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).

Alle Versuche mit Mäusen sollte in Übereinstimmung mit den Richtlinien der jeweiligen institutionellen Tierpflege und Verwendung Ausschuss durchgeführt werden. 1. Allgemeine Kommentare zu Maus Experimente <ol><li> Halten Sie die Maus unter pathogenfreien Bedingungen und ihnen Zugang zu Futter und Wasser ad libitum. HINWEIS: Es ist wichtig, Alter und Geschlecht abgestimmte Mäuse in Versuchsgruppen zu verwenden, da immunologische Muster können mit Alter und Geschlecht variieren. </li></ol> 2. Herstellung und Aktivierung von OVA-spezifischen CD8 + T-Zellen (OT-I) <ol><li> Führen Stimulation der OT-I T-Zellen, wie unter 5 Tage vor der Vorbereitung Hirnschnitten beschrieben. HINWEIS: 5 × 10 5 aktiviert Effektor CD8 + OT-I T-Zellen (CD8 + T-Zellen von transgenen Mäusen, die Anerkennung nur die OVA 257-264-Peptid im Kontext der MHC-I-Moleküle) pro Schicht benötigt Die Konzentration 5 x 10 5 wurde Experiment gewählteVerbündeter als optimal ein, um eine gute Zell-Zell-Interaktion einmal bevorzugen, dass die Zellen beginnen, in die Scheibe der Migration und, zur gleichen Zeit, ist die Menge der Zellen nicht zu hoch ist, um eine Überreaktion ermöglicht Einzelzellzählung 8,18 induzieren. </li><li> Bereiten Sie das Medium zur Kultivierung OT-I T-Zellen. Supplement 500 ml DMEM mit 5% fötalem Kälberserum (FCS), 10 mM HEPES, 2 mM L-Glutamin, 50 uM 2-Mercaptoethanol, 1% nicht-essentielle Aminosäuren und 25 &amp; mgr; g / ml Gentamicin. Lagern Sie das Medium bei 4 ° C bis zur Verwendung. </li><li> Entfernen Sie die Milz von OT-I transgenen Mäusen nach Referenz 16. Übertragen Sie sie in die vorbereitete Medium. </li><li> Generieren Einzelzellsuspension durch Maischen Milzen durch ein 70 um-Sieb und Zentrifugen Suspension bei 300 xg für 5 min, 4 ° C. </li><li> Inkubieren Zellsuspension mit 5 ml Ammoniumchlorid-Kalium (ACK) Puffer (150 mM NH 4 Cl, 10 mM KHCO 3, 0,1 mM EDTA, pH 7,3) für 5 min, um rote Blutkörperchen zu lysieren. </li><li> Stoppen incubatiauf 10 ml Medium und zentrifugiert erneut wie oben beschrieben. </li><li> Verdünnte Zellsuspension in mittleren und berechnen Zahlen. Platten Zellen in einer Dichte von 3 x 10 7 Zellen / Vertiefung in eine Platte mit 12 Vertiefungen und prime sie durch Inkubation mit OVA 257-264 (SIINFEKL; 1 nM) und Interleukin 2 (IL-2) (500 IU / ml) 5 Tage. </li><li> Nach 4 Tagen hinzuzufügen IL-2 in einer Konzentration von 500 IU / ml wieder. </li><li> Nach Stimulation reinigen OT-I-T-Zellen aus der Zellsuspension durch die Verwendung eines negativen Selektion basierend Maus CD8 + T-Zellisolations-Kit den Anleitungen des Herstellers. Die Reinigung wird auf die Verringerung von allen nicht-CD8 + -Zellen mit einem Cocktail von Antikörpern gegen CD4, CD11b, CD11c, CD19, CD45R (B220), CD49b (DX5), CD105, MHC Klasse II, Ter-119 und TCRγ basierend / δ welche dann durch Verwendung von Magnetspalten zur Elution der gereinigten CD8 + T-Zellen verworfen werden. HINWEIS: Kritische Schritte in diesem procedure werden von den Herstellern angegeben: Es ist wichtig, daß die Reaktion beinhaltet nur einzelne Zellen, weil die Anwesenheit von Klumpen könnten die Spalte während des Elutionsschritts zu blockieren; Zählen der Zellen vor der Zugabe des Byotin Cocktail ist wichtig für den Grad der Reinheit der Probe, und die Prozedur sollte auf Eis durchgeführt, um unspezifische Antikörperbindungen minimieren. </li></ol> 3. Vorbereitung der akuten Hirnschnitten und Co-Kultur mit OT-I T-Zellen <ol><li> Vor der Herstellung des akuten Hirnschnitten 19, bereiten placedine eiskalter physiologischer Kochsalzlösung mit 200 mM Saccharose, 20 mM PIPES, 2,5 mM KCl, 1,25 mM NaH 2 PO 4, 10 mM MgSO 4, 0,5 CaCl 2 und 10 mM Dextrose und künstlicher Zerebrospinalflüssigkeit (ACSF) durch Verwendung von 125 mM NaCl, 2,5 KCl, 1,25 NaH 2 PO 4, 24 mM NaHCO 3, 2 mM MgSO 4, 2 mM CaCl 2, 10 mM Dextrose. Der pH-Wert jeder Lösung auf 7,35 durch using NaOH. Vor Einstellung des pH der ACSF müssen Lösung mit einer Mischung aus 95% O 2 und 5% CO 2 gesprudelt werden. </li><li> Vorkühlung des Lösungen. </li><li> Anesthetize 8 - 10 Wochen alten Mäusen (zB C57Bl / 6 als Kontrolle oder transgenen ODC-OVA Mäusen 17 mit der Inhalation mit 5,0% Isofluran, 5,0% Halothan oder injizieren 100 mg / kg Ketamin und 10 mg / kg Körpergewicht Xylazin über ip Wait für Anästhesie und verwenden eine Zehe Prise, um das Niveau der Anästhesie zu bewerten und zu enthaupten sie sofort. Euthanize die Mäuse nach institutionellen Tierpflege und verwenden Ausschuss Kriterien für Euthanasie. </li><li> Setzen Maus in Bauchlage. Desinfizieren und schneiden Kopfhaut sagittal. Öffnen Sie die Schädelknochen sagittal, entfernen Sie das Gehirn schnell mit Hilfe einer Schaufel und befestigen Sie es auf der Platte eines Vibratom mit Hilfe von Leim. </li><li> Füllen Sie den Teller mit der vorbereiteten placedine eiskalter physiologischer Kochsalzlösung. </li><li> Cut 300 um koronalen Scheiben mit dem Vibratom. Hinweis: Diese Vorgehensweise ist weißn, um lebensfähige intakte Gewebeproben für die funktionelle zelluläre Studien innerhalb eines Zeitintervalls von mindestens 8 h 19 bis 21 entsprechende ergeben. In der Tat, nach dieser Zeit, schon nach 6 Stunden ab der Zubereitung hat ergeben, verminderter Qualität, nämlich Veränderungen in einfachen und funktionalen Eigenschaften wie Aktionspotential Erzeugung und Ausbreitung. </li><li> Nach dem Schneiden, übertragen eine Scheibe sofort in jede Vertiefung einer 12-Well-Platte mit ACSF gefüllt. </li><li> Fügen Sie sorgfältig 5 × 10 5 OT-I T-Zellen pro Scheibe. </li><li> Scheiben Inkubieren für bis zu 8 h in einem Brutschrank (37 ° C, 5% CO 2). </li><li> Nach der Inkubationszeit, Ernte Scheiben schneiden und mit Hilfe OCT-Verbindung Tissue-Tek betten sie und frieren sie in flüssigem Stickstoff. Im Kühlschrank bei -20 ° C für die weitere histologische Untersuchungen zu Beispiel auszuwerten neuronale Struktur (zB unter Verwendung von Anti-MAPII oder Anti-Synaptophysin-Antikörper) oder Apoptose (zB mit Anti-Caspase-3-Antikörpern und Anti-Anti NeuNStellen) nach Standardverfahren. </li></ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subpial+demyelination+in+the+cerebral+cortex+of+multiple+sclerosis+patients.">Subpial demyelination in the cerebral cortex of multiple sclerosis patients.</a> J Neuropathol Exp Neurol. 62 (7), 723-732 (2003).</li><li>Babbe, H., 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=Clonal+expansions+of+CD8(+)+T+cells+dominate+the+T+cell+infiltrate+in+active+multiple+sclerosis+lesions+as+shown+by+micromanipulation+and+single+cell+polymerase+chain+reaction.">Clonal expansions of CD8(+) T cells dominate the T cell infiltrate in active multiple sclerosis lesions as shown by micromanipulation and single cell polymerase chain reaction.</a> J Exp Med. 192 (3), 393-404 (2000).</li><li>Junker, 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=Multiple+sclerosis:+T-cell+receptor+expression+in+distinct+brain+regions.">Multiple sclerosis: T-cell receptor expression in distinct brain regions.</a> Brain. 130 (11), 2789-2799 (2007).</li><li>Skulina, C., 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=Multiple+sclerosis:+brain-infiltrating+CD8++T+cells+persist+as+clonal+expansions+in+the+cerebrospinal+fluid+and+blood.">Multiple sclerosis: brain-infiltrating CD8+ T cells persist as clonal expansions in the cerebrospinal fluid and blood.</a> Proc Natl Acad Sci U S A. 101 (8), 2428-2433 (2004).</li><li>Friese, M. 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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.

Verschiedene Tiermodelle haben in den letzten Jahrzehnten, die pathologischen Merkmale der entzündlichen demyelinisierenden Erkrankungen wie MS-Adresse beschrieben. In-vivo-Maus- und Rattenmodelle sind weit verbreitet, um pathophysiologische Merkmale der Krankheit nachahmen, nämlich Analyse der Folgen der Demyelinisierung und Remyelinisierung Prozesse und der vermischten Episoden von Entzündungen und Neurodegeneration. Trotzdem nur eine ex vivo Ansatz erlaubt es, die genauen zugrunde liegenden Mecha...

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.

Nach einer Inkubation von Gehirnschnitten mit Oligodendrozyten gerichtete CD8 + T-Zellen, Oligodendrozyten und Neuronen Apoptose (2A bzw. 1C). Histologische Anzeichen von Apoptose (zB Caspase-3, Tunel) kann frühestens nach 3 h Inkubation nachgewiesen werden. Inkubationszeit sollte, um eine gute Qualität der Vorbereitung und reproduzierbare Ergebnisse garantieren nicht länger als 8 Stunden sein. Apoptotischen Zellen kann auf der ganzen Scheibe mit Übergewicht in m...

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.

Ein<em&gt; Ex-vivo-</em&gt; Modell eines Oligodendrozyten-gerichtete T-Cell Attack in akuten Hirnschnitten

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

an-ex-vivo-model-an-oligodendrocyte-directed-t-cell-attack-acute

Research

JoVE Journal

Immunology and Infection

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

6.8K Aufrufe.  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.

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

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

Ex-vivo-Imaging von T-Zellen in Murine Lymph Node Slices mit Weitfeld und konfokale Mikroskope

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

Forschung

Immunologie und Infektion

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Induktion der Graft-versus-Host-Krankheit und<em&gt; In Vivo</em&gt; T-Zell-Überwachung mit einem MHC-matched Mausmodell

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&amp;#948;1+CD4+&amp;#947;&amp;#948; T Cells, an Extrathymic &amp;#945;&amp;#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

Isolation und<em&gt; Ex Vivo</em&gt; Kultur des Vδ1<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt; γδ T-Zellen, eine extrathymic αβT-Zell-Vorläufer

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Einrichtung eines Setup-Hochdurchsatz-Screening für kleine Moleküle, die Modulate c-di-GMP-Signalisierung in<em&gt; Pseudomonas aeruginosa</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

Ein<em&gt; In Vitro</em&gt; Modell der Blut-Hirn-Schranke Mit Impedanzspektroskopie: Ein Fokus auf T-Zell-Endothelzellinteraktion

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

Isolation of the Side Population in Myc-induced T-cell Acute Lymphoblastic Leukemia in Zebrafish

Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to efflux the DNA-binding dye Hoechst 33342. This "side population" of cells can be identified using flow cytometric methods after the Hoechst 33342 dye is excited by an ultraviolet (UV) laser. The side population of many cell types contains stem- or progenitor-like cells. However, not all cell types have an identifiable side population. Danio rerio, zebrafish, have a robust in vivo model of T-cell acute lymphoblastic leukemia (T-ALL), but whether these zebrafish T-ALLs have a side population is unknown. The method described here outlines how to isolate the side population cells in zebrafish T-ALL. To begin, the T-ALL in zebrafish is generated via the microinjection of tol2 plasmids into one-cell stage embryos. Once the tumors have grown to a stage at which they expand into more than half of the animal's body, the T-ALL cells can be harvested. The cells are then stained with Hoechst 33342 and examined by flow cytometry for side population cells. This method has broad applications in zebrafish T-ALL research. While there are no known cell surface markers in zebrafish that confirm whether these side population cells are cancer stem cell-like, in vivo functional transplantation assays are possible. Furthermore, single-cell transcriptomics could be applied to identify the genetic features of these side population cells.

Here, we describe a technique to isolate the side population cells from a zebrafish model of myc-induced T-cell acute lymphoblastic leukemia (T-ALL). This side population assay is highly sensitive and is described for zebrafish T-ALL, but it may be applicable to other malignant and non-malignant zebrafish cell types.

Die Isolierung der Seiten Bevölkerung in Myc-induzierte T-Zell akute lymphatische Leukämie in Zebrabärbling

Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

In Vivo Test zum Nachweis von Antigen-spezifischen T-Zell Zytolyse-Funktion mit einer Impfung-Modell

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

An &lt;em&gt;In Vivo&lt;/em&gt; Mouse Model to Measure Na&amp;#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

Eine In Vivo Mausmodell Maßnahme naive CD4-T-Zell-Aktivierung, Proliferation und Th1 Differenzierung induziert durch Knochenmark-abgeleitete Dendritische Zellen

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

Ein Ex-Vivo Huhn Primärkultur Bursitis-Zelle Modell zur Bursitis Infektionskrankheit Virus Pathogenese

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

Multicolor Flow Cytometry-basierte Quantifizierung der Mitochondrien, Lysosomen in T-Zellen

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...