In vitro characterization of subventricular zone isolated neural stem cells, from adult monkey and rat brain.

Neural stem cells (NSCs) are multipotent, self-renewable cells who are capable of differentiating into neurons, astrocytes, and oligodendrocytes. NSCs reside at the subventricular zone (SVZ) of the adult brain permanently to guarantee a lifelong neurogenesis during neural network plasticity or undesirable injuries. Although the specious inaccessibility of adult NSCs niche hampers their in vivo identification, researchers have been seeking ways to optimize adult NSCs isolation, expansion, and differentiation, in vitro. NSCs were isolated from rhesus monkey SVZ, expanded in vitro and then characterized for NSCs-specific markers expression by immunostaining, real-time PCR, flow cytometry, and cell differentiation assessments. Moreover, cell survival as well as self-renewal capacity were evaluated by TUNEL, Live/Dead and colony assays, respectively. In the next step, to validate SVZ-NSCs identity in other species, a similar protocol was applied to isolate NSCs from adult rat's SVZ as well. Our findings revealed that isolated SVZ-NSCs from both monkey and rat preserve proliferation capacity in at least nine passages as confirmed by Ki67 expression. Additionally, both SVZ-NSCs sources are capable of self-renewal in addition to NESTIN, SOX2, and GFAP expression. The mortality was measured meager with over 95% viability according to TUNEL and Live/Dead assay results. Eventually, the multipotency of SVZ-NSCs appraised authentic after their differentiation into neurons, astrocytes, and oligodendrocytes. In this study, we proposed a reliable method for SVZ-NSCs in vitro maintenance and identification, which, we believe is a promising cell source for therapeutic approach to recover neurological disorders and injuries condition.

Neural Stem Cells of the Subventricular Zone Contribute to Neuroprotection of the Corpus Callosum after Cuprizone-Induced Demyelination.

Myelin loss occurring in demyelinating diseases, including multiple sclerosis, is the leading cause of long-lasting neurological disability in adults. While endogenous remyelination, driven by resident oligodendrocyte precursor cells (OPCs), might partially compensate myelin loss in the early phases of demyelinating disorders, this spontaneous reparative potential fails at later stages. To investigate the cellular mechanisms sustaining endogenous remyelination in demyelinating disorders, we focused our attention on endogenous neural precursor cells (eNPCs) located within the subventricular zone (SVZ) since this latter area is considered one of the primary sources of new OPCs in the adult forebrain. First, we fate mapped SVZ-eNPCs in cuprizone-induced demyelination and found that SVZ endogenous neural stem/precursor cells are recruited during the remyelination phase to the corpus callosum (CC) and are capable of forming new oligodendrocytes. When we ablated SVZ-derived eNPCs during cuprizone-induced demyelination in female mice, the animals displayed reduced numbers of oligodendrocytes within the lesioned CC. Although this reduction in oligodendrocytes did not impact the ensuing remyelination, eNPC-ablated mice experienced increased axonal loss. Our results indicate that, in toxic models of demyelination, SVZ-derived eNPCs contribute to support axonal survival.SIGNIFICANCE STATEMENT One of the significant challenges in MS research is to understand the detrimental mechanisms leading to the failure of CNS tissue regeneration during disease progression. One possible explanation is the inability of recruited oligodendrocyte precursor cells (OPCs) to complete remyelination and to sustain axonal survival. The contribution of endogenous neural precursor cells (eNPCs) located in the subventricular zone (SVZ) to generate new OPCs in the lesion site has been debated. Using transgenic mice to fate map and to selectively kill SVZ-derived eNPCs in the cuprizone demyelination model, we observed migration of SVZ-eNPCs after injury and their contribution to oligodendrogenesis and axonal survival. We found that eNPCs are dispensable for remyelination but protect partially from increased axonal loss.

Subventricular zone-derived extracellular vesicles promote functional recovery in rat model of spinal cord injury by inhibition of NLRP3 inflammasome complex formation.

Spinal cord injury (SCI) is the destruction of spinal cord motor and sensory resulted from an attack on the spinal cord, which can cause significant physiological damage. The inflammasome is a multiprotein oligomer resulting in inflammation; the NLRP3 inflammasome composed of NLRP3, apoptosis-associated speck-like protein (ASC), procaspase-1, and cleavage of procaspase-1 into caspase-1 initiates the inflammatory response. Subventricular Zone (SVZ) is the origin of neural stem/progenitor cells (NS/PCs) in the adult brain. Extracellular vesicles (EVs) are tiny lipid membrane bilayer vesicles secreted by different types of cells playing an important role in cell-cell communications. The aim of this study was to investigate the effect of intrathecal transplantation of EVs on the NLRP3 inflammasome formation in SCI rats. Male wistar rats were divided into three groups as following: laminectotomy group, SCI group, and EVs group. EVs was isolated from SVZ, and characterized by western blot and DLS, and then injected into the SCI rats. Real-time PCR and western blot were carried out for gene expression and protein level of NLRP3, ASC, and Caspase-1. H&E and cresyl violet staining were performed for histological analyses, as well as BBB test for motor function. The results indicated high level in mRNA and protein level in SCI group in comparison with laminectomy (p < 0.001), and injection of EVs showed a significant reduction in the mRNA and protein levels in EVs group compared to SCI (p < 0.001). H&E and cresyl violet staining showed recovery in neural cells of spinal cord tissue in EVs group in comparison with SCI group. BBB test showed the promotion of motor function in EVs group compared to SCI in 14 days (p < 0.05). We concluded that the injection of EVs could recover the motor function in rats with SCI and rescue the neural cells of spinal cord tissue by suppressing the formation of the NLRP3 inflammasome complex.

Evolutionary dynamics of residual disease in human glioblastoma.

BACKGROUND: Glioblastoma is the most common and aggressive adult brain malignancy against which conventional surgery and chemoradiation provide limited benefit. Even when a good treatment response is obtained, recurrence inevitably occurs either locally (∼80%) or distally (∼20%), driven by cancer clones that are often genomically distinct from those in the primary tumour. Glioblastoma cells display a characteristic infiltrative phenotype, invading the surrounding tissue and often spreading across the whole brain. Cancer cells responsible for relapse can reside in two compartments of residual disease that are left behind after treatment: the infiltrated normal brain parenchyma and the sub-ventricular zone. However, these two sources of residual disease in glioblastoma are understudied because of the difficulty in sampling these regions during surgery. PATIENT AND METHODS: Here, we present the results of whole-exome sequencing of 69 multi-region samples collected using fluorescence-guided resection from 11 patients, including the infiltrating tumour margin and the sub-ventricular zone for each patient, as well as matched blood. We used a phylogenomic approach to dissect the spatio-temporal evolution of each tumour and unveil the relation between residual disease and the main tumour mass. We also analysed two patients with paired primary-recurrence samples with matched residual disease. RESULTS: Our results suggest that infiltrative subclones can arise early during tumour growth in a subset of patients. After treatment, the infiltrative subclones may seed the growth of a recurrent tumour, thus representing the 'missing link' between the primary tumour and recurrent disease. CONCLUSIONS: These results are consistent with recognised clinical phenotypic behaviour and suggest that more specific therapeutic targeting of cells in the infiltrated brain parenchyma may improve patient's outcome.

An easy and cost-effective method for the isolation and culturing of neural stem/progenitor cells from the subventricular (SVZ) and dentate gyrus (DG) of adult mouse brain.

BACKGROUND: Isolation of adult Neural Stem/Progenitor Cells (NSPCs) from their neurogenic niches, is a prerequisite for studies involving culturing of NSPCs as neurospheres or attached monolayers in vitro. The currently available protocols involve the use of multiple animals and expensive reagents to establish the NSPCs culture. NEW METHOD: This unit describes a method to isolate and culture NSPCs from the two neurogenic niches in the mouse brain, the Subventricular Zone (SVZ) and Dentate gyrus (DG)/subgranular zone (SGZ), in an easy and cost-effective manner. RESULTS: NSPCs from SVZ and DG regions of adult mouse brains were isolated and cultured up to passage 15 without losing their stem/progenitor characteristics. These NSPCs could be differentiated into neurons, astrocytes, and oligodendrocytes, revealing its trilineage potential. COMPARISON WITH EXISTING METHODS: This protocol eliminates the need for multiple animals as well as the use of many expensive reagents mentioned in previous protocols, adding to the cost-effectiveness of experiments. In addition, we have effectively reduced the number of steps involved in isolation and propagation, thereby minimizing the chances of contamination. CONCLUSION: Our simplified protocol for the isolation and culturing of adult NSPCs from the SVZ and DG demonstrates a cost-effective and efficient alternative to existing methods, reducing the need for sacrificing many animals and the usage of expensive reagents. This method permits the long-term maintenance of NSPCs' stem/progenitor characteristics and their effective differentiation into the major types of cells in the brain, making it a valuable resource for researchers in the field. BASIC PROTOCOL: Isolation and Culturing of Neural Stem/Progenitor cells from the Sub ventricular Zone and the Dentate Gyrus of the adult mouse brain. SUPPORT PROTOCOL 1: Cryopreservation, and revival of frozen NSPCs. SUPPORT PROTOCOL 2: Preparation of adherent monolayer cultures of neural stem/progenitor cells for the differentiation into multiple lineages SUPPORT PROTOCOL 3: Differentiation of NSPCs to neuronal and glial lineages SUPPORT PROTOCOL 4: Characterization of differentiated cells by immunocytochemistry.

Neural stem cells and oligodendrocyte progenitor cells compete for remyelination in the corpus callosum.

A major therapeutic goal in demyelinating diseases, such as Multiple Sclerosis, is to improve remyelination, thereby restoring effective axon conduction and preventing neurodegeneration. In the adult central nervous system (CNS), parenchymal oligodendrocyte progenitor cells (pOPCs) and, to a lesser extent, pre-existing oligodendrocytes (OLs) and oligodendrocytes generated from neural stem cells (NSCs) in the sub-ventricular zone (SVZ) are capable of forming new myelin sheaths. Due to their self-renewal capabilities and the ability of their progeny to migrate widely within the CNS, NSCs represent an additional source of remyelinating cells that may be targeted to supplement repair by pOPCs. However, in demyelinating disorders and disease models, the NSC contribution to myelin repair is modest and most evident in regions close to the SVZ. We hypothesized that NSC-derived cells may compete with OPCs to remyelinate the same axons, with pOPCs serving as the primary remyelinating cells due to their widespread distribution within the adult CNS, thereby limiting the contribution of NSC-progeny. Here, we have used a dual reporter, genetic fate mapping strategy, to characterize the contribution of pOPCs and NSC-derived OLs to remyelination after cuprizone-induced demyelination. We confirmed that, while pOPCs are the main remyelinating cells in the corpus callosum, NSC-derived cells are also activated and recruited to demyelinating lesions. Blocking pOPC differentiation genetically, resulted in a significant increase in the recruitment NSC-derived cells into the demyelinated corpus callosum and their differentiation into OLs. These results strongly suggest that pOPCs and NSC-progeny compete to repair white matter lesions. They underscore the potential significance of targeting NSCs to improve repair when the contribution of pOPCs is insufficient to affect full remyelination.

Ependyma: a new target for autoantibodies in neuromyelitis optica?

Neuromyelitis optica (NMO) is an autoimmune demyelinating disease of the central nervous system characterized by the presence of autoantibodies (called NMO-IgG) targeting aquaporin-4. Aquaporin-4 is expressed at the perivascular foot processes of astrocytes, in the glia limitans, but also at the ependyma. Most studies have focused on studying the pathogenicity of NMO-IgG on astrocytes, and NMO is now considered an astrocytopathy. However, periependymal lesions are observed in NMO suggesting that ependymal cells could also be targeted by NMO-IgG. Ependymal cells regulate CSF-parenchyma molecular exchanges and CSF flow, and are a niche for sub-ventricular neural stem cells. Our aim was to examine the effect of antibodies from NMO patients on ependymal cells. We exposed two models, i.e. primary cultures of rat ependymal cells and explant cultures of rat lateral ventricular wall whole mounts, to purified IgG of NMO patients (NMO-IgG) for 24 hours. We then evaluated the treatment effect using immunolabelling, functional assays, ependymal flow analysis and bulk RNA sequencing. For each experiment, the effects were compared with those of purified IgG from a healthy donors and non-treated cells. We found that: (i) NMO-IgG induced aquaporin-4 agglomeration at the surface of ependymal cells and induced cell enlargement in comparison to controls. In parallel, it induced an increase in gap junction connexin-43 plaque size; (ii) NMO-IgG altered the orientation of ciliary basal bodies and functionally impaired cilia motility; (iii) NMO-IgG activated the proliferation of sub-ventricular neural stem cells; (iv) treatment with NMO-IgG up-regulated the expression of pro-inflammatory cytokines and chemokines in the transcriptomic analysis. Our study showed that NMO-IgG can trigger an early and specific reactive phenotype in ependymal cells, with functional alterations of intercellular communication and cilia, activation of sub-ventricular stem cell proliferation and the secretion of pro-inflammatory cytokines. These findings suggest a key role for ependymal cells in the early phase of NMO lesion formation.

ADAM10 facilitates rapid neural stem cell cycling and proper positioning within the subventricular zone niche via JAMC/RAP1Gap signaling.

The mechanisms that regulate neural stem cell (NSC) lineage progression and maintain NSCs within different domains of the adult neural stem cell niche, the subventricular zone are not well defined. Quiescent NSCs are arranged at the apical ventricular wall, while mitotically activated NSCs are found in the basal, vascular region of the subventricular zone. Here, we found that ADAM10 (a disintegrin and metalloproteinase 10) is essential in NSC association with the ventricular wall, and via this adhesion to the apical domain, ADAM10 regulates the switch from quiescent and undifferentiated NSC to an actively proliferative and differentiating cell state. Processing of JAMC (junctional adhesion molecule C) by ADAM10 increases Rap1GAP activity. This molecular machinery promotes NSC transit from the apical to the basal compartment and subsequent lineage progression. Understanding the molecular mechanisms responsible for regulating the proper positioning of NSCs within the subventricular zone niche and lineage progression of NSCs could provide new targets for drug development to enhance the regenerative properties of neural tissue.

Testosterone and Adult Neurogenesis.

It is now well established that neurogenesis occurs throughout adulthood in select brain regions, but the functional significance of adult neurogenesis remains unclear. There is considerable evidence that steroid hormones modulate various stages of adult neurogenesis, and this review provides a focused summary of the effects of testosterone on adult neurogenesis. Initial evidence came from field studies with birds and wild rodent populations. Subsequent experiments with laboratory rodents have tested the effects of testosterone and its steroid metabolites upon adult neurogenesis, as well as the functional consequences of induced changes in neurogenesis. These experiments have provided clear evidence that testosterone increases adult neurogenesis within the dentate gyrus region of the hippocampus through an androgen-dependent pathway. Most evidence indicates that androgens selectively enhance the survival of newly generated neurons, while having little effect on cell proliferation. Whether this is a result of androgens acting directly on receptors of new neurons remains unclear, and indirect routes involving brain-derived neurotrophic factor (BDNF) and glucocorticoids may be involved. In vitro experiments suggest that testosterone has broad-ranging neuroprotective effects, which will be briefly reviewed. A better understanding of the effects of testosterone upon adult neurogenesis could shed light on neurological diseases that show sex differences.

Cognitive impairments in adult mice with RIP140 overexpression in neural stem cells.

Receptor-interacting protein 140 (RIP140) is a transcription co-regulator of several transcription factors and a signal transduction regulator. RIP140 was recently implicated in the regulation of cognitive functions. The gene that encodes RIP140 is located on chromosome 21. An increase in RIP140 expression was observed in the fetal cerebral cortex and hippocampus in Down syndrome patients who exhibited strong cognitive disabilities. We hypothesized that RIP140 overexpression affects cognitive function in adult neural development. The present study used a Cre-dependent adeno-associated virus to selectively overexpress RIP140 in neural stem cells using nestin-Cre mice. RIP140 overexpression efficiency was evaluated at the subgranular zone (SGZ) of the dorsal dentate gyrus (dDG) and the subventricular zone (SVZ) of the lateral ventricles (LVs). Mice with RIP140 overexpression in the SGZ exhibited deficits in cognitive function and spatial learning and memory, measured in the Morris water maze, object-place recognition test, and novel object recognition test. However, overexpression of RIP140 in SVZ only impaired performance in the Morris water maze and novel object recognition test but not in the object-place recognition test. Altogether, these results indicated defects in cognitive functions that were associated with RIP140 overexpression in neural stem cells and revealed a behavioral phenotype that may be used as a framework for further investigating the neuropathogenesis of Down syndrome.

Static Magnetic Field Exposure In Vivo Enhances the Generation of New Doublecortin-expressing Cells in the Sub-ventricular Zone and Neocortex of Adult Rats.

Static magnetic field (SMF) is gaining interest as a potential technique for modulating CNS neuronal activity. Previous studies have shown a pro-neurogenic effect of short periods of extremely low frequency pulsatile magnetic fields (PMF) in vivo and pro-survival effect of low intensity SMF in cultured neurons in vitro, but little is known about the in vivo effects of low to moderate intensity SMF on brain functions. We investigated the effect of continuously-applied SMF on subventricular zone (SVZ) neurogenesis and immature doublecortin (DCX)-expressing cells in the neocortex of young adult rats and in primary cultures of cortical neurons in vitro. A small (3 mm diameter) magnetic disc was implanted on the skull of rats at bregma, producing an average field strength of 4.3 mT at SVZ and 12.9 mT at inner neocortex. Levels of proliferation of SVZ stem cells were determined by 5-ethynyl-2'-deoxyuridine (EdU) labelling, and early neuronal phenotype development was determined by expression of doublecortin (DCX). To determine the effect of SMF on neurogenesis in vitro, permanent magnets were placed beneath the culture dishes. We found that low intensity SMF exposure enhances cell proliferation in SVZ and new DCX-expressing cells in neocortical regions of young adult rats. In primary cortical neuronal cultures, SMF exposure increased the expression of newly generated cells co-labelled with EdU and DCX or the mature neuronal marker NeuN, while activating a set of pro neuronal bHLH genes. SMF exposure has potential for treatment of neurodegenerative disease and conditions such as CNS trauma and affective disorders in which increased neurogenesis is desirable.

Increasing Progenitor Cell Proliferation in the Sub-Ventricular Zone: A Therapeutic Treatment for Progressive Multiple Sclerosis?

INTRODUCTION: The purpose of this study was to determine if pharmacological treatment could increase progenitor cell proliferation in the Sub-ventricular Zone of aged rats. Previous work had shown that increasing progenitor cell proliferation in this region correlated well (R2=0.78; p= 0.0007) with functional recovery in a damaged corpus callosum (white matter tract), suggesting that progenitor cell proliferation results in oligodendrocytes in this region. METHODS: 10 month old male and female Sprague Dawley rats were fed the drugs for 30 days in cookie dough, then immunocytochemistry was performed on coronal brain sections, using Ki67 labeling to determine progenitor cell proliferation. RESULTS: Female rats showed low endogenous (control) progenitor cell proliferation, significantly different from male rats (P<0.0001), at this age. Ascorbic Acid (20 mg/kg, daily for 30 days) increased progenitor cell proliferation overall, but maintained the innate gender difference in stem cell proliferation (P=0.001). Prozac (5 mg/kg, daily for 30 days) increased progenitor cell proliferation for females but decreased stem cell proliferation for males, again showing a gender difference (P<0.0001). Simvastatin (1 mg/kg for 30 days) also increased progenitor cell proliferation in females and decreased progenitor cell proliferation in males, leading to a significant gender difference. DISCUSSION: The three drug combinations (fluoxetine, simvastatin, and ascorbic acid, patent # 9,254,281) led to ~ 4 fold increase in progenitor cell proliferation in females, while male progenitor cell proliferation was highest with 50 mg/kg ascorbic acid. However, the ascorbic acid increase in proliferation appears to be only on the sides of the ventricles, which is not the region that normally gives rise to oligodendrocytes. CONCLUSION: There are innate gender differences in progenitor cell proliferation at the Sub-Ventricular Zone at middle age in rats, possibly due to the loss of estrogen in females. We also see notable gender differences in progenitor cell proliferation in the Sub ventricular Zone in response to common drugs, such as fluoxetine, simvastatin and Vitamin C (ascorbic acid).

Mild myelin disruption elicits early alteration in behavior and proliferation in the subventricular zone.

Myelin, the insulating sheath around axons, supports axon function. An important question is the impact of mild myelin disruption. In the absence of the myelin protein proteolipid protein (PLP1), myelin is generated but with age, axonal function/maintenance is disrupted. Axon disruption occurs in Plp1-null mice as early as 2 months in cortical projection neurons. High-volume cellular quantification techniques revealed a region-specific increase in oligodendrocyte density in the olfactory bulb and rostral corpus callosum that increased during adulthood. A distinct proliferative response of progenitor cells was observed in the subventricular zone (SVZ), while the number and proliferation of parenchymal oligodendrocyte progenitor cells was unchanged. This SVZ proliferative response occurred prior to evidence of axonal disruption. Thus, a novel SVZ response contributes to the region-specific increase in oligodendrocytes in Plp1-null mice. Young adult Plp1-null mice exhibited subtle but substantial behavioral alterations, indicative of an early impact of mild myelin disruption.

Isolation of Neural Stem and Progenitor Cells from the Adult Brain and Live Imaging of Their Cell Cycle with the FUCCI System.

Neural stem cells (NSCs) enter quiescence in early embryonic stages to create a reservoir of dormant NSCs able to enter proliferation and produce neuronal precursors in the adult mammalian brain. Various approaches of fluorescent-activated cell sorting (FACS) have emerged to allow the distinction between quiescent NSCs (qNSCs), their activated counterpart (aNSCs), and the resulting progeny. In this article, we review two FACS techniques that can be used alternatively. We also show that their association with transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice allows an unprecedented overlook on the cell cycle dynamics of adult NSCs.

miR-148b Regulates Proliferation and Differentiation of Neural Stem Cells via Wnt/ß-Catenin Signaling in Rat Ischemic Stroke Model.

Stroke is the second leading cause of death worldwide. Stroke induced proliferation and differentiation of neural stem cells (NSCs) that have been proven to participate in ischemic brain repair. However, molecular mechanisms that regulate neurogenesis have not been fully investigated. MicroRNAs play an important role in the neurological repairing process and impact stroke recovery outcome. MiRNA-148b has been reported to regulate cell proliferation in tumor cells, but its role in NSCs after ischemic stroke remains unknown. Here, we found an overexpression of MiRNA-148b in subventricular zone (SVZ) of rat ischemic brain. In original cultured ischemic NSCs, transfection of MiRNA-148b mimic or inhibitor could suppress or enhance the expression of Wnt-1, ß-catenin, and Cyclin D1, hence effected wnt/ß-catenin signaling. MiRNA-148b inhibitor promoted NSCs proliferation and differentiation into newborn neural and astrocytes, and this action could be silenced with knockdown of Wnt-1. In middle cerebral artery occlusion (MCAo) rats, injection of MiRNA-148b inhibitor could reduce ischemic lesion volume and improve neurological function outcome. Collectively, our data suggest that MiRNA-148b suppressed wnt/ß-catenin signaling attenuates proliferation and differentiation of neural stem cells, these findings shed new light on the role of MiRNA-148b in the recovery process during the stroke and contribute to the novel therapy strategy.

Neural Stem Cells and Human Induced Pluripotent Stem Cells to Model Rare CNS Diseases.

BACKGROUND & OBJECTIVE: Despite the great effort spent over recent decades to unravel the pathological mechanisms underpinning the development of central nervous system disorders, most of them still remain unclear. In particular, the study of rare CNS diseases is hampered by the lack of postmortem samples and of reliable epidemiological studies, thus the setting of in vitro modeling systems appears essential to dissect the puzzle of genetic and environmental alterations affecting neural cells viability and functionality. The isolation and expansion in vitro of embryonic (ESC) and fetal neural stem cells (NSC) from human tissue have allowed the modeling of several neurological diseases "in a dish" and have also provided a novel platform to test potential therapeutic strategies in a pre-clinical setting. In recent years, the development of induced pluripotent stem cell (iPS) technology has added enormous value to the aforementioned approach, thanks to their capability for generating diseaserelevant cell phenotypes in vitro and to their perspective use in autologous transplantation. However, while the potentiality of ESC, NSC and iPS has been widely sponsored, the pitfalls related to the available protocols for differentiation and the heterogeneity of lines deriving from different individuals have been poorly discussed. Here we present pro and contra of using ESC, NSC or iPS for modeling rare diseases like Lysosomal Storage disorders and Motor Neuron Diseases. CONCLUSION: In this view, the advent of gene editing technologies is a unique opportunity to standardize the data analysis in preclinical studies and to tailor clinical protocols for stem cell-mediated therapy.

In vitro differentiation of neural stem cells into noradrenergic-like cells.

Neural stem cells (NSCs) as a heterogeneous multipotent and self- renewal population are found in different areas in the developing mammalian nervous system, as well as the sub-ventricular zone (SVZ) and the hippocampus of the adult brain. NSCs can give rise to neurons, astrocytes and oligodendrocytes. The aim of this study was to differentiate neural stem cells into noradrenergic-like cells in vitro. Neural stem cells were harvested from SVZ of newborn rat brains. The cells were cultured in DMEM12, B-27 supplemented with 20 ng/ ml (hFGF) and 20 ng/ ml (EGF) for 2 weeks. Neurospheres were differentiated in neurobasal medium, B-27 supplemented with BDNF (50 ng/ ml) and GDNF (30 ng/ ml) for 3 and 5 days. Cell culture techniques and immunocytochemistry were applied to examine neurospheres and tyrosine hydroxylase positive cells. The number of neurites was counted 3 and 5 days after the induction of differentiation. Nestin and Sox2 were expressed in NSCs and neurospheres. NSCs were differentiated into noradrenergic- like cells (NACs). Tyrosine hydroxylase was detected in these cells. The results of NSCs differentiation for 5 days culture had a significant decrease (P≤0.05) in the number of TH positive cells with one or two neurite per cell, and a significant increase (P≤0.05) in the number of TH positive cells with three, four or more neurites per cell, compared with 3 days culture. Based on these results, NSCs have the ability to differentiate into noradrenergic cells in the presence of BDNF and GDNF growth factors.

Tenascin-C: Form versus function.

Tenascin-C is a large, multimodular, extracellular matrix glycoprotein that exhibits a very restricted pattern of expression but an enormously diverse range of functions. Here, we discuss the importance of deciphering the expression pattern of, and effects mediated by, different forms of this molecule in order to fully understand tenascin-C biology. We focus on both post transcriptional and post translational events such as splicing, glycosylation, assembly into a 3D matrix and proteolytic cleavage, highlighting how these modifications are key to defining tenascin-C function.

Temporal features of adult neurogenesis: differences and similarities across mammalian species.

Production of new neurons continues throughout life in most invertebrates and vertebrates like crustaceans, fishes, reptiles, birds, and mammals including humans. Most studies have been carried out on rodent models and demonstrated that adult neurogenesis is located mainly in two structures, the dentate gyrus (DG) of the hippocampus and the sub-ventricular zone (SVZ). If adult neurogenesis is well preserved throughout evolution, yet there are however some features which differ between species. The present review proposes to target similarities and differences in the mechanism of mammalian adult neurogenesis by comparing selected species including humans. We will highlight the cellular composition and morphological organization of the SVZ in primates which differs from that of rodents and may be of functional relevance. We will particularly focus on the dynamic of neuronal maturation in rodents, primates, and humans but also in sheep which appears to be an interesting model due to its similarities with the primate brain.

Citalopram enhances neurovascular regeneration and sensorimotor functional recovery after ischemic stroke in mice.

Recent clinical trials have demonstrated that treatment with selective serotonin reuptake inhibitors after stroke enhances motor functional recovery; however, the underlying mechanisms remain to be further elucidated. We hypothesized that daily administration of the clinical drug citalopram would produce these functional benefits via enhancing neurovascular repair in the ischemic peri-infarct region. To test this hypothesis, focal ischemic stroke was induced in male C57/B6 mice by permanent ligation of distal branches of the middle cerebral artery to the barrel cortex and 7-min occlusion of the bilateral common carotid arteries. Citalopram (10mg/kg, i.p.) was injected 24h after stroke and daily thereafter. To label proliferating cells, bromo-deoxyuridine was injected daily beginning 3 days after stroke. Immunohistochemical and functional assays were performed to elucidate citalopram-mediated cellular and sensorimotor changes after stroke. Citalopram treatment had no significant effect on infarct formation or edema 3 days after stroke; however, citalopram-treated mice had better functional recovery than saline-treated controls 3 and 14 days after stroke in the adhesive removal test. Increased expression of brain-derived neurotrophic factor was detected in the peri-infarct region 7 days after stroke in citalopram-treated animals. The number of proliferating neural progenitor cells and the distance of neuroblast migration from the sub-ventricular zone toward the ischemic cortex were significantly greater in citalopram-treated mice at 7 days after stroke. Immunohistochemical staining and co-localization analysis showed that citalopram-treated animals generated more new neurons and microvessels in the peri-infarct region 21 and 28 days after stroke. Taken together, these results suggest that citalopram promotes post-stroke sensorimotor recovery likely via enhancing neurogenesis, neural cell migration and the microvessel support in the peri-infarct region of the ischemic brain.