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、乾燥紙タオルの上にチャンバー内にマウスを置き、チャンバーが覆われていることを確認してください。マウスを安楽死されることを保証するために呼吸停止後、少なくとも1分待ってください。 </li><li>マウスを安楽死されていることを確認するために、首の脊髄離断を実行します。これは脊髄を損傷する可能性がある頸椎脱臼を実行しないでください。 </li><li>毛髪を湿らせ、70％エタノールでマウススプレー。 </li><li> 1（C1約頚椎椎弓から脊柱を露出するために、マウスの背面から髪や皮膚を除去するために、鋭いファインはさみを使用してください））仙骨ラミナ4（S4へ。 </li><li> ＃10ブレードを用いてメスを用いて、筋肉および脂肪からそれを分離するために、脊柱の左右に切開を行う。これは、脊髄の除去がより簡単になります。 </li><li>オプション：湾曲したルアー骨鉗子は、追加の肉を離れてすくうために使用することができます。 </li><li>鋸歯状グレーフェ鉗子と脊椎骨の上部を押しながら、脊髄に触れないように注意しながら、C1で露出脊柱に湾曲した面を上にしてチタン湾曲Vannasはさみを挿入します。 </li><li>すべての道を右にVannasはさみ湾曲したチタンをスライドさせ、一つの小さなカットを作る。小さなクランチ音が聞こえとハサミは脊椎骨を切るように感じられるはずです。コー​​ドの左端にあるこのカットを繰り返します。これはハサミでコードを傷つけ危険にさらすようにあまりにも速く行くか、カットの大きすぎ行うことがないように注意してください。 </li><li>シングルラミナは、脊椎の側がカットされたら、ピンセットで持ち上げてすることができるはずです。 R保持し、脊髄脊椎骨を引き戻すために続けて、S4まで、各ラミナのために、この手順をEPEAT。 注：それは移植部位に削減されると、移植部位上記の脊椎骨はおそらく外れます。ただ移植部位の下に開始する手順を続行します。 </li><li>オプション：移植部位への吻側および尾側20ミリメートル〜移植部位がどこにある見て、測定し、カットする上下の椎骨を築く。 注：より一般的には15ミリメートルよりも遠くに移動しない移植したNPCとして必要されることはありませんのNPCを移植する。必要な脊髄の量は、移植された細胞型の実験および移動特性に依存する。移植部位に等距離で吻側および尾側のカットを作ることは撮像中に、より容易に位置するように移植部位を可能にします。 </li><li> ＃11ブレードとメスを用いて慎重に右の神経節をカットし、吻端から始まる脊髄の腹側の左。このW病気より簡単に腹側の椎骨から削除する脊髄を可能にする。 注：迅速に行う（1.7から1.11ステップ）、脊髄が完全に乾くことはありません。しかし、1×PBSで湿ったそれを維持するコードに投与することができる。 </li><li>マウスを反転ので、脊髄を下に向けているマウスをホールドアップ。慎重に閉じられた鋸歯状グレーフェ鉗子を使用して脊髄をはがします。脊髄をニッキングすることなく、慎重に脊髄が単一の完全な部分で持ち上げることができるように、残りの神経節をカット。 </li><li>脊髄はRPMI-1640であることを確認しますと2P顕微鏡への輸送のために氷の上に置きます。 </li></ol>イメージングのための脊髄の調製<ol><li>アガロースで脊髄を埋め込む<ol><li>イメージングの前に氷上で冷却RPMI-1640中で孤立した脊髄を保管してください。 </li><li>アガロース（低ゲル化温度）を秤量し、1×PBS、5mlの5％溶液を調製する。 agに溶解させる15秒間の5％アガロース溶液をマイクロ波起きて、37℃に冷却ソリューションを聞かせて。 </li><li>腹側を上に向けてパラフィルムのシート上に配置することによって脊髄を準備します。 </li><li>ピペット脊髄の腹側の上の5％アガロース溶液の約5 mlである。アガロースは室温に冷却することにより固化するましょう。完全な凝固は約5分かかります。 </li><li> 22ミリメートルの正方形カバースリップに組織接着剤の薄手のコートを適用します。 </li><li>パラフィルム上の腹側を配置、埋め込み脊髄を反転。背側は今まで直面するであろう。背側にカバースリップを接着し、接着剤を固化するRMPIアガロース埋め込み脊髄/カバースリップの準備を沈める。過剰にカミソリの刃を用いてアガロースを固化し削除します。 </li></ol></li><li>顕微鏡ステージ上の脊髄のマウント<ol><li>カバースリップの底にワセリンを適用します。これは、灌流中に準備を安定します。 </li><li>脊髄調製物を置く腹側は25X浸漬目標に向かって上向きにして顕微鏡ステージでよく撮影する。特注のイメージングウェルが約20ミリメートル、50ミリメートル、深長く、そして全体で50ミリメートルである。 </li><li>画像、温めた（37℃）で酸素準備をsuperfusingながら、血清を含まないRPMI-1640培地（95：二酸化炭素、カルボゲン：5酸素）。 <ol><li>水浴中で37℃に前加温メディアおよびメディアボトルにチューブを挿入することにより、チューブポンプに接続する。 </li><li>チャンバーへのチューブの接続部で37℃、加熱装置における媒体温度を保持します。ウェルで3 ml /分を通してSuperfuse媒体はチューブポンプ（ 図1B）に接続されたチューブを使用。 </li></ol></li></ol></li></ol>腹側脊髄の3 2Pイメージング<ol><li>顕微鏡のセットアップ<ol><li> 900nmのチューニングサファイアレーザー：カメレオンウルトラチタンを使用してはThy1-のeYFP脊髄から画像を取得する。レーザパワーを減衰させる&lt;5％の試料は、最小限の毒性16を確保する。安定した画像形成を確実にするために、組織ドリフト及び細胞の損傷を防止するために、単一のインライン液ヒーターを用いて一定の37℃での酸素灌流RPMI-1640を灌流することによって温度を維持する。 </li><li>のeGFPとのeYFP蛍光信号を分離するために、意図的に視認性を高めるために緑色発光を分割する3つのチャネルに2P発光を分離するために直列に520 nmの単一エッジダイクロと560nmの単一エッジダイクロイックビームスプリッタを配置する。 注：これらのチャネルは、青緑色（発光&lt;520 nm）を、（520〜560 nm）の黄緑色、および赤色（発光&gt; 560nmの）と呼ばれる。光電子増倍管は、各チャネルに放出された光を検出する。 </li></ol></li><li>関心の撮像領域の検索<ol><li>接眼レンズと明視野光源を用いて、基準点を設定するために、脊髄の腹側縁に浸漬対物レンズの焦点を合わせる。 </li><li>必ず周囲の光源がオフとSWであることを確認してくださいレーザシャッタを開くことによって2P励起かゆみ。 </li><li>利用可能な場合は関心のある分野のための組織を検索する際に、より低い解像度の設定、デジタルズームのない高いボリューム、および最終的な画像を取得するときよりも高いスキャンレートを使用しています。関心領域のための組織を検索このシステムの典型的な設定は次のとおりです。解像度：256ピクセル。ボリューム：X = 600ミクロン、Y =600μmで、Z = 0〜300程度である。 </li><li>脊髄の腹側縁の近くのEYFP軸索を観察します。コラーゲン（青）からの第2高調波信号は、脊髄組織のエッジで最も明るくなります。ちょうどコラーゲンからの第2高調波信号に背とのeYFP信号が黄緑色のチャンネルで可視化されたEYFP軸索の位置を確認します。 </li><li>脊髄準備14の長手方向の中央に移植部位の位置を確認します。のeGFP-NPC移植後のマウスの1日の場合は、深部組織へのz平面内のビーム経路を中心に移植部位を見つけます。移植サイトのWiLL動物間で若干異なるが、一般的には腹側の縁から〜200μmの位置しています。 1日目移植後後のマウスにおける腹側と横方向縁部に近い、白質路におけるEGFP-のNPCのクラスターを観察します。 </li></ol></li><li>最終的な画像を取得する<ol><li> 512ピクセルの画像の解像度を取得。ボリューム：X = 270ミクロン、Y = 212ミクロン、及びSlidebook 6ソフトウェアを使用して、Z =100μmである。 2.5ミクロン単位でシーケンシャル焦点面を取得することで、Zスタックをコンパイルします。 </li><li>脊髄内の任意の移行オブジェクトの迅速な定量化を確実にするためにオフセット適切なインターレースとの双方向スキャンを実行します。脊髄内の細胞の速度を決定するのに理想的な取得フレームレートは約1フレーム/秒であることを確認してください。 注：イメージング設定は、取得フレームレート、撮像分解能と撮像ボリュームとして個々のユーザの好みに変更することができる。より大きな画像ボリュームは、一般的にGIVで取得するために時間がかかります解像度EN。 </li><li>そのようなビットプレーンIMARIS 7.7などの画像解析ソフトウェアを使用して、分析する作物、滑らか、かつ疑似Slidebook画像ファイル。 SlidebookとIMARISで自動化されたプロセスを使用して、連続した画像化ボリュームをコーミングすることにより、最終的なタイムラプスビデオを制作。 </li></ol></li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+the+immune+system.">Two-photon imaging of the immune system.</a> Curr Protoc Cytom. Chapter 12 (Unit12 26), (2012).</li><li>Matheu, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+4-activated+B+cells+out-compete+Toll-like+receptor+9-activated+B+cells+to+establish+peripheral+immunological+tolerance.">Toll-like receptor 4-activated B cells out-compete Toll-like receptor 9-activated B cells to establish peripheral immunological tolerance.</a> Proc Natl Acad Sci U S A. 109 (20), E1258-E1266 (2012).</li><li>Matheu, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+phases+of+CD8+T+cell+response+in+the+lung+following+H1N1+influenza+infection+and+sphingosine+1+phosphate+agonist+therapy.">Three phases of CD8 T cell response in the lung following H1N1 influenza infection and sphingosine 1 phosphate agonist therapy.</a> PLoS One. 8 (3), e58033 (2013).</li></ol>

The authors have no competing interests.

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。

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			<td height="21" style="height:21px;width:381px;">Name of Reagent/ Equipment</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>
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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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			<td height="21" style="height:21px;width:381px;">scalpel blade #10</td>
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			<td style="width:136px;">10010-00</td>
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			<td height="21" style="height:21px;width:381px;">scalpel handle</td>
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			<td height="21" style="height:21px;width:381px;">Vannas scissors</td>
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			<td style="width:136px;">15615-08</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>
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			<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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			<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>
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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>
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			<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>
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			<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>
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			<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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			<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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			<td height="21" style="height:21px;width:381px;">photomultiplier tubes</td>
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			<td height="21" style="height:21px;width:381px;">C/L variable-speed tubing pump</td>
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			<td height="21" style="height:21px;width:381px;">digital thermometer</td>
			<td style="width:175px;">Comar Instruments</td>
			<td style="width:136px;">3501</td>
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			<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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			<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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	</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 ...

two-photon-imaging-of-cellular-dynamics-in-the-mouse-spinal-cord

Research

JoVE Journal

Neuroscience

10.1K ビュー.  University of California, Irvine. 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。 

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

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

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 significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

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

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

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 ...

神経科学

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

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

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

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

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

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 diagnostic and prognostic tool in cases of disease or injury. Diffusion weighted imaging (DWI), is sensitive to the thermal motion of water molecules and allows for inferences of tissue microstructure. This report describes a protocol to acquire and analyze DWI of the rat cervical spinal cord on a small-bore animal system. It demonstrates an imaging setup for the live anesthetized animal and recommends a DWI acquisition protocol for high-quality imaging, which includes stabilization of the cord and control of respiratory motion. Measurements with diffusion weighting along different directions and magnitudes (b-values) are used. Finally, several mathematical models of the resulting signal are used to derive maps of the diffusion processes within the spinal cord tissue that provide insight into the normal cord and can be used to monitor injury or disease processes noninvasively.

The goal of this protocol is to obtain high-quality diffusion weighted magnetic resonance imaging (DWI) of the rat spinal cord for noninvasive characterization of tissue microstructure. This protocol describes optimizations of the MRI sequence, radiofrequency coil, and analysis methods to enable DWI images free from artifacts.

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 ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

イメージングカルシウム<sup&gt; 2+</supマウス網膜の光受容コーン軸索末端で&gt;ダイナミクス

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse 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 microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

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

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

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

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

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

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

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

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

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

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues throughout the acute injury phase. In fact, endothelial cells appear to be the first to die after a contusive SCI. The early vascular events, including increased permeability of the blood-spinal cord barrier (BSCB), induce vasogenic edema and contribute to detrimental secondary injury events caused by complex injury mechanisms. Targeting the vascular disruption, therefore, could be a key strategy to reduce secondary injury cascades that contribute to histological and functional impairments after SCI. Previous studies were mostly performed on postmortem samples and were unable to capture the dynamic changes of the vascular network. In this study, we have developed an in vivo duo-color two-photon imaging method to monitor acute vascular dynamic changes following contusive SCI. This approach allows detecting blood flow, vessel diameter, and other vascular pathologies at various sites of the same rat pre- and post-injury. Overall, this method provides an excellent venue for investigating vascular dynamics.

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

We introduce an in vivo imaging method using two different fluorescent dyes to track dynamic spinal vascular changes following a contusive spinal cord injury in adult Sprague-Dawley rats.

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

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

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

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 methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

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

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

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 ...