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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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. 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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><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.

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 ...

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Universidad de Cartagena UDEC

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Immunology and Infection

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

6.9K 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

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.

Exzitotoxische Stimulation der Hirn Microslices als<em&gt; In-vitro-</em&gt; Modell der Stroke

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

Forschung

Medizin

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.

Hirnschnitt Biotinylierung: Eine<em&gt; Ex Vivo</em&gt; Ansatz zur Region spezifische Plasma Membrane Protein Handel Erwachsene Neuronen messen

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

Neurowissenschaften

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.

Organotypischen Kulturen zu Oligodendrozyten Dynamik und Myelinisierung Studieren

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.

Organotypischen Hippocampal Slice Kulturen als Modell zur Studie Neuroprotektion und Invasivität der Tumorzellen

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.

Visualisierung und Live Imaging Oligodendrozyt Organellen in organotypischen Gehirnscheiben mit Adeno-assoziierten Virus und konfokale Mikroskopie

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.

Ein alternativer Ansatz zur primären Ereignissen in Neurodegeneration mit Ex Vivo Ratte Gehirnscheiben studieren

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.

Zwei-Photon Imaging der Mikroglia Prozesse Anziehung in Richtung ATP oder Serotonin im akuten Gehirnscheiben

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.

Ein Modell der Epileptogenese in rhinalen Kortex-Hippocampus Organotypischen Schnittkulturen

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 ...

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Exzitotoxische Stimulation der Hirn Microslices als

Hirnschnitt Biotinylierung: Eine