著者らは、ダートマスの比較医学研究センター(CCMR)のスタッフに、これらの研究に使用されたマウスの専門家によるケアに感謝しています。ボルンシュタイン研究基金はこの研究に資金を提供しました。

著者は開示するものは何もありません。

ホメオスタシスおよび疾患中のCNSコンパートメント内の細胞組成を評価する方法は、CNSの生理学的および病理学的状態を理解するために不可欠である。しかし、CNSの重要なバリアとして機能し、多様な免疫細胞を収容しているにもかかわらず、脳および脊髄の多くの従来の組織抽出方法ではこれらの膜の収集が不可能であるため、髄膜は分析から除外されることがよくあります。この省略は?...

中枢神経系(CNS)は免疫学的に特殊な部位です。CSF腔、髄膜、および血管系を除くCNS実質は、古典的に免疫特権部位1,2,3,4,5と見なされており、恒常性状態の間は免疫細胞が比較的ありません2,6,7。対照的に、硬膜、くも膜、および軟膜層で構成される髄膜は、CNSコンパートメントの重要な構成要素であり、疾患の病因中の恒常性免疫監視および炎症プロセスに積極的に関与しています3,6,7,8。定常状態の間、髄膜は、自然リンパ系細胞(ILC)、マクロファージ、樹状細胞(DC)、肥満細胞、T細胞、および程度は低いがB細胞を含む多数の免疫センチネル細胞をサポートする9,10,11。
髄膜は高度に血管新生した構造であり、CNSとその末梢との間にリンパ管接続を提供するリンパ管を含む8,12,13,14。CNS損傷、感染症、自己免疫、さらには神経変性によって誘発される炎症状態では、末梢由来の免疫細胞が実質に浸潤し、髄膜内の免疫ランドスケープを変化させます。細胞浸潤に続いて、髄膜は末梢由来の免疫細胞の機能的ニッチを表し、免疫細胞の凝集、局所免疫細胞の活性化、およびCNSコンパートメントでの長期生存を促進します。顕著な髄膜炎症は、多発性硬化症(MS)15、16、17、18、19、脳卒中20、21、無菌損傷22、23(すなわち、脊髄損傷および外傷性脳損傷)、片頭痛24、および微生物感染25、26を含む、CNSに影響を及ぼす複数の疾患において観察され、27、28、29。したがって、髄膜コンパートメント内の常在細胞および末梢由来免疫細胞の特性評価は、定常状態および疾患の病因におけるこれらの細胞の役割を理解するために不可欠です。
頭蓋体と椎体からの脳、脊髄、髄膜の抽出は、技術的に困難で時間がかかります。現在、3つの髄膜層すべてを無傷のままにして脳を迅速に抽出するために利用できる技術はありません。椎弓切除術は優れた脊髄組織の形態をもたらし、髄膜層を保存しますが、それは非常に時間がかかり、複雑です30,31。逆に、頭蓋骨からの脳の除去および脊髄の水圧押し出しなどのより従来の抽出方法は、CNS組織の迅速な抽出を容易にするが、くも膜および硬膜髄膜の両方がこれらの技術で失われる30,31。脳および脊髄組織の従来の単離中に硬膜およびくも膜層が省略されると、CNSコンパートメント内の細胞の不完全な分析がもたらされる。したがって、無傷の髄膜を有するCNS組織の迅速な抽出に焦点を当てた新しい技術の同定は、CNSコンパートメントの最適な分析にとって重要です。
この原稿は、マウスから脳、脊髄、髄膜を迅速に抽出するための2つの方法を提示し、CNS実質および髄膜の常在細胞および末梢由来免疫細胞の下流分析を容易にします。これらの最適化されたプロトコルは、1)ダウンストリーム分析用の単一細胞懸濁液の単離、および2)組織学的処理のための組織の準備に焦点を当てています。脳、脊髄組織、硬膜およびくも膜髄膜32から単一細胞懸濁液を得ることは、実質および髄膜コンパートメントの両方に存在する細胞の同時分析を可能にする。シングルセル懸濁液は、in vitro刺激33、酵素結合イムノスポット(ELISpot)28、34、35、フローサイトメトリー36、33、シングルセル37またはバルクトランスクリプトミクスを実行するための細胞培養アッセイなど、さまざまなアプリケーションで使用できます。さらに、無傷の頭蓋骨または脊柱を備えた脳全体と脊髄の脱灰に最適化されたプロトコルにより、周囲の骨を穏やかに脱灰し、髄膜を無傷のままにし、組織の形態を維持することができます。この方法では、実質空間と髄膜腔の両方で免疫組織化学(IHC)またはin situハイブリダイゼーション(ISH)技術を使用してタンパク質またはRNAを選択的に同定できます。CNS内の常在細胞および末梢由来免疫細胞の表現型、活性化状態、および局在の特性評価は、CNSコンパートメント内の個々の細胞タイプが恒常性と疾患の病因にどのように寄与するかを理解するために不可欠な情報を提供する可能性があります。

<table><tbody><tr><td>Aluminum foil</td><td>any</td><td>N/A</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>ThermoFisher Scientific</td><td>37002D</td><td></td></tr><tr><td>Centrifuge</td><td>Beckman Coulter</td><td>Allegra X-12R centrifuge</td><td></td></tr><tr><td>Collagenase I</td><td>Worthington</td><td>LS004196</td><td></td></tr><tr><td>Conical tube, 15 mL</td><td>VWR</td><td>525-1069</td><td></td></tr><tr><td>Conical tube, 50 mL</td><td>VWR</td><td>89039-658</td><td></td></tr><tr><td>Cover glass</td><td>Hauser Scientific</td><td>5000</td><td></td></tr><tr><td>Cryomold</td><td>VWR</td><td>18000-128</td><td></td></tr><tr><td>Curved forceps</td><td>Fine Science Tools</td><td>11003-14</td><td></td></tr><tr><td>Disposable polystyrene tube, 14 mL</td><td>Fisher Scientific</td><td>14-959-1B</td><td></td></tr><tr><td>Disposable Scalpel</td><td>Fisher Scientific</td><td>NC0595256</td><td></td></tr><tr><td>DNAse I</td><td>Worthington</td><td>LS002139</td><td></td></tr><tr><td>Dry ice</td><td>Airgas</td><td>N/A</td><td></td></tr><tr><td>Durmont #7Forceps</td><td>Fine Science Tools</td><td>11271-30</td><td></td></tr><tr><td>EDTA disodium salt dihydrate</td><td>Amresco</td><td>0105-500g</td><td></td></tr><tr><td>Ethanol, 100%</td><td>any</td><td>N/A</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Hyclone</td><td>SH30910.03</td><td></td></tr><tr><td>Filter top tube, 5 mL</td><td>VWR</td><td>352235</td><td></td></tr><tr><td>Fixable viability stain 780</td><td>Becton Dickinson</td><td>565388</td><td></td></tr><tr><td>Flow cytometer</td><td>Beckman Coulter</td><td>Gallios</td><td></td></tr><tr><td>Glucose</td><td>Fisher Chemical</td><td>D16-500</td><td></td></tr><tr><td>Goat anti-mouse IgG (488 conjugate)</td><td>Jackson immunoresearch</td><td>115-546-146</td><td></td></tr><tr><td>Goat anti-mouse IgG (594 conjugate)</td><td>Jackson immunoresearch</td><td>115-586-146</td><td></td></tr><tr><td>Goat anti-rabbit 488</td><td>Jackson immunoresearch</td><td>111-545-144</td><td></td></tr><tr><td>Goat anti-rat 594</td><td>Jackson immunoresearch</td><td>112-585-167</td><td></td></tr><tr><td>Goat anti-rat 650</td><td>Jackson immunoresearch</td><td>112-605-167</td><td></td></tr><tr><td>Hank&#039;s Balnced Salt Solution (HBSS)</td><td>Corning</td><td>21-020-CV</td><td></td></tr><tr><td>Hemacytometer</td><td>Andwin Scientific</td><td>02-671-51B</td><td></td></tr><tr><td>Hemostat</td><td>Fine Science Tools</td><td>13004-14</td><td></td></tr><tr><td>HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid)</td><td>ThermoFisher Scientific</td><td>15630080</td><td></td></tr><tr><td>KCl</td><td>Fisher chemical</td><td>BP366-500</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; (anhydrous)</td><td>Sigma Aldrich</td><td>P5655-100G</td><td></td></tr><tr><td>Liquid Nitrogen</td><td>Airgas</td><td>N/A</td><td></td></tr><tr><td>Mouse FC block (CD16/32)</td><td>Becton Dickinson</td><td>553141</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HP0&lt;sub&gt;4&lt;/sub&gt; (anhydrous)</td><td>Fisher Chemical</td><td>S374-500</td><td></td></tr><tr><td>NaCl</td><td>Fisher chemical</td><td>S671-500</td><td></td></tr><tr><td>Needle, 25 gauge</td><td>Becton Dickinson</td><td>305122</td><td></td></tr><tr><td>Normal mouse serum</td><td>ThermoFisher Scientific</td><td>31881</td><td></td></tr><tr><td>Nylon mesh strainer</td><td>VWR</td><td>352350</td><td></td></tr><tr><td>OCT</td><td>Sakura</td><td>4583</td><td></td></tr><tr><td>Paraformaldehyde, 20%</td><td>Electron Microscopy Sciences</td><td>15713-S</td><td>Diluted to 4% using 1 x PBS</td></tr><tr><td>Pasteur pipette, 9 inch, unplugged</td><td>Fisher Scientific</td><td>13-678-20C</td><td></td></tr><tr><td>PBS (1x)</td><td>Corning</td><td>21-040-CV</td><td></td></tr><tr><td>PE Rat Anti-Mouse CD4</td><td>Becton Dickinson</td><td>553730</td><td></td></tr><tr><td>PE-CF594 Rat Anti-Mouse CD19</td><td>Becton Dickinson</td><td>562329</td><td></td></tr><tr><td>Percoll density gradient media</td><td>GE healthcare</td><td>17-0891-01</td><td></td></tr><tr><td>PerCP-Cy5.5 Rat Anti-Mouse CD45</td><td>Becton Dickinson</td><td>550994</td><td></td></tr><tr><td>Petri dish, 100 mm</td><td>VWR</td><td>353003</td><td></td></tr><tr><td>pH meter</td><td>Fisher Scientific</td><td>13-636-AB150</td><td></td></tr><tr><td>Pipet-Aid</td><td>Drummond Scientific Corporation</td><td>4-000-101</td><td></td></tr><tr><td>Pipette 200 &amp;micro;l</td><td>Gilson</td><td>FA10005M</td><td></td></tr><tr><td>Pipette tips, 1 mL</td><td>USA Scientific</td><td>1111-2831</td><td></td></tr><tr><td>Pipette tips, 200 &amp;micro;l</td><td>USA Scientific</td><td>1111-1816</td><td></td></tr><tr><td>Pipette, 1 mL</td><td>Gilson</td><td>FA10006M</td><td></td></tr><tr><td>Prolong Diamond mountant with DAPI</td><td>ThermoFisher Scientific</td><td>P36962</td><td></td></tr><tr><td>Purified Rat Anti-Mouse CD16/CD32</td><td>Becton Dickinson</td><td>553141</td><td></td></tr><tr><td>Rabbit anti-mouse CD3 (SP7 clone)</td><td>Abcam</td><td>ab16669</td><td></td></tr><tr><td>Rabbit anti-mouse laminin</td><td>Abcam</td><td>ab11575</td><td></td></tr><tr><td>Rat anti-mouse ERT-R7</td><td>Abcam</td><td>ab51824</td><td></td></tr><tr><td>RPMI 1640</td><td>Corning</td><td>10-040-CV</td><td></td></tr><tr><td>Serological pipet, 1 mL</td><td>VWR</td><td>357521</td><td></td></tr><tr><td>Serological pipet, 10 mL</td><td>VWR</td><td>357551</td><td></td></tr><tr><td>Serological pipet, 5 mL</td><td>VWR</td><td>357543</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Fisher Scientific</td><td>S318-100</td><td></td></tr><tr><td>Sucrose</td><td>Fisher chemical</td><td>S5-500</td><td></td></tr><tr><td>Surgical scissors</td><td>Fine Science Tools</td><td>14001-16</td><td></td></tr><tr><td>Surgical scissors, extra fine</td><td>Roboz</td><td>RS-5882</td><td></td></tr><tr><td>Syringe, 10 mL</td><td>Becton Dickinson</td><td>302995</td><td></td></tr><tr><td>Syringe, 5 mL</td><td>Becton Dickinson</td><td>309646</td><td></td></tr><tr><td>Trypan blue</td><td>Gibco</td><td>15250-061</td><td></td></tr><tr><td>Vacuum filter system</td><td>Millipore</td><td>20207749</td><td></td></tr><tr><td>Vacuum flask</td><td>Thomas Scientific</td><td>5340-2L</td><td></td></tr><tr><td>Vacuum in-line filter</td><td>Pall Corporation</td><td>4402</td><td></td></tr><tr><td>Vacuum line</td><td>Cole Palmer</td><td>EW-06414-20</td><td></td></tr><tr><td>Water bath</td><td>ThermoFisher Scientific</td><td>Versa bath</td><td></td></tr></tbody></table>

isolating central nervous system tissues and associated meninges for the downstream analysis of immune cells

中枢神経系(CNS)は脳と脊髄で構成され、末梢と中枢神経系の間の障壁として機能する膜層である髄膜に包まれています。CNSは免疫学的に特殊な部位であり、定常状態では、免疫特権はCNS実質で最も明白です。対照的に、髄膜には、自然免疫細胞や適応免疫細胞など、多様な常在細胞があります。CNS損傷、自己免疫、感染、さらには神経変性によって引き起こされる炎症状態の間、末梢由来の免疫細胞が実質に入り、髄膜内に居住する可能性があります。これらの細胞は、CNS疾患の病因の間に有益な作用と有害な作用の両方を行うと考えられています。この知識にもかかわらず、従来のCNS組織抽出方法では髄膜層が省略されているため、CNSコンパートメントを分析するときに髄膜が見落とされがちです。このプロトコルは、単一細胞技術、免疫組織化学、およびin situハイブリダイゼーション法によるダウンストリーム分析に適した、マウスCNS組織(すなわち、脳、脊髄、および髄膜)を迅速に分離するための2つの異なる方法を提示します。記載された方法は、CNS組織の包括的な分析を提供し、恒常性条件下でおよび疾患の病因の間にCNS区画を占める細胞の表現型、機能、および局在を評価するのに理想的である。

この代表的な実験は、B細胞とT細胞を定量化し、恒常性状態における髄膜および実質CNSコンパートメントおよびマウス進行性MSモデル(すなわち、TMEV-IDD)におけるB細胞およびT細胞の局在を記述することを目的としていました。TMEV-IDDは、前述のようにTMEV BeAnの5 x 106 プラーク形成ユニット(PFU)による頭蓋内感染によって5週齢の雌SJLマウスで誘導されました29。
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Watch this Scientific Journal Video about Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells at JoVE.com

Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells

この論文では、脳、脊髄、髄膜など、中枢神経系内の常在免疫細胞および末梢由来の免疫細胞を調べるための2つの最適化されたプロトコルを紹介します。これらの各プロトコルは、定常状態および炎症状態下でこれらのコンパートメントを占める細胞の機能と組成を確認するのに役立ちます。

免疫細胞の下流分析のための中枢神経系組織および関連する髄膜の単離

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier ...

isolating-central-nervous-system-tissues-associated-meninges-for

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

9.0K ビュー.  Dartmouth-Hitchcock Medical Center. この論文では、脳、脊髄、髄膜など、中枢神経系内の常在免疫細胞および末梢由来の免疫細胞を調べるための2つの最適化されたプロトコルを紹介します。これらの各プロトコルは、定常状態および炎症状態下でこれらのコンパートメントを占める細胞の機能と組成を確認するのに役立ちます。

ビデオ: Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells

Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required to define their phenotype and effector functions. Histochemical approaches are instrumental to determine the location of the infiltrating cells and to analyze the associated CNS pathology. However, in-situ histochemistry and immunofluorescent staining techniques are limited by the number of antibodies that can be used at a single time to characterize immune cell subtypes in a particular tissue. Therefore, histological approaches in conjunction with immune-phenotyping by flow cytometry are critical to fully characterize the composition of local CNS infiltration. This protocol is based on the separation of CNS cellular suspensions over discontinous percoll gradients. The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for identification of various immune cell populations in a single sample by flow cytometry.

The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for flow cytometric analyses.

パーコール勾配を用いて脳と脊髄単核細胞の単離

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required ...

免疫・感染学

Isolation of Mononuclear Cells from the Central Nervous System of Rats with EAE

Whether studying an autoimmune disease directed to the central nervous system (CNS), such as experimental autoimmune encephalomyelitis (EAE, 1), or the immune response to an infection of the CNS, such as poliomyelitis, Lyme neuroborreliosis, or neurosyphilis, it is often necessary to isolate the CNS-infiltrating immune cells.
In this video-protocol we demonstrate how to isolate mononuclear cells (MNCs) from the CNS of a rat with EAE. The first step of this procedure requires a cardiac perfusion of the rodent with a saline solution to ensure that no blood remains in the blood vessels irrigating the CNS. Any blood contamination will artificially increase the number of apparent CNS-infiltrating MNCs and may alter the apparent composition of the immune infiltrate. We then demonstrate how to remove the brain and spinal cord of the rat for subsequent dilaceration to prepare a single-cell suspension. This suspension is separated on a two-layer Percoll gradient to isolate the MNCs. After washing, these cells are then ready to undergo any required procedure. 
Mononuclear cells isolated using this procedure are viable and can be used for electrophysiology, flow cytometry (FACS), or biochemistry. If the technique is performed under sterile conditions (using sterile instruments in a tissue culture hood) the cells can also be grown in tissue culture medium. A given cell population can be further purified using either magnetic separation procedures or a FACS.

In this video we demonstrate how to isolate mononuclear cells from the central nervous system of rats with experimental autoimmune encephalomyelitis.

EAEモデルラットの中枢神経系から単核細胞の単離

Whether studying an autoimmune disease directed to the central nervous system (CNS), such as experimental autoimmune encephalomyelitis (EAE, 1), or the ...

生物学

Reliable Isolation of Central Nervous System Microvessels Across Five Vertebrate Groups

Isolation of microvessels from the central nervous system (CNS) is commonly performed by combining cortical tissue from multiple animals, most often rodents. This approach limits the interrogation of blood-brain barrier (BBB) properties to the cortex and does not allow for individual comparison. This project focuses on the development of an isolation method that allows for the comparison of the neurovascular unit (NVU) from multiple CNS regions: cortex, cerebellum, optic lobe, hypothalamus, pituitary, brainstem, and spinal cord. Moreover, this protocol, originally developed for murine samples, was successfully adapted for use on CNS tissues from small and large vertebrate species from which we are also able to isolate microvessels from brain hemisphere white matter. This method, when paired with immunolabeling, allows for quantitation of protein expression and statistical comparison between individuals, tissue type, or treatment. We proved this applicability by evaluating changes in protein expression during experimental autoimmune encephalomyelitis (EAE), a murine model of a neuroinflammatory disease, multiple sclerosis. Additionally, microvessels isolated by this method could be used for downstream applications like qPCR, RNA-seq, and Western blot, among others. Even though this is not the first attempt to isolate CNS microvessels without the use of ultracentrifugation or enzymatic dissociation, it is unique in its adeptness for the comparison of single individuals and multiple CNS regions. Therefore, it allows for investigation of a range of differences that may otherwise remain obscure: CNS portions (cortex, cerebellum, optic lobe, brainstem, hypothalamus, pituitary, and spinal cord), CNS tissue type (gray or white matter), individuals, experimental treatment groups, and species.

The goal of this protocol is to isolate microvessels from multiple regions of the central nervous system of lissencephalic and gyrencephalic vertebrates.

5つの脊椎動物群にわたる中枢神経系微小血管の信頼性の高い絶縁

Isolation of microvessels from the central nervous system (CNS) is commonly performed by combining cortical tissue from multiple animals, most often ...

神経科学

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive human diseases. Considerable advances have been made to uncover the molecular signatures of homeostatic microglia as well as alterations of their gene expression in response to environmental stimuli. With the advent and maturation of single-cell genomic methodologies, it is increasingly recognized that heterogenous microglia may underlie the diverse roles they play in different developmental and pathological conditions. Further dissection of such heterogeneity can be achieved through efficient isolation of microglia from a given region of interest, followed by sensitive profiling of individual cells. Here, we provide a detailed protocol for the rapid isolation of microglia from different brain regions in a single adult mouse brain hemisphere. We also demonstrate how to use these sorted microglia for plate-based deep single-cell RNA sequencing. We discuss the adaptability of this method to other scenarios and provide guidelines for improving the system to accommodate large-scale studies.

We provide a protocol for isolation of microglia from different dissected regions of an adult mouse brain hemisphere, followed by semi-automated library preparation for deep single-cell RNA sequencing of full-length transcriptomes. This method will help to elucidate functional heterogeneity of microglia in health and disease.

ディープシングルセルRNAシーケンシングのための1つの成人マウス脳半球からの領域特異的ミクログリアの分離

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

複数の中枢神経系領域からのマウスグリアの解剖と分離

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

自己免疫マウスからすべてのCNS常在細胞型を同時に単離

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 the still unknown etiology of MS in order to develop new treatment strategies. The myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) EAE model reproduces a self-limiting monophasic disease course with ascending paralysis within 10 days after immunization. The mice are examined daily using a clinical scoring system. MS is driven by different pathomechanisms with a specific temporal pattern, thus the investigation of the role of central nervous system (CNS)-resident cell types during disease progression is of great interest. The unique feature of this protocol is the simultaneous isolation of all principal CNS-resident cell types (microglia, oligodendrocytes, astrocytes, and neurons) applicable in adult EAE and healthy mice. The dissociation of the brain and the spinal cord from adult mice is followed by magnetic-activated cell sorting (MACS) to isolate microglia, oligodendrocytes, astrocytes, and neurons. Flow cytometry was used to perform quality analyses of the purified single-cell suspensions confirming viability after cell isolation and indicating the purity of each cell type of approximately 90%. In conclusion, this protocol offers a precise and comprehensive way to analyze complex cellular networks in healthy and EAE mice. Moreover, required mice numbers can be substantially reduced as all four cell types are isolated from the same mice.

著者スポットライト:すべての主要なCNS常在細胞タイプの同時分離による神経炎症経路の研究 

To date, protocols for the simultaneous isolation of all principal central nervous system-resident cell types from the same mouse are an unmet demand. The protocol shows a procedure applicable in na&#239;ve and experimental autoimmune encephalomyelitis mice to investigate complex cellular networks during neuroinflammation and simultaneously reduce the required mice numbers.

成体自己免疫性脳脊髄炎マウスからの主要な中枢神経系常在細胞型の同時単離

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

マウスの脳および頭蓋骨骨髄からの単一細胞懸濁液の取得

Isolating Immune Cells from Mouse Brain and Skull

Mounting evidence indicates that the immune response triggered by brain disorders (e.g., brain ischemia and autoimmune encephalomyelitis) occurs not only in the brain, but also in the skull. A key step toward analyzing changes in immune cell populations in both the brain and skull bone marrow after brain damage (e.g., stroke) is to obtain sufficient numbers of high-quality immune cells for downstream analyses. Here, two optimized protocols are provided for isolating immune cells from the brain and skull bone marrow. The advantages of both protocols are reflected in their simplicity, speed, and efficacy in yielding a large quantity of viable immune cells. These cells may be suitable for a range of downstream applications, such as cell sorting, flow cytometry, and transcriptomic analysis. To demonstrate the effectiveness of the protocols, immunophenotyping experiments were performed on stroke brains and normal brain skull bone marrow using flow cytometry analysis, and the results aligned with findings from published studies.

著者スポットライト:脳虚血後の複雑な免疫応答の調査

To investigate the immune response to brain disorders, one common approach is to analyze changes in immune cells. Here, two simple and effective protocols are provided for isolating immune cells from murine brain tissue and skull bone marrow.

マウスの脳と頭蓋骨からの免疫細胞の単離

Mounting evidence indicates that the immune response triggered by brain disorders (e.g., brain ischemia and autoimmune encephalomyelitis) occurs not only ...

免疫細胞の下流分析のための中枢神経系組織および関連する髄膜の単離

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パーコール勾配を用いて脳と脊髄単核細胞の単離

EAEモデルラットの中枢神経系から単核細胞の単離

5つの脊椎動物群にわたる中枢神経系微小血管の信頼性の高い絶縁

ディープシングルセルRNAシーケンシングのための1つの成人マウス脳半球からの領域特異的ミクログリアの分離

複数の中枢神経系領域からのマウスグリアの解剖と分離

成体自己免疫性脳脊髄炎マウスからの主要な中枢神経系常在細胞型の同時単離

成体自己免疫性脳脊髄炎マウスからの主要な中枢神経系常在細胞型の同時単離

成体自己免疫性脳脊髄炎マウスからの主要な中枢神経系常在細胞型の同時単離

マウスの脳と頭蓋骨からの免疫細胞の単離

マウスの脳と頭蓋骨からの免疫細胞の単離