Name: An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices (Video) | JoVE Journal
Uploaded: 2015-02-05T13:00:00.000+00:00
Duration: 6 min 36 s
Description: Learn an ex vivo model of oligodendrocyte-directed T-cell attack using acute brain slices and CD8+ T-cells. This method enables detailed study of immune-mediated neuronal and oligodendroglial apoptosis.

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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).

All experiments using mice should be performed in accordance with the guidelines of the respective institutional animal care and use committee.
1. General Comments for Mouse Experiments
<ol>
	<li>Keep the mice under pathogen-free conditions and enable them access to food and water ad libitum. 
	NOTE: It is important to use age- and sex-matched mice in experimental groups because immunological patterns can vary with age and gender.</li>
</ol>
2. Preparation and Activation of OVA-specific CD8+ T-cells (OT-I)
<ol>
	<li>Perform stimulation of OT-I T cells as described below 5 days before preparing brain slices. 
	NOTE: 5 x 105 activated effector CD8+ OT-I T-cells (CD8+ T-cells of transgenic mice recognizing only the OVA257-264 peptide in the context of MHC-I molecules) are needed per slice The concentration 5 x 105 was chosen experimentally as optimal one to favor a good cell-cell interaction once that the cells start migrating into the slice and, at the same time, the amount of cells is not too high to induce an overreaction allowing single cell counting8,18.</li>
	<li>Prepare the medium for culturing OT-I T-cells. Supplement 500 ml DMEM with 5% fetal calf serum (FCS), 10 mM HEPES, 2 mM L-glutamine, 50 &#181;M 2-mercaptoethanol, 1% nonessential amino acids and 25 &#181;g/ml gentamicin. Store the medium at 4 &#176;C until use.</li>
	<li>Remove the spleen of OT-I transgenic mice as per reference16. Transfer them into the prepared medium.</li>
	<li>Generate single cell suspension by mashing spleens through a 70 &#181;m strainer and centrifuge suspension at 300 x g for 5 min, 4 &#176;C.</li>
	<li>Incubate cell suspension with 5 ml ammonium-chloride-potassium (ACK) buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.3) for 5 min to lyse red blood cells.</li>
	<li>Stop incubation with 10 ml medium and centrifuge it again as described above.</li>
	<li>Dilute cell suspension in medium and calculate numbers. Plate cells at a density of 3 x 107 cells/well in a 12-well plate and prime them by incubation with OVA257&#8211;264 (SIINFEKL; 1 nM) and interleukin 2 (IL-2) (500 IU/ml) for 5 days.</li>
	<li>After 4 days, add IL-2 at a concentration of 500 IU/ml again.</li>
	<li>Subsequent to stimulation, purify OT-I T-cells from cell suspension by using a negative selection based mouse CD8+ T-cell isolation kit following manufacturer&#8217;s instructions. Purification is based on the depletion of all non-CD8+ cells by using a cocktail of antibodies against CD4, CD11b, CD11c, CD19, CD45R (B220), CD49b (DX5), CD105, MHC Class II, Ter-119, and TCR&#947;/&#948; which are then discarded by using magnetic columns for the elution of the purified CD8+ T-cells. 
	NOTE: Critical steps in this procedure are indicated by the manufactures: it is important that the reaction involves only single cells because the presence of clumps&#160;could block the column during the elution step; counting of the cell before adding the Byotin cocktail is important for the degree of purity of the sample and the procedure should be performed on ice to minimize unspecific antibody bindings.</li>
</ol>
3. Preparation of Acute Brain Slices and Co-culture with OT-I T-cells
<ol>
	<li>Prior to the preparation of acute brain slices19, prepare placedine ice-cold physiological saline solution using 200 mM sucrose, 20 mM PIPES, 2.5 mM KCL, 1.25 mM NaH2PO4, 10 mM MgSO4, 0.5 CaCl2 and 10 mM dextrose and artificial cerebrospinal fluid (ACSF) by using 125 mM NaCl, 2.5 KCl, 1.25 NaH2PO4, 24 mM NaHCO3, 2 mM MgSO4, 2 mM CaCl2, 10 mM dextrose. Adjust pH of each solution to 7.35 by using NaOH. Before adjusting pH of ACSF, solution must be bubbled with a mixture of 95% O2 and 5% CO2.</li>
	<li>Pre-cool the solutions.</li>
	<li>Anesthetize 8 - 10 week old mice (e.g., C57Bl/6 as control or transgenic ODC-OVA mice17 with inhalation using 5.0% isoflurane, 5.0% halothane or inject 100 mg/kg ketamine and 10 mg/kg body weight xylazine via i.p. Wait for anesthesia and use a toe pinch to assess the level of anesthesia and decapitate them at once. Euthanize the mice as per institutional animal care and use committee criteria for euthanasia.</li>
	<li>Put mouse in ventral position. Disinfect and cut scalp sagittally. Open the cranial bone sagitally, remove the brain quickly with the help of a scoop and fix it on the plate of a vibratome by using glue.</li>
	<li>Fill the plate with the prepared placedine ice-cold physiological saline solution.</li>
	<li>Cut 300 &#181;m coronal slices with the vibratome. 
	NOTE: This procedure is known to yield viable intact tissue specimens appropriate for functional cellular studies within a time interval of at least 8 hr19-21. Indeed, after this time period, already starting after 6 hr, the preparation shows reduced quality, namely changes in basic and functional properties like action potential generation and propagation.</li>
	<li>After sectioning, transfer one slice immediately into each well of a 12-well plate filled with ACSF.</li>
	<li>Add carefully 5 x 105 OT-I T cells per slice.</li>
	<li>Incubate slices for up to 8 hr in an incubator (37 &#176;C, 5% CO2).</li>
	<li>After the incubation period, harvest slices and embed them by using OCT compound tissue-tek and freeze them in liquid nitrogen. Store them at -20 &#176;C for further histological studies to e.g., evaluate neuronal structure (e.g., using anti-MAPII or anti-synaptophysin antibodies) or apoptosis (e.g., using anti-Caspase-3 antibodies and anti-NeuN antibodies) according to standard procedures.</li>
</ol>

<ol><li>Moll, N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cortical+demyelination+in+PML+and+MS%3A+Similarities+and+differences%5BTitle%5D)+AND+%22Neurology%22%5BJournal%5D)">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/pubmed?term=((Transected+neurites%2C+apoptotic+neurons%2C+and+reduced+inflammation+in+cortical+multiple+sclerosis+lesions%5BTitle%5D)+AND+%22Ann+Neurol%22%5BJournal%5D)">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/pubmed?term=((Subpial+demyelination+in+the+cerebral+cortex+of+multiple+sclerosis+patients%5BTitle%5D)+AND+%22J+Neuropathol+Exp+Neurol%22%5BJournal%5D)">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/pubmed?term=((Clonal+expansions+of+CD8%28%2B%29+T+cells+dominate+the+T+cell+infiltrate+in+active+multiple+sclerosis+lesions+as+shown+by+micromanipulation+and+single+cell+polymerase+chain+reaction%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">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/pubmed?term=((Multiple+sclerosis%3A+T-cell+receptor+expression+in+distinct+brain+regions%5BTitle%5D)+AND+%22Brain%22%5BJournal%5D)">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/pubmed?term=((Multiple+sclerosis%3A+brain-infiltrating+CD8%2B+T+cells+persist+as+clonal+expansions+in+the+cerebrospinal+fluid+and+blood%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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/pubmed?term=((Autoreactive+CD8%2B+T+cells+in+multiple+sclerosis%3A+a+new+target+for+therapy%5BTitle%5D)+AND+%22Brain%22%5BJournal%5D)">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/pubmed?term=((CD8%2B+T+cells+and+neuronal+damage%3A+direct+and+collateral+mechanisms+of+cytotoxicity+and+impaired+electrical+excitability%29%5BTitle%5D)+AND+%22FASEB+J%22%5BJournal%5D)">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/pubmed?term=((Immunohistological+analysis+of+T+lymphocyte+subsets+in+the+central+nervous+system+in+chronic+progressive+multiple+sclerosis%5BTitle%5D)+AND+%22J+Neurol+Sci%22%5BJournal%5D)">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/pubmed?term=((Immunohistochemical+analysis+of+the+cellular+infiltrate+in+multiple+sclerosis+lesions%5BTitle%5D)+AND+%22Ann+Neurol%22%5BJournal%5D)">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/pubmed?term=((Expression+of+major+histocompatibility+complex+class+I+molecules+on+the+different+cell+types+in+multiple+sclerosis+lesions%5BTitle%5D)+AND+%22Brain+Pathol%22%5BJournal%5D)">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>Göbel, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Collateral+neuronal+apoptosis+in+CNS+gray+matter+during+an+oligodendrocyte-directed+CD8%28%2B%29+T+cell+attack%5BTitle%5D)+AND+%22Glia%22%5BJournal%5D)">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/pubmed?term=((Excitotoxic+neuronal+cell+death+during+an+oligodendrocyte-directed+CD8%2B+T+cell+attack+in+the+CNS+gray+matter%5BTitle%5D)+AND+%22J+Neuroinflammation%22%5BJournal%5D)">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/pubmed?term=((A+pathogenic+role+for+myelin-specific+CD8%28%2B%29+T+cells+in+a+model+for+multiple+sclerosis%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">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/pubmed?term=((Bystander+killing+of+neurons+by+cytotoxic+T+cells+specific+for+a+glial+antigen%5BTitle%5D)+AND+%22Glia%22%5BJournal%5D)">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/pubmed?term=((T+cell+receptor+antagonist+peptides+induce+positive+selection%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((Induction+of+experimental+autoimmune+encephalomyelitis+in+transgenic+mice+expressing+ovalbumin+in+oligodendrocytes%5BTitle%5D)+AND+%22Eur+J+Immunol%22%5BJournal%5D)">Induction of experimental autoimmune encephalomyelitis in transgenic mice expressing ovalbumin in oligodendrocytes.</a> Eur J Immunol. 36 (1), 207-215 (2006).</li>
<li>Göbel, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CD4%28%2B%29+CD25%28%2B%29+FoxP3%28%2B%29+regulatory+T+cells+suppress+cytotoxicity+of+CD8%28%2B%29+effector+T+cells%3A+implications+for+their+capacity+to+limit+inflammatory+central+nervous+system+damage+at+the+parenchymal+level%5BTitle%5D)+AND+%22J+Neuroinflammation%22%5BJournal%5D)">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/pubmed?term=((A+thin+slice+preparation+for+patch+clamp+recordings+from+neurones+of+the+mammalian+central+nervous+system%5BTitle%5D)+AND+%22Pflugers+Arch%22%5BJournal%5D)">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/pubmed?term=((Contribution+of+TWIK-related+acid-sensitive+K%2B+channel+1+%28TASK1%29+and+TASK3+channels+to+the+control+of+activity+modes+in+thalamocortical+neurons%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Dependence+of+the+viability+of+neurons+in+hippocampal+slices+on+oxygen+supply%5BTitle%5D)+AND+%22Brain+Res+Bull%22%5BJournal%5D)">Dependence of the viability of neurons in hippocampal slices on oxygen supply.</a> Brain Res Bull. 8 (1), 95-100 (1982).</li>
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The authors declare that they have no competing financial interests.

Different animal models have been described over the last decades to address the pathological features of inflammatory demyelinating disorders like MS. In vivo mouse and rat models are widely used to mimic pathophysiological features of the disease, namely, analysis of the consequences of demyelination and remyelination processes and of intermingled episodes of inflammation and neurodegeneration. Nevertheless, only an ex vivo approach allows to dissect the exact underlying mechanisms.
<p class="jove...

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

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.

After incubation of brain slices with oligodendrocyte-directed CD8+ T-cells, oligodendrocytes as well as neurons undergo apoptosis (Figures 2A and 1C, respectively). Histological signs of apoptosis (e.g., Caspase-3, Tunel) can be earliest detected after 3 hr of incubation. Incubation period should not be longer than 8 hr in order to guarantee a good quality of the preparation and reproducible results. Apoptotic cells can be found all over the slice with preponderance in myelinated are...

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.

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

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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.

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

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.

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

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.

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

We introduce a protocol for measuring real-time drug and radiation response of breast cancer brain metastatic cells in an organotypic brain slice model. The methods provide a quantitative assay to investigate the therapeutic effects of various treatments on brain metastases from breast cancer in an ex vivo manner within the brain microenvironment interface.

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

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

Learn an ex vivo model of oligodendrocyte-directed T-cell attack using acute brain slices and CD8+ T-cells. This method enables detailed study of immune-mediated neuronal and oligodendroglial apoptosis.

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

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