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

Råtta "hjärnmjölkning" för isolering av neurala stam- och oligodendrocytstamceller

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.

Författare i fokus: Insamling av neurala stam- och stamceller från levande djur med hjälp av ett nytt protokoll för hjärnmjölkning 

"Brain Milking"-metoden för isolering av neurala stamceller och oligodendrocytstamceller från levande råttor

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   

Frisättning av murina neurala stamceller genom stereotaktisk intracerebroventrikulär injektion av frisättningscocktail 

Börja med att administrera 0,3 milligram per milliliter buprenorfin subkutant till den sövda råttan. Montera råttan på den stereotaktiska ramen. Använd sedan en rakhyvel för att raka bort pälsen från huvudet.Desinficera huden med tre omväxlande omgångar av 10 % povidonjod och alkohol, med sterila bomullspinnar, gnugga i olika riktningar i tre till fem sekunder varje gång. Applicera ögonsalva för att förhindra torrhet. Gör nu ett två centimeter långt snitt längs huvudets mittlinje med en steril skalpell och rengör sedan skallen noggrant med sterila svabbar och koksaltlösning.Identifiera bregman med hjälp av en 10 mikroliters Hamilton sprutnål monterad på den stereotaktiska enheten och ställ in den som 00. Flytta enheten främre, bakre och laterala koordinater. Skapa ett borrhål på en millimeter med hjälp av en tandläkarborr.Ladda fyra mikroliter av frigöringscocktailen i 10 mikroliters Hamilton-sprutan och sänk sedan försiktigt nålen med en trubbig eller konisk kant för att komma i kontakt med duran. Stick in nålen till önskat djup på 3,5 millimeter. Infundera två mikroliter av frigöringscocktailen med en hastighet av en mikroliter per minut.Låt nålen sitta kvar i ytterligare två minuter och dra sedan långsamt upp den för att förhindra att cocktailen kommer upp till ytan. Suturera snittet med 5/0 nylon och rengör det suturerade området med antiseptiskt medel. Överför sedan djuret till övervakningsområdet efter operationen.Injektion av mer än tre mikroliter av frisättningscocktailen ledde till ospecifika mekaniska skador. Injektion av två mikroliter av cocktailen ledde till uppkomsten av dubbelkortinimmunpositiva kluster som var synliga även efter åtta månader. Immunfärgning av hjärnan avslöjade denudationsställen på det ependymala skiktet, nära endast nivån av injektioner.En gradvis minskning av störningarna i det ependymala lagret sågs i mer bakre och mer främre områden av den subependymala zonen.

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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 ...