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.).

OBS: Etik Uttalande: Protokollet för djurhantering godkändes av Institutional Animal Care och användning kommittén (IACUC) vid University of California, Irvine, protokoll # 2010-2943. 1. Borttagning av ryggmärgs <ol><li> Placera pappershanddukar fuktade med ~ 100% flytande isofluran, USP i euthanizing kammaren och placera torra pappershanddukar ovanpå. Placera musen i kammaren på toppen av torra pappershanddukar så musen inte vidrör isofluran och se till kammaren är täckt. Vänt...

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Realtids 2P avbildning av intakt vävnad krävs för att utreda NPC kinetik och interaktioner efter transplantation till demyelinerad musen ryggmärgen. Intravital 2P avbildning används ofta för att bestämma cellulära dynamiken på ryggsidan av ryggmärgen i levande möss, och har använts för att studera rygg demyelinisering i demyeliniserande sjukdom 17-19. Men eftersom transplanterade NPCs migrera till den ventrala vita substansen, som ligger för djupt för att bilden på plats med hjälp av 2P mikro...

Musmodeller av demyelinisering, inklusive experimentell autoimmun encefalomyelit (EAE) och intrakraniell infektion med neuroadapted mus hepatitvirus (MHV), är utmärkta verktyg för att studera molekylära vägar och cellulära interaktioner förknippade med sjukdomen. De har lett till och stött effektivitet FDA godkända läkemedelsbehandlingar, främst inriktade upphörande av autoimmunitet och inflammation 1. Men när endogen remyelinisering har misslyckats, de närvarande godkända behandlingar inte effektivt reparera demyelinerade lesioner i det centrala nervsystemet. Därför reparationsfokuserade terapier i detta skede av sjukdomen är kritiska för lindring av kroniska symptom och förbättring av livskvaliteten. Nyligen har neurala prekursorceller (NPC) kommit i förgrunden som en potentiell regenerativ terapeutisk modalitet att rikta områden av inflammation och demyelinisering. Flera studier har visat på förmågan hos NPCs att inducera endogenooss remyelinisering och delta direkt i remyelinisering 2-8. Eftersom NPCs är involverade i direkt remyelinisering, är det absolut nödvändigt att förstå deras kinetik och interaktioner med endogena celler efter transplantation. Efter transplantation, NPCs migrera ventralt till områden av vit substans skada, sedan rostrally och kaudalt i förhållande till transplantation platsen 5,9. Kinetiken för migrationen skiljer som svar på miljösignaler; NPC transplanteras in i en icke-skadad ryggmärg har större hastigheter än NPC transplanteras till en skadad ryggmärg 6. Efter en vandrande period, överfört NPCs föröka i stor utsträckning, i högre takt i en skadad ryggmärg relativt en intakt ryggmärgen 6. Slutligen, majoriteten av NPC differentierar till oligodendrocyter och initiera direkt remyelinisering 4,6,9. Den demyelinerad lesionen är komplex och kan innehålla en skiftande population av celler i olika skeden av enctivation. Till exempel kan en aktiv multipel skleros (MS) lesion omfattar en betydande population av aktiverade T-celler, M1 mikroglia och M1 makrofager, men en kronisk tyst MS lesionen kan bestå primärt av reaktiva astrocyter med få inflammatoriska celler 10-13. På grund av mångfalden av effektorceller, är två-foton (2P) avbildning i musmodeller av demyelinisering ett mycket användbart verktyg för att förstå lokala cellulära interaktioner inom lesionen. Vid MS och många utsträckning används MS forskningsmodeller, är majoriteten av lesioner som ligger på den ventrala sidan av ryggmärgen, en region oåtkomlig för intravital 2P avbildning på grund av skada djup och den höga fetthalten i ryggmärgen. För att kringgå dessa problem och studie cell-cell interaktioner inom lesioner längs den ventrala ryggmärgen har vi utvecklat en enkel ex vivo 2P avbildning förberedelse 6. Denna studie följer upp en tidigare metoder publikation, vilket visadeförfarandet för omplantering förbättrade grönt fluorescerande protein (EGFP) uttryckande NPC in i ryggmärgen av möss efter JHMV stam av MHV-inducerad demyelinisering 14. Fem veckor gamla möss infekterade med JHMV och transplanteras med eGFP-NPCs på thorax nivå 9 på dag 14 efter infektion. Protokollet presenteras här ger detaljerade steg för hur du extrahera ryggmärgen, gör en ex vivo -ig förberedelser, och bild transplanterade eGFP-NPC interaktioner med förstärkt gula fluorescerande protein (EYFP) uttryckande axoner. Möss som uttrycker EYFP under neuronala specifika Thy1 promotor användes i detta förfarande 15. Endast en del av de axoner uttrycka EYFP, vilket gör den användbar för avbildning enskilda axoner. Här visar vi ryggmärg avlägsnades vid 7 dagar efter transplantation; emellertid kan ryggmärg utvinnas vid någon tidpunkt efter transplantation. Medan vi visar interaktioner av NPCs med skadade axoner, kan våra protokoll användas i kombination med genetiska fluorescerande markörerav andra celltyper för att undersöka en mängd cellulära interaktioner inträffar hela musen ryggmärgen.

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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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		<tr height="25">
			<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>
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			<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>
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			<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>
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			<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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			<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>
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			<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>
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		<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>
			<td></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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			<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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			<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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			<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>
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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> Medan den explanterade ryggmärgen avbildning protokoll kan användas för att visualisera något fluorescens inom ryggmärgen, våra representativa resultat visar EGFP-NPC interaktioner med EYFP-axoner. Först visar vi den inbäddade ventrala ryggmärgen förberedelser i figur 1A. Därefter visar vi 2P mikroskop setup och nyckelkomponenter i figur 1B. Figur 2 visar eGFP och EYFP fluorescens i en enda z-stack inom den ventrala ryggmärgen. Förvärv av z-stackar konsekutiva kan ko...

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

En ny ex vivo förberedelse för avbildning musen ryggmärgen. Detta protokoll möjliggör två-foton-avbildning av levande cellulära interaktioner hela ryggmärgen.

Två-foton Imaging av Cellular Dynamics i ryggmärgen hos möss

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

In vivo avbildning av Mouse Spinal Cord Använda Två-foton mikroskopi

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 ...

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

Ett förfarande för att implantera en Spinal avdelningen för Längs<em&gt; In Vivo</em&gt; Avbildning av Mouse Spinal Cord

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 i Rat Cervical Spinal Cord

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

Samtidig Två-foton<em&gt; In Vivo</em&gt; Avbildning av synaptiska inmatningar och postsynaptiska mål i mus Retrosplenial Cortex

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 Serotonerga Fibrer i ryggmärgen hos möss med hjälp av CLARITY / CUBIC teknik

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 Neural aktivitet i den primära somatosensoriska Cortex med Thy1-GCaMP6s transgena möss

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

En In Vivo Duo-färg metod för Imaging vaskulär Dynamics efter Contusive ryggmärgsskada

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

Implantation av laminektomi och Ryggmärgs fönster i musen

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 Två-foton avbildning av kortikala nervceller hos neonatala möss

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

Intra-arteriell leverans av neurala stamceller till råtta och mus hjärnan: Ansökan till cerebral ischemi

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