This work was supported by the Interdisciplinary Center for Clinical Research (IZKF) M&#252;nster (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, M&#252;nster (I-BI111316, SB and SGM).

Todos os experimentos com ratos deve ser realizado em conformidade com as orientações do respectivo comitê de cuidados com os animais e uso institucional. 1. Comentários gerais para mouse Experiments <ol><li> Mantenha os ratos em condições livres de patógenos e permitir-lhes o acesso a comida e água ad libitum. NOTA: É importante o uso de ratos da mesma idade e sexo em grupos experimentais porque os padrões imunológicos podem variar de acordo com idade e sexo. </li></ol> 2. As células T específicas de OVA-preparação e activação de células CD8 + (OT-I) <ol><li> Realize a estimulação das células T OT-I, conforme descrito abaixo cinco dias antes de preparar fatias de cérebro. NOTA: 5 x 10 5 efectoras activadas CD8 + OT-I de células T (CD8 + T-células de ratinhos transgénicos que reconhecem apenas o peptídeo OVA 257-264, no contexto das moléculas MHC-I) são necessários por fatia Os concentração de 5 x 10 5 foi escolhido experimentoaliado como uma óptima para favorecer uma interacção célula-célula boa uma vez que as células começam a migrar para o corte e, ao mesmo tempo, a quantidade de células não é demasiado elevada para induzir uma reacção excessiva permitindo que uma única célula contando 8,18. </li><li> Preparar o meio para a cultura de células T OT-I. Suplemento 500 ml de DMEM com 5% de soro fetal de vitelo (FCS), 10 mM de HEPES, 2 mM de L-glutamina, 50 uM de 2-mercaptoetanol, 1% de aminoácidos não essenciais e 25 ug / ml de gentamicina. Armazenar o meio a 4 ° C até à sua utilização. </li><li> Retire o baço de OT-I camundongos transgênicos como por referência 16. Transferi-los para o meio preparado. </li><li> Gerar suspensão de células individuais triturando os baços através de uma peneira de centrifugação e suspensão de 70 mm a 300 xg durante 5 min, 4 ° C. </li><li> Incubar suspensão de células com 5 ml de cloreto de amónio-potássio (ACK) de tampão (150 mM de NH4Cl, 10 mM de KHCO3, 0,1 mM de EDTA, pH 7,3) durante 5 minutos para lisar as células vermelhas do sangue. </li><li> Pare incubaticom 10 ml de meio e centrifuga-se novamente conforme descrito acima. </li><li> Diluir suspensão de células em médio e calcular números. As células em placas a uma densidade de 3 x 10 7 células / poço numa placa de 12 poços e eles privilegiada por incubação com OVA 257-264 (SIINFEKL; 1 nM) e a interleucina 2 (IL-2) (500 UI / ml) durante 5 dias. </li><li> Após 4 dias, adicionar IL-2 a uma concentração de 500 UI / ml, novamente. </li><li> Após a estimulação, purificar as células T OT-I a partir de suspensão de células usando um mouse negativo seleção baseada kit de isolamento de células T CD8 +, seguindo as instruções do fabricante. A purificação é baseado na depleção de todas as células não-CD8 +, utilizando um cocktail de anticorpos contra CD4, CD11b, CD11c, CD19, CD45R (B220), CD49b (DX5), CD105, MHC de Classe II, Ter-119, e TCRγ / δ que são então descartados utilizando colunas magnéticas para a eluição das células T CD8 + purificadas. NOTA: As etapas críticas neste procedure são indicados pelos fabricantes: é importante que a reacção envolve apenas as células individuais, pois a presença de aglomerados pode bloquear a coluna durante o passo de eluição; contagem da célula antes de adicionar o cocktail Byotin é importante para o grau de pureza da amostra e o procedimento deve ser realizado em gelo para minimizar vinculações anticorpos inespecíficos. </li></ol> 3. Preparação de fatias do cérebro do aguda e Co-cultura com células T OT-I <ol><li> Antes da preparação de fatias cerebrais agudas 19, preparar uma solução fisiológica gelada placedine salina utilizando 200 mM de sacarose, PIPES 20 mM, KCl 2,5 mM, 1,25 mM NaH 2 PO 4, 10 mM de MgSO4, dextrose 0,5 CaCl 2 e 10 mM e fluido cerebrospinal artificial (ACSF), utilizando NaCl a 125 mM, KCl 2,5, 1,25 NaH 2 PO 4, mM de NaHCO 3 24, 2 mM MgSO4, 2 mM de CaCl2, 10 mM de dextrose. Ajustar o pH de cada solução a 7,35 por using de NaOH. Antes de ajustar o pH da ACSF, solução deve ser borbulhado com uma mistura de 95% de O2 e 5% de CO 2. </li><li> Pré-resfriar as soluções. </li><li> Anestesiar 8 - camundongo de 10 semanas de idade (por exemplo, C57BL / 6 como controlo ou ratinhos transgénicos ODC OVA-17 com a inalação usando 5,0% de isoflurano, 5,0% de halotano ou injectar 100 mg / kg de cetamina e 10 mg / kg de peso corporal de xilazina via ip Espera para anestesia e usar uma pitada de igual para avaliar o nível de anestesia e decapitá-los de uma só vez. Euthanize os ratos como por cuidados com os animais institucional e utilizar critérios da comissão para a eutanásia. </li><li> Coloque o mouse em posição ventral. Desinfetar e corte sagital couro cabeludo. Abra o osso craniano sagitalmente, retire o cérebro rapidamente com a ajuda de uma colher e corrigi-lo sobre a placa de um vibratome usando cola. </li><li> Encher o prato com a solução salina preparada placedine gelada fisiológico. </li><li> Corte 300 mm cortes coronais com o vibratome. NOTA: Este procedimento é sabern, para se obter amostras de tecido intactas viáveis ​​adequados para estudos celulares funcionais dentro de um intervalo de tempo de pelo menos 8 h 19-21. De fato, após este período de tempo, já começando após 6 horas, a preparação mostra qualidade reduzida, ou seja, mudanças nas propriedades básicas e funcionais, como ação potencial de geração e propagação. </li><li> Após a secção, transferir uma fatia imediatamente em cada poço de uma placa de 12 poços preenchidos com ACSF. </li><li> Adicione cuidadosamente 5 x 10 5 células OT-I T por fatia. </li><li> Incubar fatias de até 8 horas, numa incubadora (37 ° C, 5% CO 2). </li><li> Após o período de incubação, as fatias de colheita e de incorporá-los usando outubro composto tecido-tek e congelá-los em nitrogênio líquido. Armazená-las a -20 ° C durante mais estudos histológicos para, por exemplo, avaliar a estrutura neuronal (por exemplo, utilizando anticorpo anti-MAPII ou anticorpos anti-sinaptofisina) ou apoptose (por exemplo, utilizando caspase 3 anti-anti-anticorpos e de anti-NeuNorganismos) de acordo com procedimentos padrão. </li></ol>

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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pathogenic+role+for+myelin-specific+CD8(+)+T+cells+in+a+model+for+multiple+sclerosis.">A pathogenic role for myelin-specific CD8(+) T cells in a model for multiple sclerosis.</a> J Exp Med. 194 (5), 669-676 (2001).</li><li>McPherson, S. W., Heuss, N. D., Roehrich, H., Gregerson, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bystander+killing+of+neurons+by+cytotoxic+T+cells+specific+for+a+glial+antigen.">Bystander killing of neurons by cytotoxic T cells specific for a glial antigen.</a> Glia. 53 (5), 457-466 (2006).</li><li>Hogquist, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+receptor+antagonist+peptides+induce+positive+selection.">T cell receptor antagonist peptides induce positive selection.</a> Cell. 76 (1), 17-27 (1994).</li><li>Cao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+experimental+autoimmune+encephalomyelitis+in+transgenic+mice+expressing+ovalbumin+in+oligodendrocytes.">Induction of experimental autoimmune encephalomyelitis in transgenic mice expressing ovalbumin in oligodendrocytes.</a> Eur J Immunol. 36 (1), 207-215 (2006).</li><li>G&#246;bel, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4(+)+CD25(+)+FoxP3(+)+regulatory+T+cells+suppress+cytotoxicity+of+CD8(+)+effector+T+cells:+implications+for+their+capacity+to+limit+inflammatory+central+nervous+system+damage+at+the+parenchymal+level.">CD4(+) CD25(+) FoxP3(+) regulatory T cells suppress cytotoxicity of CD8(+) effector T cells: implications for their capacity to limit inflammatory central nervous system damage at the parenchymal level.</a> J Neuroinflammation. 9, 41 (2012).</li><li>Edwards, F. A., Konnerth, A., Sakmann, B., Takahashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+thin+slice+preparation+for+patch+clamp+recordings+from+neurones+of+the+mammalian+central+nervous+system.">A thin slice preparation for patch clamp recordings from neurones of the mammalian central nervous system.</a> Pflugers Arch. 414 (5), 600-612 (1989).</li><li>Meuth, S. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+TWIK-related+acid-sensitive+K++channel+1+(TASK1)+and+TASK3+channels+to+the+control+of+activity+modes+in+thalamocortical+neurons.">Contribution of TWIK-related acid-sensitive K+ channel 1 (TASK1) and TASK3 channels to the control of activity modes in thalamocortical neurons.</a> J Neurosci. 23 (16), 6460-6469 (2003).</li><li>Misgeld, U., Frotscher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dependence+of+the+viability+of+neurons+in+hippocampal+slices+on+oxygen+supply.">Dependence of the viability of neurons in hippocampal slices on oxygen supply.</a> Brain Res Bull. 8 (1), 95-100 (1982).</li><li>Ling, C., Verbny, Y. I., Banks, M. 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The authors declare that they have no competing financial interests.

Diferentes modelos animais foram descritos nas últimas décadas para abordar as características patológicas de doenças desmielinizantes inflamatórias como MS. Modelos in vivo em ratinhos e ratos são amplamente utilizados para imitar aspectos fisiopatológicos da doença, ou seja, a análise das consequências de processos de desmielinização e remielinização e de episódios de permeio de inflamação e neurodegeneração. No entanto, só uma abordagem ex vivo permite dissecar os mecanismos exa...

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 &#8216;&#8216;collateral&#8217;&#8217; 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 &#8216;&#8216;collateral&#8217;&#8217; 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.

<table border="0" cellpadding="0" cellspacing="0" width="786">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:187px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:264px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">12-Well-plate</td>
			<td style="width:187px;">Corning</td>
			<td style="width:119px;">3513</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">2-Mercaptoethanol</td>
			<td style="width:187px;">Gibco</td>
			<td style="width:119px;">31350-010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">2-Methylbutan</td>
			<td style="width:187px;">Roth</td>
			<td style="width:119px;">3927.1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">70 &#181;m strainer</td>
			<td style="width:187px;">Falcon</td>
			<td style="width:119px;">352350</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">CaCl2</td>
			<td style="width:187px;">Merck</td>
			<td style="width:119px;">1.02382.0500</td>
			<td>calcium chloride</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">CD8+-isolation kit</td>
			<td style="width:187px;">Miltenyi Biotech</td>
			<td style="width:119px;">130-090-859</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">D(+)-glukose</td>
			<td style="width:187px;">Merck</td>
			<td style="width:119px;">1.08337.1000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">DMEM</td>
			<td style="width:187px;">Gibco</td>
			<td style="width:119px;">31966-021</td>
			<td>warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">EDTA</td>
			<td style="width:187px;">Sigma</td>
			<td style="width:119px;">E5134</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">FCS</td>
			<td style="width:187px;">PAA Laboratories</td>
			<td style="width:119px;">A15-151</td>
			<td>fetal calve serum</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">gentamicin</td>
			<td style="width:187px;">Gibco</td>
			<td style="width:119px;">15750-060</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">HEPES 1M</td>
			<td style="width:187px;">Gibco</td>
			<td style="width:119px;">15630-050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">IL-2</td>
			<td style="width:187px;">Peprotech</td>
			<td style="width:119px;">212-12</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">Isofluran</td>
			<td style="width:187px;">Abbott</td>
			<td style="width:119px;">05260-05</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">KCl</td>
			<td style="width:187px;">Merck</td>
			<td style="width:119px;">1.04933.0500</td>
			<td>potassium chloride</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">KHCO3</td>
			<td style="width:187px;">Sigma</td>
			<td style="width:119px;">P9144</td>
			<td>potassium hydrogen carbonate</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">L-Glutamine</td>
			<td style="width:187px;">Gibco</td>
			<td style="width:119px;">35050-038</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">MgSO4</td>
			<td style="width:187px;">Merck</td>
			<td style="width:119px;">1.05886.0500</td>
			<td>magnesium sulfate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">NaCl</td>
			<td style="width:187px;">Sigma</td>
			<td style="width:119px;">31434</td>
			<td>sodium chloride</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">NaH2PO4 * H2O</td>
			<td style="width:187px;">Merck</td>
			<td style="width:119px;">1.06346.0500</td>
			<td>sodium hydrogen phosphate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">NaHCO3</td>
			<td style="width:187px;">Merck</td>
			<td style="width:119px;">1.06329.0500</td>
			<td>sodium hydrogen carbonate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">NaOH</td>
			<td style="width:187px;">Merck</td>
			<td style="width:119px;">1.09137.1000</td>
			<td>sodium hydroxide</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">NH4Cl</td>
			<td style="width:187px;">Sigma</td>
			<td style="width:119px;">213330</td>
			<td>ammonium chloride</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Non Essential Amino Acid</td>
			<td style="width:187px;">Gibco</td>
			<td style="width:119px;">11140-050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">OVA (257-264)</td>
			<td style="width:187px;">Genscript</td>
			<td style="width:119px;">RP10611</td>
			<td>ovalbumin</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">PIPES</td>
			<td style="width:187px;">Sigma</td>
			<td style="width:119px;">P6757</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">Sukrose</td>
			<td style="width:187px;">Merck</td>
			<td style="width:119px;">1.07687.1000</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">Tissue-Tek OCT</td>
			<td style="width:187px;">Sakura</td>
			<td style="width:119px;">4583</td>
			<td></td>
		</tr>
	</tbody>
</table>

an ex vivo model of an oligodendrocyte-directed t-cell attack in acute brain slices

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 &#8216;&#8216;collateral&#8217;&#8217; 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.

Após a incubação de fatias de cérebro com as células T CD8 dirigida-oligodendrócitos +, oligodendrócitos, bem como neurónios, sofrem apoptose (Figuras 2A e 1C, respectivamente). Sinais histológicos de apoptose (por exemplo, caspase-3, Tunel) pode ser mais antigo detectado após 3 horas de incubação. O período de incubação não deve ser maior do que 8 horas, a fim de garantir uma boa qualidade da preparação e reprodutibilidade dos resultados. Células em apoptose pode ...

Watch this Scientific Journal Video about An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices at JoVE.com

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

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.

Um<em&gt; Ex vivo</em&gt; Modelo de um Ataque de T-Cell dirigiu-Oligodendrocyte em Agudos fatias de cérebro

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

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Research

JoVE Journal

Immunology and Infection

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

6.8K visualizações.  University of Münster. 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.