Trabalho apoiado pelo NIH conceder HD-32116, da Família Sandler Apoio Foundation, The John Bowes Fundo de Células-Tronco, MEXT, MHLW e HFSP. ZM apoiado pela Fundação Carlos Baldoceda e Krevans UCSF Fellowship. AA-B. detém a Heather e Melanie Muss cadeira dotada.

<ol>
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USA. 93, 14895-14900 (1996).</li><li>Shen, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+SVZ+stem+cells+lie+in+a+vascular+niche:+a+quantitative+analysis+of+niche+cell-cell+interactions.">Adult SVZ stem cells lie in a vascular niche: a quantitative analysis of niche cell-cell interactions.</a> Cell Stem Cell. 3, 289-300 (2008).</li><li>Tavazoie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+specialized+vascular+niche+for+adult+neural+stem+cells.">A specialized vascular niche for adult neural stem cells.</a> Cell Stem Cell. 3, 279-288 (2008).</li><li>Mirzadeh, Z., Merkle, F. T., Soriano-Navarro, M., Garcia-Verdugo, J. 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USA. 96, 11619-11624 (1999).</li><li>Sawamoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+neurons+follow+the+flow+of+cerebrospinal+fluid+in+the+adult+brain.">New neurons follow the flow of cerebrospinal fluid in the adult brain.</a> Science. 311, 629-632 (2006).</li><li>Coskun, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD133++neural+stem+cells+in+the+ependyma+of+mammalian+postnatal+forebrain.">CD133+ neural stem cells in the ependyma of mammalian postnatal forebrain.</a> Proc. Natl. Acad. Sci. USA. 105, 1026-1031 (2008).</li><li>Merkle, F. T., Mirzadeh, Z., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosaic+organization+of+neural+stem+cells+in+the+adult+brain.">Mosaic organization of neural stem cells in the adult brain.</a> Science. 317, 381-384 (2007).</li><li>Young, K. M., Fogarty, M., Kessaris, N., Richardson, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subventricular+zone+stem+cells+are+heterogeneous+with+respect+to+their+embryonic+origins+and+neurogenic+fates+in+the+adult+olfactory+bulb.">Subventricular zone stem cells are heterogeneous with respect to their embryonic origins and neurogenic fates in the adult olfactory bulb.</a> J Neurosci. 27, 8286-8296 (2007).</li><li>Waclaw, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zinc+finger+transcription+factor+Sp8+regulates+the+generation+and+diversity+of+olfactory+bulb+interneurons.">The zinc finger transcription factor Sp8 regulates the generation and diversity of olfactory bulb interneurons.</a> Neuron. 49, 503-516 (2006).</li><li>Kohwi, M., Osumi, N., Rubenstein, J. L., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pax6+is+required+for+making+specific+subpopulations+of+granule+and+periglomerular+neurons+in+the+olfactory+bulb.">Pax6 is required for making specific subpopulations of granule and periglomerular neurons in the olfactory bulb.</a> J Neurosci. 25, 6997-7003 (2005).</li><li>Kohwi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+subpopulation+of+olfactory+bulb+GABAergic+interneurons+is+derived+from+Emx1-+and+Dlx5/6-expressing+progenitors.">A subpopulation of olfactory bulb GABAergic interneurons is derived from Emx1- and Dlx5/6-expressing progenitors.</a> J Neurosci. 27, 6878-6891 (2007).</li><li>Hack, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+fate+determinants+of+adult+olfactory+bulb+neurogenesis.">Neuronal fate determinants of adult olfactory bulb neurogenesis.</a> Nat Neurosci. , (2005).</li><li>Ventura, R. E., Goldman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal+radial+glia+generate+olfactory+bulb+interneurons+in+the+postnatal+murine+brain.">Dorsal radial glia generate olfactory bulb interneurons in the postnatal murine brain.</a> J Neurosci. 27, 4297-4302 (2007).</li><li>Kelsch, W., Mosley, C. P., Lin, C. W., Lois, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+mammalian+precursors+are+committed+to+generate+neurons+with+defined+dendritic+projection+patterns.">Distinct mammalian precursors are committed to generate neurons with defined dendritic projection patterns.</a> PLoS Biol. 5, e300 (2007).</li><li>Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+centrosome+inheritance+maintains+neural+progenitors.">Asymmetric centrosome inheritance maintains neural progenitors.</a> Nature. 461, 947-955 (2009).</li></ol>

A maioria dos estudos de neurogênese em zonas ventricular e subventricular têm contado com técnicas clássicas de corte para examinar a microanatomia e relacionamentos celular nestas regiões. Aqui nós descrevemos uma técnica alternativa, usada pela primeira vez para analisar a rede de cadeias migratórias dos neuroblastos gerados no SVZ 1, então utilizado para estudo da regeneração da população progenitora SVZ seguintes anti-mitótico tratamento 6, e, mais recentemente utilizado para estudar a apical precisa basal e interações célula-célula de adulto SVZ células-tronco neurais 2,3,4. Curiosamente, esta técnica revelou que as células-tronco neurais, ou células de tipo B1, do SVZ adultos fazem parte de um neuroepitélio misturado com não-diferenciada dividindo células ependimárias. En face-imagem usando wholemounts mostrou que este neuroepitélio mista tem arquitetura pinwheel constituído das terminações apical de células do tipo B1, rodeados de grandes superfícies apicais das células ependimárias 4. Esta análise en-cara esclareceu a nossa compreensão da linhagem de células-tronco neurais no cérebro embrionárias e adultas como consistindo de células com terminações apical na superfície do ventrículo e processos basal entrar em contato com um nicho vascular. Essas descobertas teria sido quase impossível utilizando técnicas clássicas de corte. Wholemounts também facilitar a identificação de células-tronco neurais através de seu processo de ventrículo-contactando apical. Como marcadores mais específicos para estas células-tronco são encontradas, wholemounts será parte integrante de identificar e analisar o comportamento das células-tronco neurais. Wholemounts das paredes do ventrículo lateral também apresentar a perspectiva ideal para estudar a polaridade planar de células ependimárias. Células ependimárias são células que revestem os ventrículos multiciliated que a função de impulsionar CSF de forma coordenada. Com a técnica wholemount, o epitélio toda ependimárias é exposta en-face e pode ser manchado e estudado de forma abrangente a partir do seu anterior para posterior e dorsal com as fronteiras ventral. Além disso, ensaios realizados em fluxo ependimárias intensamente dissecado, wholemounts viver robustamente demonstrar o fluxo planar polarizada gerados por cílios ependimárias. Trabalhos recentes usando abordagens wholemount descobriu determinantes celulares dessa polaridade planar ependimárias 5. Curiosamente, estudos wholemount também sugeriram que gerou um fluxo de ependimárias-CSF estabelece gradientes de chemorepellents que guiam a migração dos neurônios jovens na SVZ 7. Abordagens Wholemount que inicialmente identificou a rede de cadeias migratórias neuronal são, portanto, continuar a fornecer insights sobre os mecanismos que regulam a migração em cadeia. Análise da VZ e SVZ por wholemount imagem acrescenta uma nova abordagem para os dois futuros estudos e uma forma de esclarecer nossa compreensão dos estudos existentes. Por exemplo, um estudo recente sugere que as células-tronco neurais no SVZ adultos foram CD133 + / CD24-células em contato com o 8 ventrículo. Com base em suas imunocoloração em seções, esses autores afirmaram que essas células foram uma subpopulação de células ependimárias multiciliated. No entanto, em nosso estudo utilizando a abordagem wholemount, o que dá uma visão mais abrangente de todo o epitélio ependimárias, descobrimos que todas as células ependimárias expressar CD24 eo único ventrículo entrar em contato com as células que foram CD133 + / CD24-se um subconjunto do tipo B1 células 4. Além disso, a técnica wholemount promete ser útil em futuros estudos examinando a organização mosaico recentemente descrito de células-tronco neurais no cérebro adulto 9. Vários estudos têm mostrado que as células-tronco neurais no cérebro adulto não são uma população homogênea, mas são regionalmente especificada e, normalmente, produzem apenas subtipos específicos de interneurônios bulbo olfatório. Estes estudos têm proposto que diferentes subpopulações de células-tronco neurais pode ser distinguido, quer pela expressão dos fatores de transcrição específicos 10,11,12,13,14 e / ou por sua localização regional ao longo das extensões dorsal-ventral e antero-posterior do parede lateral 9,15,16. Como mais marcadores moleculares do subpopulações regional específico de células-tronco adultas neurais são identificados, imagem wholemount deve fornecer uma visão abrangente do parcelation destes domínios progenitor diferentes ao longo da parede ventricular. A dissecção wholemount e técnicas de imagem aqui apresentados também podem ser usados ​​para analisar as paredes ventricular no embrião. A dissecção da parede lateral é realizada embrionárias, passo-a-passo, da mesma maneira. Existem apenas pequenas diferenças no nível de dificuldade, os ventrículos embrionárias são relativamente maiores tornando a dissecção mais fácil, mas o tecido é mais macio fazendo a manipulação mais difícil. Em particular, uma exposição similar do ventrículo lateral pode ser usado em EmbryOS para dissecar a parede cortical do ventrículo para estudar a neurogênese cortical. Evidências recentes sugerem que a herança assimétrica centrossoma mantém glia radial na superfície ventricular durante a neurogênese cortical 17. En-face radial glial imagem de superfícies apical pode fornecer insights sobre como centrossomas dentro dessas células em divisão são assimetricamente herdada. Como a maioria das técnicas, especialmente aquelas que envolvem destreza precisa, o domínio requer prática. Há, no entanto, alguns elementos na dissecção que são fundamentais para obter melhores resultados: 1) de iluminação ajustar a iluminação da amostra para criar sombras fornece o contraste de valor inestimável durante a dissecção do tecido que é outra forma relativamente homogênea, 2) usando o fórceps como dois pinos de insetos a pinça nesta técnica nunca são usadas para comprimir juntos ou pegar o tecido, mas são usados ​​como pinos manobrável que podem ser continuamente reajustada para estabilizar o tecido durante o corte, 3) um balanço de retração gentil e corte a faca não deve só serão utilizadas para cortar, mas também para fornecer retração gentil para separar as paredes medial e lateral, lembrando que a maioria desta dissecção é realmente realizado pela retração gentil com apenas corte intermitente.

the subventricular zone en-face: wholemount staining and ependymal flow

As paredes dos ventrículos laterais contêm a maior região germinal no cérebro adulto de mamíferos. A zona subventricular (SVZ) nessas paredes é um sistema modelo estudado extensivamente para a compreensão do comportamento das células-tronco neurais e regulação da neurogênese adulta. Tradicionalmente, estes estudos basearam-se em técnicas clássicas de corte para análise histológica. Aqui nós apresentamos uma abordagem alternativa, a técnica wholemount, que fornece uma visão completa e en face desta região germinal. Em comparação com seções, wholemounts preservar a citoarquitetura completa e relações celular dentro do SVZ. Esta abordagem revelou recentemente que o adulto de células-tronco neurais, ou células B1 tipo, fazem parte de um neuroepitélio misturado com diferenciados células ependimárias que revestem os ventrículos laterais. Além disso, esta abordagem tem sido utilizada para estudar a polarização planar de células ependimárias e do fluxo de líquido cefalorraquidiano que geram no ventrículo. Com evidências recentes de que células-tronco adultas neurais são uma população heterogênea que é regionalmente especificado, a abordagem wholemount provavelmente será uma ferramenta essencial para a compreensão da organização e divisão em parcelas deste nicho de células-tronco.

Watch this Scientific Journal Video about The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow at JoVE.com

The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow

As paredes ventrículo lateral contêm a maior região germinal no cérebro adulto de mamíferos. Tradicionalmente, os estudos sobre a neurogênese na região têm contado com técnicas clássicas de corte para análise histológica. Aqui nós apresentamos uma abordagem alternativa, a técnica wholemount, que fornece uma visão completa e en face desta região germinal.

A zona subventricular En face: Coloração Wholemount e Fluxo ependimárias

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an ...

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Research

JoVE Journal

Neuroscience

41.6K visualizações.  University of California, San Francisco - UCSF. As paredes ventrículo lateral contêm a maior região germinal no cérebro adulto de mamíferos. Tradicionalmente, os estudos sobre a neurogênese na região têm contado com técnicas clássicas de corte para análise histológica. Aqui nós apresentamos uma abordagem alternativa, a técnica wholemount, que fornece uma visão completa e en face desta região germinal.

Vídeo: The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow

Wholemount Immunohistochemistry for Revealing Complex Brain Topography

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its relatively uniform cytoarchitecture is a complex array of parasagittal domains of gene and protein expression. The molecular compartmentalization of the cerebellum is mirrored by the anatomical and functional organization of afferent fibers. To fully appreciate the complexity of cerebellar organization we previously refined a wholemount staining approach for high throughput analysis of patterning defects in the mouse cerebellum. This protocol describes in detail the reagents, tools, and practical steps that are useful to successfully reveal protein expression patterns in the adult mouse cerebellum by using wholemount immunostaining. The steps highlighted here demonstrate the utility of this method using the expression of zebrinII/aldolaseC as an example of how the fine topography of the brain can be revealed in its native three-dimensional conformation. Also described are adaptations to the protocol that allow for the visualization of protein expression in afferent projections and large cerebella for comparative studies of molecular topography. To illustrate these applications, data from afferent staining of the rat cerebellum are included.

Neural circuits are topographically organized into functional compartments with specific molecular profiles. Here, we provide the practical and technical steps for revealing global brain topography using a versatile wholemount immunohistochemical staining approach. We demonstrate the utility of the method using the well-understood cytoarchitecture and circuitry of cerebellum.

Imuno-histoquímica para Topografia Wholemount cerebrais, Complexo

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its ...

Neurociência

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

Preparação de fatias agudas zona subventricular for Imaging Cálcio

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Neonatal Subventricular Zone Electroporation

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal cells1. Understanding the molecular pathways responsible for NSC self-renewal, commitment, and differentiation is critical for harnessing their unique potential to repair the brain and better understand central nervous system disorders. Previous methods for the manipulation of mammalian systems required the time consuming and expensive endeavor of genetic engineering at the whole animal level2. Thus, the vast majority of studies have explored the functions of NSC molecules in vitro or in invertebrates.
Here, we demonstrate the simple and rapid technique to manipulate neonatal NPCs that is referred to as neonatal subventricular zone (SVZ) electroporation. Similar techniques were developed a decade ago to study embryonic NSCs and have aided studies on cortical development3,4 . More recently this was applied to study the postnatal rodent forebrain5-7. This technique results in robust labeling of SVZ NSCs and their progeny. Thus, postnatal SVZ electroporation provides a cost and time effective alternative for mammalian NSC genetic engineering.

We demonstrate a minimally invasive technique referred to as neonatal subventricular zone electroporation. The technique consists of injecting plasmid DNA into the lateral ventricles of neonatal pups and applying electrical current to deliver and genetically manipulate neural stem cells

Eletroporação Zona Neonatal subventricular

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal ...

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

Os métodos de imagem de imuno-histoquímica e cálcio em Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

Imagens ao vivo da ependimária Cilia nas laterais ventrículos do cérebro do rato

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

Seleção de células-tronco neurais de células progenitoras e do rato adulto subventricular Zone e Live-imagem de seus Dynamics com o ciclo celular

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

Isolamento e Cultura de células estaminais adultas neurais do Subcallosal Zona Rato

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke involves middle cerebral artery occlusion (MCAO). Infarct zone, brain edema and blood-brain barrier (BBB) breakdown are measured as parameters that reflect the extent of brain injury after MCAO. A significant limitation to this method is that these measurements are normally obtained in different rat brain samples, leading to ethical and financial burdens due to the large number of rats that need to be euthanized for an appropriate sample size. Here we present a method to accurately assess brain injury following MCAO by measuring infarct zone, brain edema and BBB permeability in the same set of rat brains. This novel technique provides a more efficient way to evaluate the pathophysiology of stroke.

This protocol describes a novel technique of measuring the three most important parameters of ischemic brain injury on the same set of rodent brain samples. Using only one brain sample is highly advantageous in terms of ethical and economic costs.

Medição de edema cerebral pós-acidente vascular cerebral, zona de infarto e quebra da barreira hematoencefálica em um único conjunto de amostras cerebrais de roedores

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke ...

Cryo-section Dissection of the Adult Subependymal Zone for Accurate and Deep Quantitative Proteome Analysis

The subependymal neurogenic niche consists of a paraventricular ribbon of the lateral ventricular wall of the lateral ventricle. The subependymal zone (SEZ) is a thin and distinct region exposed to the ventricles and cerebrospinal fluid. The isolation of this niche allows the analysis of a neurogenic stem cell microenvironment. However, extraction of small tissues for proteome analysis&#160;is challenging, especially for the maintenance of considerable measurement depth and the achievement of reliable robustness. A new method termed cryo-section-dissection (CSD), combining high precision with minimal tissue perturbation, was developed to address these challenges. The method is compatible with state-of-the-art mass spectrometry (MS) methods that allow the detection of low-abundant niche regulators. This study compared the CSD and its proteome data to the method and data obtained by laser-capture-microdissection (LCM) and a standard wholemount dissection. The CSD method resulted in twice the quantification depth in less than half the preparation time compared to the LCM and simultaneously clearly outperformed the dissection precision of the wholemount dissection. Hence, CSD is a superior method for collecting the SEZ for proteome analysis.

Cryo-section-dissection allows fresh, frozen preparation of the largest neurogenic niche in the murine brain for deep quantitative proteome analysis. The method is precise, efficient, and causes minimal tissue perturbation. Therefore, it is ideally suited for studying the molecular microenvironment of this niche, as well as other organs, regions, and species.

Dissecção crio-seção da zona subependímal adulta para análise proteome quantitativa precisa e profunda

The subependymal neurogenic niche consists of a paraventricular ribbon of the lateral ventricular wall of the lateral ventricle. The subependymal zone ...

<meta charset="utf8"></head><body>Nucleofecção eficiente de NSCs isoladas da zona subventricular murina

Isolation, Expansion, and Nucleofection of Neural Stem Cells from Adult Murine Subventricular Zone

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) as mitogens, producing clonal aggregates known as neurospheres. This in vitro system is a valuable tool for studying NSC potential. Transfection of siRNAs or genes carried in plasmids can be used to induce perturbations to gene expression and study NSC biology. However, the exogenous nucleic acid delivery to NSC cultures is challenging due to the low efficiency of central nervous system (CNS) cells transfection. Here, we present an improved nucleofection system that achieves high efficiency of gene delivery in expanded NSCs from adult murine SVZ. We demonstrate that this relatively simple method enhances gene perturbation in adult NSCs, surpassing traditional transfection protocols with survival rates exceeding 80%. Moreover, this method can also be applied in primary isolated NSCs, providing a crucial advancement in gene function studies through gene expression manipulation via knockdown or overexpression in neurosphere cultures.

<meta charset="utf8"></head><body>Autor em destaque: Nucleofecção aprimorada para entrega de genes de alta eficiência em culturas de células-tronco neurais derivadas da zona subventricular murina</

Here, we describe a nucleofection system designed to enhance gene delivery efficiency in expanded neural stem cells (NSCs) isolated from the adult murine subventricular zone. The findings demonstrate that this method significantly improves gene perturbation in NSCs, surpassing the effectiveness of traditional transfection protocols and enhancing cell survival rate.

Isolamento, expansão e nucleofecção de células-tronco neurais da zona subventricular murina adulta

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented ...