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  • 摘要
  • 摘要
  • 引言
  • 研究方案
  • 结果
  • 讨论
  • 披露声明
  • 致谢
  • 材料
  • 参考文献
  • 转载和许可

摘要

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.

摘要

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.

引言

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.

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研究方案

使用小鼠所有实验应根据各自的机构动物护理和使用委员会的指导方针进行。

1.一般注释小鼠实验

  1. 保持小鼠无病原体的条件下，使它们获得食物和水随意。
    注:由于免疫模式可以与年龄和性别的不同是非常重要的使用年龄和性别匹配的小鼠实验组。

2.制备和活化的OVA特异性CD8 + T细胞（OT-I）

  1. 为准备脑片之前描述如下5天执行OT-I T细胞的刺激。
    注:5×10 5活化的效应的CD8 + OT-I的T细胞（CD8 + T细胞的转基因小鼠识别只有OVA 257-264肽的MHC-I类分子的上下文中），需要每片的浓度为5× 10 5被选为实验同盟作为最佳的一种以有利于一个良好的细胞-细胞相互作用，一旦这些细胞开始迁移到切片，并在同一时间，细胞的量不太高，以诱发过度反应，允许单细胞计数8,18。
  2. 备的OT-I的T细胞培养的培养基。补充500毫升DMEM，5％胎牛血清（FCS），10mM的HEPES，2mM的L-谷氨酰胺，50μM2-巯基乙醇，1％非必需氨基酸和25微克/毫升庆大霉素。储存在4℃直至使用的介质。
  3. 除去的OT-I转基因小鼠的脾按照参考16。他们转移到准备好的媒介。
  4. 通过一个70微米的过滤器和离心机悬浮糖化脾脏，在300×g离心5分钟，4℃产生单细胞悬浮液。
  5. 孵育细胞悬浮液用5ml铵氯化物-钾（ACK），缓冲液（150mM的氯化铵 ，10mM的KHCO 3，0.1mM的EDTA，pH7.3）中进行5分钟，以裂解红细胞。
  6. 停止incubati上用10毫升培养基并离心再次如上所述。
  7. 稀释细胞悬浮在培养基中，并计算数字。板的细胞以3×10 7个细胞的密度/孔在12孔板和素它们温育以OVA 257 - 264（SIINFEKL;为1nM）和白细胞介素-2（IL-2）（500国际单位/毫升），用于5天。
  8. 4天后，/毫升再次添加的IL-2在500国际单位的浓度。
  9. 随后，以刺激，纯化的OT-I的T细胞，从细胞悬浮液通过使用负选择基于小鼠的CD8 + T细胞分离试剂盒按照生产商的说明。纯化是通过用抗CD4，细胞CD11b，CD11c的，CD19，CD45R（B220），CD49b（DX5），CD105，MHC II类，泰尔-119，和TCRγ的鸡尾酒基于所有非CD8 +细胞的耗竭/δ其中通过使用磁列，用于纯化的CD8 + T细胞的洗脱然后丢弃。
    注意:在此程序，可以关键步骤再指示由所述生产:重要的是，该反应仅涉及单个细胞，因为团块的存在可能在洗脱步骤阻止柱;小区添加Byotin鸡尾酒之前计数是用于样品的纯度程度重要并且过程应在冰上进行，以尽量减少非特异性抗体绑定。

3.准备急性脑切片和共培养与OT-I T细胞

  1. 现有急性脑的制备切片19，制备用200mM的蔗糖，20毫摩尔PIPES，2.5mM的氯化钾，1.25毫的NaH 2 PO 4，10mM的硫酸镁 ，0.5 氯化钙和10mM葡萄糖placedine冰冷的生理盐水溶液和人工脑脊液（ACSF）通过使用125 mM氯化钠，2.5氯化钾，1.25的NaH 2 PO 4，24 mM的碳酸氢钠 ，2毫硫酸镁 ，2毫的CaCl 2，10mM的葡萄糖。由全光照pH调节各溶液，以7.35克NaOH。前调节pH值ACSF的，溶液必须通入95％O 2和5％ 的 CO 2的混合物。
  2. 预冷却的解决方案。
  3. 麻醉8 - 10周大的小鼠（ 例如，C57BL / 6对照或转基因的ODC-OVA小鼠17与吸入用5.0％异氟烷，5.0％氟烷或通过IP注射100mg / kg的氯胺酮和10mg / kg体重甲苯噻嗪等待麻醉，并使用脚趾捏来评估麻醉水平和斩首一次他们。安乐死小鼠按照机构动物护理和使用委员会标准安乐死。
  4. 把鼠标腹位置。消毒，切头皮矢状。打开颅骨矢，迅速用瓢的帮助下取出脑并用胶水将其固定到一个的vibratome的板块。
  5. 填充板与制备placedine冰冷的生理盐水溶液。
  6. 削减300微米冠状切片用的vibratome。
    注意:这个程序是知道n至得到适合于至少8小时19-21的时间间隔内功能性细胞的研究活完整的组织标本。事实上，在这一段时间内，6小时后已经开始，该制剂显示质量降低的，即改变像动作电位的产生和传播的基本和功能性质。
  7. 切片后，立即传送一个片成12孔板填充ACSF的每个孔中。
  8. 每片加小心5×10 5的OT-I的T细胞。
  9. 孵育切片在培养箱多达8小时（37℃，5％的CO 2）。
  10. 潜伏期，收获切片后嵌入他们通过使用十月复合组织-TEK，并冻结他们在液氮。存储它们在-20℃用于进一步的组织学研究，以例如评估神经结构（ 例如，使用抗MAPII或抗突触抗体）或细胞凋亡（ 例如，使用抗Caspase-3的抗体和抗的NeuN反体）按照标准方法。

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结果

脑切片与少突胶质细胞定向的CD8 + T细胞的培养后，少突胶质细胞以及神经元凋亡（ 图2A和图1C，分别）。 （ 例如，胱天蛋白酶-3，TUNEL）可以最早3小时孵育后检测细胞凋亡的组织学迹象。潜伏期不应该长于8小时，以保证在制备和可再现的结果的一个良好的品质。细胞凋亡，可以发现全国各地有优势的领域髓切片。片的结构的完整性和凋亡神经元的示例性组织染色中描?...

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讨论

不同的动物模型已经描述在过去的几十年来解决的炎性脱髓鞘疾病如MS的病理特征。 体内小鼠和大鼠模型被广泛用于模拟疾病的病理生理特征，即，脱髓鞘和髓鞘再生过程的后果分析和炎症和神经退行性疾病的混合体发作。然而，只有一个离体方法允许解剖确切基本机制。

也是急性脑切片制备一个广泛分布的模型，并可以被认为是可靠的和可复制的。在与隔离?...

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披露声明

The authors declare that they have no competing financial interests.

致谢

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).

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材料

NameCompanyCatalog NumberComments
12-Well plateCorning3513
2-MercaptoethanolGibco31350-010
2-MethylbutanRoth3927.1
70 µm strainerFalcon352350
CaCl2Merck1.02382.0500calcium chloride
CD8+-isolation kitMiltenyi Biotech130-090-859
D(+)-glucoseMerck1.08337.1000
DMEMGibco31966-021warm in 37 °C water bath before use
EDTASigmaE5134
FCSPAA LaboratoriesA15-151fetal calve serum
GentamicinGibco15750-060
HEPES 1 MGibco15630-050
IL-2Peprotech212-12
IsofluranAbbott05260-05
KClMerck1.04933.0500potassium chloride
KHCO3SigmaP9144potassium hydrogen carbonate
L-GlutamineGibco35050-038
MgSO4Merck1.05886.0500magnesium sulfate
NaClSigma31434sodium chloride
NaH2PO4 * H2OMerck1.06346.0500sodium hydrogen phosphate
NaHCO3Merck1.06329.0500sodium hydrogen carbonate
NaOHMerck1.09137.1000sodium hydroxide
NH4ClSigma213330ammonium chloride
Non essential amino acidGibco11140-050
OVA (257-264)GenscriptRP10611ovalbumin
PIPESSigmaP6757
SucroseMerck1.07687.1000
Tissue-Tek OCTSakura4583

参考文献

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  16. Hogquist, K. A., et al. T cell receptor antagonist peptides induce positive selection. Cell. 76 (1), 17-27 (1994).
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