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>
	<li>Moll, N. M., 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=Cortical+demyelination+in+PML+and+MS:+Similarities+and+differences.">Cortical demyelination in PML and MS: Similarities and differences.</a> Neurology. 70 (5), 336-343 (2008).</li><li>Peterson, J. W., Bo, L., Mork, S., Chang, A., Trapp, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transected+neurites,+apoptotic+neurons,+and+reduced+inflammation+in+cortical+multiple+sclerosis+lesions.">Transected neurites, apoptotic neurons, and reduced inflammation in cortical multiple sclerosis lesions.</a> Ann Neurol. 50 (3), 389-400 (2001).</li><li>Bo, L., Vedeler, C. A., Nyland, H. I., Trapp, B. D., Mork, S. 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. A., Fugger, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoreactive+CD8++T+cells+in+multiple+sclerosis:+a+new+target+for+therapy.">Autoreactive CD8+ T cells in multiple sclerosis: a new target for therapy.</a> Brain. 128 (8), 1747-1763 (2005).</li><li>Melzer, N., Meuth, S. G., Wiendl, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD8++T+cells+and+neuronal+damage:+direct+and+collateral+mechanisms+of+cytotoxicity+and+impaired+electrical+excitability).">CD8+ T cells and neuronal damage: direct and collateral mechanisms of cytotoxicity and impaired electrical excitability).</a> FASEB J. 23 (11), 3659-3673 (2009).</li><li>Booss, J., Esiri, M. M., Tourtellotte, W. W., Mason, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistological+analysis+of+T+lymphocyte+subsets+in+the+central+nervous+system+in+chronic+progressive+multiple+sclerosis.">Immunohistological analysis of T lymphocyte subsets in the central nervous system in chronic progressive multiple sclerosis.</a> J Neurol Sci. 62 (1-3), 219-232 (1983).</li><li>Hauser, S. L., 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=Immunohistochemical+analysis+of+the+cellular+infiltrate+in+multiple+sclerosis+lesions.">Immunohistochemical analysis of the cellular infiltrate in multiple sclerosis lesions.</a> Ann Neurol. 19 (6), 578-587 (1986).</li><li>Hoftberger, R., 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=Expression+of+major+histocompatibility+complex+class+I+molecules+on+the+different+cell+types+in+multiple+sclerosis+lesions.">Expression of major histocompatibility complex class I molecules on the different cell types in multiple sclerosis lesions.</a> Brain Pathol. 14 (1), 43-50 (2004).</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=Collateral+neuronal+apoptosis+in+CNS+gray+matter+during+an+oligodendrocyte-directed+CD8(+)+T+cell+attack.">Collateral neuronal apoptosis in CNS gray matter during an oligodendrocyte-directed CD8(+) T cell attack.</a> Glia. 58, 469-480 (2010).</li><li>Melzer, N., 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=Excitotoxic+neuronal+cell+death+during+an+oligodendrocyte-directed+CD8++T+cell+attack+in+the+CNS+gray+matter.">Excitotoxic neuronal cell death during an oligodendrocyte-directed CD8+ T cell attack in the CNS gray matter.</a> J Neuroinflammation. 10, 121 (2013).</li><li>Huseby, E. 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. I., Sandor, M., Fabry, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+activation+of+antigen-specific+CD8++T+cells+in+the+presence+of+antigen+in+organotypic+brain+slices.">In situ activation of antigen-specific CD8+ T cells in the presence of antigen in organotypic brain slices.</a> J Immunol. 180, 8393-8399 (2008).</li></ol>

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><tbody><tr><td>12-Well plate</td><td>Corning</td><td>3513</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Gibco</td><td>31350-010</td><td></td></tr><tr><td>2-Methylbutan</td><td>Roth</td><td>3927.1</td><td></td></tr><tr><td>70 &amp;micro;m strainer</td><td>Falcon</td><td>352350</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Merck</td><td>1.02382.0500</td><td>calcium chloride</td></tr><tr><td>CD8&lt;sup&gt;+&lt;/sup&gt;-isolation kit</td><td>Miltenyi Biotech</td><td>130-090-859</td><td></td></tr><tr><td>D(+)-glucose</td><td>Merck</td><td>1.08337.1000</td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>31966-021</td><td>warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>EDTA</td><td>Sigma</td><td>E5134</td><td></td></tr><tr><td>FCS</td><td>PAA Laboratories</td><td>A15-151</td><td>fetal calve serum</td></tr><tr><td>Gentamicin</td><td>Gibco</td><td>15750-060</td><td></td></tr><tr><td>HEPES 1 M</td><td>Gibco</td><td>15630-050</td><td></td></tr><tr><td>IL-2</td><td>Peprotech</td><td>212-12</td><td></td></tr><tr><td>Isofluran</td><td>Abbott</td><td>05260-05</td><td></td></tr><tr><td>KCl</td><td>Merck</td><td>1.04933.0500</td><td>potassium chloride</td></tr><tr><td>KHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>P9144</td><td>potassium hydrogen carbonate</td></tr><tr><td>L-Glutamine</td><td>Gibco</td><td>35050-038</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Merck</td><td>1.05886.0500</td><td>magnesium sulfate</td></tr><tr><td>NaCl</td><td>Sigma</td><td>31434</td><td>sodium chloride</td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; * H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Merck</td><td>1.06346.0500</td><td>sodium hydrogen phosphate</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Merck</td><td>1.06329.0500</td><td>sodium hydrogen carbonate</td></tr><tr><td>NaOH</td><td>Merck</td><td>1.09137.1000</td><td>sodium hydroxide</td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl</td><td>Sigma</td><td>213330</td><td>ammonium chloride</td></tr><tr><td>Non essential amino acid</td><td>Gibco</td><td>11140-050</td><td></td></tr><tr><td>OVA (257-264)</td><td>Genscript</td><td>RP10611</td><td>ovalbumin</td></tr><tr><td>PIPES</td><td>Sigma</td><td>P6757</td><td></td></tr><tr><td>Sucrose</td><td>Merck</td><td>1.07687.1000</td><td></td></tr><tr><td>Tissue-Tek OCT</td><td>Sakura</td><td>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 ...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

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

6.9K 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.

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

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Eccitotossica stimolazione del cervello Microslices come<em&gt; In vitro</em&gt; Modello di Corsa

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Ricerca

Medicina

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

Slice Cervello biotinilazione: An<em&gt; Ex Vivo</em&gt; Approccio alla Misura Regione-specifica proteina Tratta plasma membrana in neuroni adulti

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

Neuroscienze

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

Culture Organotipica Slice per studiare Oligodendrocyte Dynamics e mielinizzazione

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

Colture Hippocampal Fetta organotipiche come modello di studio Neuroprotection e l'invasività delle cellule del tumore

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced tools currently used to study neurons, have only partly been taken on by oligodendrocyte researchers. Cell type-specific staining by viral transduction is a useful approach to study live organelle dynamics. This paper describes a protocol for visualizing oligodendrocyte mitochondria in organotypic brain slices by transduction with adeno-associated virus (AAV) carrying genes for mitochondrial targeted fluorescent proteins under the transcriptional control of the myelin basic protein promoter. It includes the protocol for making organotypic coronal mouse brain slices. A procedure for time-lapse imaging of mitochondria then follows. These methods can be transferred to other organelles and may be particularly useful for studying organelles in the myelin sheath. Finally, we describe a readily available technique for visualization of unstained myelin in living slices by Confocal Reflectance microscopy (CoRe). CoRe requires no extra equipment and can be useful to identify the myelin sheath during live imaging.

Myelinating oligodendrocytes promote rapid action potential propagation and neuronal survival. Described here is a protocol for oligodendrocyte-specific expression of fluorescent proteins in organotypic brain slices with subsequent time-lapse imaging. Further, a simple procedure for visualizing unstained myelin is presented.

Visualizzazione e Live Imaging del Oligodendrocyte organelli in fettine di cervello Organotypic utilizzando Virus Adeno-associati e microscopia confocale

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Un approccio alternativo per studiare eventi primari nella neurodegenerazione utilizzando fettine di cervello di ratto Ex Vivo

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of homeostasis, undergo rapid morphological changes. For example, a laser lesion induces in a few minutes an oriented growth of microglial processes, also called &#34;directional motility&#34;, toward the site of injury. A similar effect can be obtained by delivering locally ATP or serotonin (5-hydroxytryptamine [5-HT]). In this article, we describe a protocol to induce a directional growth of microglial processes toward a local application of ATP or 5-HT in acute brain slices of young and adult mice and to image this attraction over time by multiphoton microscopy. A simple method of quantification with free and open-source image analysis software is proposed. A challenge that still characterizes acute brain slices is the limited time, decreasing with age, during which the cells remain in a physiological state. This protocol, thus, highlights some technical improvements (medium, air-liquid interface chamber, imaging chamber with a double perfusion) aimed at optimizing the viability of microglial cells over several hours, especially in slices from adult mice.

Microglia, the resident immune cells of the brain, respond quickly with morphological changes to modifications of their environment. This protocol describes how to use two-photon microscopy to study the attraction of microglial processes toward serotonin or ATP in acute brain slices of mice.

Imaging del due-fotone di attrazione dei processi Microglial verso ATP o serotonina nelle fette del cervello acuto

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of ...

Ex Vivo Myelination and Remyelination in Cerebellar Slice Cultures as a Quantitative Model for Developmental and Disease-Relevant Manipulations

Studying myelination&#160;in vitro&#160;and&#160;in vivo poses numerous challenges. The differentiation of oligodendrocyte precursor cells (OPCs)&#160;in vitro, although scalable,&#160;does not recapitulate axonal myelination. OPC-neuron cocultures and OPC-fiber cultures allow for the examination of in vitro myelination, but they lack additional cell types that are present in vivo, such as astrocytes and microglia. In vivo&#160;mouse models, however, are less amenable to chemical, environmental, and genetic manipulation and are much more labor intensive. Here, we describe&#160;an&#160;ex vivo mouse cerebellar slice culture (CSC) quantitative system that is useful for: 1) studying developmental myelination, 2) modeling demyelination and remyelination, and 3) conducting translational research. Sagittal sections of the cerebellum and hindbrain are isolated from postnatal day (P) 0&#8211;2 mice, after which they myelinate&#160;ex vivo&#160;for 12 days. During this period, slices can be manipulated in various ways, including the addition of compounds to test for an effect on developmental myelination. In addition, tissue can be fixed for electron microscopy to assess myelin ultrastructure and compaction. To model disease, CSC can be subjected to acute hypoxia to induce hypomyelination. Demyelination in these explants can also be induced by lysolecithin, which allows for the identification of factors that promote remyelination. Aside from chemical and environmental modifications, CSC can be isolated from transgenic mice and are responsive to genetic manipulation induced with Ad-Cre adenoviruses and tamoxifen. Thus, cerebellar slice cultures are a fast, reproducible, and quantifiable model for recapitulating myelination.

Presented is a protocol for an ex vivo quantitative model of demyelination and remyelination using mouse cerebellar slice cultures. This method closely recapitulates an in vivo model with its full complement of CNS cell types in intact tissue, while maintaining the chemical, genetic, and environmental amenability of an in vitro system.

Studying myelination&#160;in vitro&#160;and&#160;in vivo poses numerous challenges. The differentiation of oligodendrocyte precursor cells (OPCs)&#160;in ...

A Model of Epileptogenesis in Rhinal Cortex-Hippocampus Organotypic Slice Cultures

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s neuroprotective and therapeutic potential. Organotypic slices are prepared from explanted tissue and represent a complex multicellular ex vivo environment. They preserve the three-dimensional cytoarchitecture and local environment of brain cells, maintain the neuronal connectivity and the neuron-glia reciprocal interaction. Hippocampal organotypic slices are considered suitable to explore the basic mechanisms of epileptogenesis, but clinical research and animal models of epilepsy have suggested that the rhinal cortex, composed of perirhinal and entorhinal cortices, play a relevant role in seizure generation.
Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Recordings of spontaneous activity from the CA3 area under perfusion with complete growth medium, at physiological temperature and in the absence of pharmacological manipulations, showed that these slices depict evolving epileptic-like events throughout time in culture. Increased cell death, through propidium iodide uptake assay, and gliosis, assessed with fluorescence-coupled immunohistochemistry, was also observed. The experimental approach presented highlights the value of rhinal cortex-hippocampus organotypic slice cultures as a platform to study the dynamics and progression of epileptogenesis and to screen potential therapeutic targets for this brain pathology.

Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Under a gradual and controlled deprivation of serum, these slices depict evolving epileptic-like events and can be considered an ex vivo model of epileptogenesis. This system represents an excellent tool for monitoring the dynamics of spontaneous activity, as well as for assessing the progression of neuroinflammatory features throughout the course of epileptogenesis.

Un modello di epileptogenesi nelle culture delle fette organotipiche della corteccia rinale-ippocampo

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s ...

Confocal Imaging of Mitochondrial Movement within Oligodendrocytes in Organotypic Brain Slice Cultures

<a href='https://appjove.unicartagenaproxy.elogim.com/v/56237/visualization-live-imaging-oligodendrocyte-organelles-organotypic'>Source: Kennedy, L. H., et. al. Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy. J. Vis. Exp. (2017)</a>This video demonstrates the imaging of oligodendrocyte mitochondria in organotypic mouse brain slices. The slice is placed inside a bath chamber with continuous fluid circulation and controlled temperature. After setting up various parameters of the confocal microscope, time-lapse images of mitochondria with red fluorescence are captured, followed by Z-stack images of the oligodendrocyte with green fluorescence.

Source: Kennedy, L. H., et. al. Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and ...

Un

Riepilogo

Esplora Altri Video

Capitoli in questo video

Eccitotossica stimolazione del cervello Microslices come

Slice Cervello biotinilazione: An