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PURPOSE@#Mannitol is one of the first-line drugs for reducing cerebral edema through increasing the extracellular osmotic pressure. However, long-term administration of mannitol in the treatment of cerebral edema triggers damage to neurons and astrocytes. Given that neural stem cell (NSC) is a subpopulation of main regenerative cells in the central nervous system after injury, the effect of mannitol on NSC is still elusive. The present study aims to elucidate the role of mannitol in NSC proliferation.@*METHODS@#C57 mice were derived from the animal house of Zunyi Medical University. A total of 15 pregnant mice were employed for the purpose of isolating NSCs in this investigation. Initially, mouse primary NSCs were isolated from the embryonic cortex of mice and subsequently identified through immunofluorescence staining. In order to investigate the impact of mannitol on NSC proliferation, both cell counting kit-8 assays and neurospheres formation assays were conducted. The in vitro effects of mannitol were examined at various doses and time points. In order to elucidate the role of Aquaporin 4 (AQP4) in the suppressive effect of mannitol on NSC proliferation, various assays including reverse transcription polymerase chain reaction, western blotting, and immunocytochemistry were conducted on control and mannitol-treated groups. Additionally, the phosphorylated p38 (p-p38) was examined to explore the potential mechanism underlying the inhibitory effect of mannitol on NSC proliferation. Finally, to further confirm the involvement of the p38 mitogen-activated protein kinase-dependent (MAPK) signaling pathway in the observed inhibition of NSC proliferation by mannitol, SB203580 was employed. All data were analyzed using SPSS 20.0 software (SPSS, Inc., Chicago, IL). The statistical analysis among multiple comparisons was performed using one-way analysis of variance (ANOVA), followed by Turkey's post hoc test in case of the data following a normal distribution using a Shapiro-Wilk normality test. Comparisons between 2 groups were determined using Student's t-test, if the data exhibited a normal distribution using a Shapiro-Wilk normality test. Meanwhile, data were shown as median and interquartile range and analyzed using the Mann-Whitney U test, if the data failed the normality test. A p < 0.05 was considered as significant difference.@*RESULTS@#Primary NSC were isolated from the mice, and the characteristics were identified using immunostaining analysis. Thereafter, the results indicated that mannitol held the capability of inhibiting NSC proliferation in a dose-dependent and time-dependent manner using cell counting kit-8, neurospheres formation, and immunostaining of Nestin and Ki67 assays. During the process of mannitol suppressing NSC proliferation, the expression of AQP4 mRNA and protein was downregulated, while the gene expression of p-p38 was elevated by reverse transcription polymerase chain reaction, immunostaining, and western blotting assays. Subsequently, the administration of SB203580, one of the p38 MAPK signaling pathway inhibitors, partially abrogated this inhibitory effect resulting from mannitol, supporting the fact that the p38 MAPK signaling pathway participated in curbing NSC proliferation induced by mannitol.@*CONCLUSIONS@#Mannitol inhibits NSC proliferation through downregulating AQP4, while upregulating the expression of p-p38 MAPK.
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Humans , Animals , Mannitol/pharmacology , Brain Edema , Neural Stem Cells/metabolism , MAP Kinase Signaling System , p38 Mitogen-Activated Protein Kinases/pharmacology , Cell ProliferationABSTRACT
Background Cadmium (Cd) is a ubiquitous and toxic heavy metal that can accumulate in human body. Previous studies have shown that Cd exposure can induce neurotoxicity, but the underlying mechanism remains unclear. Objective To investigate the metabolic impacts of multiple doses of Cd on mouse neural stem cells (NSCs), and to explore the potential mechanism and biomarkers of its neurotoxicity. Methods The NSCs were obtained from the subventricular zone (SVZ) of 1-day-old neonatal C57BL/6 mice. The passage 3 (P3) NSCs were exposed to CdCl2 at designed doses (0, 0.5, 1.0, and 1.5 μmol·L−1). The cells were treated with seven replicates, of which one plate was for cell counting. After 24 h of exposure, the intracellular and extracellular metabolites were extracted respectively and then detected by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS). The orthogonal partial least-squares discriminant analysis (OPLS-DA) was applied to visualize the alterations of metabolomic profiles and to identify the differential metabolites (DMs) based on their variable importance for the projection (VIP) value >1 and P<0.05. The metabolite set enrichment analysis (MSEA) and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis were performed to recognize the significantly altered metabolite sets and pathways. The dose-response relationships were established and the potential biomarkers of Cd exposure were identified by 10% up-regulated or 10% down-regulated effective concentration (EC) of target metabolites. Results A total of 1201 metabolites were identified in the intracellular metabolomic samples and 1207 for the extracellular metabolomic samples. The intracellular and extracellular metabolome of Cd-treated NSCs were distinct from that of the control group, and the difference grew more distant as the Cd dosage increased. At 0.5, 1.0, and 1.5 μmol·L−1 dosage of Cd, 87, 83, and 185 intracellular DMs and 161, 176, and 166 extracellular DMs were identified, respectively. Within the significantly changed metabolites among the four groups, 176 intracellular DMs and 167 extracellular DMs were identified. Both intracellular and extracellular DMs were enriched in multiple lipid metabolite sets. Intracellular DMs were mainly enriched in taurine and hypotaurine metabolism, glycerophospholipid metabolism, and glycerolipid metabolism pathways. Extracellular DMs changed by Cd were mainly enriched in glycerophospholipid metabolism, steroid hormone biosynthesis, and cysteine and methionine metabolism pathways. Among intracellular DMs, 125 metabolites were fitted with dose-response relationships, of which 108 metabolites showed linear changes with the increase of Cd dosage. And 134 metabolites were fitted with dose-response relationships among extracellular DMs, of which 86 metabolites showed linear changes. The intracellular DMs with low EC values were hypotaurine, ethanolamine, phosphatidylethanolamine, and galactose, while the extracellular DMs with low EC values were acetylcholine and 1,5-anhydrosorbitol. Conclusion Cd treatment can significantly alter the intracellular and extracellular metabolome of mouse NSCs in a dose-dependent manner. The neurotoxicity of Cd may be related to glycerophospholipid metabolism. Acetylcholine, ethanolamine, and phosphatidylethanolamine involved in glycerophospholipid metabolism pathway might be potential biomarkers of Cd-induced neurotoxicity.
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Central nervous system (CNS) injuries, including stroke, traumatic brain injury, and spinal cord injury, are essential causes of death and long-term disability and are difficult to cure, mainly due to the limited neuron regeneration and the glial scar formation. Herein, we apply extracellular vesicles (EVs) secreted by M2 microglia to improve the differentiation of neural stem cells (NSCs) at the injured site, and simultaneously modify them with the injured vascular targeting peptide (DA7R) and the stem cell recruiting factor (SDF-1) on their surface via copper-free click chemistry to recruit NSCs, inducing their neuronal differentiation, and serving as the nanocarriers at the injured site (Dual-EV). Results prove that the Dual-EV could target human umbilical vascular endothelial cells (HUVECs), recruit NSCs, and promote the neuronal differentiation of NSCs in vitro. Furthermore, 10 miRNAs are found to be upregulated in Dual-M2-EVs compared to Dual-M0-EVs via bioinformatic analysis, and further NSC differentiation experiment by flow cytometry reveals that among these miRNAs, miR30b-3p, miR-222-3p, miR-129-5p, and miR-155-5p may exert effect of inducing NSC to differentiate into neurons. In vivo experiments show that Dual-EV nanocarriers achieve improved accumulation in the ischemic area of stroke model mice, potentiate NSCs recruitment, and increase neurogenesis. This work provides new insights for the treatment of neuronal regeneration after CNS injuries as well as endogenous stem cells, and the click chemistry EV/peptide/chemokine and related nanocarriers for improving human health.
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Objective To investigate the effect of cholesterol on the proliferation and differentiation of neural stem cells (NSCs) in ob/ob obese mice, and to explore the possible mechanism of central nervous systym dysfunction caused by obesity. Methods Selected 64-month-old ob/ob and wild type (WT) mice, and cell proliferation antigen (Ki67) and doublecortin (DCX) immunofluorescenct staining were used to detect ob/ob mice lateral ventricle subventricular zone (SVZ) neurogenesis level. Cultured SVZ NSCs isolated from 184-month-old ob/ob and WT mice, and BrdU incorporation experiment and β-III-tubulin (Tuj1) immunofluorescent staining were employed to detect the self-renewal and differentiation ability of NSCs. Matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI- MS)was used to detect the lipid distribution in 4-month-old ob/ob and WT mice brain tissues, and measure the changes of cholesterol(ST) content and the expression genes related to cholesterol synthesis. Cultured 15 WT postnatal day 0(P0) mouse SVZ NSCs in vitro and electrotransfected with the small interfering RNA(siRNA) sequence of cholesterol synthesis rate-limiting enzyme 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (Hmgcr) verified the knockdown efficiency, to detecte the effect of Hmgcr gene knockdown on NSCs by BrdU incorporation experiment and Tuj1 immunofluorescent staining. Results Compared with the WT mice, the number of Ki67
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Objective To investigate the expression and role of roundabout guidance receptor 1 (Robo1) in the neuronal differentiation of neural stem cells (NSCs) induced by valproate (VPA). Methods The hippocampus NSCs of SD rats were isolated and cultured. Normal NSCs and VPA-treated NSCs were extracted from 10 SD rats. After VPA treatment, the proportion of neuron-specific marker β-tubulin III (Tuj1) positive neurons differentiated from NSCs were detected by immunofluorescence. The differentially expressed mRNA in normal NSCs and VPA-induced NSCs were detected by gene chip technology. After VPA treatment, the expression levels of Robo1 mRNA and protein were detected by Real-time PCR and Western blotting. The dynamic changes of Robo1 mRNA were detected by Real-time PCR after the differentiation of NSCs. After the expression of Robo1 was down-regulated in NSCs by small interfering RNA, the expression of Robo1 protein was detected by Western blotting, and the expression levels of neuron-specific markers Tuj1 and microtubule associated protein-2 (MAP-2) were detected by Real-time PCR and immunofluorescence. Results VPA induced NSCs to differentiate into neurons. Compared with the control group, the expression levels of Robo1 mRNA and protein in the differentiation of NSCs were significantly up-regulated during valproate treatment. After interference of Robo1 expression, not only Robo1 upregulation was inhibited during the differentiation of NSCs induced by VPA, but also the proportion of NSCs differentiated into neurons decreased. Conclusion VPA may promote the differentiation of NSCs into neurons by up-regulating the expression of Robo1.
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Objective To investigate the effect and mechanism of astrocytes on the proliferation of neural stem cells (NSCs) in adult and juvenile hippocampus microenvironment. Methods Hippocampal astrocytes were isolated and cultured from 5 female SD rats at day 1 and week 30 postnatal, respectively; Embryonic hippocampus NSCs was isolated and cultured from 1 SD rat at day 15 of gestation; Conditioned astrocyte culture medium(CM) was collected for NSCs culture; Flow cytometry and CCK-8 were used to detect the proliferation of NSCs cultured in CM. Colony stimulating factor 1 (CSF-1) with differential expression was screened by mass spectrometry after cultured astrocyte CM. Western blotting and ELISA were used to verify the result of mass spectrometry. Immunofluorescence, flow cytometry and CCK-8 were used to detect the proliferation of NSCs treated with different concentrations of CSF-1 recombinant protein (20 μg/ L, 100 μg/ L, 1 mg/ L and 5 mg/ L). Results Compared with the adult group, the CM of hippocampal astrocytes in the young group could promote the proliferation of NSCs(P<0. 01); Compared with the conditioned medium of hippocampal astrocytes in the juvenile group, the expression of CSF-1 in the hippocampus of the elder group was significantly up-regulated(P<0. 01); At 20 μg/ L, CSF-1 promoted the proliferation of NSCs(P<0. 01), and 5 mg/ L CSF-1 inhibited significantly the proliferation of NSCs(P<0. 01). Conclusion The secretion of CSF-1 by astrocytes in hippocampal microenvironment can regulate the proliferation of NSCs with the development of the times.
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【Objective】 To investigate the improvement of motor function recovery and the activation of endogenous neural stem cells (eNSCs) via voluntary exercise in mice with hyperlipidemia after intracerebral hemorrhage (ICH). 【Methods】 Four-month-old male Nestin-CreERT2: tdTomato transgenic mice were fed with high-fat diet (HFD) for eight weeks. Type Ⅳ collagenase was micro-injected into the corpus striatum to construct mouse ICH model with the help of stereotaxic apparatus. Voluntary exercise (wheel running) was initiated on the second day after ICH and monitored daily for seven days. Neurological severity score (NSS) and beam walking test were applied to evaluate motor function and coordination. Liver and brain tissues were collected at day 9 after ICH and sliced for staining. Then the Nestin-labeled cells, Ki67+, and doublecortin (DCX)+ in subventricular zone (SVZ) were counted to evaluate eNSCs activation. 【Results】 ① Compared with those of mice fed by chow diet (CD), the body weight, blood glucose level, concentration of lipid metabolism factors and the number of Nile Red positive cells in liver tissue were significantly higher in HFD-fed mice, confirming hyperlipidemia. ② Compared with the sham group, NSS score increased and the distance of cross-beam walking of ICH mice significantly decreased, showing the deficiency of motor function. It could be rescued by 7-day wheel running, as shown by a lower NSS score and a longer cross-beam walking distance. ③ Compared with the sham group, the number of Nestin+/Ki67+ cells decreased and Nestin+/DCX+ cells increased after ICH. After 7-day voluntary exercise, the number of Nestin+/Ki67+ cells decreased but that of Nestin+/DCX+ cells further increased significantly. However, compared with ICH, the increase of Nestin+/DCX+ cells in ICH+Ex was not significant. 【Conclusion】 Short-term voluntary exercise during the acute stage of ICH improved the recovery of motor function and enhance the proliferation of eNSCs in mice with hyperlipidemia. This provides a new idea for further developing ICH accelerated rehabilitation strategy based on eNSCs.
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OBJECTIVE@#To study the effects of total ginsenosides (TG) extract from Panax ginseng on neural stem cell (NSC) proliferation and differentiation and their underlying mechanisms.@*METHODS@#The migration of NSCs after treatment with various concentrations of TG extract (50, 100, or 200 µ g/mL) were monitored. The proliferation of NSCs was examined by a combination of cell counting kit-8 and neurosphere assays. NSC differentiation mediated by TG extract was evaluated by Western blotting and immunofluorescence staining to monitor the expression of nestin and microtubule associated protein 2 (MAP2). The GSK-3β/β-catenin pathway in TG-treated NSCs was examined by Western blot assay. The NSCs with constitutively active GSK-3β mutant were made by adenovirus-mediated gene transfection, then the proliferation and differentiation of NSCs mediated by TG were further verified.@*RESULTS@#TG treatment significantly enhanced NSC migration (P<0.01 or P<0.05) and increased the proliferation of NSCs (P<0.01 or P<0.05). TG mediation also significantly upregulated MAP2 expression but downregulated nestin expression (P<0.01 or P<0.05). TG extract also significantly induced GSK-3β phosphorylation at Ser9, leading to GSK-3β inactivation and, consequently, the activation of the GSK-3β/β-catenin pathway (P<0.01 or P<0.05). In addition, constitutive activation of GSK-3β in NSCs by the transfection of GSK-3β S9A mutant was found to significantly suppress TG-mediated NSC proliferation and differentiation (P<0.01 or P<0.05).@*CONCLUSION@#TG promoted NSC proliferation and neuronal differentiation by inactivating GSK-3β.
Subject(s)
Animals , Rats , Cell Differentiation , Cell Proliferation , Ginsenosides/pharmacology , Glycogen Synthase Kinase 3 beta/metabolism , Neural Stem Cells/metabolism , Panax , Plant Extracts/pharmacology , beta Catenin/metabolismABSTRACT
OBJECTIVE@#To observe the effect of electroacupuncture (EA) on the proliferation of endogenous neural stem cells in the hippocampus of young mice with Alzheimer's disease (AD), so as to explore its mechanisms underlying improvement of AD.@*METHODS@#Forty 1.5-month-old APP/PS1 transgenic male mice were randomly divided into an EA group and a model group, 20 mice in each group, and other 20 C57BL/6J male mice of the same age were used as the normal control group. EA (intermittment wave 10 Hz, 2 mA) was applied to "Baihui" (GV 20), "Fengfu" (GV 16) and "Shenshu" (BL 23) for 20 min, once a day, 6 days a week for 16 weeks. H.E. staining was used to assess histopathological changes of neurons of the hippocampal dentate gyrus. Immunohistochemical stain was used to detect the expression of 5-bromodeoxyuridine (BrdU)-positive in the hippocampus, and immunofluorescence double-labeled technique was used to detect the number of proliferated positive neurons of hippocampal neural stem cells. The expression levels of brain derived neurotrophic factor (BDNF) and Nestin mRNA and protein were detected by using real-time PCR and Western blot, separately.@*RESULTS@#The immunoactivity of BrdU, and the expression levels of BDNF and Nestin mRNA and protein in the hippocampus in the model group were significantly lower than in the normal control group (P<0.01, P<0.05), and considerably higher in the EA group than in the model group (P<0.01, P<0.05). The number of BrdU/NeuN dual labeled neurons was slightly increased in the model group than in the normal control group (P>0.05), and evidently increased in the EA group relevant to the model group (P<0.05), suggesting a proliferation of hippocampal neural stem cells. After modeling, the neurons of hippocampal dentate gyrus were arranged loosely and irregularly and their structure was fuzzy, with an appearance of different degrees of nuclear pyknosis, whereas in the EA group, the neuronal contour was clear and the nuclear structure was relatively distinct.@*CONCLUSION@#EA can activate the proliferation of neural stem cells in the hippocampus in AD mice, which may contribute to its function in improving the neuronal structure by upregulating the expression of BDNF.
Subject(s)
Animals , Male , Mice , Alzheimer Disease/therapy , Cell Proliferation , Electroacupuncture , Hippocampus , Mice, Inbred C57BL , Neural Stem CellsABSTRACT
Background Paraquat (PQ) is a widely used herbicide that exerts neurotoxicity. The effects of PQ on neural stem cells (NSCs) through microglia mediated neuroinflammation remain limitedly studied. Objective To investigate the effects of PQ on the proliferation and neurogenesis of NSCs through neuroinflammation mediated by microglia. Methods Microglial cell lines (BV2 cells) and primary NSCs were used. BV2 cells were exposed to 0, 1, 3.3, 10, 33, and 100 μmol·L−1 of PQ for 6 h followed by viability assessment. The highest PQ concentration that had no effect on cell viability was selected as the final exposure concentration (33 μmol·L−1). To exclude the direct effect of PQ on NSCs, after the BV2 cells were cultured in complete medium containing 33 μmol·L−1 PQ for 6 h, the BV2 culture medium was replaced by NSCs complete medium without PQ for 24 h. The concentration of interleukin-1β (IL-1β) in supernatant was detected by enzyme-linked immune sorbent assay. Besides, in order to detect the effects of IL-1β on NSCs proliferation and neurogenesis, NSCs isolated from hippocampus of adult mice were cultured in the supernatant obtained above and divided into four groups: control supernatant + control antibody, control supernatant + IL-1β neutralizing antibody (10 ng·mL−1), PQ supernatant + control antibody, PQ supernatant + IL-1β neutralizing antibody (10 ng·mL−1). Proportion of Ki67-positive NSCs was detected by flow cytometry (FCS) and immunofluorescence after 24 h culture, and neurogenesis was detected by FCS and immunofluorescence after 3-7 d of culture. Results The IL-1β concentration in the supernatant of BV2 cells was significantly increased after the 33 μmol·L−1 PQ exposure compared with the control group (t=3.020, P<0.05). After the NSCs were cultured with the supernatant of PQ-treated BV2 cells, the proportion of Ki67-positive NSCs (t=9.129, P<0.01) and the proportion of newborn neurons (t=4.638, P<0.01) were significantly decreased compared to the control group. After neutralizing IL-1β, the proportion of Ki67-positive NSCs (t=22.05, P<0.01) and the proportion of newborn neurons (t=11.09, P<0.01) were significantly higher than those in the un-neutralized group. The results of immunofluorescence detection also showed that after neutralizing IL-1β secreted by 33 μmol·L−1 PQ-treated BV2 cells, the number of Ki67-positive NSCs and the number of newborn neurons were significantly higher than those in the un-neutralized group. Conclusion The secretion of IL-1β by microglia is increased after PQ treatment, resulting in a decrease in the proliferation and neurogenesis of NSCs. These results suggest that neuroinflammation is involved in NSCs damage caused by PQ.
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Objective To explore the effect of 18α-glycyrrhetinic acid (18α-GA) on the proliferation of adult mice neural stem cells (NSCs) and its underlying mechanism. Methods One hundred 6-month BALB/c mice were randomly divided into DMSO control group and 18α-GA group (mice were intraperitoneally injected with 40 mg/kg 18α-GA every day for 2 months). The proliferation capability, oxidative status and nuclear factor E2-related factor 2 (Nrf2) level of NSCs in the adult mice subventricular zone (SVZ) were measured through both in vivo and in vitro experiments, including Ki-67 staining, neurosphere formation assay, BrdU incorporation, CCK-8 assay, reactive oxygen species (ROS) detection, superoxide dismutase 1 (SOD1) determination, Real-time PCR and Western blotting. Results Elevated Ki-67 positive cells were observed in SVZ of mice with 18α-GA application. Meanwhile, ROS level attenuated but SOD1 mRNA and protein level increased significantly in the SVZ of 18α-GA group mice, the latter of which were (3. 17 ± 0. 073) and (2. 12±0. 02) times respectively than that of the control group (P<0. 05 and P<0. 001). Likewise, the similar changes were exhibited in vitro data. NSCs of 18α-GA group mice displayed higher proliferation potency confirmed by accelerated neurosphere formation and increased neurosphere number (P<0. 001), as well as higher BrdU positive ratio (P<0. 01) and NSCs vitality (P<0. 001). NSCs of mice with 18α-GA injection exhibited decreased ROS level by 18. 91%±4. 33% (P<0. 05) and enhanced SOD1 level, compared with those in NSCs of DMSO group mice. Furtherly, the Nrf2 expression in SVZ and NSCs of 18α-GA group mice was higher than that of the control group. Conclution 18α-GA administration plays a vital role in the maintainence and amelioration of adult mice NSCs proliferation through activating SOD1 and diminishing ROS aggregations.
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Objective To investigate the role of ataxia-telangiectasia mutated gene (ATM) in maintaining quiescence of neural stem cells (NSCs) from subgranular zone (SGZ) of hippocampal dentate gyms in mice. Methods We constructed 1-month-old and 4-month-old mice ATM knockout mice, with 12 mice in each group. The NSCs in SGZ of ATM knockout mice were isolated, cultured and identified in vitro. The proliferation ability of NSCs in SGZ of 1-month-old ATM
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Objective To investigate the expression and role of Huntingtin-associated protein-1 (HAP-1) in the process of valproate acid (VP A) inducing neural stem cells (NSCs) into neurons. Methods The hippocampus NSCs of SD rats were isolated and cultured, Real-time PCR and Western blotting were used to detect HAP-1 mRNA and protein expression at day 0, day 1, day 3 and day 5 during the induction of VPA on NSCs differentiation into neurons ; Real-time PCR was used to detect the expression level of HAP-1 mRNA in multiple tissues of adult SD rats, as well as NSCs, neurons and astrocytes. After applying small interfering RNA technology to down-regulate the expression of HAP-1 mRNA in NSCs, Real-time PCR was used to detect the mRNA expression levels of neuron-specific molecules stathmin-2 ( Stmn-2), neuronal differentiation-1 (Neurod-1), microtubule-associated protein-2 (Map-2) and synapsin-1 (Syn-1), and Western blotting was used to detect the protein expression levels of neuron-specific marker β-tubulin III (Tuj-1). Immunofluorescence was used to detect the proportion of NSCs differentiated into Tuj-1 positive neurons, and to observe the development of neurons. Results At day 1 and day 3 after VPA treatment, the expression of HAP-1 mRNA and protein in the VPA group was significantly up-regulated; HAP-1 mRNA was predominantly expressed in the hippocampus, and its expression was higher in neurons, followed by NSCs, and minimally in astrocytes. After down-regulating HAP-1 with small interference technology, the proportion of NSCs differentiated into Tuj-1 positive neurons reduced, and neuron development became worse. Conclusion VPA may promote the differentiation of NSCs into neurons by up-regulating HAP-1 expression.
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Objective To observe the effect of edaravone on endogenous neural stem cells in rats with permanent cerebral ischemia. Methods The rat model of permanent cerebral ischemia was established by electrocoagulation. Thirty SD rats were randomly divided into three groups; sham operation group, brain injury group and edaravone group. Six hours after the establishment of the model, the edaravone group was intraperitoneally injected with 1.5 g/L edaravone (10 ml/kg) once a day. The sham operation group and the cerebral ischemia group were intraperitoneally injected with saline of equal volume for 7 days. 24 hours after the last administration, BrdU positive cells, Nestin/BrdU positive cells, neuronal class III β-tubulin (Tuj1)/BrdU positive cells and glial fibrillary acidic protein (GFAP)/BrdU positive cells were observed by immunofluorescent staining, Tuj1 and GFAP protein expressions were detected by Western blotting. Results Compared with the cerebral ischemia group, the BrdU positive cells, Nestin/BrdU positive cells, Tuj1/BrdU positive cells and GFAP/BrdU positive cells increased significantly in the infraventricular area and the cortex area around the ischemia in the edaravone group (P<0.05). Compared with the cerebral ischemia group, the expression of Tuj1 and GFAP protein in the cerebral cortex of edaravone group increased ( P<0.05). Conclusion Edaravone can promote the proliferation of endogenous neural stem cells and astrocytes in the subventricular area and the cortex around ischemia, and promote the differentiation of endogenous neural stem cells into neurons.
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Objective To investigate whether Danhong injection can enhance the therapeutic effect of neural stem cell (NSC) transplantation in repairing cerebral ischemia injury by regulating the nuclear factor E2-related factor 2 (Nrf2) signaling pathway. Methods Forty male SD rats were randomly divided into the NSC transplantation group (NSC group), Danhong injection group (DH group), NSC+ Danhong injection group (N+D group), NSC+ Danhong injection group +ML385 group(N+D+M group) and PBS control group (PBS group), 8 rats in each group. All rat models of cerebral ischemia were established by embolization of the middle cerebral artery. Reperfusion was performed at 1.5 h after embolization. All rats in each group received corresponding interventions at 3 d after reperfusion. The neurological function score was evaluated before and 1, 2, 4 weeks after NSC transplantation. All rats were sacrificed at 4 weeks after NSC transplantation. The parameters related to oxidative stress were detected. The expression levels of neuron-specific nuclear protein (NeuN) and von Willebrand factor (vWF) were determined by immunofluorescence staining. Results Before NSC transplantation, the neurological function scores did not significantly differ among different groups (all P > 0.05). At postoperative 1, 2 and 4 weeks, the neurological function scores in the NSC, DH and N+D groups were significantly lower than those in the PBS and N+D+M groups (all P < 0.05). Compared with the PBS and N+D+M groups, the malondialdehyde (MDA) levels were significantly decreased, whereas the superoxide dismutase (SOD) and glutathione peroxidase (GPX) levels were considerably increased in the NSC, DH and N+D groups (all P < 0.05). The GPX level in the N+D+M group was significantly lower than that in the PBS group (P < 0.05). Immunofluorescence staining showed that the transplant NSC in the rat brain migrated to the surrounding area of cerebral infarction and survived, and expressed neuronal marker NeuN and neovascularization marker vWF. However, the number of living NSC in the N+D+M group was significantly lower compared with those in the remaining groups. Conclusions Danhong injection may improve the microenvironment of stem cell transplantation, enhance the survival rate of transplant NSC and improve the therapeutic effect of NSC transplantation for cerebral ischemia injury probably by regulating the Nrf2 signaling pathway.
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Objective To investigate the mechanism of inhibiting the Notch signaling pathway in promoting neural regeneration after neural stem cells (NSCs) transplantation in rats with cerebral ischemia. Methods The model of focal cerebral ischemia was established by middle cerebral artery occlusion (MCAO) method . NSCs were cultured in vitro and transplanted into the striatum ischemic area. In the experiment, 40 SD rats were divided into sham operation group, model group, transplantation group (transplanted neural stem cells), and N-[N-(3, 5-difluorohenacetyl )-L-alanyl ]-S-phenylglycinet-butyl ester( DAPT) + transplantation group. The degree of neuronal damage in each group was observed by HE staining. The expressions of Notchl, Hesl and Hes5 in the brain tissue of each group were detected by immunohistochemistry and Western blotting. Results Compared with the sham operation group, the neuron injury in the model group was severe, and nuclear pyknosis and nuclear lysis were observed. The Notchl, Hesl and Hes5 positive cells increased significantly, and the expression of Notchl, Hesl and Hes5 proteins were significantly up-regulated (P<0. 05). Compared with the model group, the neuronal damage in the transplantation group and the DAPT+ transplantation group were all relieved, and the Notchl, Hesl and Hes5 positive cells were partially expressed, and the expression of each protein decreased (P<0. 05). The DAPT+ transplantation group neurons were compared. The damage was obviously restored, and the expression of protein-positive cells and protein further decreased (P<0. 05). Conclusion Inhibition of Notch signaling pathway can promote nerve regeneration after neural stem cell transplantation in rats with cerebral ischemia. The mechanism is mainly related to down-regulation of Notch 1 , Hesl and Hes5 expression.
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OBJECTIVE: To observe the effect of penetrative needling from "Baihui" (GV20) to "Qubin" (GB7) on neural stem cell proliferation and sonic hedgehog (Shh) signaling in subventricular zone (SVZ) in intracerebral hemorrhage (ICH) rats so as to explore its mechanisms underlying improvement of ischemic injury of brain. METHODS: Male SD rats were randomly divided into blank control, model, acupuncture and agonist (Purmorphamine, an activator of Shh signaling pathway) groups (n＝18 in each group, 6 for H．E. stain, 6 for examining neuronal cell proliferation, and 6 for immunohistochemistry). The ICH model was established by injecting autogenous blood (50 µL) into the right caudate nucleus. The neurological defect was scored with refe-rence to Bederson's method. Penetrative needling from GV20 to GB7 was performed by manipulating the needle for 6 min (repeated 3 times in 30 min), once daily for 7 days. Intraperitoneal injection of Purmorphamine (1 mg/mL, 1 mg/kg) was performed, once daily for 7 days. Histopathological changes of the hemorrhagic penumbra region were observed under microscope after H．E. stain, the newborn neural stem cell proliferation (BrdU＋/Nestin＋ double labeled cells) in the SVZ was observed by immunofluorescence after intraperitoneal injection of BrdU (50 mg/kg), and the expression of Shh and glioma-associated hemolog-1 (Gli1) detected by immunohistochemistry. RESULTS: After modeling, the neurological score and expression levels of Shh and Gil1 proteins were significantly increased in the model group relevant to the blank control group (P0.05). Outcomes of H．E. stain showed obvious edema, disordered arrangement of cells, infiltration of inflammatory cells and red blood cells with glial cell hyperplasia around the hematoma area in the model group, which was relatively milder in both acupuncture and agonist groups such as in basic disappearance of edema and inflammatory reaction. CONCLUSION: Penetrative needling from GV20 to GB7 can obviously improve neurological function in ICH rats, which is related to its effects in activating Shh/Gil1 signaling and in further promoting neural stem cell proliferation in the SVZ region.
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MicroRNA-132 (miR-132), a small RNA that regulates gene expression, is known to promote neurogenesis in the embryonic nervous system and adult brain. Although exposure to psychoactive substances can increase miR-132 expression in cultured neural stem cells (NSCs) and the adult brain of rodents, little is known about its role in opioid addiction. So, we set out to determine the effect of miR-132 on differentiation of the NSCs and whether this effect is involved in opioid addiction using the rat morphine self-administration (MSA) model. We found that miR-132 overexpression enhanced the differentiation of NSCs in vivo and in vitro. Similarly, specific overexpression of miR-132 in NSCs of the adult hippocampal dentate gyrus (DG) during the acquisition stage of MSA potentiated morphine-seeking behavior. These findings indicate that miR-132 is involved in opioid addiction, probably by promoting the differentiation of NSCs in the adult DG.
Subject(s)
Animals , Male , Cell Differentiation , Cell Line, Tumor , Dentate Gyrus , Metabolism , Gene Expression Regulation , MicroRNAs , Metabolism , Neural Stem Cells , Metabolism , Opioid-Related Disorders , Metabolism , Rats, Sprague-DawleyABSTRACT
Neural stem cell is a kind of stem cells that can differentiate into neural and glial cells. While Müller cells, the main endogenous neural stem cell in retina,have the features to reentry into the cell cycle and differentiate into neural cells after retinal damage. Although it is highly effective for retinal Müller cell differentiation spontaneously after retinal injury in vertebrates, this feature is rigorous restricted in mammals. Recently, some transcription factors,such as Ascl1, Sox2, Lin28, Atoh7, are sufficient to drive quiescent Müller cells back in proliferation to generate new retinal neurons. Moreover, combining Ascl1 expression with a histone deacetylase inhibitor can bypass the limitation and increase the generation of new neurons in the adult retina. These regenerated neurons integrate the existing neuronal network and are able to respond to light, indicating that they can likely be used to restore vision. While these results are extremely promising, the regenerative response is still limited, likely because the proliferative capacity of mammalian Müller cells is low compared to their zebrafish counterparts. It is indeed necessary to identify new factors increasing the efficiency of the regenerative response.
ABSTRACT
The olfactory epithelium is capable of structural and functional recovery after injury through neurogenesis. Neurogenesis occurs via stem cells in the olfactory epithelium. Horizontal basal cells and globose basal cells in the basal layer of the epithelium have the characteristics of stem cells and progenitor cells of olfactory neurons. In order for the horizontal basal cells and globose basal cells to differentiate into olfactory neurons, distinct transcriptional factors are required at each stage. These transcription factors inhibit or synergize with each other or cells at each differentiation stage, regulating olfactory neurogenesis. Recently, the regulation of neurogenesis and development through epigenetic controls that change gene expression without changing the gene sequence have been studied. Studies of olfactory epithelium have helped to elucidate complex neurological systems including spinal cord and brain. In particular, features of neurogenesis will lead to medical advances in the treatment of central nervous diseases, which until this time have been considered impossible.