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BACKGROUND:Cel replacement therapy as an effective strategy for reconstruction of the central nervous system has very broad application prospects. OBJECTIVE:To investigate the effect of stereotactic transplantation of neural stem cels into the brain on the neuromotor function of craniocerebral trauma rats. METHODS:Twenty male Sprague-Dawley rats were equivalently randomized into study and control groups. Animal models of craniocerebral trauma were made using the improved free-fal method in the rats. Then, model rats in the study and control groups were given parenchymal transplantation of embryonic neural stem cels and the same volume of culture medium with no stem cels at 1 day after injury, respectively. Neuromotor function of rats was assessed based on the neurological severity scores. At 2 weeks after transplantation, brain tissues were taken for hematoxylin-eosin staining, anti-BrdU, glial fibrilary acidic protein, β-tubulin III and tyrosine hydroxylase immunohistochemistry staining. RESULTS AND CONCLUSION:The neurological severity scores in the study group were significantly lower than those in the control group at 1 and 2 weeks after injury (P< 0.05). In the study group, there were many BrdU-positive neural stem cels in the brain tissues, some of which were positive for glial fibrilary acidic protein, β-tubulin III and tyrosine hydroxylase; while in the control group, there was no BrdU-positive cel in the brain tissues. Experimental findings show that neural stem cels stereotacticaly transplanted into the brain can proliferate and differentiate in the brain lesion, and thereby notably improve the neuromotor function of rats with craniocerebral trauma.
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BACKGROUND:Isoflurane cannot only induce a wide range of large neuronal apoptosis, but also inhibit hippocampal neurogenesis in neonatal rats, thereby resulting in hippocampus-dependent learning and memory defects. OBJECTIVE:To investigate the isoflurane effect on proliferation and differentiation of the hippocampal neural stem cels. METHODS:Twenty-six Sprague-Dawley rats were randomly divided into air group and isoflurane group (n=13 per group). Rats in the isoflurane group were subjected to 2.5% isoflurane inhalation for 3 minutes folowed by 1.5% isoflurane inhalation for 4 hours. Rats in the air group only breathed in air. After the intervention, blood glucose and arterial blood gas changes were detected in the two groups. Additionaly, rats in the two groups were given intraperitoneal injection of 5-bromodeoxyuridine before and after intervention. At 24 hours after the last injection of 5-bromodeoxyuridine, brain tissues were taken to make frozen sections for immunofluorescence staining. RESULTS AND CONCLUSION:There were no significant difference in pH, PaO2, PaCO2, HCO3, BE and SaO2 levels between the two groups (P> 0.05). Compared with the air group, the number of BrdU+ cels was significantly less in the isoflurane group (P < 0.05), while the number of NeuroD+/BrdU+ cels was significantly higher in the isoflurane group (P < 0.05). The incidence of adverse reactions was 23% in the isoflurane group, which was significantly higher than that in the air group (7.7%;P < 0.05). These findings indicate that isoflurane can inhibit the proliferation of neural stem cels in the hippocampal dentate gyrus, and promote their differentiation into neurons.
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BACKGROUND:Isoflurane is an anesthesia drug that has a certain effect on the nervous system. It possibly causes neurologic disorders through impacting nerve stem cel function or morphology. OBJECTIVE:To investigate the effects of isoflurane on the proliferation and differentiation of neural stem cels in the hippocampus of rats. METHODS:Neural stem cels from the hippocampus of neonatal Sprague-Dawley rats, aged 7 days, were induced and differentiated. Passage 3 cels were obtained and divided into two groups: isoflurane group (a mixture gas of 2.8% isoflurane, 5% CO2 and 95% O2) and control group (a mixture of 5% CO2 and 95% O2). After intervention of 6 hours folowed by 2 hours of routine culture, anti-BrdU monoclonal antibody immunofluorescent staining was used to detect cel proliferation, and western blot assay to detect the expression of β3-tubulin and glial fibrilary acidic protein. RESULTS AND CONCLUSION:Compared with the control group, the number of BrdU positive cels in the isoflurane group reduced significantly, indicating that isoflurane inhibits the proliferation of neural stem cels. Compared with the control group, the expression of glial fibrilary acidic protein in the isoflurane group up-regulated, but the expression of β3-tubulin had no changes, indicating isoflurane promotes the differentiation of neural stem cels into astrocytes. Cite this article:Min N, Hu QF, Li XP, Nie XH, Yang LL.Isoflurane effects on the proliferation and differentiation of neural stem cels in the hippocampus of neonatal rats. Zhongguo Zuzhi Gongcheng Yanjiu. 2016;20(1):118-122.
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BACKGROUND:Neural stem cel transplantation provides an important way to treat sequela of traumatic brain injury, but the timing for treatment is inconclusive. OBJECTIVE:To explore the clinical effect of neural stem cel transplantation in the treatment of sequela of traumatic brain injury and the choice of the best treatment time. METHODS: Totaly 178 patients with sequela of traumatic brain injury who underwent neural stem cel transplantation were divided into three groups as per the timing for neural stem cel transplantation: group A (with 6 months after injury,n=60), group B (6-12 months after injury,n=59), and group C (over 12 months after injury,n=59). Improvement in clinical symptoms and scores on function independent measure (FIM) were recorded and compared in the three groups. RESULTS AND CONCLUSION:The total effective rate of group A was significantly higher than that in groups B and C (P < 0.05). FIM scores were significantly improved in the three groups after cel transplantation (P < 0.05). At 3 months after the fourth transplantation, the FIM score in the group A was significantly higher than that in the other two groups, and the incidence of adverse reactions in the group A was significantly lower than that in the other two groups (P < 0.05). These findings indicate that neural stem cel transplantation at different timing can al harvest certain clinical effects, but the best timing for neural stem cel transplantation is within 6 months after injury.
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BACKGROUND:Abnormal hippocampal neurogenesis during aging has been reported to result in learning and memory dysfunction. But its mechanism is unclear. OBJECTIVE: To understand the changes of mouse neurogenesis in the hippocampal subgranular zone during aging. METHODS:C57BL/6 mice 2, 6 and 20 months of age were used. Immunochemistry was used to count the number of neural stem cels (nestin+), neuroblasts (Doublecortin+, DCX+), and proliferative cels (proliferating cel nuclear antigen+, PCNA+) in the hippocampal subgranular zone. mRNA expressions of aging-related genes, p19Arf and p21Cip1/Waf1, in the hippocampus were detected by reverse transcription-PCR. RESULTS AND CONCLUSION: Compared with the young and middle age groups, the number of PCNA+ cels, nestin+ and DCX+ cels in the hippocampal subgranular zone of the aged group decreased dramaticaly; the expression of p19Arf and p21Cip1/Waf1 mRNA increased in the aged group. Proliferation activity, the number of neural stem cels and neuronal differentiation al decreased. These findings indicate that the decline of hippocampal neurogenesis may be associated with increased expression of aging-related genes p19Arf and p21Cip1/Waf1 in the p19Arf-Mdm2-p53-p21Cip1/Waf1pathway.
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BACKGROUND:Chinese herb extracts can restore and protect the nervous system of rats through intervention of neural stem cels. OBJECTIVE:To explore the role of ginsenosides Rg1 in the proliferation and protection of neural stem cels. METHOD:Sprague-Dawley rats at pregnant 19 days were dissected to take out fetal rats, and then the hippocampal tissues from fetal rats were isolated to extract neural stem cels. Neural stem cels were co-cultured with DMEM/F12 medium containing 50 g/L ginsenosides Rg1 as intervention group, with DMEM/F12 medium as blank control group, and with DMEM/F12 containing 0.64% phenol as positive control group, respectively. MTT assay was used to detect the proliferation of neural stem cels in each group, and western blot method to detect the protein expression of brain-derived neurotrophic factor and transforming growth factor-β in neural stem cels. RESULTS AND CONCLUSION:Rat neural stem cels were round single cels with clear border at early period after isolation but at 2 days after inoculation, the cels were adherent and aggregated into smal cel spheres. Compared with the blank control group, the proliferative rate of neural stem cels was significantly increased in the ginsenosides Rg1 group (P < 0.05), but decreased in the positive control group (P < 0.05). Compared with the blank control group, in the ginsenosides Rg1 group, the expression of brain-derived neurotrophic factor was elevated, and the expression of transforming growth factor-β was reduced, indicating ginsenosides Rg1 has a certain effect to promote the proliferation of neural stem cels as wel as to protect the neural stem cels.
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BACKGROUND:Organism in the hypoxic state can produce hypoxia-inducible factor, but nitric oxide generated from inducible nitric oxide synthase can inhibit the activity of hypoxia-inducible factor 1α. OBJECTIVE:To discuss the relationship of hypoxia-inducible factor 1α and inducible nitric oxide synthase with hypoxia/reoxygenation injury in neural stem cels. METHODS:Under sterile conditions, Wistar rats born within 24 hours were sacrificed to separate the rat hippocampus that was used to prepare a cel suspension of brain tissue. After culture and passage, neural stem cels were divided into normoxia, hypoxia and hypoxia/reoxygenation groups. In the latter two groups, 150 μmol/L cobalt chloride solution was used to prepare hypoxia models, and in the hypoxia/reoxygenation group, the cels were reoxygenated after 4-hour hypoxia. RESULTS AND CONCLUSION:Under hypoxic conditions, a significant increase in mRNA expressions of hypoxia-inducible factor 1α and inducible nitric oxide synthase as wel as the number of apoptotic neural stem cels. Compared with the hypoxia group, the number of apoptotic neural stem cels was higher in the hypoxia/reoxygenation group, but the mRNA expressions of hypoxia-inducible factor 1α and inducible nitric oxide synthase were lower. These findings indicate that these two factors are involved in the hypoxia/reoxygenation injury of neural stem cels.
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BACKGROUND:In recent years, neural stem cels are considered to be ideal for the treatment of spinal cord injury, but the proportion of its natural differentiation into neurons in the host body is relatively low, which severely restricts the therapeutic effect on spinal cord injury. OBJECTIVE:To investigate the effect of erythropoietin on the differentiation of neural stem cels in vitro. METHODS:Under sterile condition, neural stem cels from the hippocampus of neonatal Wistar rats were isolated, cultured and identified by immunofluorescencein vitro. The third generation of neural stem cels were randomly divided into 0.5, 5, 50 U/mL erythropoietin groups and control group (with no erythropoietin). RESULTS AND CONCLUSION:Compared with the control group, the differentiation rate of neural stem cels was significantly improved in the 0.5, 5, 50 U/mL erythropoietin groups (P 0.05). These findings indicate that erythropoietin can effectively induce the differentiation of neural stem cels into neurons in vitro, and moreover, it can significantly improve the differentiation rate of neural stem cels into neurons.
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each group were sacrificed, respectively. Distributions of BrdU positive cels and ChAT positive cels were detected by S-P immunohistochemical method. The learning and memory abilities of rats were detected by Morris water maze system. RESULTS AND CONCLUSION:BrdU positive cels were mainly distributed in the cortex and hippocampus, especialy around the blood vessels, and there was the presence of focal aggregation. A smal amount of BrdU positive cels were observed in the basal ganglia and thalamus as wel as in the ependyma. BrdU positive cels were counted at different time after operation. The number of BrdU positive cels decreased with time, and only a smal number of BrdU positive cels were observed at 60 days after transplantation. The number of ChAT positive cels at different time after transplantation was ranked as folows: neural stem cel transplantation group > model group > sham operated group (P < 0.05). Compared with the model group, the time for searching the platform was significantly lower in the neural stem transplantation group and sham operated group, but the number of crossing the platform was significantly higher in the neural stem cel transplantation group and sham operated group (P < 0.05). The results show that neural stem cels could be transplanted into the rats with vascular dementia, and the cels could survive and migrate in the brain of rats and significantly improve the learning and memory ability. This mechanism may be related to the differentiation and growth of cholinergic neurons in the hippocampus.
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BACKGROUND:Neural stem cel transplantation alone has achieved unsatisfactory outcomes in the repair of damaged spinal cord tissues. To promote the survival, proliferation and neuronal differentiation of transplanted cels in vivo, it is necessary to further improve the micro-environment of spinal cord injury. OBJECTIVE:To investigate the effect of neural stem cel transplantation plus electroacupuncture on the hindlimb function and electrophysiological changes of rats with spinal cord injury. METHODS: Animal models of spinal cord injury were made in 72 Sprague-Dawley rats and randomized into four groups: control group with injection of culture mediumvia the tail vein; neural stem cel group with injection of neural stem cel suspensionvia the tail vein; electroacupuncture group given 1-week electroacupuncture atDu meridian and body points starting from 6 hours after modeling; combined group given injection of neural stem cel suspension via the tail vein+1-week electroacupuncture atDu meridian and body points starting from 6 hours after modeling. Motor functional recovery in rats was assessed by Basso-Beattie-Bresnahan score and inclined plane test before and at 1, 3 days and 1-4 weeks after modeling. At 4 weeks after modeling, hematoxylin-eosin staining was performed for pathological observation; fluorescence microscope was used to observe the survival and distribution of CM-Dil-labeled neural stem cels; horseradish peroxidase tracer was used to observe nerve fiber regeneration; rat neurophysiological recovery was assessed by determining motor evoked potentials and somatosensory evoked potentials. RESULTS AND CONCLUSION:At 2-4 weeks after modeling, the hindlimb function was better in the combined group than the neural stem cel group and electroacupuncture group; while it was better in the neural stem cel group and electroacupuncture group than the control group. At 4 weeks after modeling, there were few nerve axon-like structures and smal voids in the spinal cord of the neural stem cel group and electroacupuncture group; however, in the combined group, there were more nerve axon-like structures and no void in the spinal cord. At 4 weeks after modeling, the number of nerve fibers positive for CD-Dil and horseradish peroxidase was ranked as folows: combined group > neural stem cel group and electroacupuncture group > control group, and there were significant differences between groups (P < 0.05). The latencies of motor and somatosensory evoked potentials were significantly lower in the combined group than the neural stem cel group and electroaucpuncture group folowed by the control group (P < 0.05), whereas the amplitudes of motor and somatosensory evoked potentials were significantly higher in the combined group than the neural stem cel group and electroacupuncture group folowed by the control group (P < 0.05). In conclusion, these findings indicate that neural stem cel transplantation combined with electroacupuncture can promote synaptic regeneration and improve the motor and electrophysiological functions of rats.
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BACKGROUND:Until now, there is yet no complete recovery from spinal cord injury in terms of structure and functional recoveries. Neurotrophic factors have limited effects on nerve regeneration. Currently, stem cel transplantation may be an effective way to repair spinal cord injury. OBJECTIVE:To separate, cultivate and purify mouse spinal cord-derived neural stem cels using serum-free suspension method folowed by morphological observation, immunofluorescence technology and multi-lineage differentiation experiments. METHODS:By using the suspension culture method, mouse spinal cord-derived neural stem cels at embryonic day 13.5 were cultured and purified. Cel morphology changes were observed under inverted microscope. Cel proliferation ability was detected using cel counting kit-8. Nestin and Sox2 expression was detected by immunofluorescence technology. Multilineage differentiation of spinal cord-derived neural stem cels at passage 4 was detected by natural differentiation method in order to prove the differentiation ability. RESULTS AND CONCLUSION: Serum-free medium suspension culture method was successfuly applied to separate spinal cord-derived neural stem cels. Cultured cels had good proliferative ability and highly expressed Nestin and Sox2 that was in accordance with the results of DAPI nucleus staining, suggesting the high purity of cels. After induction, the cels could express both Tuj1 and GFAP, indicating the cels had good differentiation potential. This experiment has successfuly established the isolation, culture, identification system of spinal cord-derived neural stem cels, providing experimental basis for subsequent studies of neural stem cels.
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BACKGROUND:The use of neural stem cels provides a new approach for nervous system functional reconstruction and nerve regeneration. How to solve the induced differentiation of neural stem cels is stil a research hotspot. OBJECTIVE:To investigate the differentiation of rat neural stem cels into neurons and dopaminergic neurons under induction of glial cel-line derived neurotrophic factor gene. METHODS:PcDNA3-GDNF-GFP plasmids were constructed and transferredvia lipidosome into rat embryonic neural stem cels. Differentiation of neural stem cels after transfection was identified under a fluorescence microscope. Immunofluorescent staining was used to detect expression of β-tubulin III and tyrosine hydroxylase. RESULTS AND CONCLUSION:At 3 days after transfection, green fluorescence was observed in the transfected cels that were globular. At 7 days after transfection, the proportion of neural stem cels differentiating into neurons and dopaminergic neurons were significantly increased. These results indicate that the glial cel-line derived neurotrophic factor gene can induce the differentiation of neural stem cels into neurons and dopaminergic neurons.
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BACKGROUND:Neural stem cels have the potential to differentiate into neurons and glial cels to replace the injured brain cels, so as to achieve the purpose of repairing nerve injury. OBJECTIVE:To observe the neuronal differentiation ability of cel subsets with stem cel characteristics in the adult rat meningeal tissues. METHODS:Under anesthesia, the meningeal tissues were obtained from adult Sprague-Dawley rats to make cel suspension folowed by inoculation and subculture. Then, the Nestin immunofluorescence staining was performed. The third generation cels were culturedin vitro with complete culture medium containing trichostatin A. After 7 days of induction, western blot assay was used to detect the expression of NF-200 and BM88 proteins in neural cels. RESULTS AND CONCLUSION: At 24 hours of culture, some spherical cels were suspended and some cels adherent. In addition, some spherical cels scattered gradualy formed the clone spheres, and the growth rate decreased with the increasing volume. The positive expression of Nestin was detected by immunocytochemistry staining, and the cel nucleus was stained blue by Hoechst staining. BM88 and NF-200 proteins were al expressed at 7 days of neural induction. These findings indicate that the cel subsets with stem cel characteristics in the adult rat meningeal tissues can differentiate into neurons after in vitro induction.
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BACKGROUND:CT perfusion technology is a common non-invasive detection method, which can be used to quantitatively determine the ischemia severity and range at early stage of cerebral infarction and then judge whether ischemic brain tissues can survive or recover. OBJECTIVE:To assess the neurological function recovery of cerebral infarction rats undergoing neural stem cel transplantation using CT perfusion imaging. METHODS:A total of 60 Sprague-Dawley rats were randomly divided into control group, cerebral infarction group, transplantation group, with 20 rats in each group. Rat model of middle cerebral artery occlusion was made in the latter two groups. After 24 hours of modeling, PBS and 8×105 neural stem cels were administratedvia the tail vein into the rats in the cerebral infarction and transplantation groups, respectively. CT perfusion-weighted imaging was performed at 1, 3, 7, 14, 28 days after transplantation. Modified neurological severity scores were recorded at 1, 2, 3, 4 weeks after transplantation. Triphenyltetrazolium chloride staining was used to calculate infarct volume at 4 weeks after transplantation. Hematoxylin- eosin staining was adopted to observe pathological changes of brain tissues at 2 weeks after transplantation. RESULTS AND CONCLUSION: There were no abnormal hemodynamic changes in the control group at different time points. The transplantation group exhibited an increasing CT value with time, and the increased cerebral blood flow could improve the survival rate of neurons in the ischemic penumbra. The modified neurological severity score and infract volume in the transplantation group were both significantly lower than those in the cerebral infarction group (P < 0.05). Cel necrosis was improved obviously in the transplantation group. These results show that CT perfusion imaging can be used to observe the neurologic function recovery of cerebral infarction rats in aspects of morphology and hemodynamics.
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BACKGROUND:Adipose-derived mesenchymal stem cels are a group of pluripotent stem cels, and under certain conditions can differentiate into neural stem celsin vitro. OBJECTIVE:To investigate the proliferative and differentiation ability of different passage mesenchymal stem cellfrom adipose tissue into neurospheres. METHODS:The adipose-derived mesenchymal stem cels from Sprague-Dawley rats were separated and culturedin vitro, and morphology and proliferation rate of cels were observed and compared respectively at passages 3, 6, 10 and 20. The cellsurface antigens and cels cycle were identified by flow cytometry. Furthermore, adipose-derived mesenchymal stem cels were induced into neurospheres, and the neurosphere rate was counted. RESULTS AND CONCLUSION: Adipose-derived mesenchymal stem cels were mainly in long spindle shape, and cels at different passages had highly proliferative capacity in vitro. Except passage 3, adipose-derived mesenchymal stem cels strongly expressed CD29, CD44, CD73 and lowly expressed CD45 and CD34. The proportion of G0/G1 phase in cellcycle was 93.4% at passage 3, 92.7% at passage 6, 92.4% at passage 10, 86.0% at passage 20. Adipose-derived mesenchymal stem cels at passages 6 and 10 were easier to differentiate into neurospheres than those at passage 20 (P < 0.05), but cels at passage 3 were difficult to differentiate into neurospheres. Therefore, when using adipose-derived mesenchymal stem cels as seed cels, we should pay attention to choose the appropriate amplification passage in order to obtain the cels with best differentiation potential and cellpurity.
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BACKGROUND:Nerve growth factor (NGF) belongs to a biological macromolecule that is difficult to pass through the blood-brain barrier. However, a retroviral vector carrying exogenous gene can be stably inserted and integrated into the host cellgenome, which is suitable for gene therapy. OBJECTIVE:To study the gene expression of recombinant retroviral vector carrying rat NGF gene in neural stem cels. METHODS: The rat NGF gene was inserted into a retroviral vector pLEGFP-N1, which was transferred into packaging cels PT67 by Lipofectamine 2000. The positive clones in virus supernatant were colected by G418 selection and used to infect neural stem cels. After that, the NGF expression was tested by enzyme linked immunosorbent assay and the biological competence by PC12 cels, and then morphological change of neural stem cels and celltyping were examined by fluorescent microscope. RESULTS AND CONCLUSION:The neural stem cels could express extrinsic source NGF protein after the recombinant plasmid was infected into neural stem cels. The PC12 cels increased in the experimental group and stretched out long neurites. And the NGF protein could maintain the neural stem cellsurvival and stimulate their differentiation. These findings suggest that the neural stem cels carrying extrinsic source NGF gene could express NGF successfuly, and the NGF protein induced the survival and differentiation of neural stem cels.
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BACKGROUND:Poly(lactide-co-glycolide) (PLGA) scaffold is widely used in tissue engineering, but its poor cel adhesion ability and strong hydrophobicity limit its further development and application. OBJECTIVE: To study the biocompatibility of electrospun poly (lactide-co-glycolide)/polyethylene glycol (PLGA-PEG) nanofibrous scaffolds with mouse neural stem celsin vitro. METHODS:Neural stem cels were isolated from embryos of CD-1 mice at 15 embryonic days. Electrospinning was used to prepare PLGA and PLGA-PEG nanofibrous scaffolds. Scanning electron microscope was used for scanning observation of scaffolds. The 5th passage neural stem cels were seeded onto PLGA and PLGA-PEG scaffolds respectively, and culturedin vitro. RESULTS AND CONCLUSION: Interconnected porous network structure was observed in both two kinds of scaffolds under the scanning electron microscope. Fiber diameters and porosities of PLGA and PLGA-PEG scaffolds showed no significant differences (P > 0.05). Cel Counting Kit-8 detection showed neural stem cels grew wel on both two kinds of scaffolds and the absorbance value of two groups increased continuously with incubation time (1, 3, 5, 7, 9, 11 days). And there were statisticaly significant differences in the absorbance values between two groups at each time point (P < 0.05). Moreover, the cel adhesion rate was significantly higher in the PLGA-PEG group than in the PLGA group at 3, 6, 9 hours of culture (P < 0.05). Hoechst 33342 staining showed normal morphology and quality of the nuclei, and significantly more cels were observed in the PLGA-PEG group than the PLGA group (P < 0.05). Under the scanning electron microscope, compared with the PLGA scaffold, the PLGA-PEG scaffold was better for growth and matrix secretion of neural stem cels. In conclusion, PLGA-PEG nanofibrous scaffolds prepared by electrospinning are safe, non-toxic and suitable for neural stem cels growth with wel biocompatibility, appropriate aperture and porosity.