Summary

アン<em>エクスビボ</em急性脳スライスにおけるオリゴデンドロサイト指向T細胞攻撃の>モデル

Published: February 05, 2015
doi:

Summary

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Abstract

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of ‘‘collateral’’ neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

Introduction

Death of oligodendrocytes and destruction of the myelin sheath accompanied by loss of neurons and axons are typical pathological findings in grey matter lesions in individuals suffering from multiple sclerosis (MS)1,2. Cortical lesions can be divided so far in three different subtypes2: subpial, intracortical and leukocortical lesions. In comparison to white matter plaques, infiltrates are characterized by a predominance of CD8+ T-cells, suggesting their possible decisive role in grey matter inflammation3. Furthermore, oligoclonal expansions in blood, cerebrospinal fluid (CSF) and within inflammatory lesions can be found for CD8+ T-cells themselves4-6.

In line with this, it is assumed that CD8+ T-cells may be specific for different myelin proteins7,8. Indeed, CD8+ T-cells are found near oligodendrocytes and myelin sheaths9,10 that show MHC I expression11 and might therefore be responsible for the loss of the myelin sheath. This process is often seen together with extensive ‘‘collateral’’ neuronal and axonal damage within the central nervous system (CNS) grey matter1,2. In fact, direct and indirect death of oligodendrocytes and neurons is induced by CD8+ T-cells via two different mechanism: (i) cell membrane swelling and rupture due to the formation of cytotoxic granules following the release of perforins and granzymes and (ii) ligation to the Fas receptors or exposition of FasL on their surface8,12,13.

Different types of animal models are established to study the underlying mechanism of the mentioned processes. In this respect, primed CD8+ T-cells specific for autoantigens with induced expression in CNS glial cells, like oligodendrocytes or astrocytes, can be adoptively transferred to analyse ‘‘collateral’’ neuronal and axonal death in grey matter subsequently14,15. To perform such in vivo experiments is a big help to mimic some pathophysiological aspects of MS, however, this approach is not suited to resolve the underlying mechanism and kinetics of axonal damage and neuronal apoptosis.

To overcome these restrictions, an ex vivo approach was established to study the mechanisms and time course of neuronal cell death following a oligondendrocytes-directed CD8+ T-cell attack. Since only oligodendrocytes and therefore myelin sheath production should be targeted by immune cells, MHC class-I-restricted, ovalbumin (OVA)-reactive OT-I Tcells are used16. These cells are subsequently transferred into brain slices obtained from mice selectively expressing OVA in oligodendrocytes (ODC-OVA mice)17.

Protocol

マウスを用いた実験はすべて、それぞれの機関の動物管理使用委員会のガイドラインに従って行われるべきである。 マウスの実験のために1。一般的なコメント病原体を含まない条件下でマウスを維持し、それらに食物と水を自由にアクセスできるようにする。 注:免疫学的パターンは、年齢や性別に応じて変化することができますので、それは実験群にお?…

Representative Results

オリゴデンドロサイト特異的CD8 + T細胞による脳切片をインキュベートした後、希突起膠細胞、ならびに神経細胞(それぞれ図2Aおよび図1C)、アポトーシスを受ける。アポトーシス( 例えば、カスパーゼ-3、TUNEL)の組織学的徴候が早いインキュベーションの3時間後に検出することができる。潜伏期間は準備と再現性のある結果の良い品質を保証するために、8時間…

Discussion

別の動物モデルは、MSのような炎症性脱髄性疾患の病理学的特徴に対処するために、最後の数十年にわたって記載されている。 インビボでのマウス及びラットモデルは広く疾患の病態生理学的特徴を模倣するために使用される、すなわち、脱髄及び髄鞘再形成のプロセスの結果の分析炎症および神経変性の混在エピソードの。それにもかかわらず、唯一のex vivoでのアプローチは…

Offenlegungen

The authors have nothing to disclose.

Acknowledgements

This work was supported by the Interdisciplinary Center for Clinical Research (IZKF) Münster (SEED 03/12, SB), Deutsche Forschungsgemeinschaft (SFB TR128, TP B6 to S.G.M. ME3283/2-1 to S.G.M.) and by Innovative Medizinische Forschung, Münster (I-BI111316, SB and SGM).

Materials

Name of Material/ Equipment Company Catalog Number Comments/Description
12-Well-plate Corning 3513
2-Mercaptoethanol Gibco 31350-010
2-Methylbutan Roth 3927.1
70 µm strainer Falcon 352350
CaCl2 Merck 1.02382.0500 calcium chloride
CD8+-isolation kit Miltenyi Biotech 130-090-859
D(+)-glukose Merck 1.08337.1000
DMEM Gibco 31966-021 warm in 37 °C water bath before use
EDTA Sigma E5134
FCS PAA Laboratories A15-151 fetal calve serum
gentamicin Gibco 15750-060
HEPES 1M Gibco 15630-050
IL-2 Peprotech 212-12
Isofluran Abbott 05260-05
KCl Merck 1.04933.0500 potassium chloride
KHCO3 Sigma P9144 potassium hydrogen carbonate
L-Glutamine Gibco 35050-038
MgSO4 Merck 1.05886.0500 magnesium sulfate
NaCl Sigma 31434 sodium chloride
NaH2PO4 * H2O Merck 1.06346.0500 sodium hydrogen phosphate
NaHCO3 Merck 1.06329.0500 sodium hydrogen carbonate
NaOH Merck 1.09137.1000 sodium hydroxide
NH4Cl Sigma 213330 ammonium chloride
Non Essential Amino Acid Gibco 11140-050
OVA (257-264) Genscript RP10611 ovalbumin
PIPES Sigma P6757
Sukrose Merck 1.07687.1000
Tissue-Tek OCT Sakura 4583

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Göbel, K., Bittner, S., Cerina, M., Herrmann, A. M., Wiendl, H., Meuth, S. G. An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices. J. Vis. Exp. (96), e52205, doi:10.3791/52205 (2015).

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