Neural Stem Cells Group Research Articles

Brain-derived neurotrophic factor and retinal ganglion cells in the retina of diabetic rat after intravitreal injection of human umbilical cord mesenchymal stem cells-induced neural stem cells

Objective To investigate the effect of intravitreal injection of neural stem cells (NSC) derived from human umbilical cord mesenchymal stem cells (hUCMSC) on the expression of brain-derived neurotrophic factor (BDNF) and the number of retinal ganglion cells (RGC). Methods Fifty-two adult male Sprague-Dawley rats were randomly divided into normal group (group A) and diabetes mellitus group which received intraperitoneal injection of streptozocin to make diabetic rat models. One month after the diabetic rat models were confirmed successfully, diabetic rats were randomly divided into diabetic group (group B), hUCMSC group (group C) and hUCMSC-induced NSC group (group D). And thirteen diabetic rats were included in each group. Immuno-cytochemistry was applied to observe BDNF and thymosin-1(Thy-1) staining in the retina. Then mean integrated absorbance of the staining region on the retina slices were analyzed by Image-Pro Plus 6.0. The number of Thy-1 labeled RGC was record. Results BDNF and Thy-1 were positive on the retina slices from group A. The staining intensity from group B became weak and the expression of BDNF and Thy-1 gradually decrease with time (P 0.05) at 2 weeks after injection, but were significant different for other time points (P<0.05). Significant positive correlation between the expression of BDNF and the number of RGC were found by the Pearson correlation analysis (r=0.964, P<0.05). Conclusion Intravitreal injection of hUCMSC-derived NSC to diabetic rat may protect the retina by promoting the expression of BDNF and increasing the number of RGC. Key words: Diabetic retinopathy/therapy; Neural stem cells/transplantation; Brain-derived neurotrophic factor; Retinal ganglion cells; Animal experimentation

Neural stem cell transplantation combined with erythropoietin for the treatment of spinal cord injury in rats

Spinal cord injury (SCI) comprises nerve and motor function disorders that may be caused by a variety of damaging factors and is challenging to treat. The aim of the present study was to investigate the regenerative effects of neural stem cell (NSC) transplantation combined with intraperitoneal injection of erythropoietin (EPO) on cross-sectional SCI in rats. A model of SCI was induced in 40 adult Wistar rats via the complete transection of the 10th thoracic vertebra (T10). The rats were allocated at random into 4 groups: Control, NSC, EPO and NSC + EPO groups (n=10 per group). Morphological alterations associated with axonal regeneration were detected using neurofilament (NF)-200 immunohistochemistry and immunofluorescence staining after 8 weeks. Basso, Beattie and Bresnahan (BBB) scoring was used to evaluate the recovery of hindlimb function. A total of 5 rats died following surgery, including 2 control rats and 1 rat each in the EPO, NSC and NSC + EPO groups. NSCs labeled with bromodeoxyuridine were observed to have survived and migrated in the spinal cord tissue after 8 weeks. Significant histomorphological differences were observed in the NSC and NSC + EPO groups compared with the EPO and control groups. Furthermore, the rats of the NSC + EPO group exhibited significantly enhanced axonal regeneration in the SCI area compared with the NSC group rats. The rats of the NSC and NSC + EPO groups exhibited significantly improved BBB scores compared with the EPO and control group rats at 7 days after treatment (P<0.05). In addition, the BBB scores of the NSC + EPO group were significantly improved compared with those of the three other groups at 7 days after surgery (P<0.05). Therefore, the results of the present study suggest that NSC transplantation combined with intraperitoneal injection of EPO may benefit the survival and regeneration of injured axons, and accelerate the repair of injured spinal cord tissue, thus facilitating the functional recovery of hindlimb locomotor function in rats.

Transplantation of hypoxia-inducible factor-1α gene modified neural stem cells increases cell survival and angiogenesis after cerebral ischemia

Previous studies have indicated the beneficial effects of transplanted neural stem cells (NSCs) on cerebral ischemia. Hypoxia-inducible factor-1α (HIF-1α) is a master transcription factor of cellular hypoxic gene expression, and its signal pathway might be the primary mechanism through which hypoxia promotes the growth of NSCs. To test the hypothesis that HIF-1α contributes to the therapeutic effect of NSCs transplantation in cerebral ischemia, we compared the efficacy of transplanting PBS, NSCs infected with recombinant adenovirus, HIF-1α gene and NSCs infected with a recombinant adenovirus vector with HIF-1α gene (HIF-1α-NSCs). A transient middle cerebral artery occlusion (tMCAO) was used in this study. PBS, HIF-1α gene, NSCs and HIF-1α-NSCs were respectively injected into cortical peri-infarction of the rat brain at 24 h after MCAO. Neurological deficits were assessed using the modified neurological severity score (NSS). Immunohistochemistry for BrdU, VEGF, Von Willebrand Factor and Nissl staining were performed. Compared with other groups HIF-1α-NSCs showed better behavioral recovery at 7, 14, 21and 28 days, and lesser degree of brain atrophy in cortex and hemisphere. More BrdU-positive cells in HIF-1α-NSCs group than that in NSCs group. Expression of VEGF and Von Willebrand Factor are both higher in HIF-1α-NSCs than those in HIF-1α or in NSCs group. Thus, we concluded that during the early period after transplantation HIF-1α infected NSCs expressed gene products，which reduce brain injury by improving the survival of NSCs and protecting the vascular system．

Using primate neural stem cells cultured in self-assembling peptide nanofiber scaffolds to repair injured spinal cords in rats.

Transplanted primates' neural stem cells (NSCs) tissue engineering complex into spinal cord injury (SCI) model rats, analyze and evaluate the long-term effects of repairing. Primate NSCs were cultured in self-assembling peptide nanofiber scaffolds to repair SCI. Sun Yat-sen Memorial Hospital, Guangzhou, China. Primate NSCs were isolated and cultured in self-assembling peptide nanofiber scaffolds. T10 SCI model was established; the rats were randomly divided into four groups: NSC plus self-assembling peptide scaffold group; NSC group; self-assembling peptide scaffold group; and control group. Immunohistochemical staining and electronic microscope were used to investigate the growth and differentiation of transplanted NSCs. The motor function of the hind limbs of rats was evaluated (P<0.05 was considered as statistically significant). NSCs and NSCs cultured in self-assembling peptide nanofiber scaffolds could be induced to differentiation into neurons, glial cells and oligodendrocytes in vitro. The primate NSC culture was established in self-assembling peptide scaffolds. No significant difference was seen in the differentiation rate between primate NSCs cultured in self-assembling peptide nanofiber scaffolds and primate NSCs cultured in regular medium. The motor function of the hind limbs in the NSC plus self-assembling peptide scaffold group was significantly better than that of the other three groups. In addition, the NSCs of the NSC group mainly differentiated into astrocytes. Transplantation of primate NSCs cultured in self-assembling peptide scaffolds is efficient for repairing the injured spinal cord and for improving the motor function of spinal cord in rats. The National Natural Science Foundation of China; Science and Technology Office of Guangdong Province.

Characterization of Neurogenic Potential of Dental Pulp Stem Cells Cultured in Xeno/Serum-Free Condition: In Vitro and In Vivo Assessment.

Neural stem cells (NSCs) have a high potency for differentiation to neurons and glial cells for replacement of damaged cells and paracrine effects for the regeneration and remyelination of host axons. Dental pulp is known to have a potential to differentiate into neural-like cells; therefore, dental pulp may be used as an autologous cell source for neural repair. In this study, we selectively expanded stem cells from human dental pulp in an initial culture using NSC media under xeno- and serum-free conditions. At the initial step of primary culture, human dental pulp was divided into two groups according to the culture media: 10% fetal bovine serum medium group (FBS group) and NSC culture medium group (NSC group). In the NSC group relative to the FBS group, the expression of NSC markers and the concentrations of leukemia inhibitory factor, nerve growth factor, and stem cell factor were higher, although their expression levels were lower than those of human fetal NSCs. The transplanted cells of the NSC group survived well within the normal brain and injured spinal cord of rats and expressed nestin and Sox2. Under the xeno- and serum-free conditions, autologous human dental pulp-derived stem cells might prove useful for clinical cell-based therapies to repair damaged neural tissues.

BDNF Pretreatment of Human Embryonic-Derived Neural Stem Cells Improves Cell Survival and Functional Recovery after Transplantation in Hypoxic–Ischemic Stroke

Intra-arterial neural stem cell (NSC) therapy has the potential to improve long-term outcomes after stroke. Here we evaluate if pretreatment of NSCs with brain-derived neurotrophic factor (BDNF) prior to transplantation improves cell engraftment and functional recovery following hypoxic-ischemic (HI) stroke. Human embryonic-derived NSCs with or without BDNF pretreatment (1 h, 100 ng/ml) were transplanted 3 days after HI stroke. Functional recovery was assessed using the horizontal ladder test. Cell engraftment was evaluated using bioluminescence imaging (BLI) and histological counts of SC121(+) cells. Fluoro-Jade C (FJC) and NeuN stains were used to evaluate neuroprotection. The effect of BDNF on NSCs was analyzed using a migration assay, immunocytochemistry, Luminex proteomic assay, and RT-qPCR.BLI analysis demonstrated significantly higher photon flux in the BDNF-treated NSC group compared to untreated NSC (p = 0.049) and control groups (p = 0.0021) at 1 week after transplantation. Immunohistochemistry confirmed increased transplanted cell survival in the cortex (p = 0.0126) and hippocampus (p = 0.0098) of animals injected with BDNF-treated NSCs compared to untreated NSCs. Behavioral testing revealed that the BDNF-treated NSC group demonstrated increased sensorimotor recovery compared to the untreated NSC and control groups (p < 0.001) over the 1-month period (p < 0.001) following transplantation. A significant improvement in performance was found in the BDNF-treated NSC group compared to the control group at 14, 21, and 28 (p < 0.05) days after transplantation. The cortex and hippocampus of the BDNF-treated NSC group had significantly more SC121(+) NSCs (p = 0.0125, p = 0.0098), fewer FJC(+) neurons (p = 0.0370, p = 0.0285), and a higher percentage of NeuN(+) expression (p = 0.0354) in the cortex compared to the untreated NSC group. BDNF treatment of NSCs resulted in significantly greater migration to SDF-1, secretion of M-CSF, VEGF, and expression of CXCR4, VCAM-1, Thrombospondins 1 and 2, and BDNF. BDNF pretreatment of NSCs results in higher initial NSC engraftment and survival, increased neuroprotection, and greater functional recovery when compared to untreated NSCs.

Effect of neural stem cells transplantation on protein expressions of doublecortin and nestin in hippocampal regions in aPP/PS1 mice

Objective To explore the effect of neural stem cells (NSCs) transplantation on neurogenesis in an alzheimer'disease mouse model and its related mechanism. Methods A total of 16 12-month-old aPP/PS1 double transgenic aD mice were randomly divided into two groups: NSC group (receiving NSCs transplantation in the bilateral hippocampi, n=8) and PBS group as the negative control group (receiving an equal quantity of 0.01M/L phosphate buffer saline, n=8). another 8 wild type mice without any treatment were selected as the positive control group (Wt group). at five weeks after transplantation, the expressions of doublecortin (DCX) and nestin in the hippocampal dentate gyrus (DG) and subgranular zone (SGZ) were analyzed by immunofluorescence staining, the number of positive cells in these regions were counted by confocal microscopy, and Morris water maze (MWM) test was used to assess cognitive function in all mice. Results NSC group showed the enhanced spatial learning and memory ability in Morris water maze (MWM) as compared to PBS group, but it was still lower than that in Wt mice (both P 0.05). Conclusions NSCs transplantation can promote endogenous neurogenesis via the increased expression of DCX- and Nestin-positive cells in the hippocampal DG/SGZ in aD mice, which improves the cognitive abilities in aD mice in some extent. Key words: Alzheimer disease; Stem cells transplantation

Synergistic effects of Buyang Huanwu decoction and embryonic neural stem cell transplantation on the recovery of neurological function in a rat model of spinal cord injury.

The aim of the present study was to investigate the therapeutic effect of a combined treatment of Buyang Huanwu decoction (BYHWD), a well-known formula of traditional Chinese medicine, and neural stem cells (NSCs) on spinal cord injury (SCI) and the associated underlying mechanisms. A SCI model was established by surgery via a complete transection of the T10 vertebra of female Sprague-Dawley rats. Gelatin sponges were used to absorb NSCs labeled with the thymidine analog, 5-bromo-2-deoxyuridine (BrdU), and were transferred into the transected spinal cords. BYHWD was administered once a day by introgastric infusion. Motor functions of the hind limbs were evaluated using the 21-point locomotor rating scale developed by Basso, Beattie and Bresnahan (BBB). The fate of the transplanted NSCs under the various conditions was examined by double immunofluorescence staining, using markers for neurons, astrocytes and oligodendrocytes, with BrdU. Ultrastructural changes of the SCI site following the various treatments were examined under a transmission electron microscope. The number of double positive cells for glial fibrillary acidic protein and BrdU in the BYHWD + NSC group was significantly decreased when compared with that in the NSC group (P<0.05). However, the number of cells that were labeled double positive for myelin basic protein and BrdU, as well as neuron specific enolase and BrdU, was greater in the BYHWD + NSC group when compared with the NSC group. Electron microscopy demonstrated that treatment with BYHWD combined with NSCs significantly alleviated demyelination. Results from the BBB motor function test exhibited a significant improvement in the BYHWD + NSC group when compared with the SCI, BYHWD and NSC only groups. In conclusion, the results demonstrated that the traditional Chinese medicine formula, BYHWD, exerted an effect on the differentiation and migration of NSCs. Combining the administration of BYHWD with NSCs was shown to have a synergistic effect on the recovery of neurological function, mitigating the progress of demyelination or ameliorating the recovery of myelination.

Effectiveness of muscle basal lamina carrying neural stem cells and olfactory ensheathing cells in spinal cord repair.

We examined the effect of muscle basal lamina (MBL) with neural stem cells (NSCs) and olfactory ensheathing cells (OECs) on spinal cord injury repair. Seventy-two Sprague-Dawley rats were subjected to spinal cord hemisection and divided into 6 groups. In blank control group (group A), the ends of the spinal cord hemisection model were flushed with physiological saline. In NSC transplantation group (B), OEC transplantation group (C), MBL with NSC transplantation group (D), MBL with OEC transplantation group (E), and MBL with NSC and OEC transplantation group (F), NSCs, OECs, MBL with NSCs, MBL with OECs, and MBL with NSCs and OECs were implanted into the ends of the hemisection model. Survival and migration of transplanted cells were detected by immunohistochemistry and immunofluorescence after 4 and 8 weeks. Hind limb function repair was evaluated by Bundle branch block score at various time points before and after surgery. MBL could promote NSC growth along its lumen and promote host cell advancement in the lumen, reducing local inflammatory responses. Using MBL with NSCs and/or OECs for spinal cord repair shows advantages over simple cell transplantation. Group F contained more nerve cells in muscle basal lamina than group E. This method is useful for forming more axons, synaptic connections, and signal transduction pathways. However, these new axons showed nerve demyelination, which may greatly limit nerve signal conduction. In group F, OECs could induce neural stem cells, axonal growth, and synaptic connection formation, but its role is limited.

CB-09 * THE CELL OF ORIGIN FOR GLIOBLASTOMA CONTRIBUTES TO THE PHENOTYPIC HETEROGENEITY OF GLIOMA STEM CELLS

Glioblastoma Multiforme (GBM) is the most frequent adult primary malignant brain tumor that remains incurable despite aggressive treatment. The cell of origin (COO) for GBM is unknown but assumed to be a glial stem or progenitor cell. GBM harbours hierarchical tumor cells called glioma stem cells (GSCs) that maintain tumor growth, drive tumor progression and cause tumor relapse due to their increased resistance to therapy. We have analyzed the significance of cellular origin for GBM development and GSC properties by comparing mouse GBMs and GSCs derived thereof induced in neural stem cells (NSCs), glial-restricted precursor cells (GPCs) or oligodendrocyte precursor cells (OPCs) by identical mutations. There were striking differences in GBM development and the phenotypes of GSCs and their response to drugs owing to the COO. Global gene expression analysis of mouse GSC lines displayed a clear separation due to COO and differential gene expression analysis identified a COO gene signature of 175 genes. Cross-species bioinformatics analyses were performed. First we analyzed the human cancer genome atlas (TCGA) GBM tissue samples and the mouse GSC expression data for a collection of TCGA GBM subtype signature genes. This showed that we could model both Proneural and Mesenchymal GBMs in mice by merely switching the COO. Next, we used the mouse COO gene signature to stratify a large number of newly established human glioma stem cell lines. This produced two groups of human GSCs; the NSC origin group and the progenitor cell (PC) origin group in which the mouse GPC- and OPC-derived genes were combined. Importantly, patient survival was significantly different between the NSC and PC COO groups with a better prognosis for the PC group patients. Thus, the cell of origin is essential for GBM biology and needs to be considered for more accurate patient stratification, target identification and drug discovery.

The Effects of Harvesting Media on Biological Characteristics and Repair Potential of Neural Stem Cells after Traumatic Brain Injury

Various solutions are utilized widely for the isolation, harvesting, sorting, testing and transplantation of neural stem cells (NSCs), whereas the effects of harvesting media on the biological characteristics and repair potential of NSCs remain unclear. To examine some of these effects, NSCs were isolated from cortex of E14.5 mice and exposed to the conventional harvesting media [0.9% saline (Saline), phosphate-buffered saline (PBS) or artificial cerebrospinal fluid (ACSF)] or the proliferation culture medium (PCM) for different durations at 4°C. Treated NSCs were grafted by in situ injection into the lesion sites of traumatic brain injury (TBI) mice. In vitro, harvesting media-exposed NSCs displayed time-dependent reduction of viability and proliferation. S phase entry decreased in harvesting media-exposed cells, which was associated with upregulation of p53 protein and downregulation of cyclin E1 protein. Moreover, harvesting media exposure induced the necrosis and apoptosis of NSCs. The levels of Fas-L, cleaved caspase 3 and 8 were increased, which suggests that the death receptor signaling pathway is involved in the apoptosis of NSCs. In addition, exposure to Saline did not facilitate the neuronal differentiation of NSCs, suggesting that Saline exposure may be disadvantageous for neurogenesis. In vivo, NSC-mediated functional recovery in harvesting media-exposed NSC groups was notably attenuated in comparison with the PCM-exposed NSC group. In conclusion, harvesting media exposure modulates the biological characteristics and repair potential of NSCs after TBI. Our results suggest that insight of the effects of harvesting media exposure on NSCs is critical for developing strategies to assure the successful long-term engraftment of NSCs.

The Effects of Controlled Release of Neurotrophin-3 from PCLA Scaffolds on the Survival and Neuronal Differentiation of Transplanted Neural Stem Cells in a Rat Spinal Cord Injury Model

Neural stem cells (NSCs) have emerged as a potential source for cell replacement therapy following spinal cord injury (SCI). However, poor survival and low neuronal differentiation remain major obstacles to the use of NSCs. Biomaterials with neurotrophic factors are promising strategies for promoting the proliferation and differentiation of NSCs. Silk fibroin (SF) matrices were demonstrated to successfully deliver growth factors and preserve their potency. In this study, by incorporating NT-3 into a SF coating, we successfully developed NT-3-immobilized scaffolds (membranes and conduits). Sustained release of bioactive NT-3 from the conduits for up to 8 weeks was achieved. Cell viability was confirmed using live/dead staining after 14 days in culture. The efficacy of the immobilized NT-3 was confirmed by assessing NSC neuronal differentiation in vitro. NSC neuronal differentiation was 55.2±4.1% on the NT-3-immobilized membranes, which was significantly higher than that on the NT-3 free membrane. Furthermore, 8 weeks after the NSCs were seeded into conduits and implanted in rats with a transected SCI, the conduit+NT-3+NSCs group achieved higher NSC survival (75.8±15.1%) and neuronal differentiation (21.5±5.2%) compared with the conduit+NSCs group. The animals that received the conduit+NT-3+NSCs treatment also showed improved functional outcomes, as well as increased axonal regeneration. These results indicate the feasibility of fabricating NT-3-immobilized scaffolds using the adsorption of NT-3/SF coating method, as well as the potential of these scaffolds to induce SCI repair by promoting survival and neuronal differentiation of transplanted NSCs.

Graft of the gelatin sponge scaffold containing genetically-modified neural stem cells promotes cell differentiation, axon regeneration, and functional recovery in rat with spinal cord transection.

Biological materials combined with genetically-modified neural stem cells (NSCs) are candidate therapy targeting spinal cord injury (SCI). Based on our previous studies, here we performed gelatin sponge (GS) scaffold seeded with neurotrophin-3 (NT-3) and its receptor TrkC gene modifying NSCs for repairing SCI. Eight weeks later, compared with other groups, neurofilament-200 and 5-hydroxytryptamine positive nerve fibers were more in the injury site of the N+T-NSCs group. Immunofluorescence staining showed the grafted NSCs could differentiate into microtubule associated protein (Map2), postsynaptic density (PSD95), and mouse oligodendrocyte special protein (MOSP) positive cells. The percentage of the Map2, PSD95, and MOSP positive cells in the N+T-NSCs group was higher than the other groups. Immuno-electron microscopy showed the grafted NSCs making contact with each other in the injury site. Behavioral analysis indicated the recovery of hindlimbs locomotion was better in the groups receiving cell transplant, the best recovery was found in the N+T-NSCs group. Electrophysiology revealed the amplitude of cortical motor evoked potentials was increased significantly in the N+T-NSCs group, but the latency remained long. These findings suggest the GS scaffold containing genetically-modified NSCs may bridge the injury site, promote axon regeneration and partial functional recovery in SCI rats.

E-cadherin-transfected neural stem cells transplantation for spinal cord injury in rats.

The effects of E-cadherin-transfected neural stem cells (NSCs) transplantation for spinal cord injury (SCI) in rats were investigated. Sixty SD rats were randomly divided into model control group, NSCs group, empty plasmid group and E-cadherin overexpression group (n=15 each). The animal SCI model was established by using the modified Allen's method. NSCs were cultured. Rats in NSCs group were subjected to NSCs transplantation. E-cadherin gene eucaryotic expression vector and pcDNA3.1-E-cadherin were respectively transfected into cultured NSCs, serving as empty plasmid group and E-cadherin overexpression group respectively. At 7th day after transplantation, neurological function of all rats was assessed by Tarlov score. After rats were sacrificed in each group, the number of BrdU and Nestin positive cells was counted by immunohistochemistry. Immumofluorescence method was used to detect the expression of neurofilament protein (NF) and glial fibrillary acidic protein (GFAP). As compared with model control group, the Tarlov score and the number of of BrdU and Nestin positive cells, and the expression of NF and GFAP in NSCs group, empty plasmid group, and E-cadherin overexpression group were increased significantly (P<0.05), and those in the E-cadherin overexpression group were increased more significantly than the other transplantation groups (P<0.05). It was suggested that E-cadherin could be conductive to nerve regeneration and repair probably by promoting the proliferation and differentiation of NSCs.

Co-grafting of neural stem cells with olfactory en sheathing cells promotes neuronal restoration in traumatic brain injury with an anti-inflammatory mechanism.

BackgroundWe sought to investigate the effects of co-grafting neural stem cells (NSCs) with olfactory ensheathing cells (OECs) on neurological behavior in rats subjected to traumatic brain injury (TBI) and explore underlying molecular mechanisms.MethodsTBI was established by percussion device made through a weight drop (50 g) from a 30 cm height. Cultured NSCs and OECs isolated from rats were labeled by Hoechst 33342 (blue) and chloromethyl-benzamidodialkyl carbocyanine (CM-Dil) (red), respectively. Then, NSCs and/or OECs, separately or combined, were transplanted into the area surrounding the injury site. Fourteen days after transplantation, neurological severity score (NSS) were recorded. The brain tissue was harvested and processed for immunocytochemistry, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and reverse transcription-polymerase chain reaction (RT-PCR).ResultsSignificant neurological function improvement was observed in the three transplant groups, compared to the TBI group, and co-transplantation gave rise to the best improvement. Morphological evaluation showed that the number of neurons in cortex from combination implantation was more than for other groups (P <0.05); conversely, the number of apoptotic cells showed a significant decrease by TUNEL staining. Transplanted NSCs and OECs could survive and migrate in the brain, and the number of neurons differentiating from NSCs in the co-transplantation group was significantly greater than in the NSCs group. At the molecular level, the expressions of IL-6 and BAD in the co-graft group were found to be down regulated significantly, when compared to either the NSC or OEC alone groups.ConclusionThe present study demonstrates for the first time the optimal effects of co-grafting NSCs and OECs as a new strategy for the treatment of TBI via an anti-inflammation mechanism.

Effects of transplanted neural stem cells on synaptogenesis in APP/PS1 mice

To explore the effects of transplanted neural stem cells (NSCs) on synaptogenesis in an Alzheimer' disease (AD) murine model and related mechanism. Twenty 9-month-old APP/PS1 double transgenic mice were randomly divided into 2 groups. One group received NSCs transplantation (NSC group) in bilateral hippocampi while another group received an equal volume of 0.01 mol/L phosphate buffer saline (PBS group) as a negative control group. Ten wild-type mice were selected as the positive control group (WT group) without any treatment. After 8-week transplantation, the expressions of synaptophysin (SYN) and growth associated protein-43 (GAP-43) proteins in hippocampal areas were analyzed by immunofluorescence and Western blot. The number and structure of synapses in transplanted regions were observed by electron microscopy. (1) Immunofluorescence staining showed that NSC-induced neurons highly expressed SYN and GAP-43 at the protein levels; (2) the expression of SYN and GAP-43 significantly increased in the NSC group versus the PBS group (F = 58.367, P < 0.01; F = 75.296, P < 0.01). No difference existed in the SYN level between NSC and WT groups (P > 0.05). However, the GAP-43 expression was significantly higher than that of the WT group (P < 0.01); (3) ultrastructure showed that the number of synapses in the NSC group with normal morphology (12.1 ± 2.1) increased than that in the PBS group (6.5 ± 2.2) (F = 15.981, P < 0.01). No difference existed between NSC and WT groups (11.0 ± 1.4) (P > 0.05). NSC-induced neurons increase the number of synapses by an up-regulation of synaptic proteins, SYN and GAP-43. Thus synaptogenesis may be a key factor in improving the symptom of AD mice.

The experimental research between magnetic resonance spectroscopy and changes of behavior for neural stem cell transplantation in APP/PS1 transgenic mice

Objective To explore the effect of neural stem cell(NSCs) transplantation on proton magnetic resonance spectroscopy (1H-MRS) and the behavior in APP/PS1 double transgenic AD mice. Methods NSCs from C57BL/6 mice were cultured and amplified. APP/PS1 double transgenic AD mice (n=30) aged 12 months were used as the study group, and mild-type mice (n=15) were used as the control group(group C ). Animals in the study group were randomly divided into two subgroups: one receiving NSCs (group A) and the other receiving PBS transplantation (group B) in bilateral hippocampal CA1 of the AD model mice. Animals in the group C were not treated. 1H-MRS and Morris water maze (MWM) were performed before transplantation and 4 weeks after transplantation, and compared with the histopathological results. Results 1H-MRS showed that there was no significant change in NAA/ Cr(1.01±0.08 and 1.03±0.05) and mI/Cr (0.69±0.05 and 0.71±0.06) ratios between group A and group B before transplantation (P> 0.05), but the changes were significant compared with the group C(NAA/ Cr: 1.21±0.05; mI/Cr: 0.58±0.06) (P<0.05). Four weeks after transplantation, NAA/ Cr ratio(1.18±0.09) was increased and mI/ Cr ratio (0.53±0.04) was decreased in group A. The difference was significant compared with the group B at the same time points (P<0.05). MWM showed the escape latency in group A was significantly shorter than that in group B after transplantation (P<0.05). In addition, group A also showed an exclusive preference for the target quadrant, and spent more time((35.21±5.44)s) in the 3rd quadrant compared with group B (P<0.05). For number of platform crossings, similar results were also shown(5.75±3.23). Nissl's staining showed that the number of neurons in the hippocampal area increased more significantly in group A than those in group B(P<0. 05). Conclusion NSCs transplantation can improve spatial learning and memory via neurons regeneration in APP/PS1 double transgenic AD mice, and 1H-MRS is able to display intracranial metabolite changes after NSCs transplantation. Key words: Neural stem cell; Alzheimer disease; Magnetic resonance spectroscopy; Transplantation

Migration of neural stem cells to ischemic brain regions in ischemic stroke in rats

An established rat model of ischemic stroke, produced by temporary middle cerebral artery occlusion and reperfusion (MCAO/R), was used in the evaluation of organ migration of intra-arterial (IA) transplantation of neural stem cells (NSCs). Immediately after transplantation, ischemic rats (n=8) transplanted with either NSCs (MCAO/R+NSC group) or NSC growth medium (MCAO/R+medium group) exhibited neurological dysfunction but rats in a sham+NSCs group (n=5) did not. During the post-operative period, neurological function improved to a similar extent in both MCAO/R groups. At 10 and 14 days post-transplantation, neurological function in the MCAO/R+NSC group was superior to that in the MCAO/R+medium group (p<0.001). Hematoxylin–eosin staining showed neuronal degeneration and necrosis in ischemic rats. Immunofluorescence staining revealed that NSCs had migrated to the frontal and parietal lobes, caudate, and putamen. Some cells had begun differentiating into neurons and astrocytes. Rat NSCs can migrate into the ischemic region, survive, and differentiate into astrocytes and neurons, and thereby potentially improve neurologic function after cerebral ischemia.

Role of Wnt/β-catenin signaling pathway in proliferation of neural stem cells of neonatal rats in vitro after hyperbaric oxygen therapy

Objective To investigate the role of Wnt/β-catenin signaling pathway in hyperbaric oxygen (HBO)therapy for hypoxic-ischemic brain damage(HIBD). Methods The neural stem cells (NSCs) of neonatal rats were randomly divided into control group and 8 treatment groups, then the treatment groups were cultured in the supernatant of brain homogenate to simulate micro-environment of HIBD and normal brain respectively.Through electroporation β-catenin siRNA and negative control plasmid was transfected into NSCs respectively, then HBO therapy was performed.Immunocytochemical staining was performed simultaneously in the 9 groups of NSCs on precoated chamber slides to detect the differentiation of NSCs.Quantitative reverse transcriptase(RT)-PCR was used to detect the relative content of Ngn1 mRNA and bone morphogenetic protein(BMP4) mRNA in the of NSCs.Western blot was used to detect the relative content of Ngn1 protein and BMP4 protein in the NSCs. Results In vitro, HBO alone promoted NSCs infected with negative control siRNA (ncNSCs) to differentiate into neurons and oligodendrocytes and depressed astrogliosis; HIBD or normal brain tissue extract cultures promoted ncNSCs to differentiate into neurons and oligodendrocytes but depressed astrogliosis, and the effect of HIBD brain extract cultures was superior to the latter and could be further increased by HBO; β-catenin siRNA decreased the NSE-positive neurons and increased glial fibrillary acidic protein-po-sitive astrocytes in the siNSCs (NSCs infected with β-catenin siRNA) in vitro, the effect could not be inversed by HBO, but could be alleviated; HBO increased the level of Ngn1 mRNA and Ngn1 protein, decreased BMP4 mRNA and BMP4 protein of ncNSCs, transfection of β-catenin siRNA could down-regulate the expression of Ngn1 mRNA and up-regulate BMP4 mRNA of NSCs in vitro respectively. Conclusions HBO can promote NSCs cultured with HIBD brain extract cultures to differentiate into neuronal or Oligodendrocyte, and inhibit them to differentiate into astrocytes.HBO therapy promotes the proliferation of NSCs in vitro, an effect which is correlated with β-catenin protein.HBO therapy can promote neurogenesis by β-catenin-induced activated Ngn1, and repress astrocytogenesis by β-catenin-induced down-regulated BMP4.There are potential cooperative actions of BMP4 and Ngn1 on differentiating rat NSCs in cerebral ischemic brain.The ability of Ngn1 to promote neurogenesis may allow Ngn1 to act as a potent neuronal commitment factor. Key words: β-catenin; RNA interference; Hyperbaric oxygen therapy; Hypoxic-ischemic brain damage; Electroporation

Transplantation of N-Acetyl Aspartyl-Glutamate Synthetase-Activated Neural Stem Cells after Experimental Traumatic Brain Injury Significantly Improves Neurological Recovery

Background/Aims: Neural stem cells (NSCs) hold considerable potential as a therapeutic tool for repair of the damaged nervous system. In the current study, we examined whether transplanted N-acetyl aspartyl-glutamate synthetase (NAAGS)-activated NSCs (NAAGS/NSCs) further improve neurological recovery following traumatic brain injury (TBI) in Sprague-Dawley rats. Methods: Animals received TBI and stereotactic injection of NSCs, NAAGS/NSCs or phosphate buffered saline without cells (control) into the injured cortex. NAAGS protein expression was detected through western blot analysis. Dialysate NAAG levels were analyzed with radioimmunoassay. Cell apoptosis was detected via TUNEL staining. The expression levels of specific pro-inflammatory cytokines were detected with enzyme-linked immunosorbent assay. Results: Groups with transplanted NSCs and NAAGS/NSCs displayed significant recovery of the motor behavior, compared to the control group. At 14 and 21 days post-transplantation, the motor behavior in NAAGS/NSC group was significantly improved than that in NSC group (p<0.05). Additionally, transplanted NAAGS/NSCs inhibited cell apoptosis and the expression levels of specific pro-inflammatory cytokines, including interleukin-1β, interleukin-6 and tumor necrosis factor-α. Conclusion: Our results collectively demonstrate that NAAGS/NSCs provide a more powerful autoplastic therapy for the injured nervous system.