﻿releasing murine neural stem cells by stereotaxic intracerebroventricular injection of release cocktail

"Ordenha cerebral" de rato para isolamento de células-tronco neurais e oligodendrócitos

Tissue-specific stem cells are partially committed cells that can give rise to all cell populations that constitute the respective tissues. Apart from being multipotent, they are self-renewing cells and crucial for maintaining the homeostasis and the regenerative capacity of tissues1. Some tissue-specific stem cells remain in an active, strongly proliferative state, such as intestinal or hematopoietic stem cells. Others, such as brain stem cells, remain largely quiescent or dormant2. In the adult brain, neural stem cells (NSCs) can be found in specialized areas, often called niches. Two such well described areas exist in the subependymal zone (SEZ) of the lateral ventricles and in the dentate gyrus of the hippocampus. The SEZ niche generates the highest numbers of cells, primarily neuroblasts that migrate toward the olfactory bulbs and contribute to the local interneuron population; in contrast, generated oligodendroblasts migrate to the adjacent corpus callosum (CC)3. Oligodendrocyte progenitor cells (OPCs) are mitotically active cells, widely distributed throughout the central nervous system, that: i) are committed to the oligodendroglial lineage, ii) can migrate to sites of demyelination, and iii) can differentiate into myelinating oligodendrocytes. OPCs also exhibit self-renewal potential and quiescence4.
Until now, the isolation and study of NSCs and OPCs required postmortem dissociation of the dissected brain and spinal cord tissue. To circumvent this experimental limitation, we established a method that allows, for the first time, the isolation of brain NSCs and OPCs from live animals. We call this method &#34;milking&#34;, because it enables multiple collections of cells as their pools are not depleted. The protocol was developed in rats, due to their large brain size, targeting mainly the SEZ, or the CC, and includes two major steps. First, NSCs or OPCs are &#34;removed&#34; from the tissue via i.c.v. injection of a &#34;release cocktail&#34; containing neuraminidase, a toxin that induces ventricular wall denudation, an integrin-&#946;1-blocking antibody, and fibroblast growth factor 2 (FGF2). The cocktail is stereotaxically injected bilaterally within the lateral ventricles. If the intended use is the isolation of NSCs, rostral areas of the lateral ventricles are targeted. If the aim is to isolate OPCs more purely, the cocktail is injected caudally in the area of the hippocampal fimbria. At a second &#34;collection&#34; step, liquid biopsies of cerebrospinal fluid (CSF) are performed from the cisterna magna of anesthetized rats, without the need of an incision. The liquid biopsy is mixed with NSC culture medium and can be kept at 4 &#176;C until plating.

Autor em destaque: Coletando células-tronco neurais e progenitoras de animais vivos usando um novo protocolo de ordenha cerebral 

Método "Brain Milking" para o Isolamento de Células-Tronco Neurais e Células Progenitoras de Oligodendrócitos de Ratos Vivos

We are working with brain neural stem cells, very sparse cells that cluster within specialized microenvironments called niches. Although they contribute to olfaction, memory, and learning, they are extremely inefficient in driving regeneration. Thus, we investigate their properties and ways to manipulate them to increase the healing of the nervous tissue.We have shown that the contribution of brain neural stem cells to regeneration is limited by endogenous and endogenous factors, such as their restricted differentiation potential, and their dependence on microenvironment factors. Equally, we have identified key properties that can be used to devise strategies to modulate their capacity. The milking protocol allows the collection of brain neural stem and oligodendrocyte progenitor cells from live experimental animals.Thus, it allows a more uninterrupted analysis of their properties and the performance of longitudinal studies. Currently, there are no alternative techniques for the isolation of brain murine stem and progenitor cells from live experimental animals. The present standard of comparison is the postmortem isolation of such cells, which introduces various experimental uncertainties and biological bias.We are now investigating how brain neural stem cells become activated by existing quiescence. We are also comparing the properties of different progenitor populations of the brain. For example, neurogenic versus oligodendrogenic, in order to define their differential properties and their adaptations to varied microenvironment.

Watch this Scientific Journal Video about ﻿Releasing Murine Neural Stem Cells by Stereotaxic Intracerebroventricular Injection of Release Cocktail at JoVE.com

Releasing Murine Neural Stem Cells by Stereotaxic Intracerebroventricular Injection of Release Cocktail   

Liberação de células-tronco neurais murinas por injeção intracerebroventricular estereotáxica de coquetel de liberação 

Comece administrando 0,3 miligramas por mililitro de buprenorfina por via subcutânea ao rato anestesiado. Monte o rato na estrutura estereotáxica. Em seguida, use uma navalha para raspar o pelo da cabeça.Desinfetar a pele com três rodadas alternadas de iodopovidona a 10% e álcool, usando cotonetes estéreis, esfregando em direções diferentes por três a cinco segundos cada vez. Aplique pomada ocular para evitar o ressecamento. Agora, faça uma incisão de dois centímetros ao longo da linha média da cabeça usando um bisturi estéril e, em seguida, limpe o crânio meticulosamente usando cotonetes estéreis e soro fisiológico.Identifique o bregma usando uma agulha de seringa Hamilton de 10 microlitros montada no dispositivo estereotáxico e coloque-a como 00. Movimente as coordenadas anteroposterior e lateral do dispositivo. Crie um furo de rebarba de um milímetro usando uma broca dental.Coloque quatro microlitros do coquetel de liberação na seringa Hamilton de 10 microlitros e, em seguida, abaixe cuidadosamente a agulha com uma borda romba ou cônica para entrar em contato com a dura-máter. Insira a agulha na profundidade desejada de 3,5 milímetros. Infundir dois microlitros do cocktail de libertação a uma taxa de um microlitro por minuto.Deixe a agulha no lugar por mais dois minutos e, em seguida, recupere-a lentamente para evitar o surgimento do coquetel de liberação. Sutura da incisão com náilon 5/0 e limpeza da área suturada com antisséptico. Em seguida, transfira o animal para a área de monitoramento pós-operação.A injeção de mais de três microlitros do coquetel de liberação levou a lesões mecânicas inespecíficas. A injeção de dois microlitros do coquetel levou ao surgimento de clusters imunopositivos de doublecortin que eram visíveis mesmo após oito meses. A imunomarcação cerebral revelou locais de desnudamento na camada ependimária, próximos apenas ao nível das injeções.Um declínio gradual nas perturbações da camada ependimária foi observado nas áreas mais posteriores e anteriores da zona subependimária.

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﻿Releasing Murine Neural Stem Cells by Stereotaxic Intracerebroventricular Injection of Release Cocktail

The "Brain Milking" Method for the Isolation of Neural Stem Cells and Oligodendrocyte Progenitor Cells from Live Rats

Tissue-specific neural stem cells (NSCs) remain active in the mammalian postnatal brain. They reside in specialized niches, where they generate new ...