Increased Expression of Sema3C Indicates a Poor Prognosis and Is Regulated by miR-142-5p in Glioma.

Sema3C has been reported to promote glioma stem cells self-renewal and glioblastoma growth. However, the prognostic value and the regulatory mechanism for its abnormal expression in glioma remain poorly understood. In the current study, the immunohistochemistry results demonstrated that Sema3C was overexpressed in 169 of 216 (78.2%) interpretable glioma patients compared with 3 of 15 (20.0%) interpretable non-neoplastic brain cases (p = 0.0001). Sema3C overexpression was significantly associated with histologic type (p = 0.008), high Ki67 labeling index (p = 0.02), tumor grade (p = 0.002) and wild type IDH1 (p = 0.0001). Importantly, its overexpression predicts the shorter overall survival of glioma patients (p = 0.0017), especially the ones with high grade (p = 0.0124). Functionally, Sema3C silencing significantly reduced the proliferation and invasion of glioma cells, indicating an oncogenic role of Sema3C in glioma in vitro. To elucidate the reason accounting for its overexpression, it is identified miR-142-5p as a tumor suppressor that directly targets Sema3C in glioma cells. miR-142-5p and Sema3C were co-regulators of epithelial-mesenchymal transition. Clinically, miR-142-5p expression was conversely related with Sema3C expression in glioma samples. Together, we identified that Sema3C could promote the progression of glioma and its expression was negatively regulated by miR-142-5p in vitro. Thus, the miR-142-5p-Sema3C axis plays importantly in glioma and holds potential to be therapeutic targets as well.

Chinese Angelica Polysaccharide (CAP) Alleviates LPS-Induced Inflammation and Apoptosis by Down-Regulating COX-1 in PC12 Cells.

BACKGROUND/AIMS: Chinese angelica polysaccharide (CAP) is the main effective ingredient of angelica sinensis and exerts anti-inflammatory and anti-apoptotic effects on many diseases. This study aimed to explore the pharmacological potential of CAP on spinal cord injury (SCI). METHODS: PC12 cells were pretreated by CAP and were subjected to LPS. Transfection was performed to alter the expression of COX-1. Cell viability and apoptotic cell rate were measured by CCK-8 and flow cytometry respectively. qRT-PCR and western blot analysis were performed to assess the expression changes of pro-inflammatory cytokines, apoptosis-related factor and core kinases in PI3K/AKT pathway. RESULTS: LPS stimulation induced significant cell damage in PC12 cells as cell viability was repressed, apoptosis was induced and the expression levels of IL-1ß, IL-6, IL-8, and TNF-α were increased. CAP pretreatment protected PC12 cells against LPS-induced cell damage. Meanwhile CAP treatment reduced the expression of COX-1 even in LPS-stimulated PC12 cells. More importantly, COX-1 overexpression abolished the protective effects of CAP on LPS-injured PC12 cells. Finally, Western blot analytical results showed that CAP activated PI3K/AKT pathway also in a COX-1-dependent manner. CONCLUSION: CAP exerted anti-apoptotic and anti-inflammatory effects on LPS-injured PC12 cells via down-regulation of COX-1.

Insulin-like growth factor binding protein 4 inhibits proliferation of bone marrow mesenchymal stem cells and enhances growth of neurospheres derived from the stem cells.

Insulin-like growth factor binding protein 4 (IGFBP-4) was reported to trigger cellular senescence and reduce cell growth of bone marrow mesenchymal stem cells (BMSCs), but its contribution to neurogenic differentiation of BMSCs remains unknown. In the present study, BMSCs were isolated from the femur and tibia of young rats to investigate effects of IGFBP-4 on BMSC proliferation and growth of neurospheres derived from BMSCs. Bone marrow mesenchymal stem cell proliferation was assessed using CCK-8 after treatment with IGFBP-4 or blockers of IGF-IR and ß-catenin. Phosphorylation levels of Akt, Erk, and p38 in BMSCs were analysed by Western blotting. Bone marrow mesenchymal stem cells were induced into neural lineages in NeuroCult medium; the number and the size of BMSC-derived neurospheres were counted after treatment with IGFBP-4 or the blockers. It was shown that addition of IGFBP-4 inhibited BMSC proliferation and immunodepletion of IGFBP-4 increased the proliferation. The blockade of IGF-IR with AG1024 increased BMSC proliferation and reversed IGFBP-4-induced proliferation inhibition; however, blocking of ß-catenin with FH535 did not. p-Erk was significantly decreased in IGFBP-4-treated BMSCs. IGFBP-4 promoted the growth of neurospheres derived from BMSCs, as manifested by the increases in the number and the size of the derived neurospheres. Both AG1024 and FH535 inhibited the formation of NeuroCult-induced neurospheres, but FH535 significantly inhibited the growth of neurospheres in NeuroCult medium with EGF, bFGF, and IGFBP-4. The data suggested that IGFBP-4 inhibits BMSC proliferation through IGF-IR pathway and promotes growth of BMSC-derived neurospheres via stabilizing ß-catenin.

Malignant transformation of bone marrow stromal cells induced by the brain glioma niche in rats.

Normal human embryonic stem cells (hESCs) can develop neoplastic cancer stem cell (CSC) properties after coculture with transformed hESCs in vitro. In the present study, the influence of the tumor microenvironment on malignant transformation of bone marrow stromal cells (BMSCs) was studied after allografting a mixture of enhanced green fluorescent protein (EGFP)-labeled BMSCs and C6 glioma cells into the rat brain to understand the influence of the cellular environment, especially the tumor environment, on the transformation of grafted BMSCs in the rat brain. We performed intracerebral transplantation in the rat brain using EGFP-labeled BMSCs coinjected with C6 tumor cells. After transplantation, the EGFP-labeled cells were isolated from the tumor using fluorescence-activated cell sorting, and the characteristics of the recovered cells were investigated. Glioma-specific biomarkers of the sorted cells and the biological characteristics of the tumors were analyzed. The BMSCs isolated from the cografts were transformed into glioma CSCs, as indicated by the marked expression of the glioma marker GFAP in glioma cells, and of Nestin and CD133 in neural stem cells and CSCs, as well as rapid cell growth, decreased level of the tumor suppressor gene p53, increased level of the oncogene murine double minute gene 2 (MDM2), and recapitulation of glioma tissues in the brain. These data suggest that BMSCs can be transformed into CSCs, which can be further directed toward glioma formation under certain conditions, supporting the notion that the tumor microenvironment is involved in transforming normal BMSCs into glial CSCs.

Subthalamic hGAD65 gene therapy and striatum TH gene transfer in a Parkinson's disease rat model.

The aim of the present study is to detect a combination method to utilize gene therapy for the treatment of Parkinson's disease (PD). Here, a PD rat model is used for the in vivo gene therapy of a recombinant adeno-associated virus (AAV2) containing a human glutamic acid decarboxylase 65 (rAAV2-hGAD65) gene delivered to the subthalamic nucleus (STN). This is combined with the ex vivo gene delivery of tyrosine hydroxylase (TH) by fibroblasts injected into the striatum. After the treatment, the rotation behavior was improved with the greatest efficacy in the combination group. The results of immunohistochemistry showed that hGAD65 gene delivery by AAV2 successfully led to phenotypic changes of neurons in STN. And the levels of glutamic acid and GABA in the internal segment of the globus pallidus (GPi) and substantia nigra pars reticulata (SNr) were obviously lower than the control groups. However, hGAD65 gene transfer did not effectively protect surviving dopaminergic neurons in the SNc and VTA. This study suggests that subthalamic hGAD65 gene therapy and combined with TH gene therapy can alleviate symptoms of the PD model rats, independent of the protection the DA neurons from death.

A comprehensive study on long-term injury to nigral dopaminergic neurons following intracerebroventricular injection of lipopolysaccharide in rats.

Parkinson's disease (PD) is characterized by selective and progressive degeneration of dopaminergic neurons in the substantia nigra (SN). Lipopolysaccharide (LPS) can induce chronic inflammation and has been widely used to study the pathogenesis of PD. In this study, a single intracerebroventricular injection of LPS was used to induce neurotoxic effects on dopaminergic neurons in Sprague-Dawley rats. The long-term neurotoxic effects of LPS were evaluated at different time points. Microglia were activated in the hippocampus and striatum at 4 weeks, and in the SN at 24 weeks. Astrocytes were activated in the hippocampus and nigrostriatal system at 2 and 24 weeks. The expression of brain-derived neurotrophic factor in the SN increased at 4 weeks and decreased after 12 weeks, and tyrosine hydroxylase-positive neurons in the SN were shown to have an atrophic appearance, with cell loss evident after 24 weeks. Phospho-α-synuclein expression, a reflection of parkinsonian pathogenesis, increased at 12 weeks, and peaked at 24 weeks. Abnormal motor behavior appeared at 16 weeks and lasted up to 48 weeks. These results indicate that microglia are activated for several months after a single, low dose injection of LPS, which eventually results in progressive and selective damage to dopaminergic neurons in the SN.

Human umbilical cord blood-derived non-hematopoietic stem cells suppress lymphocyte proliferation and CD4, CD8 expression.

One concern in the use of transplantation of non-hematopoietic stem cells from human umbilical cord blood (CB-nHSCs) is the possibility of rejection by the host's immune system. This study shows that both CB-nHSCs and their progenies after passaging, neuronal differentiation or IFN-gamma treatment have no significant effects on proliferation of xenogenic T lymphocytes. CB-nHSCs transplanted into the striatum of SD rat are shown to induce a lower level of CD4 and CD8 expression in the brain and in the peripheral blood and to survive better in the brain than SH-SY5Y cells. The results indicate that both undifferentiated and differentiated CB-nHSCs all have weak immunogenicity.

Establishment of an immortalized GABAergic neuronal progenitor cell line from embryonic ventral mesencephalon in the rat.

Effective cell replacement therapies for neurological disease require neuron-restricted precursors as grafted cells. The problem of obtaining sufficient grafts for transplantation can be resolved by creating an appropriate immortalized cell line. In the present study, a thermally controlled immortalized GABAergic neuronal progenitor cell line (RMNE6) was established from E13 rat ventral mesencephalon cells immortalized using the temperature-sensitive mutant of SV40 large T antigen (ts-TAg). RMNE6 cells proliferated rapidly and expressed a neuron-like phenotype at the permissive temperature (33 degrees C), but eventually stopped growing at the non-permissive temperature (39 degrees C). Expression of the neuronal markers PSA-NCAM, beta-tubulin III and MAP2 by RMNE6 cells was confirmed by RT-PCR or immunocytochemistry. Furthermore, these cells exhibited functional GABAergic neuron properties, as evidenced by the expression of glutamate decarboxylase (GAD) as well as the synthesis and release of the neurotransmitter GABA in a calcium-dependent manner. Moreover, RMNE6 cells spontaneously expressed and secreted several neurotrophic factors, such as NGF, BDNF, NT-3, NT-4/5, and GDNF. The cells survived well and kept expression of SV40 Tag, GAD65/67 and GABA in the striatum, at least 28 days after being transplanted in the rat brain. Tumorigenesis assays confirmed the safety of the immortalized cell line in vivo. Taken together, the results support the use of RMNE6 cells as an ideal cell model for transplantation research aimed at the treatment and prevention of neurodegenerative disease.

Recombinant insulin-like growth factor binding protein-4 inhibits proliferation and promotes differentiation of neural progenitor cells.

Insulin-like growth factor (IGF) is involved in regulating many processes during neural development, and IGF binding protein-4 (IGFBP4) functions as a modulator of IGF actions or in an IGF-independent manner (e.g., via inhibiting Wnt/ß-catenin signaling). In the present study, neural progenitor cells (NPCs) were isolated from the forebrain of newborn mice to investigate effects of IGFBP4 on the proliferation and differentiation of NPCs. The proliferation of NPCs was evaluated using Cell Counting Kit-8 (CCK-8) after treatment with or without IGFBP4 as well as blockers of IGF-IR and ß-catenin. Phosphorylation levels of Akt, Erk1, 2 and p38 were analyzed by Western blotting. The differentiation of NPCs was evaluated using immunofluorescence and Western blotting. It was shown that exogenous IGFBP4 significantly inhibited the proliferation of NPCs and it did not induce a more pronounced inhibition of cell proliferation after blockade of IGF-IR but it did after antagonism of ß-catenin. Akt phosphorylation was significantly decreased and phosphorylation levels of Erk1, 2 and p38 were not significantly changed in IGFBP4-treated NPCs. Excessive IGFBP4 significantly promoted NPCs to differentiate into astrocytes and neurons. These data suggested that exogenous IGFBP4 inhibits proliferation and promotes differentiation of neural progenitor cells mainly through IGF-IR signaling pathway.

RNAi-mediated silencing of HLA A2 suppressed acute rejection against human fibroblast xenografts in the striatum of 6-OHDA lesioned rats.

Major histocompatibility complex class l (MHC I) molecules play a role in determining whether transplanted cells will be accepted or rejected, and masking of MHC I on donor cells has been found useful for immunoprotection of neural xenografts. In the present study, primary human embryonic lung fibroblasts (HELF), HELF treated with lentivirus-mediated small interfering RNAs (siRNAs) targeting human leukocyte antigen A2 (HLA A2, MHC I in humans) (siHELF), and rat embryonic lung fibroblasts (RELF) were stereotaxically grafted into the striatum of 6-hydroxydopamine lesioned rats to explore whether knockdown of HLA A2 could reduce host immune responses against xenografts. Before lentiviral infection, the cells were transduced with retroviruses harboring tyrosine hydroxylase cDNA. Knockdown of HLA A2 protein was examined by Western blotting. The immune responses (the number of CD4 and CD8 T-cells in the brain and peripheral blood), glial reaction, and survival of human fibroblasts were quantitatively evaluated by flow cytometry and immunohistochemistry at 4d, 2w, and 6w post-graft. Animal behaviors were assessed by counting apomorphine-induced rotations pre- and post-grafts. It was shown that a lower level of HLA A2 was observed in siHELF grafts than in HELF grafts, and knockdown of HLA A2 decreased rat immune responses, as indicated by less remarkable increases in the number of CD8 and CD4 T-cells in the brain and the ratio of CD4:CD8 T-cells in the peripheral blood in rats grafted with siHELF. Rats grafted with siHELF exhibited a significant improvement in motor asymmetry post-transplantation and a better survival of human fibroblasts at 2w. The increasing number of activated microglia and the decreasing number of astrocytes were found in three groups of rats post-implantation. These data suggested that RNAi-mediated knockdown of HLA A2 could suppress acute rejection against xenogeneic human cell transplants in the rat brain.

Long-term expansion of human neural progenitor cells by epigenetic stimulation in vitro.

Human neural progenitor cells (hNPCs) are currently believed to have important potential for clinical application and basic neuroscience research. In the present study, we have developed a new technique for expansion of human neural progenitor cells in vitro. We showed that the cultures of hNPCs in monolayer could keep the same features with that growing in neurospheres. These cells expressed the typical protein of neural progenitors, nestin, and could form neurons and astrocytes upon differentiation. Using this method, we achieved an exponential increase in cells number over a period of 240 days in vitro. We also confirmed these cells expressed the orphan nuclear receptor-related factor 1 (Nurr1). Furthermore, we acquired the GFP-expressing human neural progenitor cells using retroviral-mediated transgenic system. The results of present study indicate the feasibility of long-term in vitro expansion of human neural progenitor cells using the monolayer culture technique, which may be of value as vehicles for ex vivo gene transfer to the CNS and as a potential source for basic research of dopaminergic (DA) neurons development.

[Clinical analysis of 2643 cases of trigeminal neuralgia treated by microvascular decompression].

OBJECTIVE: To evaluate the clinical effects of microvascular decompression in treating trigeminal neuralgia. METHODS: Surgical experience and operative findings of 2643 cases of trigeminal neuralgia treated by microvascular decompression were analyzed retrospectively. RESULTS: Two thousand four hundred and eighty-seven of 2643 cases were cured, 76 cases were ineffectiveness, 48 cases were effectiveness and 31 were ineffective. One patient died. Two thousand one hundred and thirty-six cases were followed up in 3-240 months, 1918 cases were cured, 85 cases were obviously effective, 39 cases were effective and 30 were ineffective. Sixty-four cases were pain relapsed and 37 cases were cured by second operation among them. CONCLUSION: The etiology of trigeminal neuralgia is that the unusual vascular oppress the root entry zone, and arachnoid membrane circling the nerve is thickened and sticked. To untie the arachnoid membrane and decompress the offending vascular is the effective methods in treating trigeminal neuralgia.

[Therapeutic evaluation of microvascular decompression in patients with trigeminal neuralgia associated with hypertension].

OBJECTIVE: The aim of work was to evaluate the efficacy of microvascular decompression (MVD) in patients with trigeminal neuralgia (TN) associated with hypertension. METHODS: Five hundred and thirty-eight cases of TN associated with hypertension treated with MVD were retrospectively analyzed, all the cases were treated with MVD in the zone of the ventrolateral medulla oblongata and inspected postoperative blood pressure. RESULTS: Among 538 cases, 341 cases (63.4%) had cure result, 71 cases (13.2%) had obvious effectiveness, 53 cases (9.8%) had effectiveness, and 73 cases had no effectiveness. Four hundred and eighty-three cases have been followed 1.0 approximately 15.8 years, mean 6.3 years. At the time of their follow-up, 313 cases had cure result, 63 cases had obvious effectiveness, 42 cases had effectiveness and 65 cases had no effectiveness. CONCLUSIONS: It was pathogenesis of nedulla oblongata by abnormal vascular tab, long-term aching stimulation and emotional stress. It was an effective method to treat TN associated with hypertension with MVD in this zone.

Lesion of the locus coeruleus aggravates dopaminergic neuron degeneration by modulating microglial function in mouse models of Parkinson׳s disease.

The degeneration of noradrenergic neurons in the locus coeruleus (LC) commonly occurs in patients with Parkinson's disease (PD), which is characterized by a selective injury of dopaminergic neurons in the substantia nigra (SN). The pathological impact of the LC on the SN in the disease is unknown. In the present study, we used a noradrenergic toxin, N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4), to deplete noradrenaline (NA) derived from the LC to explore its influence on degeneration or injury of dopaminergic neurons in the SN in mouse model produced by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or lipopolysaccharide (LPS). Our results demonstrated that lesion of the LC could change microglial function in the brain, which led to enhanced or prolonged expression of pro-inflammatory cytokines, diminished neurotrophic factors, and weakened ability of anti-oxidation in the SN. The in vitro experiments further confirmed that NA could reduce the inflammatory reaction of microglia. The selective injury of dopaminergic neurons by inflammation, however, was due to the inflammation in different brain regions rather than the depletion of NA. Our results indicate that the lesion in the LC is an important factor in promoting dopaminergic neuron degeneration by impacting the function of microglia in the midbrain.

Spatiotemporal expression patterns of Pax6 in the brain of embryonic, newborn, and adult mice.

The transcription factor Pax6 has been reported to specify neural progenitor cell fates during development and maintain neuronal commitments in the adult. The spatiotemporal patterns of Pax6 expression were examined in sagittal and horizontal sections of the embryonic, postnatal, and adult brains using immunohistochemistry and double immunolabeling. The proportion of Pax6-immunopositive cells in various parts of the adult brain was estimated using the isotropic fractionator methodology. It was shown that at embryonic day 11 (E11) Pax6 was robustly expressed in the proliferative neuroepithelia of the ventricular zone in the forebrain and hindbrain, and in the floor and the mesencephalic reticular formation (mRt) in the midbrain. At E12, its expression emerged in the nucleus of the lateral lemniscus in the rhombencephalon and disappeared from the floor of the midbrain. As neurodevelopment proceeds, the expression pattern of Pax6 changes from the mitotic germinal zone in the ventricular zone to become extensively distributed in cell groups in the forebrain and hindbrain, and the expression persisted in the mRt. The majority of Pax6-positive cell groups were maintained until adult life, but the intensity of Pax6 expression became much weaker. Pax6 expression was maintained in the mitotic subventricular zone in the adult brain, but not in the germinal region dentate gyrus in the adult hippocampus. There was no obvious colocalization of Pax6 and NeuN during embryonic development, suggesting Pax6 is found primarily in developing progenitor cells. In the adult brain, however, Pax6 maintains neuronal features of some subtypes of neurons, as indicated by 97.1% of Pax6-positive cells co-expressing NeuN in the cerebellum, 40.7% in the olfactory bulb, 38.3% in the cerebrum, and 73.9% in the remaining brain except the hippocampus. Differentiated tyrosine hydroxylase (TH) neurons were observed in the floor of the E11 midbrain where Pax6 was also expressed, but no obvious colocaliztion of TH and Pax6 was detected. No Pax6 expression was observed in TH-expressing areas in the midbrain at E12, E14, and postnatal day 1. These results support the notion that Pax6 plays pivotal roles in specifying neural progenitor cell commitments and maintaining certain mature neuronal fates.

Projections from the paralemniscal nucleus to the spinal cord in the mouse.

The present study investigated the projection from the paralemniscal nucleus (PL) to the spinal cord in the mouse by injecting the retrograde tracer fluoro-gold to different levels of the spinal cord and injecting the anterograde tracer biotinylated dextran amine into PL. We found that PL projects to the entire spinal cord with obvious contralateral predominance--420 neurons projected to the contralateral cervical cord and 270 to the contralateral lumbar cord. Fibers from PL descended in the dorsolateral funiculus on the contralateral side and terminated in laminae 5, 6, 7, and to a lesser extent in the dorsal and ventral horns. A smaller number of fibers also descended in the ventral funiculus on the ipsilateral side and terminated in laminae 7, 8 and, to a lesser extent in lamina 9. The present study is the first demonstration of the PL fiber termination in the spinal cord in mammals. The PL projection to the spinal cord may be involved in vocalization and locomotion.

Long-term restoration of nigrostriatal system function by implanting GDNF genetically modified fibroblasts in a rat model of Parkinson's disease.

The motor behavior and levels of dopamine and its metabolites in the striatum were studied in rats that received a unilateral injection of 6-OHDA and underwent grafting of rat-derived primary fibroblasts that had been genetically modified to express lacZ and human glial cell line-derived neurotrophic factor (GDNF). Rotation behavior tests were performed each week and striatal levels of DA and its metabolites were measured every 4 weeks after grafting of fibroblasts that expressed lacZ, with or without additional transfection of the GDNF transgene. Rats grafted with GDNF-producing fibroblasts showed a significant improvement in motor behavior as determined by the rotation test, with a less pronounced reduction in the levels of dopamine and its metabolites in the striatum as compared with those in the control animals or brain parts. In addition, there was a lower decrease in the number of TH immunoreactive neurons in the substantia nigra ipsilateral to the lesion in rats with GDNF-producing fibroblasts than in rats with lacZ-expressing fibroblasts. These results support the notion that intracerebral grafting of fibroblasts that express GDNF is a potentially useful therapeutic strategy for treating Parkinson's disease.

[Clinical study on the removal of sellar region tumors by pterional approach (report of 119 cases)].

BACKGROUND & OBJECTIVE: Sellar region tumors are common intracranial tumor. This study was designed to investigate the methods of the removal of tumors in sellar region by pterional approach. METHODS: A total of 119 cases with sellar region tumors were performed operation. Before the operation, the relationships between the tumors and their periphery were determined according to CT, MR and the cerebral angiography and then the tumors were removed by pterional approach microsurgery in 4 different spaces. RESULTS: The number of the patients treated with total, subtotal, and partial resection were 72, 36, and 11, respectively; with 85 cases significantly improved, 24 getting better, 8 without changes and 2 dead. Follow-up half a year, 92 cases were survival in the half a year of follow-up, in which 61 cases were able to work with normal life, 15 could do light work, 9 could take care of themselves but 6 patients were disabled, and 1 died of heart disease. CONCLUSION: Pterional approach can be used to remove many kinds of tumors in sellar region and with better exploration, therefore, increase the total removal and cure rate of such tumors.