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.).

NOT: Etik Açıklama: hayvan taşıma için protokol California, Irvine, protokol # 2010-2943 Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Komitesi (IACUC) tarafından onaylandı. Omurilik 1. Kaldırma <ol><li> Bölmesine euthanizing içinde ~% 100 sıvı izofluran, USP ile ıslatılmış kağıt havlu yerleştirin ve üstüne kuru kağıt havlu yerleştirin. Fare izofluran dokunmadan değil çok kuru kağıt havlu üstüne odasında fare yerleştirin ve oda kapalı olduğundan emin o...

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Gerçek zamanlı sağlam doku 2P görüntüleme demiyelinize fare omuriliğe nakli sonrası NPC kinetik ve etkileşimleri araştırmak için gereklidir. Intravital 2P görüntüleme yaygın yaşayan farelerde omurilik dorsal tarafında hücresel dinamikleri belirlemek için kullanılır, ve hastalık 17-19 demiyelinizan dorsal demiyelinizasyonu incelemek için kullanılır olmuştur. Transplant NPC 2P mikroskopi kullanılarak yerinde görüntüye çok derin yatıyor ventral beyaz cevher, göç Ancak, bir ...

Deneysel otoimmün ensefalomyelit (EAE) ve neuroadapted fare hepatit virüsü (Geminin) ile intrakranial enfeksiyon dahil demiyelinizasyon Fare modelleri, moleküler yollar ve hastalıkla ilişkili hücresel etkileşimleri çalışmak için mükemmel araçlardır. Onlar yol ve FDA etkinliği ağırlıklı olarak otoimmünite ve inflamasyon 1 durması hedefleyen, ilaç tedavileri kabul desteklemiştir. Endojen remiyelinizasyon başarısız sonra, ancak, şu anda onaylanan terapiler, etkili bir şekilde, merkezi sinir sisteminde miyelin kaybetmiş lezyonlar, onarım yapmaz. Bu nedenle, hastalığın bu aşamada onarım odaklı tedaviler kronik semptomlar ve yaşam kalitesinin iyileştirilmesi giderilmesi için kritik öneme sahiptir. Son zamanlarda, nöral öncü hücreler (NPC) inflamasyon ve demiyelinizasyon alanları hedef potansiyel rejeneratif tedavi yöntemi olarak ön plana çıkmıştır. Çeşitli çalışmalar, endogeno indükleme NPC yeteneğini ortaya çıkarmıştırBize remiyelinizasyon ve remiyelinizasyon 2-8 doğrudan katılmak. NPC doğrudan remiyelinizasyon dahil Çünkü, bu nakli sonrası endojen hücreler ile kinetik ve etkileşimleri anlamak şarttır. Transplantasyon sonrası, NPC sonra rostrally ve nakli sitesine 5,9 kaudal göreli beyaz cevher hasarı alanları ile ventral göç. göç kinetik çevresel uyaranlara yanıt olarak farklılık; Olmayan bir hasar omuriliğe nakledilen NPC hasarlı omurilik 6 nakledilen NPC daha büyük hızlara sahip. Bir göç süre sonra, bir sağlıklı omurilik 6 bozuk bir omurilik nispetle daha yüksek bir hızda, NPC yaygın olarak çoğalırlar transfer edilir. Son olarak, NPC çoğunluğu oligodentrositlere farklılaştırmak ve doğrudan remiyelinizasyon 4,6,9 başlatmak. miyelin kaybetmiş lezyon karmaşıktır ve çeşitli aşamalarında bir hücre, farklı popülasyon ihtiva edebilirctivation. Örneğin, bir aktif multipl skleroz (MS), lezyon aktive edilmiş T hücreleri, M1, mikroglia ve M1 makrofajlar, ancak kronik sessiz MS lezyon temel olarak birkaç enflamatuar hücreler 10-13 ile reaktif astrositler içerebilir önemli bir nüfus içerebilir. Çünkü efektör hücrelerin çeşitliliği, demiyelinizasyon fare modellerinde iki-foton (2P) görüntüleme lezyon içinde yerel hücresel etkileşimleri anlamak için son derece yararlı bir araçtır. MS ve birçok yaygın olarak kullanılan MS araştırma modellerinde, lezyonların çoğunluğu nedeniyle lezyon derinliği ve omurilik yüksek yağ içeriği omurilik, intravital 2P görüntüleme erişilemez bir bölge ventral tarafında yer almaktadır. Ventral spinal kord boyunca lezyonlarında bu konuları ve çalışma hücre-hücre etkileşimleri aşmak için biz ex vivo 2P görüntüleme hazırlık 6 basit geliştirdik. Bu çalışma gösterdi Önceki yöntemler yayın, takipMHV kaynaklı demiyelinasyon 14 JHMV suşu ardından fare omuriliğe gelişmiş yeşil floresan protein (EGFP) -expressing NPC nakli için prosedür. Beş haftalık fareler JHMV ile enfekte ve gün 14 sonrası enfeksiyon torakal seviyede 9&#39;da eGFP-NPC ile nakledilen. Burada sunulan protokol bir ex vivo agaroz hazırlık yapmak, omuriliği ayıklamak ve gelişmiş sarı floresan protein (EYFP) -expressing akson görüntü nakledilen eGFP-NPC etkileşimleri konusunda ayrıntılı adımları sağlar. Nöronal spesifik Thy1 promotörün kontrolü altında EYFP sentezleyen Fareler Bu prosedür 15 kullanılmıştır. Sadece akson bazı bireysel aksonlar görüntüleme için yararlı hale EYFP ifade eder. Burada 7 gün transplantasyon sonrası çıkarılmış omurilikleri göstermektedir; Bununla birlikte, omurilik transplantasyonundan sonra herhangi bir zaman noktasında elde edilebilir. Zarar görmüş aksonlar NPC etkileşimlerini gösterirken, protokol genetik floresan işaretler ile kombinasyon halinde de kullanılabilirdiğer hücre türleri fare omurilik oluşan hücresel etkileşimler çok sayıda araştırmak.

<table border="0" cellpadding="0" cellspacing="0" width="858">
	<tbody>
		<tr height="21">
			<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>
		</tr>
		<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>
			<td></td>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
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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>
		</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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		</tr>
	</tbody>
</table>

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> Çıkarılan omurilik görüntüleme protokolü omurilik içinde herhangi bir floresan görselleştirmek için kullanılabilir iken, bizim temsilcisi sonuçları EYFP-akson ile eGFP-NPC etkileşimleri göstermek. Öncelikle, biz Şekil 1A gömülü ventral spinal kord hazırlanmasını göstermektedir. Sonra, Şekil 1B 2P mikroskop kurulum ve anahtar bileşenleri göstermektedir. 2 ventral spinal kord içinde bir tek z-yığın içinde eGFP ve EYFP floresan göste...

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

Fare omuriliği görüntülenmesi için yeni bir ex vivo hazırlık. Bu protokol, omurilik boyunca canlı hücresel etkileşimlerin iki foton görüntüleme için izin verir.

Fare Omurilik Hücresel Dynamics İki foton Görüntüleme

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

Fare Omurilik İki foton Mikroskop kullanarak in vivo Görüntüleme

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

Boyuna için bir Spinal Odası implantasyon için bir Prosedürü<em&gt; İn Vivo</em&gt; Fare Omurilik Görüntüleme

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

Sıçan Servikal Spinal Kord difüzyon Görüntüleme

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

Eşzamanlı İki foton<em&gt; İn Vivo</em&gt; Fare Retrosplenial kortekste sinaptik Girişlerin Görüntüleme ve postsinaptik Hedefler

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

Kullanımı Netlik Fare Omurilik içinde Serotonerjik Elyaf Görüntüleme / KÜBİK Tekniği

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

Sinirsel aktivite Thy1kullanarak birincil somatosensor kortekste Imaging-GCaMP6s transgenik fareler

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

İçinde bir Vivo Vasküler Dynamics Contusive Medulla Spinalis Yaralanmalarında takip görüntüleme için duo-renk yöntemi

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

Farede Laminektomi ve Omurilik Penceresi İmplantasyonu

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

Vivo Yenidoğan farelerde kortikal nöronların iki fotonlu görüntüleme

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

Nöral Kök Hücrelerin Sıçan ve Fare Beynine İntra-Arteriyel Teslimatı: Serebral İskemiye Uygulama

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