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

Nöral Kök ve Oligodendrosit Progenitör Hücre İzolasyonu için Sıçan "Beyin Sağım"

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.

Yazar Spotlight: Yeni Bir Beyin Sağım Protokolü Kullanarak Canlı Hayvanlardan Nöral Kök ve Progenitör Hücrelerin Toplanması 

Canlı Sıçanlardan Nöral Kök Hücrelerin ve Oligodendrosit Progenitör Hücrelerin İzolasyonuna Yönelik "Beyin Sağım" Yöntemi

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.

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

Murin Nöral Kök Hücrelerin Stereotaksik İntraserebroventriküler Enjeksiyonu ile Serbest Bırakma Kokteyli 

Anestezi uygulanan sıçana mililitre başına 0.3 miligram buprenorfin subkutan olarak uygulanarak başlayın. Fareyi stereotaksik çerçeveye monte edin. Ardından, kürkü kafadan tıraş etmek için bir tıraş bıçağı kullanın.Steril pamuklu çubuklar kullanarak, her seferinde üç ila beş saniye boyunca farklı yönlere sürtünerek, cildi üç alternatif tur% 10 povidon iyot ve alkol ile dezenfekte edin. Kuruluğu önlemek için göz merhemi sürün. Şimdi, steril bir neşter kullanarak başın orta hattı boyunca iki santimetrelik bir kesi yapın, ardından steril bezler ve salin kullanarak kafatasını titizlikle temizleyin.Stereotaksik cihaza monte edilmiş 10 mikrolitrelik bir Hamilton şırınga iğnesi kullanarak bregmayı tanımlayın ve 00 olarak ayarlayın. Cihazı ön, arka ve yan koordinatları hareket ettirin. Bir diş matkabı kullanarak bir milimetre çapak deliği oluşturun.10 mikrolitrelik Hamilton şırıngasına dört mikrolitre serbest bırakma kokteyli yükleyin, ardından dura ile temas etmek için iğneyi keskin veya konik bir kenarla dikkatlice indirin. İğneyi istenen 3,5 milimetre derinliğe kadar yerleştirin. Dakikada bir mikrolitre oranında iki mikrolitre serbest bırakma kokteyli demleyin.İğneyi iki dakika daha yerinde bırakın, ardından serbest bırakma kokteylinin yüzeye çıkmasını önlemek için yavaşça geri alın. Kesiyi 5/0 naylon kullanarak dikin ve dikişli bölgeyi antiseptik ile temizleyin. Daha sonra hayvanı operasyon sonrası izleme alanına aktarın.Üç mikrolitreden fazla serbest bırakma kokteylinin enjeksiyonu, spesifik olmayan mekanik yaralanmalara yol açtı. Kokteylin iki mikrolitresinin enjeksiyonu, sekiz ay sonra bile görülebilen doublecortin immüno-pozitif kümelerin ortaya çıkmasına neden oldu. Beyin immün boyama, ependimal tabakada sadece enjeksiyon seviyesine yakın denudasyon bölgeleri ortaya çıkardı.Subependimal zonun daha arka ve daha ön bölgelerinde ependimal tabaka pertürbasyonlarında kademeli bir azalma görüldü.

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JoVE Journal

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 ...