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

Rotte "Brain Milking" til neural stam og oligodendrocyt stamcelleisolering

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.

Forfatter Spotlight: Indsamling af neurale stam- og stamceller fra levende dyr ved hjælp af en ny hjernemalkeprotokol 

"Hjernemalkning" -metoden til isolering af neurale stamceller og oligodendrocytstamceller fra levende rotter

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   

Frigivelse af neurale stamceller fra murin ved stereotaksisk intracerebroventrikulær injektion af frigivelsescocktail 

Begynd med at administrere 0,3 milligram pr. milliliter buprenorphin subkutant til den bedøvede rotte. Monter rotten på den stereotaksiske ramme. Brug derefter en barbermaskine til at barbere pelsen fra hovedet.Desinficer huden med tre vekslende runder af 10% povidon jod og alkohol ved hjælp af sterile vatpinde, gnid i forskellige retninger i tre til fem sekunder hver gang. Påfør øjensalve for at forhindre tørhed. Lav nu et to centimeter snit langs hovedets midterlinje ved hjælp af en steril skalpel, og rengør derefter kraniet omhyggeligt ved hjælp af sterile vatpinde og saltvand.Identificer bregma ved hjælp af en 10 mikroliter Hamilton sprøjtenål monteret på den stereotaksiske enhed, og indstil den som 00. Flyt enheden anterior, bageste og laterale koordinater. Opret et hul på en millimeter ved hjælp af en tandboremaskine.Læg fire mikroliter af frigivelsescocktailen i 10 mikroliter Hamilton-sprøjten, og sænk derefter nålen forsigtigt med en stump eller konisk kant for at komme i kontakt med duraen. Indsæt nålen til den ønskede dybde på 3,5 millimeter. Tilsæt to mikroliter af frigivelsescocktailen med en hastighed på en mikroliter pr. Minut.Lad kanylen sidde i yderligere to minutter, og tag den derefter langsomt op for at forhindre, at frigørelsescocktailen kommer op til overfladen. Sutur snittet ved hjælp af 5/0 nylon og rengør det suturerede område med antiseptisk middel. Overfør derefter dyret til overvågningsområdet efter operationen.Injektion af mere end tre mikroliter frigivelsescocktail førte til ikke-specifikke mekaniske skader. Injektion af to mikroliter af cocktailen førte til fremkomsten af dobbeltcortinimmunopositive klynger, der var synlige selv efter otte måneder. Hjerneimmunfarvning afslørede denudationssteder på det ependymale lag, tæt på kun niveauet af injektioner.Et gradvist fald i forstyrrelserne i ependymalaget blev set i mere bageste og mere forreste områder af den subependymale zone.

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Neuroscience

﻿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 ...