Subcellular distribution of N-methyl-D-aspartic acid receptor subunit 1 in neural stem cells within subventricular zone of adult rats

The subcellular localization of N-methyI-D-aspartic acid receptor subunit 1 in neural stem cells of the subventricular zone of adult rats was detected using electron microscopy, following immunohistochemistry and immunogold-silver double staining. Results confirmed the presence of neural stem cells in the subventricular zone, which is a key neurogenic region in the central nervous system of adult mammals. The expression of N-methyI-D-aspartic acid receptor subunit 1 was higher than that of nestin and mainly distributed in the cell membrane, cytoplasm, rough endoplasmic reticulum and Golgi complex of neural stem cells.

Identification and culture of neural stem cells isolated from adult rat subventricular zone following fluid percussion brain injury

Objective To analyze proliferation and differentiation of glial fibrillary acid protein(GFAP)-and nestin-positive(GFAP+/nestin+)cells isolated from the subventricular zone following fluid percussion brain injury to determine whether GFAP+/nestin+ cells exhibit characteristics of neural stem cells.Methods Male Sprague-Dawley rats,aged 12 weeks and weighing 200-250 g,were randomly and evenly assigned to normal control group and model group.In the model group,a rat model of fluid percussion brain injury was established.Five days later,subventricular zone tissue was resected from each group and made into single cell suspension.After serum-free neural stem cell medium culture and subsequent serum-induced differentiation,cell type,proliferation and differentiation capacities were determined by immunofluorescence staining and flow cytometry.Results At 3-7 days after fluid percussion brain injury,nestin+/GFAP+ cells in the single cell suspension from the model group significantly outnumbered those from the normal control group(P<0.01).In the model group,an increased number of small neurospheres with smooth cell edge and bulged center formed after primary culture,and were clearly visible with the increase of culture time and medium replacement.After several passages,many clonal spheres were obtained,suggesting strong self-proliferatiing capacity.Neurospheres from the model group differentiated into astrocytes,neurons and oligodendrocytes.Conclusion GFAP+/nestin+ cells isolated from the adult rat subventricular zone after fluid percussion brain injury are thought to be neural stem cells because of their self-renewal and multi-differentiation capacities.

Hyperbaric oxygen treatment promotes neural stem cell proliferation in the subventricular zone of neonatal rats with hypoxic-ischemic brain damage

Hyperbaric oxygen therapy for the treatment of neonatal hypoxic-ischemic brain damage has been used clinically for many years, but its effectiveness remains controversial. In addition, the mechanism of this potential neuroprotective effect remains unclear. This study aimed to investigate the influence of hyperbaric oxygen on the proliferation of neural stem cells in the subventricular zone of neonatal Sprague-Dawley rats （7 days old） subjected to hypoxic-ischemic brain damage. Six hours after modeling, rats were treated with hyperbaric oxygen once daily for 7 days. Immunohistochemistry revealed that the number of 5-bromo-2＇-deoxyuridine positive and nestin positive cells in the subventricular zone of neonatal rats increased at day 3 after hypoxic-ischemic brain damage and peaked at day 5. After hyperbaric oxygen treatment, the number of 5-bromo-2＇- deoxyuddine positive and nestin positive cells began to increase at day 1, and was significantly higher than that in normal rats and model rats until day 21. Hematoxylin-eosin staining showed that hyperbaric oxygen treatment could attenuate pathological changes to brain tissue in neonatal rats, and reduce the number of degenerating and necrotic nerve cells. Our experimental findings indicate that hyperbaric oxygen treatment enhances the proliferation of neural stem cells in the subventricular zone of neonatal rats with hypoxic-ischemic brain damage, and has therapeutic potential for promoting neurological recovery following brain injury.

Adult neural stem cell dysfunction in the subventricular zone of the lateral ventricle leads to diabetic olfactory defects

Sensitive smell discrimination is based on structural plasticity of the olfactory bulb,which depends on migration and integration of newborn neurons from the subventricular zone.In this study,we examined the relationship between neural stem cell status in the subventricular zone and olfactory function in rats with diabetes mellitus.Streptozotocin was injected through the femoral vein to induce type 1 diabetes mellitus in Sprague-Dawley rats.Two months after injection,olfactory sensitivity was decreased in diabetic rats.Meanwhile,the number of Brd U-positive and Brd U＋/DCX＋double-labeled cells was lower in the subventricular zone of diabetic rats compared with agematched normal rats.Western blot results revealed downregulated expression of insulin receptorβ,phosphorylated glycogen synthase kinase 3β,and β-catenin in the subventricular zone of diabetic rats.Altogether,these results indicate that diabetes mellitus causes insulin deficiency,which negatively regulates glycogen synthase kinase 3β and enhances β-catenin degradation,with these changes inhibiting neural stem cell proliferation.Further,these signaling pathways affect proliferation and differentiation of neural stem cells in the subventricular zone.Dysfunction of subventricular zone neural stem cells causes a decline in olfactory bulb structural plasticity and impairs olfactory sensitivity in diabetic rats.

A ginkgo biloba extract promotes proliferation of endogenous neural stem cells in vascular dementia rats

The ginkgo biloba extract EGb761 improves memory loss and cognitive impairments in patients with senile dementia. It also promotes proliferation of neural stem cells in the subventricular zone in Parkinson＇s disease model mice and in the hippocampal zone of young epileptic rats. However, it remains unclear whether EGb761 enhances proliferation of endogenous neural stem cells in the brain of rats with vascular dementia. In this study, a vascular dementia model was established by repeatedly clipping and reperfusing the bilateral common carotid arteries of rats in combination with an intraperitoneal injection of a sodium nitroprusside solution. Seven days after establishing the model, rats were intragastrically given EGb761 at 50 mg/kg per day. Learning and memory abilities were assessed using the Morris water maze and proliferation of endogenous neural stem cells in the subventricular zone and dentate gyrus were labeled by 5-bromo-2-deoxyuridine immunofluorescence in all rats at 15 days, and 1, 2, and 4 months after model establishment. The escape latencies in Morris water maze tests of rats with vascular dementia after EGb761 treatment were significantly shorter than the model group. Immunofluorescence staining showed that the number and proliferation of 5-bromo-2-deoxyuridine-positive cells in the subventricular zone and dentate gyrus of the EGb761-treated group were significantly higher than in the model group. These experimental findings suggest that EGb761 enhances proliferation of neural stem cells in the subventricular zone and dentate gyrus, and significantly improves learning and memory in rats with vascular dementia.

Similarity on neural stem cells and brain tumor stem cells in transgenic brain tumor mouse models

Although it is believed that glioma is derived from brain tumor stem cells, the source and molecular signal pathways of these cells are still unclear. In this study, we used stable doxycycline-inducible transgenic mouse brain tumor models （c-myc/SV40Tag＋/Tet-on＋） to explore the malignant trans- formation potential of neural stem cells by observing the differences of neural stem cells and brain tumor stem cells in the tumor models. Results showed that chromosome instability occurred in brain tumor stem cells. The numbers of cytolysosomes and autophagosomes in brain tumor stem cells and induced neural stem cells were lower and the proliferative activity was obviously stronger than that in normal neural stem cells. Normal neural stem cells could differentiate into glial fibrillary acidic protein-positive and microtubule associated protein-2-positive cells, which were also negative for nestin. However, glial fibrillary acidic protein/nestin, microtubule associated protein-2/nestin, and glial fibrillary acidic protein/microtubule associated protein-2 double-positive cells were found in induced neural stem cells and brain tumor stem cells. Results indicate that induced neural stem cells are similar to brain tumor stem cells, and are possibly the source of brain tumor stem cells.

Directional induction of dopaminergic neurons from neural stem cells using substantia nigra homogenates and basic fibroblast growth factor

To date, complex components of available reagents have been used for directional induction of neural stem cells into dopaminergic neurons, resulting in a poor ability to repeat experiments. This study sought to investigate whether a homogenate of the substantia nigra of adult rats and/or basic fibroblast growth factor could directionally induce neural stem cells derived from the subventricular zone of embryonic rats to differentiate into dopaminergic neurons. Tyrosine hydroxylase-positive cells were observed exclusively after induction with the homogenate supernatant of the substantia nigra from adult rats and basic fibroblast growth factor for 48 hours in vitro. However, in the groups treated with homogenate supernatant or basic fibroblast growth factor alone, tyrosine hydroxylase expression was not observed. Moreover, the content of dopamine in the culture medium of subventricular zone neurons was significantly increased at 48 hours after induction with the homogenate supernatant of the substantia nigra from adult rats and basic fibroblast growth factor. Experimental findings indicate that the homogenate supernatant of the substantia nigra from adult rats and basic fibroblast growth factor could directionally induce neural stem cells derived from the subventricular zone of embryonic rats to differentiate into dopaminergic neurons in the substantia nigra with the ability to secrete dopamine.

Adult brain neural stem cells: technological and translational advances

Understanding the role of adult neural stem cells in maintaining specific brain function is a rapidly expanding research field. Recent technological advances to culture and trace neural stem cells, such as stem cell isolation and expansion and inducible transgenic lineage tracing mouse models, have enabled more in-depth studies into the mechanisms governing neural stem cell homeostasis and pathophysiology in the adult brain. In this review we will briefly discuss the types and locations of adult neural stem cells in the mammalian brain, recent developments in tools used to study these cells, and the translational implications.

Review point on neural stem cells and neurogenic areas of the central nervous system

The potential applications of neural stem cells (NSC) in the therapy of degenerative and traumatic diseases of the central nervous system (CNS) have aroused great scientific interest. NSCs can be recovered from specific areas of the CNS from fetuses, embryos and from adult brain as well, and under appropriate culture conditions, may be induced to differentiate into the three major neural cell types, neurons, astrocytes and oligodendrocytes. The main neurogenic areas of mammals are the subventricular zone (SVZ) and the dentate gyrus (DG) of the hippocampus, yet other areas of neurogenic potential have been identified, including frontal and parietal cortices, hippocampus and lateral wall of the lateral ventricle. Neural stem cells and differentiated neural cells are usually identified by expression of specific markers, proteins that are expressed by different cell types and/or at different stages of differentiation. The main cell markers are nestin and Sox-2 for undifferentiated NSCs, beta-III tubulin (B-III tubulin) for neurons, Galactocerebroside (GalC) for oligodendrocytes and Glial fibrillary acid protein (GFAP) for astrocytes. In general, the main neurogenic areas, the neurogenic potential of NSC and the morphological and biological characteristics of differentiated neural cells are strikingly similar among species, yet some differences have been reported. This article presents a brief review of NSCs, neurogenic areas and techniques used for their identification and characterization in humans and experimental animals.

Buyang Huanwu Decoction regulates neural stem cell behavior in ischemic brain

The traditional Chinese medicine Buyang Huanwu Decoction has been shown to improve the neu- rological function of patients with stroke. However, the precise mechanisms underlying its effect remain poorly understood. In this study, we established a rat model of cerebral ischemia by middle cerebral artery occlusion and intragastrically administered 5 g/kg Buyang Huanwu Decoction, once per day, for 1, 7, 14 and 28 days after cerebral ischemia. Immunohistochemical staining revealed a number of cells positive for the neural stem cell marker nestin in the cerebral cortex, the subven- tricular zone and the ipsilateral hippocampal dentate gyrus in rat models of cerebral ischemia. Buyang Huanwu Decoction significantly increased the number of cells positive for 5-bromodeoxyuridine （BrdU）, a cell proliferation-related marker, microtubule-associated protein-2, a marker of neuronal differentiation, and growth-associated protein 43, a marker of synaptic plasticity in the ischemic rat cerebral regions. The number of positive cells peaked at 14 and 28 days after intragastric administration of Buyang Huanwu Decoction. These findings suggest that Buyang Huanwu Decoction can promote the proliferation and differentiation of neural stem cells and en- hance synaptic plasticity in ischemic rat brain tissue.

Are there fetal stem cells in the maternal brain?

Fetal cells can enter maternal blood during pregnancy but whether they can also cross the blood-brain barrier to enter the maternal brain remains poorly understood. Previous results suggest that fetal cells are summoned to repair damage to the mother＇s brain. If this is confirmed, it would open up new and safer avenues of treatment for brain damage caused by strokes and neural diseases. In this study, we aimed to investigate whether a baby＇s stem cells can enter the maternal brain during pregnancy. Deceased patients who had at least one male offspring and no history of abortion and blood transfusion were included in this study. DNA was extracted from brain tissue samples of deceased women using standard phenol-chloroform extraction and ethanol precipitation methods. Genomic DNA was screened by quantitative fluorescent-polymerase chain reaction amplification together with short tandem repeat markers specific to the Y chromosome, and 13, 18, 21 and X. Any foreign DNA residues that could be used to interpret the presence of fetal stem cells in the maternal brain were monitored. Results indicated that fetal stem cells can not cross the blood-brain barrier to enter the maternal brain.

PROLIFERATION AND MIGRATION OF EPENDYMAL CELLS IN THE BRAIN OF ADULT RATS AFTER PERMANENT FOCAL CEREBRAL ISCHEMIA

Objective Ependymal cells are thought to be the primary source of neural stem cells in the adult central nervous system. The purpose of this study is to examine spatial and temporal profiles of ependymal cell proliferation and migration after focal cerebral ischemia. Methods Eighty male Sprague Dawley rats underwent permanent middle cerebral artery occlusion after injection of 10 μL of 0.2% Dil into the lateral ventricle. Rats were sacrificed and brain sections were acquired for pathological evaluation and laser confocal imaging at day 1,3,7,11,14,21 and 28 after ischemia. Results The density of Dil-labeled cells in the ischemic ipsilateral subventricular zone was significantly higher than that in the control group and these labeled cells dispersed in the ischemic ipsilateral subventricular zone and/or were located in ependyma from day 1 to 11. In the ischemic ipsilateral cortex, some Dil-labeled cells occurred in peri-infarction and infarction of parietal region at day14 and peaked at day 21 when some Dil-labeled cell nodules were found in this region. During postischemic day 14-28, a significant decrease in labeled cell density in the ischemic ipsilateral subventricular zone was coincident with a significant increase in labeled cells density in the cortex (peri-infarction and infarction). Conclusion The results indicate that ependymal cells proliferate and migrate after focal cerebral ischemia in the adult rat brain.

A core scientific problem in the treatment of central nervous system diseases:newborn neurons

It has long been asserted that failure to recover from central nervous system diseases is due to the system's intricate structure and the regenerative incapacity of adult neurons.Yet over recent decades,numerous studies have established that endogenous neurogenesis occurs in the adult central nervous system,including humans'.This has challenged the long-held scientific consensus that the number of adult neurons remains constant,and that new central nervous system neurons cannot be created or renewed.Herein,we present a comprehensive overview of the alterations and regulatory mechanisms of endogenous neurogenesis following central nervous system injury,and describe novel treatment strategies that to rget endogenous neurogenesis and newborn neurons in the treatment of central nervous system injury.Central nervous system injury frequently results in alterations of endogenous neurogenesis,encompassing the activation,proliferation,ectopic migration,diffe rentiation,and functional integration of endogenous neural stem cells.Because of the unfavorable local microenvironment,most activated neural stem cells diffe rentiate into glial cells rather than neurons.Consequently,the injury-induced endogenous neurogenesis response is inadequate for repairing impaired neural function.Scientists have attempted to enhance endogenous neurogenesis using various strategies,including using neurotrophic factors,bioactive materials,and cell reprogramming techniques.Used alone or in combination,these therapeutic strategies can promote targeted migration of neural stem cells to an injured area,ensure their survival and diffe rentiation into mature functional neurons,and facilitate their integration into the neural circuit.Thus can integration re plenish lost neurons after central nervous system injury,by improving the local microenvironment.By regulating each phase of endogenous neurogenesis,endogenous neural stem cells can be harnessed to promote effective regeneration of newborn neurons.This offers a novel approach for treating central nervous system injury.

Neural stem cell heterogeneity through time and space in the ventricular-subventricular zone

BACKGROUND： The origin and classification of neural stem cells （NSCs） has been a subject of intense investigation for the past two decades. Efforts to categorize NSCs based on their location, function and expression have established that these cells are a heterogeneous pool in both the embryonic and adult brain. The discovery and additional characterization of adult NSCs has introduced the possibility of using these cells as a source for neuronal and glial replacement following injury or disease. To understand how one could manipulate NSC developmental programs for therapeutic use, additional work is needed to elucidate how NSCs are programmed and how signals during development are interpreted to determine cell fate. OBJECTIVE： This review describes the identification, classification and characterization of NSCs within the large neurogenic niche of the ventricular-subventricular zone （V-SVZ）. METHODS： A literature search was conducted using Pubmed including the keywords ＂ventricular-subventricular zone,＂ ＂neural stem cell,＂ ＂heterogeneity,＂ ＂identity＂ and/or ＂single cell＂ to find relevant manuscripts to include within the review. A special focus was placed on more recent findings using single-cell level analyses on neural stem cells within their niche（s）. RESULTS： This review discusses over 20 research articles detailing findings on V-SVZ NSC heterogeneity, over 25 articles describing fate determinants of NSCs, and focuses on 8 recent publications using distinct single-cell analyses of neural stem cells including fl0w cytometry and RNA-seq. Additionally, over 60 manuscripts highlighting the markers expressed on cells within the NSC lineage are included in a chart divided by cell type. CONCLUSIONS： Investigation of NSC heterogeneity and fate decisions is ongoing. Thus far, much research has been conducted in mice however, findings in human and other mammalian species are also discussed here. Implications of NSC heterogeneity established in the embryo for the properties of NSCs in the adult brain are explored, including how these cells may be redirected after injury or genetic manipulation.

Epigenetic control on cell fate choice in neural stem cells

Derived from neural stem cells(NSCs)and progenitor cells originated from the neuroectoderm,the nervous system presents an unprecedented degree of cellular diversity,interwoven to ensure correct connections for propagating information and responding to environ-mental cues.NSCs and progenitor cells must integrate cell-intrinsic programs and environmental cues to achieve production of appropriate types of neurons and glia at appropriate times and places during develop-ment.These developmental dynamics are reflected in changes in gene expression,which is regulated by transcription factors and at the epigenetic level.From early commitment of neural lineage to functional plas-ticity in terminal differentiated neurons,epigenetic regulation is involved in every step of neural develop-ment.Here we focus on the recent advance in our un-derstanding of epigenetic regulation on orderly genera-tion of diverse neural cell types in the mammalian nervous system,an important aspect of neural devel-opment and regenerative medicine.

Regulation of BMP4 on the proliferation and differentiation in SVZa neural stem cells

The neural stem cells in the anterior subventricula zone (SVZa) mainly generate the progenitors that will differentiate into neurons, and along a highly circumscribed migratory access-Rostral migratory stream (RMS), they migrate to the olfactory bulbs (OB). To understand the effects of BMPs on SVZa neural stem cells, in this study BMP4 at various concentrations was used to induce SVZa ,neural stem cells, aud the living cell labeling using BMP4 pronmtor conjugated with red fluorescence protein showed the expression of BMP4 dynamically. The results demonstrated that low BMP4 doses (1-5 ng/mL) promoted while high doses(10-100 ng/mL) inhibited the proliferation of SVZa neural stem cells, and BMP4 promoted neuron differentiation in the early stage (1-3 d), howeverm, it inhibited the neuron commitment after 4 d. Noggin, the antagonist of BMP4, blocked the physiological effects of BMP4. In OB, BMP4 is mainly to accelerate the progenitors to withdraw from the cell cycle and trigger the differentiation, and in RMS, it promotes the proliferation of committed progenitors and not differentiation, further in SVZa, BMP4 enhances astrocyte commitment.

头穴丛刺法调控大鼠脑梗死后内源性神经干细胞增殖迁移分化的实验研究

目的:本实验通过观察头穴丛刺法对室管膜下区(SVZ)的神经干细胞(NSC)增殖、迁移、分化的调控作用,探讨该疗法促进成年神经再生、修复和重建神经网络的理论机制。方法:大鼠注射荧光染料Dil以预标记室SVZ细胞;栓线法制备大鼠局灶性脑缺血模型。将造模成功的Wistar大鼠随机分成4组:模型组12只,头针组12只,头针丛刺法组12只,假手术组4只。采用脉冲式的BrdU标记方法标记新生细胞;采用激光共聚焦显微镜检测预标记原始SVZNSC后,再检测双重免疫荧光染色所确定的分化的细胞。结果:①模型组、头针组、头穴丛刺法组均可见Dil标记的SVZ细胞迁移至梗死周边的纹状体和皮质,并且分化成神经元或胶质细胞,头穴丛刺法组Dil/BrdU/NeuN或Dil/BrdU/GFAP标记的细胞明显增多,与其他两组比较有显著性差异(P<0.01);②头穴丛刺法组BrdU/NeuN及BrdU/GFAP阳性细胞表达均多于其他两组,组间比较有显著性差异(P<0.01)。结论:①头穴丛刺法能促进脑缺血后SVZ区神经干细胞增殖,并且随时间递增减少其增殖衰减;②此法可促进Dil标记的SVZ细胞均迁移至梗死周边的纹状体和皮质,并且分化成神经元或胶质细胞;③此法能促进局灶性脑缺血后SVZNSC的迁移和分化。

成年小鼠脑室下区神经干细胞培养与鉴定体系的建立

目的建立系统的成年小鼠脑室下区神经干细胞分离、培养及鉴定体系。方法用无血清方法分离培养成年小鼠脑室下区来源的神经干细胞;用克隆培养、BrdU整合的方法检验培养细胞的干细胞特性;用免疫荧光细胞化学方法检测BrdU、神经干细胞标记物nestin和SOX2,分化的细胞标记物Tuj1、GFAP、NG2,用Western boltting和RT-PCR方法进一步检测神经干细胞标记物nestin和SOX2的表达。结果从成年小鼠脑室下区分离培养出具有自我更新、增殖的神经球,构成神经球的细胞nestin和SOX2呈阳性,它们分化后产生Tuj1阳性的神经元、GFAP阳性的星型胶质细胞、NG2阳性的少突胶质细胞。结论建立了简单、稳定的培养成年小鼠脑室下区神经干细胞方法。

局灶性脑缺血后室管膜 /室下区细胞迁移到梗塞区周围并分化为神经元和星形胶质细胞(英文)

目的研究局灶性脑缺血后室管膜/室下区细胞的迁移分化,揭示梗塞区周围新生细胞的来源.方法大脑中动脉阻塞前,将10μl 0.2%的荧光染料DiI注射于体质量250～350 g的雄性SD大鼠侧脑室以预标记室管膜/室下区细胞.脑缺血后,采用累积式的BrdU标记方法标记新生细胞并通过双重免疫荧光染色确定细胞分化.标记的细胞通过激光共聚焦显微镜观察.结果在非缺血对照大鼠,DiI标记细胞定居于室管膜/室下区.局灶性脑缺血后,DiI标记细胞出现于胼胝体,邻近的纹状体和皮质.此外,缺血14 d后,梗塞区周围纹状体和皮质内可见一些DiI/BrdU/GFAP或DiI/BrdU/NeuN三重标记阳性细胞.结论局灶性脑缺血后,室管膜/室下区细胞迁移到梗塞区周围并分化成神经元和星形胶质细胞,这一发现对于理解成体神经干细胞的起源和开发促进脑损伤后内源性神经发生的新措施具有重要意义.

毛蕊花糖苷通过激活PI3K/AKT通路促进成年小鼠神经干细胞增殖

目的观察毛蕊花糖苷对原代成年小鼠神经干细胞增殖的影响,并探讨其可能的作用机制。方法从成年C57BL/6小鼠脑室下区分离、培养原代神经干细胞,并通过神经干细胞标志蛋白Nestin免疫荧光染色,对分离得到的神经干细胞进行鉴定。不同浓度毛蕊花糖苷(5、10、20、40μmol·L^(-1))在无有丝分裂原(EGF/bF GF)的条件下处理细胞24 h。采用CCK-8法检测细胞活力,免疫组化法计数BrdU阳性细胞率,检测细胞增殖能力,Western blot方法检测给药后神经干细胞Akt的磷酸化水平。结果毛蕊花糖苷在无有丝分裂原存在的条件下,能明显促进神经干细胞的增殖,并明显提高p-Akt的表达。而在加入PI3K/AKT信号通路阻断剂LY294002后,这一作用被明显抑制。结论毛蕊花糖苷对体外培养的神经干细胞具有明显促增殖作用,该作用机制可能与化合物激活Akt通路有关。