Multipotent mesenchymal stromal cells decrease transforming growth factor ß1 expression in microglia/macrophages and down-regulate plasminogen activator inhibitor 1 expression in astrocytes after stroke.

Multipotent mesenchymal stromal cells (MSCs) decrease the expression of transforming growth factor ß1 (TGFß1) in astrocytes and subsequently decrease astrocytic plasminogen activator inhibitor 1 (PAI-1) level in an autocrine manner. Since activated microglia/macrophages are also a source of TGFß1 after stroke, we therefore tested whether MSCs regulate TGFß1 expression in microglia/macrophages and subsequently alters PAI-1 expression after ischemia. TGFß1 and its downstream effector phosphorylated SMAD 2/3 (p-SMAD 2/3) were measured in mice subjected to middle cerebral artery occlusion (MCAo). MSC treatment significantly decreased TGFß1 protein expression in both astrocytes and microglia/macrophages in the ischemic boundary zone (IBZ) at day 14 after stroke. However, the p-SMAD 2/3 was only detected in astrocytes and decreased after MSC treatment. In vitro, RT-PCR results showed that the TGFß1 mRNA level was increased in both astrocytes and microglia/macrophages in an astrocyte-microglia/macrophage co-culture system after oxygen-glucose deprived (OGD) treatment. MSCs treatment significantly decreased the above TGFß1 mRNA level under OGD conditions, respectively. OGD increased the PAI-1 mRNA in astrocytes in the astrocyte-microglia/macrophage co-culture system, and MSC administration significantly decreased this level. PAI-1 mRNA was very low in microglia/macrophages compared with that in astrocytes under different conditions. Western blot results also verified that MSC administration significantly decreased p-SMAD 2/3 and PAI-1 level in astrocytes in astrocyte-microglia/macrophage co-culture system under OGD conditions. Our in vivo and in vitro data, in concert, suggest that MSCs decrease TGFß1 expression in microglia/macrophages in the IBZ which contribute to the down-regulation of PAI-1 level in astrocytes.

Multipotent mesenchymal stromal cells increase tPA expression and concomitantly decrease PAI-1 expression in astrocytes through the sonic hedgehog signaling pathway after stroke (in vitro study).

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-ß1 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.

Endogenous tissue plasminogen activator mediates bone marrow stromal cell-induced neurite remodeling after stroke in mice.

BACKGROUND AND PURPOSE: Bone marrow stromal cells (BMSC) decrease neurological deficits in rodents after stroke and concomitantly induce extensive neurite remodeling in the brain, which highly correlates with the improvement of neurological function. We investigated the effects of endogenous tissue plasminogen activator (tPA) on neurite remodeling after BMSC treatment. METHODS: Adult C57BL/6 wild-type (WT) mice and tPA knockout (tPA(-/-)) mice were subjected to middle cerebral artery occlusion, followed by an injection of 1×10(6) BMSC (n=18) or phosphate-buffered saline (n=18) into the tail vein 24 hours later. Behavioral tests were performed at 3, 7, and 14 days after middle cerebral artery occlusion. Animals were euthanized at 14 days after stroke. RESULTS: The effects of BMSC on functional recovery depended on presence or absence of tPA, even after adjusting for imbalanced stroke severity. BMSC significantly improve functional recovery in WT mice compared to WT controls but show no beneficial effect in the tPA(-/-) mice compared to tPA(-/-) controls. Axonal density and synaptophysin-positive areas along the ischemic boundary zone of the cortex and striatum in WT mice are significantly higher than in the tPA(-/-) mice. BMSC treatment significantly increases tPA protein level and activity only in WT mice. CONCLUSIONS: Our results suggest that endogenous tPA promotes BMSC-induced neurite outgrowth and may contribute to functional recovery after stroke.

Reduction of Pten dose leads to neoplastic development in multiple organs of Pten (shRNA) mice.

To address the impact of partial reduction of Pten on tumor initiation, we generated PtenshRNA mice, in which PTEN expression was reduced below normal levels in various tissues. PtenshRNA mice frequently developed lymphoid and prostatic hyperplasia, splenomegaly, and sebaceous adenomas. Our observations support the notion that partial reduction of the dose of Pten with shRNA is sufficient to induce neoplastic disease in multiple organ systems.

Chronic brain tissue remodeling after stroke in rat: a 1-year multiparametric magnetic resonance imaging study.

Rats subjected to 2h of transient middle cerebral artery occlusion were studied temporally over 1 year by magnetic resonance imaging (MRI) and behavioral testing. Multiparameter MRI measures of T(2), T(1), T(1) in the presence of off-resonance saturation of the bound proton signal (T(1sat)), apparent diffusion coefficient (ADC) and susceptibility-weighted imaging (SWI) were obtained at 1 day, 1, 2, 3 and 4 weeks, and 3, 6, 9 and 12 months post-ischemia. Regions of interest included: ischemic core (damaged both at 1 day and later); new lesion (normal at 1 day, but damaged later); and recovery (damaged at 1 day, but normal later) areas. Hematoxylin and eosin, Prussian blue and ED-1, a monoclonal antibody murine macrophage marker, stainings were performed for histological assessment. Core area T(2) and ADC values increased until ~6 months, and T(1) and T(1sat) until ~12 months. New lesion area MRI parameter values increased until ~6 months (T(2), T(1) and ADC), or ~1 year (T(1sat)). Lesion area was largest at 1day (mean±SD: 37.0±13.7mm(2)) and smallest at 1 year (18.1±10.5mm(2)). Recovery area was largest at 3 weeks (8.9±3.8mm(2)) and smallest at 1year (6.4±3.3mm(2)). The ipsilateral/contralateral ventricle area ratio was 0.7±0.2 at 1 day and increased significantly at 1 year (2.4±0.7). Iron-laden macrophages, histologically confirmed at 1 year, were detected in the lesion borders by SWI at 3, 6, 9 and 12 months. Our data indicate that MRI detectable changes of ischemia-damaged brain tissue continue for at least 1 year post-ischemia.

[Magnetic resonance imaging of ovarian carcinosarcoma: correlation to the clinicopathological findings].

OBJECTIVE: To investigate magnetic resonance imaging (MRI) characteristics of ovarian carcinosarcoma and the diagnostic value of MRI. METHODS: The MRI features of ovarian carcinosarcoma and clinical data of 5 patients with ovarian carcinosarcoma were reviewed. All the lesions were confirmed by surgery and pathological examination. RESULTS: MRI of ovarian carcinosarcoma in the 5 cases all showed large tumor mass and heterogeneous high-intensity on T2-weighted images and low-intensity on T1-weighted images, with laminar or stripe-like enhancement. Hemorrhage and necrosis were also displayed in some lesions. In two cases, the tumors invaded the greater omentum, sigmoid colon and the body of the uterus, with regional lymph node involvement. Pelvic effusion was observed in all the cases with pelvic hematocele in 1 case. CONCLUSION: MRI is useful in the detection and staging of ovarian carcinosarcoma.

Increasing tPA activity in astrocytes induced by multipotent mesenchymal stromal cells facilitate neurite outgrowth after stroke in the mouse.

We demonstrate that tissue plasminogen activator (tPA) and its inhibitors contribute to neurite outgrowth in the central nervous system (CNS) after treatment of stroke with multipotent mesenchymal stromal cells (MSCs). In vivo, administration of MSCs to mice subjected to middle cerebral artery occlusion (MCAo) significantly increased activation of tPA and downregulated PAI-1 levels in the ischemic boundary zone (IBZ) compared with control PBS treated mice, concurrently with increases of myelinated axons and synaptophysin. In vitro, MSCs significantly increased tPA levels and concomitantly reduced plasminogen activator inhibitor 1 (PAI-1) expression in astrocytes under normal and oxygen and glucose deprivation (OGD) conditions. ELISA analysis of conditioned medium revealed that MSCs stimulated astrocytes to secrete tPA. When primary cortical neurons were cultured in the conditioned medium from MSC co-cultured astrocytes, these neurons exhibited a significant increase in neurite outgrowth compared to conditioned medium from astrocytes alone. Blockage of tPA with a neutralizing antibody or knock-down of tPA with siRNA significantly attenuated the effect of the conditioned medium on neurite outgrowth. Addition of recombinant human tPA into cortical neuronal cultures also substantially enhanced neurite outgrowth. Collectively, these in vivo and in vitro data suggest that the MSC mediated increased activation of tPA in astrocytes promotes neurite outgrowth after stroke.

Localization of bone marrow stromal cells to the injury site after intracerebral hemorrhage in rats.

OBJECT: Previous studies demonstrated that intravascular injection of bone marrow stromal cells (BMSCs) significantly improved neurological functional recovery in a rat model of intracerebral hemorrhage (ICH). To further investigate the fate of transplanted cells, we examined the effect of male rat BMSCs administered to female rats after ICH. METHODS: Twenty-seven female Wistar rats were subjected to ICH surgery. At 24 hours after ICH, these rats were randomly divided into 3 groups and injected intravenously with 1 ml phosphate-buffered saline or 0.5 million or 1 million male rat BMSCs in phosphate-buffered saline. To evaluate the neurological functional outcome, each rat was subjected to a series of behavioral tests (modified neurological severity score and corner turn test) at 1, 7, and 14 days after ICH. The rats were anesthetized intraperitoneally and killed, and the brain tissues were processed at Day 14 after ICH. Immunohistochemistry and in situ hybridization were used to identify cell-specific markers. RESULTS: The male rat BMSCs significantly improved the neurological functional outcome and also significantly diminished tissue loss when intravenously transplanted into the rats after ICH. Immunoassay for bromodeoxyuridine (BrdU) and neuronal markers demonstrated a significant increase in the number of BrdU-positive cells, which indicated endogenous neurogenesis, and a significant increase in the number of cells positive for immature neuronal markers. In situ hybridization showed that more BMSCs resided around the hematoma of the rats treated with the 1-million-cell dose compared with the 0.5-million-cell-dose group. In addition, a subfraction of Y chromosome-positive cells were co-immunostained with the neuronal marker microtubule-associated protein-2 or the astrocytic marker glial fibrillary acidic protein. CONCLUSIONS: Male rat BMSCs improve neurological outcome and increase histochemical parameters of neurogenesis when administered to female rats after ICH. This study has shown that the intravenously administered male rat BMSCs enter the brain, migrate to the perihematomal area, and express parenchymal markers.

Down-regulation of neurocan expression in reactive astrocytes promotes axonal regeneration and facilitates the neurorestorative effects of bone marrow stromal cells in the ischemic rat brain.

The glial scar, a primarily astrocytic structure bordering the infarct tissue inhibits axonal regeneration after stroke. Neurocan, an axonal extension inhibitory molecule, is up-regulated in the scar region after stroke. Bone marrow stromal cells (BMSCs) reduce the thickness of glial scar wall and facilitate axonal remodeling in the ischemic boundary zone. To further clarify the role of BMSCs in axonal regeneration and its underlying mechanism, the current study focused on the effect of BMSCs on neurocan expression in the ischemic brain. Thirty-one adult male Wistar rats were subjected to 2 h of middle cerebral artery occlusion followed by an injection of 3 x 10(6) rat BMSCs (n = 16) or phosphate-buffered saline (n = 15) into the tail vein 24 h later. Animals were sacrificed at 8 days after stroke. Immunostaining analysis showed that reactive astrocytes were the primary source of neurocan, and BMSC-treated animals had significantly lower neurocan and higher growth associated protein 43 expression in the penumbral region compared with control rats, which was confirmed by Western blot analysis of the brain tissue. To further investigate the effects of BMSCs on astrocyte neurocan expression, single reactive astrocytes were collected from the ischemic boundary zone using laser capture microdissection. Neurocan gene expression was significantly down-regulated in rats receiving BMSC transplantation (n = 4/group). Primary cultured astrocytes showed similar alterations; BMSC coculture during reoxygenation abolished the up-regulation of neurocan gene in astrocytes undergoing oxygen-glucose deprivation (n = 3/group). Our data suggest that BMSCs promote axonal regeneration by reducing neurocan expression in peri-infarct astrocytes.

One-year follow-up after bone marrow stromal cell treatment in middle-aged female rats with stroke.

BACKGROUND AND PURPOSE: We sought to evaluate the long-term effects of bone marrow stromal cell (BMSC) treatment on retired breeder rats with stroke. METHODS: Female retired breeder rats were subjected to 2-hour middle cerebral artery occlusion (MCAO) followed by an injection of 2 x 10(6) male BMSCs (n=8) or phosphate-buffered saline (n=11) into the ipsilateral internal carotid artery at 1 day after stroke. The rats were humanely killed 1 year later. Functional tests, in situ hybridization, and histochemical and immunohistochemical staining were performed. RESULTS: Significant recovery of neurological deficits was found in BMSC-treated rats beginning 2 weeks after cell injection compared with control animals. The beneficial effects of cell transplantation persisted for at least 1 year (P<0.01). In situ hybridization for the Y chromosome showed that donor cells survived in the brains of recipient rats, among which 22.3+/-1.95% of cells expressed the astrocyte marker glial fibrillary acidic protein, 16.8+/-2.13% expressed the neuronal marker microtubule-associated protein 2, and 5.5+/-0.42% and <1% of cells colocalized with the microglial marker IB4 and the endothelial cell marker von Willebrand factor, respectively. Only very few BMSCs, however, were found in peripheral organs such as the heart, lung, liver, spleen, and kidney in recipient rats. BMSCs significantly reduced axonal loss (P<0.01), the thickness of the lesion scar wall (P<0.01), and the number of Nogo-A-positive cells (P<0.05) along the scar border; meanwhile, synaptophysin expression (P<0.05) was significantly increased in BMSC-treated ischemic brains compared with control untreated brains. CONCLUSIONS: The beneficial effects of BMSCs on ischemic brain tissue persisted for at least 1 year. Most surviving BMSCs were present in the ischemic brain, but very few were found in other organs. The long-term improvement in functional outcome may be related to the structural and molecular changes induced by BMSCs.

Therapeutic benefit of bone marrow stromal cells administered 1 month after stroke.

Bone marrow stromal cells (BMSCs) facilitate functional recovery in rats after stroke when administered acutely (1 day) or subacutely (7 days). In this study, we postponed the time of cell transplantation to 1 month after stroke. Female retired breeder rats were subjected to 2 h of middle cerebral artery occlusion (MCAo). Male BMSCs (3 x 10(6)) or phosphate-buffered saline were administered intravenously, and the animals were killed 3 months later. An additional population of nontreated rats was killed at 1 month after MCAo. Significant recovery of behavior was found in BMSC-treated rats beginning at 1 month after cell injection in the modified neurologic severity score test and the adhesive-removal test compared with control animals (P<0.05). In situ hybridization showed that BMSCs survived and preferentially localized to the ipsilateral hemisphere. Double staining revealed that approximately 13% and 6% Y-chromosome-positive cells expressed the astrocyte marker, glial fibrillary acidic protein, and the neuronal marker, microtubule-associated protein-2, respectively. In addition, BMSC treatment reduced scar thickness, and increased the number of proliferating cells and oligodendrocyte precursor cells along the subventricular zone in the ipsilateral hemisphere. Expression of the chemokine stromal-cell-derived factor-1 (SDF-1) was significantly increased along the ischemic boundary zone compared with the corresponding areas in the contralateral hemisphere at 1 month and 4 months (P<0.01) after stroke. The SDF-1 receptor, CXC-chemokine receptor-4 (CXCR4), was expressed in BMSCs both in vitro and in vivo. Our data show that the time window of BMSC therapy is at least 1 month after stroke; the interaction of SDF-1/CXCR4 may contribute to the trafficking of transplanted BMSCs.

Anti-amnestic and anti-aging effects of ginsenoside Rg1 and Rb1 and its mechanism of action.

In the present paper, we overview the discovery of new biological activities induced by ginsenoside Rg1 and Rb1 and discuss possible mechanisms of action. Both compounds could increase neural plasticity in efficacy and structure; especially Rg1, as one small molecular drug, can increase proliferation and differentiation of neural progenitor cells in dentate gyrus of hippocampus of normal adult mice and global ischemia model in gerbils. This finding has great value for treatment of Alzheimer's disease and other neurodegenerative disorders which is characterized by neurons loss. Increase of expression of brain derived neurotrophic factor, Bcl-2 and antioxidant enzyme, enhanced new synapse formation, inhibition of apoptosis and calcium overload are also important neuron protective factors. Rg1 and Rb1 have common effects, but there are some differences in pharmacology and mechanism. These differences may attribute to their different chemical structure. Rg1 is panaxtriol with two sugars, while Rb1 is panaxtriol with four sugars.

Gliosis and brain remodeling after treatment of stroke in rats with marrow stromal cells.

The long-term (4-month) responses to treatment of stroke in the older adult rat, using rat bone marrow stromal cells (MSCs), have not been investigated. Retired breeder rats were subjected to middle cerebral artery occlusion (MCAo) alone, or injected intravenously with 3 x 10(6) MSCs, at 7 days after MCAo. Functional recovery was measured using an adhesive-removal patch test and a modified neurological severity score. Bromodeoxyuridine, a cell proliferation marker, was injected daily for 14 before sacrifice. Animals were sacrificed 4 months after stroke. Double immunostaining was used to identify cell proliferation and cell types for axons, astrocytes, microglia, and oligodendrocytes. MSC treatment induced significant improvement in neurological outcome after MCAo compared with control rats. MSC treatment reduced the thickness of the scar wall (P < 0.05) and reduced the numbers of microglia/macrophages within the scar wall (P < 0.01). Double staining showed increased expression of an axonal marker (GAP-43), among reactive astrocytes in the scar boundary zone and in the subventricular zone in the treated rats. Bromodeoxyuridine in cells preferentially colocalized with markers of astrocytes (GFAP) and oligodendrocytes (RIP) in the ipsilateral hemisphere, and gliogenesis was enhanced in the subventricular zone of the rats treated with MSCs. This is the first report to show that MSCs injected at 7 days after stroke improve long-term neurological outcome in older animals. Brain tissue repair is an ongoing process with reactive gliosis, which persists for at least 4 months after stroke. Reactive astrocytes responding to MSC treatment of ischemia may also promote axonal regeneration during long-term recovery.

Ginsenoside Rg1 promotes proliferation of hippocampal progenitor cells.

Neural progenitor cells (NPCs) exist not only in the developing brain, but also in certain areas in adult brain in mammals. Recent studies suggest that promoting neurogenesis in adult mammals might provide a therapeutic way to cure age-related neurodegenerative diseases. So, it will be of great value to find out drugs that can increase the proliferation and/or differentiation ability of neural progenitors. The present study investigated the influence of ginsenoside Rg1, an active ingredient of Panax ginseng C.A. Meyer, on proliferation ability of rodent hippocampal progenitor cells both in vitro and in vivo. Incubation of NPCs with ginsenoside Rg1 resulted in significant increase in absorbency value, 3H-thymidine incorporation and the number of proliferating progenitor cell spheres; In addition, 2 weeks Rg1 administration (i.p.) led to marked enhancement of the number of dividing cells in the hippocampus of adult mice. These findings suggest that ginsenoside Rg1 is involved in the regulation of proliferation of hippocampal progenitor cells and this effect may serve as one of the elementary mechanisms underlying its nootropic and anti-aging actions.

[Culture of neural stem cells from cerebral cortex of rat embryo and effects of drugs on the proliferation ability of stem cells].

AIM: To establish culture procedures of neural stem cells from embryonic rat brain, determine their stem-cell characteristics and observe the effects of several compounds on their proliferation ability. METHODS: Firstly, a stem cell culture system was set up from embryonic rat cortex. The cells were identified as neural stem cells through immunocytochemistry, in which antibodies to neural stem cell specific protein and markers of mature neural cells were used. Then, by using MTT assay, the survival rate of neurospheres incubated with various concentrations of ginsenoside Rg1, (-)-clausenamide and salvianolic acid A were observed. Furthermore, the effect of these drugs was measured with 3[H] thymidine incorporation assay. RESULTS: In this study, a culture model of neural stem cell was successfully set up. In this model, primary cells from E16-18 rat cortex were dissected out, and cultured as floating neurospheres. The results of immunocytochemistry showed that nestin was expressed by the majority of cells within the sphere. After growing for 8 days in differentiation medium, cells from a single neurosphere were shown to differentiate into 3 main kinds of neural cells: neurons, astrocytes and oligodendrocytes. MTT assay revealed that the three drugs all enhanced the survival rate of neural stem cells, but 3[H] thymidine incorporation assay suggested that only Rg1 significantly accelerated the proliferation rate. CONCLUSION: One culture model of neural stem cell was set up successfully. Meanwhile, several drugs were found to increase the proliferation and/or survival rate of neural stem cells. It has been demonstrated that neural stem cells exist in adult mammalian brains. So, these drugs may become promising candidates for the therapy of neurodegenerative diseases; such as Alzheimer's disease and Parkinson's disease.

Identification of mammalian Sds3 as an integral component of the Sin3/histone deacetylase corepressor complex.

Silencing of gene transcription involves local chromatin modification achieved through the local recruitment of large multiprotein complexes containing histone deacetylase (HDAC) activity. The mammalian corepressors mSin3A and mSin3B have been shown to play a key role in this process by tethering HDACs 1 and 2 to promoter-bound transcription factors. Similar mechanisms appear to be operative in yeast, in which epistasis experiments have established that the mSin3 and HDAC orthologs (SIN3 and RPD3), along with a novel protein, SDS3, function in the same repressor pathway. Here, we report the identification of a component of the mSin3-HDAC complex that bears homology to yeast SDS3, physically associates with mSin3 proteins in vivo, represses transcription in a manner that is partially dependent on HDAC activity, and enables HDAC1 catalytic activity in vivo. That key physical and functional properties are also shared by yeast SDS3 underscores the central role of the Sin3-HDAC-Sds3 complex in eukaryotic cell biology, and the discovery of mSds3 in mammalian cells provides a new avenue for modulating the activity of this complex in human disease.