Name: Preparation of CNS Tissue for Isolating Cells in EAE Mice
Uploaded: 2023-10-06T13:40:00
Duration: 2 min 1 s
Description: Watch this Scientific Journal Video about Preparation of CNS Tissue for Isolating Cells in EAE Mice at JoVE.com

preparation of cns tissue for isolating cells in eae mice 

从自身免疫小鼠中同时分离所有 CNS 驻留细胞类型

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS) characterized by demyelination, axonal damage, gliosis, and neurodegeneration. Despite numerous research approaches in this field, the pathophysiology of MS is still not fully understood1,2,3,4. The most common animal model for investigating MS is the myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55)-induced experimental autoimmune encephalomyelitis (EAE) which shares many of its clinical and pathophysiological features5,6,7,8,9. It is based on the response of the immune system against CNS-specific antigens leading to inflammation, demyelination, and neuroaxonal degeneration. Experimental autoimmune encephalomyelitis (EAE) is a suitable model for the investigation of neuroinflammatory pathways and signaling cascades found in MS.
Current therapy options for MS are only partially effective and focus primarily on the initial inflammatory phase of the disease. However, the neurodegenerative component of MS seems to be the major challenge for long-term therapeutic approaches. Therefore, reproducible, and precise cell isolation protocols are required to investigate molecular and cellular mechanisms in autoimmune diseases in a comprehensive way. Even if some protocols for the isolation of one single cell type exist10,11,12,13,14,15, there is an unmet need for the simultaneous isolation of several CNS-resident cell populations at once. Previous protocols for the isolation of CNS-resident cells lack in preserving cellular functionality and purity, resulting in co-cultivation with neighboring cells16,17,18 or the unsuitability for complex analyses of intracellular networks ex vivo19,20,21,22.
The aim of this protocol was to establish a reproducible and comprehensive method for the simultaneous isolation of pure viable single-cell suspensions of all principal CNS-resident cell types applicable in adult healthy and EAE mice. The different cell types were isolated using magnetic-activated cell sorting (MACS)23. The cell separation can be accomplished either by positive selection, i.e., magnetic labeling of cell-type-specific surface markers, or by negative selection via biotinylation and depletion of all undesired cells. Flow cytometry was applied to ensure a purity of above 90% and a viability of at least 80% of the isolated single-cell suspensions.
In conclusion, the main goal was to establish a protocol for the simultaneous isolation of all main CNS-resident cell types as a versatile tool for the investigation of neuroinflammatory pathways offering a comprehensive and precise analysis of complex cellular networks and biochemical signaling cascades in healthy and EAE mice.

作者聚焦：通过同时分离所有主要的 CNS 驻留细胞类型来研究神经炎症通路 

从成年自身免疫性脑脊髓炎小鼠中同时分离主要中枢神经系统驻留细胞类型

To investigate molecular and cellular mechanisms in autoimmune diseases such as multiple sclerosis, the generation of reproducible and sophisticated cell isolation protocols is an unmet need. Most of the current technologies to isolate and analyze CNS-resident cells show serious shortcomings, such as focusing on only one cell type or only postnatal mice. The current protocol for the simultaneous isolation of all main CNS-resident cell types from one CNS replicate offers the possibility to analyze complex neuronal networks and new inflammatory pathways, ex vivo from one single cell suspension, applicable to healthy mice and those with experimental autoimmune encephalomyelitis.Additionally, mice numbers are decreased Our protocol for the simultaneous isolation of all four major CNS-resident cell types in healthy and EAE mice could be used as a helpful tool for research groups studying new inflammatory pathways, allowing a much more accurate analysis of complex biomolecular mechanisms and cellular networks ex vivo. New scientific questions that could be answered with the help of our protocol are dynamic investigations in EAE during different stages of disease course, like neuroinflammation, neurodegeneration, and remission. Also, cell-cell interactions and biochemical pathways could be studied on an individual level.Likewise, functional assays could be performed with cultivated cells. We intend to focus on dynamic studies of the EAE course for multi-omic analysis from one individual CNS homogenate per EAE time point.

Watch this Scientific Journal Video about Preparation of CNS Tissue for Isolating Cells in EAE Mice at JoVE.com

Preparation of CNS Tissue for Isolating Cells in EAE Mice   

用于分离EAE小鼠细胞的CNS组织的制备 

首先将鼠标置于仰卧位并用针固定四肢。将75%乙醇涂抹在动物的前体上。在腹部和胸部的皮肤和筋膜上做一个纵向切片。切开胸骨旁的肋骨，折叠胸腔，以便自由进入心脏。用针将胸部向上折叠固定。用剪刀打开右心房。使用套管，将 20 毫升一次性 PBS 涂入左心室，通过切开的右心房冲洗血液。通过切割小鼠头部顶部的皮肤来制作纵向切片，以露出头骨，并使用镊子将皮肤移到头部周围。用剪刀沿着矢状缝线切开颅骨。将剪刀的尖端沿着切口线插入，以打开切口。用镊子取出的其余部分，使大脑完全暴露。小心地取出大脑并将其放入鼠脑基质中。使用剃须刀片将大脑切成一毫米厚的矢状切片。在剪刀的帮助下，剪下髂嵴上方的脊柱。将连接到含有一次性 PBS 的 20 毫升注射器的 20 号针头插入椎管。将脊髓从尾部冲洗到颅骨。用手术刀将脊髓切成0.5厘米长的段。将由大脑和相应的脊髓组成的中枢神经系统细胞悬液储存在含有三毫升冷DPBS的培养皿中。

Research

JoVE Journal

Neuroscience

Preparation of CNS Tissue for Isolating Cells in EAE Mice 

Simultaneous Isolation of Principal Central Nervous System-Resident Cell Types from Adult Autoimmune Encephalomyelitis Mice

Experimental autoimmune encephalomyelitis (EAE) is the most common murine model for multiple sclerosis (MS) and is frequently used to further elucidate ...