous cellular proliferation (BrdU-positive cells) in the injury boundary zone and hippocampus (P<0.05) compared to saline controls. Significantly enhanced neurogenesis (BrdU/NeuN-positive cells) was seen in the dentate gyrus of both EPO groups compared to the saline group. Both EPO treatments significantly improved long-term sensorimotor and cognitive functional recovery after TBI. In conclusion, the beneficial effects of posttraumatic EPO treatment on injured brain persisted for at least 3 months. The long-term improvement in functional outcome may in part be related to the neurovascular remodeling induced by EPO.
angiogenesis; cell proliferation; erythropoietin; neurogenesis; functional recovery; traumatic brain injury