Wir danken Denis Flipo für die Bildverarbeitung und Analyse Beratung und David W. Silver in dessen Labor die Gata4p [5 kb]-GFP-Mauslinie generiert wurde. Die Forschung im Labor wird von Pilon CIHR, NSERC, FRQS und FRQNT finanziert.

Ethik-Erklärung Experimente mit Mäusen wurden folgende Kanadischer Rat der Tierpflege-Richtlinien für die Versorgung und Manipulation von Tieren in der medizinischen Forschung durchgeführt. Protokolle, die die Manipulation der Tiere wurden von der Ethikkommission der Universität von Quebec in Montreal genehmigt (Comité institutionnel de Protection des Animaux, Referenznummer 0512-R3-650 bis 0513). 1. Herstellung von Kollagen-Gels Arbeiten Sie in einem steril unter einer Gewebekultur Kapuze. <ol><li> Bereiten Sie komplette 5x DMEM (Eagle modifiziertem essentiellen Medium) einschließlich Standard-Antibiotika. Man löst 3,37 g DMEM-Pulver und 0,925 g NaHCO 3 in 20 ml Wasser. Sterilisiert, indem sie durch ein 0,22 um-Filter. Hinzufügen von 2,5 ml sterilem 100x Penicillin / Streptomycin und 25 ml sterile hitzeinaktiviertem fötalem Rinderserum. Lagern bei 4 ° C </li><li> Auf Eis, mischen Sie 800 ulvon Collagen-I-Lösung (3,77 mg / ml in 0,02 N Essigsäure, filtersterilisiert), 600 ul 5x vollständigen DMEM und 17 &amp;mgr; l 1 N NaOH. Verdünnt mit sterilem Wasser auf ein Endvolumen von 3 ml. Fügen Sie relevanten Wachstumsfaktoren in der Mischung. Wir verwenden in der Regel GDNF bei 10 ng / ml zu ente NCC Migration anzuregen. </li><li> Kaution ca. 480 ul in jede Vertiefung einer einzelnen Zeile in einer 24-Well-Platte. Vermeiden Sie Luftblasen. Die restlichen Zeilen verwendet werden, um die Wirkung anderer Wachstumsfaktoren auf die Zellmigration testen. </li><li> Das Kollagen zu polymerisieren lassen mindestens 1 Stunde in einem sterilen Inkubator bei 37 ° C. </li></ol> 2. Präparation der Tiere 16 <ol><li> Richten Sie Paarungen und überprüfen Vaginalpfropf am nächsten Morgen. Tag E0.5 als Uhr am Tag der vaginale Stecker gefunden wird, isolieren die weibliche und warten Sie 12 Tage bis E12.5. </li><li> Anesthetize schwangere Mäuse mit Isofluran und einschläfern durch CO 2-Inhalation. </li><li> Sprühen Sie die Maus mit 70% Ethanol. Heben Sie die Bauch skin und öffnen Sie den Bauch Hohlraum mit Dissektionsscheren. </li><li> Entfernen der Gebärmutter in eine Glaspetrischale mit eiskaltem PBS (Phosphat-gepufferte Kochsalzlösung) gefüllt. Schneiden Sie die Gebärmutter quer zwischen den einzelnen deciduum Schwellungen, jeden Embryo Implantationsstelle zu isolieren. </li><li> Die Arbeiten an jeder Implantationsstelle separat in einem anderen Glaspetrischale mit eiskaltem PBS gefüllt. Unter einem Binokular, feinen Pinzette verwenden, um die Muskelschicht der Gebärmutter zu entfernen. </li><li> Öffnen Sie die viszerale Dottersack und Amnion, um den Embryo zu offenbaren. Achten Sie beim Abtrennen der Blutgefäße Beitritt der Embryo mit der Plazenta / viszeralen Dottersack, da sie mit den Entwicklungs Darm verflochten. </li><li> Sever Kopf des Embryos am Hals. </li><li> Legen Sie eine geschlossene Zange in der Bauchhöhle des Embryos, knapp über dem dunklen rot gefärbten Leber und lassen Sie die Zange geöffnet von sich selbst (mehr anwenden Druck), um eine Queröffnung in den Bauch Hohlraum zu machen. Ziehen Sie die Zange geöffnet nach unten in Richtungdas hintere Ende des Embryos, um den Bauch vollständig zu öffnen. </li><li> Schnappen Sie sich das Bindegewebe hinter der Leber und aus dem Bauch ziehen den Mut, man aufpassen, nicht zu brechen, den Darm (der Dickdarm bis zum Anus beigefügt). </li><li> Schneiden Sie den Doppelpunkt, um die Eingeweide aus dem Rest des Embryos zu befreien. Der Schnitt kann an jeder Stelle entlang des Dickdarms werden. Reservieren Sie den Schwanzteil für später. </li><li> Herauszuarbeiten, die das Bindegewebe von der Blinddarm, dann den Rest des Darms. Achten Sie darauf, um den Darm gewickelt, während dies zu tun. </li><li> Schneiden Sie die Leber und Magen (auf dem rostralen Ende des Dünndarms), sowie die Urniere und Genitalleisten, wenn einige vorhanden sind. </li><li> Isolieren Sie den Dünndarm. Auch darauf achten, nicht um den Darm zu ersticken. Von nun an kann der rosrrocaudal Ausrichtung des Darmgewebe unter Verwendung des auf der rostralen Ende vorhanden scharfen Krümmung verfolgt werden. Lassen Sie den Dünndarm in PBS bei Raumtemperatur für so kurze Zeit wie möglich vor emBettwäsche (siehe Schritt 3.3). </li><li> Schließlich notieren die Anzahl der Schwanz Somiten für jeden Embryo das Stadium der embryonalen Entwicklung genau zu bestimmen. </li></ol> 3. Schnitte von embryonalen Darm <ol><li> Bevor Sie mit Embryo Dissektionen fortfahren, schmelzen 1,5 g Agarose in 100 ml PBS (Phosphat-gepufferte Kochsalzlösung) und halten bei 50 ° C </li><li> Gießen Agarose in eine Einbettungsform geschmolzen (zB ein geschlossener 2 ml-Mikrozentrifugenröhrchen, die längs geschnitten auf etwa 1/4 der Rohrwand herausgeschnitten). Lassen Sie die Agarose abkühlen auf ca. 42-45 ° C, sollte es nur leicht warm an. </li><li> Betten Sie die embryonalen Darm, kurz bevor die Agarose erstarrt (dies kann mit einer Pinzette Tipps ausgewertet und 36-38 ° C um auftreten). Halten Sie den Darm mit einer Zange durch die gefaltete rostralen Ende, ziehen Sie es sehr langsam durch die Agarose entlang der Länge der Form. Dies hilft, den Darm gerade, während die Agarose-Sets zu halten. Lassen Sie die Tausgeben, sobald sie beginnt, zu wider bewegt. Verfolgen Sie die rostral-kaudale Ausrichtung der Darm. </li><li> Legen Sie die Form in einem Kühlschrank 2-3 Minuten, um zu gewährleisten, die Agarose wurde komplett eingestellt. </li><li> Nehmen Sie die Agarose aus der Form (indem Sie sie aus dem geöffneten Eppendorf-Röhrchen). Mit einer Klinge, nehmen Sie die überschüssige Agarose an beiden Enden, Kürzungen senkrecht auf den Darm. </li><li> Kleben Sie den rostralen Ende des Darms / Agarose blockieren Sie auf die Metallphase eines Mikrotom mit vibrierender Klinge. Schneiden Sie die überschüssige Agarose an den Seiten des Darms / Agarose-Block. </li><li> Montieren Sie den Metall Bühne, auf der vibrierenden Mikrotom Kammer. Wenn nötig, stellen Sie den Winkel der Bühne, so ist der Darm so senkrecht wie möglich (also senkrecht zur Klinge). Decken Sie die Probe mit eiskaltem PBS. Bringen Sie die Mikrotomklinge bis auf wenige Millimeter unter der Oberfläche Puffer. </li><li> Stellen Sie 200 um Vibratoms Querschnitte der Schwanz-die meisten Dünndarm, sicherzustellen, dass die each Agarose-Scheibe enthält eine vollständige Darmabschnitt. </li></ol> 4. Kultur der Darm Explantate <ol><li> Sanft legen das frisch geschnittene Darm / Agarose-Scheiben flach auf den Kollagen Gele mit einer Pinzette, indem eine Scheibe in die Mitte von jedem gut. </li><li> Inkubiere 3 Tage bei 37 ° C in einer feuchten 5% CO 2-Atmosphäre, um die Migration von NCC aus dem Explantat ermöglichen. </li><li> Nehmen Sie die Darm / Agarose-Scheiben aus dem Kollagen-Gel sehr vorsichtig mit einer Pinzette. Achten Sie darauf, um das Gel unter beschädigen. </li><li> Vermeiden direkt während der nachfolgenden Inkubation und Waschschritte, um Berühren der Kollagen-Gel, um sicherzustellen, dass die Zellmigrationsmuster nicht gestört wird. Fix mit 500 ul 4% PFA (Paraformaldehyd) (in PBS) pro Vertiefung 1 Stunde bei Raumtemperatur. </li><li> Ersetzen Sie das Fixiermittel mit 500 ul DAPI (4 &#39;,6-Diamino-2-phenylindol)-Lösung (5 pg / ml in PBS) pro Vertiefung und Inkubation 10 Minuten bei Raumtemperatur. </li><li> Waschen Sie jede Vertiefung 3xmit 500 ul PBS für 5 min. </li><li> Fotografieren Sie die fluoreszierenden Zellen (GFP und DAPI-Kanäle) in der Kollagen-Gel in jedem gut eingebettet. </li></ol> 5. Bildanalyse Wir haben umfangreiche Verwendung von ImageJ 17 zu verarbeiten und zu quantifizieren, die nach Explantatkultur erzeugten Bilder. <ol><li> Beginnen Sie mit der Abbildung einer Mikrometer-Objektträger mit der gleichen Vergrößerung wie die fluoreszierenden Zell Fotografien. Messen Sie die Länge von einem Mikrometer in Pixeln (mit der Straight Line Tool). </li><li> Stellen Sie die Skala (Analyse / Set-Skala; Eingang Anzahl der Pixel / Mikron). </li><li> Für jedes Foto GFP-Fluoreszenz, ändern Sie das Bildformat auf 8-Bit-Graustufen (Image/Type/8-bit). </li><li> Passen Sie Intensität des Signals (Bild / Anpassen / Helligkeit / Kontrast). </li><li> Subtrahieren Sie die Hintergrundgeräusche, falls erforderlich (Prozess / Subtrahieren Hintergrund; stellen Sie den rollenden Ball Radius). </li><li> Stellen Sie eine Schwelle, um Zellen und Zellklumpen (Bild / Anpassen / Threshold), markieren Sie dieeinen Wende n anwenden, um die Klumpen (Process / Binary / Watershed) teilen. </li><li> Analysieren Teilchen Regionen von Interesse (ROI) zu spezifizieren und generieren Zellzahl-Statistik (Analyse / Analysieren Partikel; Set-Größe Mindest, um die verbleibenden Pixel auszuschließen). </li><li> Stellen Messoptionen zu Feret-Durchmesser (Analyse / Set Maße) enthalten. </li><li> Gruppe ROIs und messen als Ganzes der Feret-Durchmesser zu bestimmen, die Angabe der Zellausbreitung (Analysieren / Tools / ROI-Manager / Mehr / OR, dann ROI-Manager / Hinzufügen und ROI-Manager / Measure). </li></ol>

<ol>
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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+signals+regulate+multiple+aspects+of+gastrointestinal+development.">Hedgehog signals regulate multiple aspects of gastrointestinal development.</a> Development. 127 (12), 2763-2772 (2000).</li><li>Natarajan, D., Marcos-Gutierrez, C., Pachnis, V., de Graaf, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+of+signaling+by+receptor+tyrosine+kinase+RET+for+the+directed+migration+of+enteric+nervous+system+progenitor+cells+during+mammalian+embryogenesis.">Requirement of signaling by receptor tyrosine kinase RET for the directed migration of enteric nervous system progenitor cells during mammalian embryogenesis.</a> Development. 129 (22), 5151-5160 (2002).</li><li>Young, H. M., Hearn, C. 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Biol. 293 (1), 203-217 (2006).</li><li>Nagy, A., Gertsenstein, M., Vintersen, K., Behringer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the mouse embryo: a laboratory manual. , 209-250 (2003).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat. Methods. 9, 671-675 (2012).</li><li>Byth, K. 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Die Autoren haben nichts zu offenbaren.

Wir zeigen, wie unser Ex-vivo-Explantatkultur Technik kann verwendet werden, um genau zu quantifizieren ente NCC Migrationspotential in Gegenwart von GDNF werden. Eine solche genaue Quantifizierung wird stark durch Verwendung von 200 um dicke Vibratoms Darmstücke statt große Stücke ungefähre Größe, wie zuvor beschrieben, 11,12,15 erleichtert. Tatsächlich ermöglicht uns das mit einer angemessenen Anzahl von Zellen in einem hoch reproduzierbare Einstellung zu arbeiten. Zu beachten ist, ermögli...

Neuralleistenzellen (NCC) sind eine Übergangszelltyp spezifisch für Wirbeltiere, die während der Embryonalentwicklung viele Derivate bildet. Diese Zellpopulation entsteht am Rand des neuronalen Platte, benachbart zu nicht-neuralen Ektoderm 1. Während Neurulation, Biegen der Neuralplatte NCC Orten entlang der dorsalen Rand der bildenden Neuralrohr. NCC laufen dann eine epitheliale-mesenchymale Transition, Trennung und die Migration weg von dem Neuralrohr. NCC besiedeln verschiedenen embryonalen Strukturen, einschließlich des Verdauungstraktes, wo sie den gesamten enterische Nervensystem (ENS) zu bilden, ein zusammenhängendes Netzwerk von Nervenknoten in der Darmwand eingebettet. Wie kürzlich Bewertung 2,3 haben viele Gene in der Entwicklung dieser komplizierten Struktur beteiligt. Die meisten der ENS wird aus einem kleinen Pool von NCC mit Ursprung aus dem Vagus Neuralrohr (dh rund um die potenziellen Hinterhirn / Rückenmark-Grenze) abgeleitet 4.Diese neuronalen Vorläuferzellen erreichen die Vorderdarm um embryonalen Tag (e) 9,0 bei Mäusen und dann kaudal wandern im Darm Mesenchym bis ca. EUR 15,0 um die ganze embryonalen Darm zu kolonisieren. Eine kleinere Teilmenge von Kolon neuralen Vorläuferzellen wird auch durch sakrale NCC, die den hinteren Darm in die entgegengesetzte Richtung bis zum Blinddarm 4 eindringen vorgesehen. Sowohl Vagus-und sakralen NCC erfordern mehrere migrations, Proliferation-, Überlebens-und Differenzierungsfördernde Hinweise, um vollständige Bildung des ENS zu gewährleisten. In dieser Hinsicht Tiermodellen - vor allem gentechnisch veränderte Mäuse - haben maßgeblich bei der Identifizierung von mehreren wesentlichen extrazellulären Liganden: GDNF (Glia-Zellen-derived neurotrophic factor), Endothelin-3, Neurotrophin-3, BMPs (Bone morphogenetische Proteine), Netrin sowie Sonic und Indian Hedgehog (Shh und Ihh) 10.05. Von diesen ist GDNF Signal durch die Transmembran-Rezeptor-Tyrosin-Kinase RET (während der Transfektion Rearranged) als th erkanntE kritischen Weg für die Anziehung und Führung von NCC nach und in der embryonalen Darm. GDNF ist durch den Darm ausgeschieden und Mesenchym bildet eine raumzeitlich gesteuert rosrrocaudal Gradienten, die direkt chemoattractive zu ente NCC, die ausdrücken, RET ist 11,12. Neben anderen Funktionen steuert der ENS Bewegung im Verdauungstrakt durch seine Wechselwirkung mit der glatten Muskulatur in der Darmwand. Abwesenheit von neuralen Ganglien im Anschlussbereich der Darm Ergebnisse in Hirschsprung-Krankheit: tonische Kontraktion des betroffenen Segments führt zu Verstopfung stromauf Anhäufung von aufgeschlossenen Material und massive Ausdehnung des Darms und Bauch. Hirschsprung-Krankheit tritt in etwa einem von 5.000 Lebendgeburten. Die rostro-kaudale Migrationsmuster von magensaft NCC ist vermutlich die wichtigsten Einflussfaktor auf die Ätiologie der Hirschsprung-Krankheit sein. Der Doppelpunkt, die am weitesten von der Quelle der Migration NCC und letzte Teil Bowel besiedelt zu werden, ist besonders anfällig für Mängel in der ENS-Bildung. In Übereinstimmung mit ihrer entscheidenden Rolle bei magensaftresistenten NCC Migration ist Störung der GDNF / RET Signalisierung der Haupt bekannte genetische Ursache von Morbus Hirschsprung 13. Um besser zu studieren, NCC und ENS Entwicklung, erzielten wir eine transgene Mauslinie - mit Namen Gata4p [5 kb]-GFP 14 -, in dem Migrations NCC mit Green Fluorescent Protein (GFP) markiert. Als nächstes perfektioniert ein ex vivo Zellmigrationsassay, durch andere Gruppen 11,12,15 aus veröffentlichten Arbeiten ausgelegt, die jetzt ermöglicht eine präzise Quantifizierung der ente NCC Migrationspotential in Gegenwart von verschiedenen Wachstumsfaktoren, wie GDNF.

<table border="1">
	<tbody>
		<tr>
			<td>DMEM powder</td>
			<td>Wisent</td>
			<td>219-010-XK</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Bioshop</td>
			<td>SOB999</td>
			<td>Biotechnology grade</td>
		</tr>
		<tr>
			<td>Steriflip vacuum filtration system (0.22 micron)</td>
			<td>EMD Millipore</td>
			<td>SCGP00525</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin-Streptomycin solution, 100x</td>
			<td>Wisent</td>
			<td>450-201-EL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Wisent</td>
			<td>095-150</td>
			<td>High quality grade</td>
		</tr>
		<tr>
			<td>Collagen I</td>
			<td>BD biosciences</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Bioshop</td>
			<td>SHY700</td>
			<td>Diluted from 10 N stock then sterile-filtered</td>
		</tr>
		<tr>
			<td>GDNF</td>
			<td>Cedarlane</td>
			<td>CLCYT305</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 24-well Plate</td>
			<td>BD biosciences</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissors</td>
			<td>Fisher Scientific</td>
			<td>089515</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Petri dish</td>
			<td>VWR</td>
			<td>89000-306</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma</td>
			<td>P5493</td>
			<td>Cell culture grade</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Leica</td>
			<td>M125</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Bioshop</td>
			<td>AGA001</td>
			<td>Biotechnology grade</td>
		</tr>
		<tr>
			<td>Surgical blade</td>
			<td>Feather</td>
			<td>21</td>
			<td></td>
		</tr>
		<tr>
			<td>All Purpose Instant Krazy Glue Pen</td>
			<td>Krazy Glue</td>
			<td>KG824</td>
			<td></td>
		</tr>
		<tr>
			<td>HM 650V Vibrating-Blade Microtome</td>
			<td>Thermo Scientific</td>
			<td>920110</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9564</td>
			<td></td>
		</tr>
	</tbody>
</table>

a quantitative cell migration assay for murine enteric neural progenitors

Neuralleistenzellen (NCC) sind eine vorübergehende und multiZellPopulation, die aus dem dorsalen Neuralrohr stammt und ausgiebig wandert in den Entwicklungswirbeltierembryo. Neben der Bereitstellung von peripheren Neuronen und Glia, NCC erzeugen Melanozyten sowie die meisten der Schädel-Gesichtsskeletts. NCC Migration und Differenzierung wird durch eine Kombination ihrer axialen Ursprung entlang des Neuralrohrs und ihrer Ausrichtung auf regional unterschiedliche extrazelluläre Signale gesteuert. Ein solcher Beitrag der extrazellulären Liganden bei der Bildung des enterischen Nervensystem (ENS), einem komplexen Netzwerk von miteinander verbundenen neuralen Ganglien, die lokal steuert (unter anderem) Darmmuskelbewegung und Darmmotilität besonders deutlich. In einem rostral Schwanz fashion - - über die gesamte Länge des zukünftigen Darm meisten der ENS wird aus einer kleinen Anfangs Pool von NCC, die eine lange Reise, um zu kolonisieren verpflichten abgeleitet. Unter mehreren Signalwege bekanntbeeinflussen ente NCC Kolonisation wird GDNF / RET-Signalisierung als wichtigste anerkannt. Tatsächlich ist zeitlich und räumlich gesteuert Sekretion des RET-Liganden GDNF durch den Darm Mesenchym vor allem für die Attraktivität und Anleitung von RET-exprimierenden ente NCC nach und in der embryonalen Darm verantwortlich. Hier beschreiben wir ein ex vivo Zellmigrationsassay unter Verwendung einer transgenen Mauslinie, die über fluoreszenzmarkierten NCC, die genaue Quantifizierung von ente NCC Migrationspotential in Gegenwart von verschiedenen Wachstumsfaktoren, einschließlich GDNF ermöglicht.

Die folgenden Ergebnisse sind repräsentativ dafür, was mit dem hier beschriebenen (1)-Technik erhalten werden. Die Verwendung von Wachstumsfaktoren (dh GDNF) stimuliert die Migration der GFP-exprimierenden ente NCC aus dem Darm in das Explantat und Kollagen-Gel (Abb. 2). Obwohl einige Zellen kommen aus dem Explantat in Abwesenheit von Wachstumsfaktoren, sind diese meist nicht GFP-markierten und für passive Eintrag. Es ist notwendig, die Scheibe von dem Darm Kollagen-Gel, um ...

Watch this Scientific Journal Video about A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors at JoVE.com

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Wir stellen ein ex vivo-Assay, der die Zellmigration präzise Quantifizierung der ente Neuralleistenzellen Migrationspotential in Gegenwart von verschiedenen Wachstumsfaktoren ermöglicht.

Eine quantitative Assay für Zellmigration Murine Enteric neuralen Vorläuferzellen

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

a-quantitative-cell-migration-assay-for-murine-enteric-neural

Research

JoVE Journal

Neuroscience

9.2K Aufrufe.  UQAM.  Wir stellen ein ex vivo-Assay, der die Zellmigration präzise Quantifizierung der ente Neuralleistenzellen Migrationspotential in Gegenwart von verschiedenen Wachstumsfaktoren ermöglicht. 

Serie: A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or thinned-skull 3 preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX3CR1 GFP/+ mice 4 to visualize microglia with enhanced green fluorescent protein to &le;150 &mu;m beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

Ein Thin-Schädel Fenster-Technik für chronische Zwei-Photonen- In-vivo- Imaging von Murine Mikrogliazellen in Modelle der Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

Forschung

Neurowissenschaften

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

Gründung Embryonale Maus Neural Stem Cell Culture Mit dem Neurosphäre Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

Neural-Colony Forming Zell-Assay: Ein Test zur Bona Fide neuralen Stammzellen aus neuralen Vorläuferzellen Discriminate

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition 1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells 1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components 3. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration 4. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands 5-8. However, not until recently have any chemoattractants of trunk NCCs been identified 9. 
Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. 
Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere10). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.

An approach to analyze the migration of explanted cells (trunk neural crest cells) is described. This method is inexpensive, gentle, and capable of distinguishing chemotaxis from both chemokinesis and other influences on migratory polarity such as those derived from cell-cell interactions within the primary trunk neural crest cell culture.

Analyse der Trunk Neural Crest Cell Migration mit Hilfe eines modifizierten Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Reproduzierbare Maus Ischiasnerv Crush und anschließende Bewertung der Regeneration von Whole mount Muskelanalyse

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

Die Analyse der Neuralleiste Migration und Differenzierung von Cross-Spezies Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

Die Neuroblast Assay: Ein Test für die Erzeugung und Anreicherung von neuronalen Vorläuferzellen aus neuralen Stammzellen differenzieren Progeny mittels Durchflusszytometrie

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

Isolierung und Kultivierung von Zellen der Neuralleiste aus embryonalen murinen Neuralrohr

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

The discovery of neural stem and progenitor cells (collectively termed neural precursor cells) (NPCs) in the adult mammalian brain has led to a body of research aimed at utilizing the multipotent and proliferative properties of these cells for the development of neuroregenerative strategies. A critical step for the success of such strategies is the mobilization of NPCs toward a lesion site following exogenous transplantation or to enhance the response of the endogenous precursors that are found in the periventricular region of the CNS. Accordingly, it is essential to understand the mechanisms that promote, guide, and enhance NPC migration. Our work focuses on the utilization of direct current electric fields (dcEFs) to promote and direct NPC migration - a phenomenon known as galvanotaxis. Endogenous physiological electric fields function as critical cues for cell migration during normal development and wound repair. Pharmacological disruption of the trans-neural tube potential in axolotl embryos causes severe developmental malformations1. In the context of wound healing, the rate of repair of wounded cornea is directly correlated with the magnitude of the epithelial wound potential that arises after injury, as shown by pharmacological enhancement or disruption of this dcEF2-3. We have demonstrated that adult subependymal NPCs undergo rapid and directed cathodal migration in vitro when exposed to an externally applied dcEF. In this protocol we describe our lab's techniques for creating a simple and effective galvanotaxis assay for high-resolution, long-term observation of directed cell body translocation (migration) on a single-cell level. This assay would be suitable for investigating the mechanisms that regulate dcEF transduction into cellular motility through the use of transgenic or knockout mice, short interfering RNA, or specific receptor agonists/antagonists.

In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.

A Galvanotaxis Assay für die Analyse von neuralen Vorläuferzellen Cell Migration Kinetics in einem von außen angelegten Direct Current Electric Field

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

Entwicklung eines raffinierten Protokolls für Trans-skleralen subretinalen Transplantation von menschlichen retinalen Pigment Epithelzellen in Ratte Augen