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).

Tutti gli esperimenti che utilizzano topi devono essere effettuate in conformità con le linee guida del rispettivo cura degli animali e l&#39;uso del comitato istituzionale. 1. Osservazioni generali per esperimenti del mouse <ol><li> Tenere i topi in condizioni esenti da organismi patogeni e consentire loro l&#39;accesso a cibo e acqua ad libitum. NOTA: E &#39;importante usare i topi per età e sesso nei gruppi trattati perché i modelli immunologiche possono variare a seconda dell&#39;età e del sesso. </li></ol> 2. Preparazione e attivazione di OVA-specifiche cellule T CD8 + (OT-I) <ol><li> Eseguire la stimolazione di cellule OT-I T come descritto di seguito 5 giorni prima della preparazione fettine di cervello. NOTA: 5 x 10 5 effettrici attivato CD8 + OT-I cellule T (CD8 + T-cellule di topi transgenici che riconoscono solo l&#39;OVA 257-264 peptide nel contesto di molecole MHC-I) sono necessari per fetta della concentrazione 5 x 10 5 è stato scelto esperimentoalleati come quella ottimale per favorire una buona interazione cellula-cellula, una volta che le cellule iniziano a migrare nella fetta e, allo stesso tempo, la quantità di cellule non è troppo elevata per indurre una reazione eccessiva permettendo conteggio singola cella 8,18. </li><li> Preparare il supporto per la coltura di cellule T OT-I. Supplemento 500 ml DMEM con siero fetale bovino al 5% (FCS), 10 mM HEPES, 2 mM L-glutammina, 50 mM 2-mercaptoetanolo, 1% aminoacidi non essenziali e 25 ug / ml di gentamicina. Conservare il mezzo a 4 ° C fino al momento dell&#39;uso. </li><li> Rimuovere la milza di OT-I topi transgenici come da riferimento 16. Trasferirle nel mezzo preparato. </li><li> Generare sospensione singola cella da schiacciare milza attraverso un colino e centrifugare la sospensione di 70 micron a 300 xg per 5 minuti, 4 ° C. </li><li> Incubare sospensione cellulare con 5 ml di ammonio-cloruro di potassio (ACK) tampone (150 mM NH 4 Cl, 10 mM KHCO 3, 0.1 mM EDTA, pH 7,3) per 5 minuti per lisare i globuli rossi. </li><li> Smettere incubaticon il supporto 10 ml e centrifugare nuovamente come sopra descritto. </li><li> Diluire sospensione cellulare in media e calcolare i numeri. Cellule piastra a una densità di 3 x 10 7 cellule / pozzetto in una piastra 12 pozzetti e prime loro da incubazione con OVA 257-264 (SIINFEKL; 1 nM) e interleuchina 2 (IL-2) (500 UI / ml) per 5 giorni. </li><li> Dopo 4 giorni, aggiungere IL-2 ad una concentrazione di 500 UI di nuovo / ml. </li><li> Successivamente alla stimolazione, purificare OT-I linfociti T di sospensione cellulare utilizzando un negativo del mouse basata selezione kit di isolamento delle cellule T CD8 + seguendo le istruzioni del produttore. La purificazione si basa sulla deplezione di tutti CD8 + cellule non utilizzando un cocktail di anticorpi contro CD4, CD11b, CD11c, CD19, CD45R (B220), CD49b (DX5), CD105, MHC di classe II, Ter-119, e TCRγ / δ che vengono poi scartati utilizzando colonne magnetiche per l&#39;eluizione delle cellule T CD8 + purificate. NOTA: passaggi critici in questo Operazire sono indicati dalla fabbrica: è importante che la reazione coinvolge solo singole cellule perché la presenza di grumi potrebbe bloccare la colonna durante la fase di eluizione; contando della cella prima di aggiungere il cocktail Byotin è importante per il grado di purezza del campione e la procedura deve essere eseguita su ghiaccio per minimizzare binding anticorpi aspecifici. </li></ol> 3. Preparazione di acuta Fette cervello e co-coltura con cellule T OT-I <ol><li> Prima della preparazione di cerebrale acuto seziona 19, preparare la soluzione ghiacciata placedine fisiologica salina con il saccarosio 200 mM, 20 TUBI mM, 2,5 mM KCl, 1,25 destrosio 0,5 CaCl 2 e 10 mM mM NaH 2 PO 4, 10 mM MgSO 4, e liquido cerebrospinale artificiale (ACSF) utilizzando 125 mM NaCl, KCl 2,5, 1,25 NaH 2 PO 4, NaHCO mM 24 3, 2 mM MgSO 4, 2 mM CaCl 2, 10 mM destrosio. Regolare il pH di ogni soluzione a 7,35 per using NaOH. Prima di regolare il pH del ACSF, soluzione deve gorgogliare con una miscela di 95% O 2 e 5% CO 2. </li><li> Pre-raffreddare le soluzioni. </li><li> Anestetizzare 8 - 10 settimane di età topi (ad esempio, C57BL / 6 come controllo o transgenici ODC-OVA 17 topi con inalazione usando 5,0% isoflurano, 5,0% alotano o iniettare 100 mg / kg ketamina e 10 mg / kg di peso corporeo xilazina via ip Attendere per l&#39;anestesia e utilizzare un pizzico punta a valutare il livello di anestesia e decapitare in una volta. Euthanize topi secondo la cura degli animali istituzionale e utilizzare criteri di commissione per l&#39;eutanasia. </li><li> Mettere il mouse in posizione ventrale. Disinfettare e tagliare il cuoio capelluto sagittale. Aprire l&#39;osso cranico sagitally, rimuovere il cervello rapidamente con l&#39;aiuto di una paletta e fissarla sulla piastra di vibratome utilizzando colla. </li><li> Riempire la piastra con la soluzione salina preparata placedine ghiacciata fisiologico. </li><li> Tagliare 300 micron fette coronali con vibratome. NOTA: Questa procedura è saperen cedere campioni di tessuto intatta vitali adeguati studi cellulari funzionale all&#39;interno di un intervallo di tempo di almeno 8 ore 19-21. Infatti, dopo questo periodo di tempo, a partire già dopo 6 ore, il preparato dimostra qualità ridotta, cioè cambiamenti nelle proprietà di base e funzionali come l&#39;azione potenziale di generazione e propagazione. </li><li> Dopo il sezionamento, trasferire immediatamente una fetta in ciascun pozzetto di una 12-pozzetti pieni di ACSF. </li><li> Aggiungi attentamente 5 x 10 5 cellule OT-I T per fetta. </li><li> Incubare fette fino a 8 ore in un incubatore (37 ° C, 5% CO 2). </li><li> Dopo il periodo di incubazione, fette di raccolta e incorporare mediante ottobre composto Tissue-Tek e congelarli in azoto liquido. Conservarli a -20 ° C per ulteriori studi istologici per esempio, valutare la struttura neuronale (ad esempio, utilizzando anti-MAPII o anti-sinaptofisina) o apoptosi (ad esempio, utilizzando anti-caspasi-3 anticorpi e anti-NeuN controorganismi) secondo le procedure standard. </li></ol>

<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.

Diversi modelli animali sono stati descritti nel corso degli ultimi decenni, per affrontare le caratteristiche patologiche di disturbi demielinizzanti infiammatorie come MS. In vivo modelli di topo e ratto sono ampiamente utilizzati per simulare le caratteristiche fisiopatologiche della malattia, vale a dire, l&#39;analisi delle conseguenze dei processi di demielinizzazione e rimielinizzazione e di episodi Interlacciati di infiammazione e neurodegenerazione. Tuttavia, solo un approccio ex vivo consente...

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.

Dopo incubazione di fette di cervello con CD8 + T-cellule oligodendrociti-diretta, oligodendrociti nonché neuroni apoptosi (Figure 2A e 1C, rispettivamente). Segni istologici di apoptosi (ad esempio, caspasi-3, Tunel) possono essere rilevati prima dopo 3 ore di incubazione. Il periodo di incubazione non deve essere superiore a 8 ore per garantire una buona qualità della preparazione e risultati riproducibili. Le cellule apoptotiche possono essere trovati in tutto il fetta con prepo...

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.

Un<em&gt; Ex vivo</em&gt; Modello di un attacco T-Cell Oligodendrocyte-diretto nel acute cervello fette

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 Views.  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.