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.).

注：倫理声明：動物の取り扱いのためのプロトコルは、カリフォルニア、アーバイン、プロトコル＃2010から2943の大学の施設内動物管理使用委員会（IACUC）によって承認された。 脊髄の1の除去<ol><li>チャンバーを安楽死で約100％の液体イソフルラン、USPで湿らせた紙タオルを置き、上に乾燥紙タオルを置きます。マウスはイソフルランに触れていないので、乾燥紙タオ?...

<ol>
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無傷組織のリアルタイム2Pイメージングは​​脱髄マウスの脊髄に移植後NPC動態との相互作用を調査する必要がある。生体2Pイメージングは、一般的に生きているマウスにおける脊髄の背側の携帯動力学を決定するために使用され、疾患17-19脱髄に背脱髄を研究するために使用されてきた。移植されたNPCは、2P顕微鏡を使用してその場で画像の深すぎる位置する腹側白質への移行しかし...

neuroadaptedマウス肝炎ウイルス（MHV）による実験的自己免疫性脳脊髄炎（EAE）、および頭蓋内感染を含む脱髄のマウスモデルは、疾患に関連した分子経路と細胞の相互作用を研究するための優れたツールである。彼らはにつながったとFDAの有効性は、主に自己免疫および炎症1の停止をターゲットに、医薬品の治療法を承認しサポートしてきました。内因性の再ミエリン化が失敗したしかし、一度、現在承認されている治療法は効果的に中枢神経系の脱髄病変を修復しないでください。したがって、疾患のこの段階で修復に焦点を当てた治療は、慢性症状および生活の質の向上の軽減のために重要である。最近では、神経前駆細胞（NPCの）は、炎症および脱髄の領域をターゲットとする潜在的な再生治療法として最前線になってきた。いくつかの研究がendogenoを誘導するのNPCの能力を強調している私たちは、再ミエリン化及び再ミエリン2-8に直接参加。 NPCは、直接再ミエリン化に関与しているので、移植後の内因性の細胞とそれらの速度および相互作用を理解することが不可欠である。移植後、NPCはその後吻側および尾側移植部位5,9に比べて、白質損傷の領域に腹移行する。移行の動力学は、環境信号に応答して異なる。非損傷した脊髄に移植されたNPCは、損傷した脊髄6に移植したNPCよりも大きな速度を持っている。遊走期間の後、無傷の脊髄6に損傷した脊髄相対的に高いレートで、NPCは広範囲に増殖移した。最後に、NPCはの大半はオリゴデンドロサイトに分化し、直接再ミエリン化の4,6,9を開始する。 脱髄病変は複雑であり、種々の段階での細胞の多様な集団を含むことができctivati​​on。例えば、活性多発性硬化症（MS）、病変は、活性化T細胞、M1ミクログリア及びM1マクロファージの有意な集団を含んでもよいが、慢性の無音MS病変は、いくつかの炎症性細胞が10-13との反応性星状細胞から主に構成されてもよい。そのためエフェクター細胞の多様性、脱髄のマウスモデルにおける二光子（2P）イメージングは​​、病変内のローカルな細胞間相互作用を理解するための非常に便利なツールです。 MSと多くの広く使用MS研究モデルでは、病変の大部分が起因する病変の深さおよび脊髄の高い脂質含量に脊髄、生体内の2Pのイメージングにアクセスできない領域の腹側に配置されている。腹側脊髄に沿って病変内のこれらの問題及び試験細胞-細胞相互作用を回避するために、我々は、ex vivoでの2P撮影準備6簡単に開発した。 この研究は示した以前の方法パブリケーションを、フォローアップMHV誘発性脱髄14のJHMV株後のマウスの脊髄に強化緑色蛍光タンパク質（EGFP）発現のNPCを移植するための手順。ファイブ週齢のマウスはJHMVに感染させ、14日目、感染後に胸部レベル9でのeGFP-のNPCを移植している。ここで紹介するプロトコルは、ex vivoでアガロース準備をし、脊髄を抽出​​し、強化黄色蛍光タンパク質（EYFP）発現性軸索の画像、移植のeGFP-NPCの相互作用する方法についての詳細な手順を提供しています。神経細胞特異的はThy1プロモーター下のeYFPを発現するマウスは、この手順15で使用した。唯一の軸索のいくつかは、個々の軸索を画像化するためには有用なもの、のeYFPを発現する。ここでは、7日後に移植に除去脊髄を表示。しかし、脊髄移植後の任意の時点で抽出することができる。我々は、損傷軸索でのNPCの相互作用を示しているが、我々のプロトコルは、遺伝子の蛍光マーカーと組み合わせて使用​​することができるマウス脊髄全体に生じる細胞相互作用の多くを調査するために、他の種類の細胞。

<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>
		</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>
		</tr>
		<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>
			<td></td>
		</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>外植脊髄イメージングプロトコルは、脊髄内の任意の蛍光を可視化するために使用することができるが、我々の代表的な結果は、軸索のeYFPでのeGFP-NPCの相互作用を実証する。まず、 図1Aに埋め込 ​​まれた腹側脊髄の調製を示す。次に、2P顕微鏡のセットアップおよび図1Bにおける主要構成要素を示す。 図2は、腹側脊髄内の単一zスタックにおけるEG...

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

マウス脊髄を撮像するための新規のex vivo調製。このプロトコルは、脊髄全体生細胞の相互作用の二光子イメージングを可能にする。

マウス脊髄における細胞ダイナミクスの二光子イメージング

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

二光子顕微鏡を用いたマウス脊髄の in vivoイメージングで

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

縦断のために脊髄商工を移植するための手順<em&gt;インビボ</emマウス脊髄の&gt;イメージング

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

ラット頸部脊髄の拡散イメージング

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

同時2光子<em&gt;インビボ</emマウス脳梁膨大後部皮質におけるシナプス入力とシナプス後ターゲットの&gt;イメージング

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

CLARITY / CUBIC技術を用いたマウスの脊髄にセロトニン繊維のイメージング

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

Thy1を用いた一次体性感覚野における神経活動のイメージング-GCaMP6s トランスジェニック マウス

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デュオ カラー霊長類脊髄損傷血管動態のイメージング手法

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

マウスにおける線込みおよび脊髄窓移植

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

生体内で新生仔マウスの大脳皮質ニューロンの 2 光子イメージング

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

ラットおよびマウス脳への神経幹細胞の動脈内デリバリー:脳虚血への応用

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