Name: two-photon imaging of cellular dynamics in the mouse spinal cord
Uploaded: 2015-02-22T16:00:00
Description: Watch this Scientific Journal Video about Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord at JoVE.com

This work was supported in part by National Institutes of Health (NIH) Grants R01 GM-41514 (to M.D.C.), R39 GM-048071 (to I.P.), and R01 NS-074987 (to T.E.L.) and the National Multiple Sclerosis Society (NMSS) Collaborative Center Research Award CA1058-A-8 (to C.M.W., T.E.L. and M.D.C.), NMSS Grant RG4925, NIH Training Grant T32-AI-060573 (to M.L.G.), NMSS Postdoctoral Fellowship FG 1960-A-1 (to J.G.W.), and funding from the George E. Hewitt Foundation for Medical Research (M.P.M.).

NOTA: Etica Dichiarazione: Il protocollo per la gestione degli animali è stato approvato dalla cura e l&#39;uso degli animali Comitato Istituzionale (IACUC) della University of California, Irvine, il protocollo # 2010-2943. 1. Rimozione del midollo spinale <ol><li> Posizionare asciugamani di carta bagnati con ~ 100% di liquido isoflurano, USP di eutanasia da camera e mettere gli asciugamani di carta a secco in cima. Posizionare il mouse nella camera in cima ai tovaglioli di carta a secco ...

<ol>
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In tempo reale 2P imaging tessuto intatto è necessario per indagare cinetica NPC e interazioni seguenti trapianto nel topo demyelinated midollo spinale. L&#39;imaging intravitale 2P è comunemente usato per determinare le dinamiche cellulari sul lato dorsale del midollo spinale nei topi viventi, ed è stato utilizzato per studiare demielinizzazione dorsale in malattia demielinizzante 17-19. Tuttavia, poiché NPC trapiantate migrano alla materia bianca ventrale, che si trova troppo in profondità all&#39;imma...

Modelli murini di demielinizzazione, tra cui l&#39;encefalomielite sperimentale autoimmune (EAE) e l&#39;infezione intracranica con neuroadapted mouse virus dell&#39;epatite (MHV), sono ottimi strumenti per studiare percorsi molecolari e interazioni cellulari associati con la malattia. Essi hanno portato e sostenuto l&#39;efficacia della FDA ha approvato terapie farmacologiche, rivolti principalmente la cessazione di autoimmunità e infiammazione 1. Tuttavia, una volta che la rimielinizzazione endogena ha fallito, le terapie attualmente approvate non riparano efficacemente lesioni demielinizzati nel sistema nervoso centrale. Pertanto, le terapie di riparazione focalizzato in questa fase della malattia sono fondamentali per la riduzione dei sintomi cronici e il miglioramento della qualità della vita. Recentemente, le cellule precursori neurali (NPC) sono venuti alla ribalta come un potenziale rigenerativo modalità terapeutica per aree di infiammazione e demielinizzazione bersaglio. Diversi studi hanno evidenziato la capacità di NPC di indurre endogenonoi rimielinizzazione e partecipare direttamente rimielinizzazione 2-8. Perché NPC sono coinvolti in rimielinizzazione diretta, è fondamentale per capire le loro cinetiche e le interazioni con le cellule endogene successivi al trapianto. Dopo il trapianto, NPC migrano ventralmente ad aree di danno sostanza bianca, poi rostralmente e caudalmente relativi al sito di trapianto 5,9. La cinetica della migrazione differiscono in risposta a stimoli ambientali; NPC trapiantati in un midollo spinale danneggiato non hanno velocità maggiori rispetto NPC trapiantate in un midollo spinale danneggiato 6. Dopo un periodo migratorio, trasferiti NPC proliferano ampiamente, ad un tasso più elevato in un cavo danneggiato rispetto spinale per un midollo spinale intatto 6. Infine, la maggior parte degli NPC differenziarsi in oligodendrociti e avviare 4,6,9 rimielinizzazione diretta. La lesione demyelinated è complesso e può includere una popolazione eterogenea di cellule in diverse fasi di unctivation. Ad esempio, un sclerosi multipla (MS) lesione attiva può includere una frazione significativa di cellule T attivate, microglia e dei macrofagi M1 M1, ma un MS lesione cronica silente può essere costituito principalmente da astrociti reattivi con poche cellule infiammatorie 10-13. A causa della diversità delle cellule effettrici, a due fotoni (2P) per immagini in modelli murini di demielinizzazione è uno strumento estremamente utile per aiutare a capire le interazioni cellulari locali all&#39;interno della lesione. In MS e molti modelli di ricerca MS ampiamente utilizzati, la maggior parte delle lesioni si trovano sul lato ventrale del midollo spinale, una regione inaccessibile per l&#39;imaging intravitale 2P grazie alla profondità della lesione e l&#39;alto contenuto lipidico del midollo spinale. Per aggirare questi problemi e interazioni studio cellula-cellula all&#39;interno lesioni lungo il midollo spinale ventrale abbiamo sviluppato un semplice vivo 2P preparazione di imaging 6 ex. Questo studio fa seguito ad una precedente pubblicazione metodi, che ha mostratola procedura per il trapianto di proteine ​​maggiore fluorescente verde (eGFP) NPC esprimente nel midollo spinale dei topi dopo JHMV ceppo di demielinizzazione MHV-indotta 14. Cinque settimane di età i topi sono infettati con JHMV e trapiantati con eGFP-NPC a livello toracico 9 al giorno 14 dopo l&#39;infezione. Il protocollo presentato qui fornisce la procedura dettagliata su come estrarre il midollo spinale, fare un ex vivo agarosio preparazione, e di immagine trapiantato interazioni eGFP-NPC con proteine ​​maggiore giallo fluorescente (EYFP) assoni esprimente. Mice esprimenti EYFP sotto il promotore Thy1 specifico-neuronale sono stati utilizzati in questa procedura 15. Solo alcuni degli assoni esprimono EYFP, rendendolo utile per l&#39;imaging singoli assoni. Qui vi mostriamo midollo spinale rimossi a 7 giorni dopo il trapianto; tuttavia, midollo spinale possono essere estratti in qualsiasi punto nel tempo dopo il trapianto. Mentre noi mostriamo interazioni di NPC con assoni danneggiati, il nostro protocollo può essere utilizzato in combinazione con marcatori fluorescenti geneticidi altri tipi di cellule per indagare una moltitudine di interazioni cellulari che si verificano durante il mouse midollo spinale.

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			<td height="21" style="height:21px;width:381px;">Name of Reagent/ Equipment</td>
			<td style="width:175px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
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			<td height="25" style="height:25px;width:381px;">Isoflurane, USP</td>
			<td style="width:175px;">Piramal Critical Care, Inc</td>
			<td style="width:136px;">N/A</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Fine scissors</td>
			<td style="width:175px;">Fine Science Tools</td>
			<td style="width:136px;">14060-09</td>
			<td>sharp</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:381px;">scalpel blade #10</td>
			<td style="width:175px;">Fine Science Tools</td>
			<td style="width:136px;">10010-00</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:381px;">scalpel handle</td>
			<td style="width:175px;">Fine Science Tools</td>
			<td style="width:136px;">10003-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Luer rongeurs</td>
			<td style="width:175px;">Fine Science Tools</td>
			<td style="width:136px;">16001-15</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Graefe forceps</td>
			<td style="width:175px;">Fine Science Tools</td>
			<td style="width:136px;">11052-10</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Vannas scissors</td>
			<td style="width:175px;">Fine Science Tools</td>
			<td style="width:136px;">15615-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">scalpel blade #11</td>
			<td style="width:175px;">Fine Science Tools</td>
			<td style="width:136px;">10011-00</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:381px;">RPMI-1640</td>
			<td style="width:175px;">Gibco</td>
			<td style="width:136px;">12-115F</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:381px;">agarose, low gelling temperature</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:136px;">A9414-25G</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Parafilm</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:136px;">13-374-12</td>
			<td></td>
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			<td height="21" style="height:21px;width:381px;">Vetbond (tissue adhesive)</td>
			<td style="width:175px;">3M</td>
			<td style="width:136px;">1469SB</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:381px;">22 mm square cover slip</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:136px;">12-547</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:381px;">25x dipping objective, 1.1 NA</td>
			<td style="width:175px;">Nikon</td>
			<td style="width:136px;">CFI Apo LWD 25XW</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Single inline solution heater</td>
			<td style="width:175px;">Warner Instruments</td>
			<td style="width:136px;">64-0102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">520 nm single-edge dichroic beam splitter</td>
			<td style="width:175px;">Semrock</td>
			<td style="width:136px;">FF520-Di02-25x36</td>
			<td>Brightline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">560 nm single-edge dichroic beam splitter</td>
			<td style="width:175px;">Semrock</td>
			<td style="width:136px;">FF560-FDi01-25x36</td>
			<td>Brightline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">photomultiplier tubes</td>
			<td style="width:175px;">Hamamatsu</td>
			<td style="width:136px;">R928</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">C/L variable-speed tubing pump</td>
			<td style="width:175px;">Masterflex</td>
			<td style="width:136px;">77122-22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">digital thermometer</td>
			<td style="width:175px;">Comar Instruments</td>
			<td style="width:136px;">3501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Chameleon Ultra Ti:Sapphire laser&#160;</td>
			<td style="width:175px;">Coherent</td>
			<td style="width:136px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Slidebook 6 software</td>
			<td style="width:175px;">3i</td>
			<td style="width:136px;">N/A</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Imaris 7.7 software</td>
			<td style="width:175px;">Bitplane</td>
			<td style="width:136px;">N/A</td>
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two-photon imaging of cellular dynamics in the mouse spinal cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

<html> Mentre il protocollo di imaging midollo spinale espiantato può essere utilizzato per visualizzare alcuna fluorescenza all&#39;interno del midollo spinale, i nostri risultati dimostrano rappresentativi interazioni eGFP-NPC con EYFP-assoni. In primo luogo, noi mostriamo la preparazione ventrale del midollo spinale incorporato nella Figura 1A. Avanti, mostriamo la configurazione 2P microscopio e componenti chiave nella Figura 1B. Figura 2 dimostra eGFP e EYFP fluorescenza in una sin...

Watch this Scientific Journal Video about Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord at JoVE.com

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Un nuovo ex vivo preparazione per l&#39;imaging del midollo spinale di topo. Questo protocollo consente a due fotoni imaging interazioni cellulari vivi tutto il midollo spinale.

Due fotoni Imaging di Cellular Dynamics nel midollo spinale del mouse

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Research

JoVE Journal

Neuroscience

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

Imaging in vivo del midollo spinale di topo Utilizzo di due fotoni Microscopia

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

Ricerca

Neuroscienze

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Una procedura di impianto del Camera spinale per longitudinale<em&gt; In Vivo</em&gt; Imaging del cavo del mouse spinale

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Diffusion Imaging in the Rat Cervical Spinal Cord

Diffusion Imaging nel midollo spinale cervicale Rat

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

Simultanea a due fotoni<em&gt; In Vivo</em&gt; Imaging di Synaptic ingressi e degli obiettivi postsinaptici nella corteccia mouse retrosplenial

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Imaging Fibre serotoninergici nel midollo spinale mouse Uso del CHIAREZZA / Tecnica CUBIC

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

Imaging di attività neurale nella corteccia somatosensoriale primaria utilizzando Thy1-topi transgenici GCaMP6s

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

In Vivo Duo-colore metodo di Imaging vascolare dinamica dopo la lesione del midollo spinale contusivi

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

Laminectomia e impianto della finestra del cavo spinale nel mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

In Vivo Due-fotone Imaging dei neuroni corticali in topi neonatali

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Consegna intra-arteriosa di cellule staminali neurali al cervello di ratto e topo: applicazione all'ischemia cerebrale

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...